Diagnostic and prognostic marker of an acute pulmonary exacerbation

Related Application Data

This application claims Convention Priority from Australian Patent Application No. 2006903239 filed on June 9, 2006, the content of which is incorporated by reference.

Field of the invention

The present invention relates to a method for the diagnosis or prognosis of an inflammatory condition of the lung, a bacterial infection of the lung, a viral infection of the lung, a respiratory infection, a respiratory disease, or a lung disease in a subject. For example, the present invention relates to a method for. determining whether or not a subject suffering from cystic fibrosis has an exacerbated condition e.g., as a consequence of lung infection and/or inflammation, or alternatively, has responded to treatment for an exacerbated condition.

Background of the invention

General Information

This specification contains nucleotide and amino acid sequence information prepared using Patentln Version 3.3, presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>l, <210>2, <210>3, etc). The length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term "SEQ ID NO:", followed by the sequence identifier (e.g. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as <400>l).

The designation of nucleotide residues referred to herein are those recommended by the IUP AC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

Unless specifically stated otherwise, each feature described herein with regard to a specific embodiment of the invention, shall be taken to apply mutatis mutandis to each and every other embodiment of the invention. The features of each and every embodiment of the invention described herein for diagnosis and/or prognosis of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration are to be applied mutatis mutandis to diagnosis and/or prognosis of an acute clinical exacerbation in a subject suffering from cystic fibrosis.

The features of each and every embodiment of the invention described herein for diagnosis and/or prognosis of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration are to be applied mutatis mutandis to monitoring the efficacy of treatment of a subject suffering from cystic fibrosis and suffering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration.

The features of each and every embodiment described herein for the detection of a neutrophil derived protein or a modified form thereof or an isoform of α-1 antitrypsin or a modified form thereof or mixtures thereof shall be taken to apply mutatis mutandis to the detection of an antibody against a neutrophil derived protein or a modified form thereof or an antibody against an isoform of α-1 antitrypsin or a modified form thereof or mixtures of such antibodies.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specific examples described herein. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

All the references cited in this application are specifically incorporated by reference herein.

The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated by reference:

1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of VoIs I, II, and III;

2. DNA Cloning: A Practical Approach, VoIs. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;

3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl- 22; Atkinson et ah, pp35-81; Sproat et ah, pp 83-115; and Wu et ah, pp 135- 151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text;

5. Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text;

6. Perbal, B., A Practical Guide to Molecular Cloning (1984); 7. Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series;

8. J.F. Ramalho Ortigao, "The Chemistry of Peptide Synthesis" In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany);

9. Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R.L. (1976). Biochem. Biophys. Res. Commun. 73 336-342 lO.Merrifield, R.B. (1963). J. Am. Chem. Soc. 85, 2149-2154. l l.Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and

Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York. 12.Wunsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Mϋler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart.

13.Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag,

Heidelberg. 14.Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis,

Springer-Verlag, Heidelberg. 15. Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.

16. Handbook of Experimental Immunology, VoIs. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications).

2. Description of the related art Cystic fibrosis (CF) is one of the most common fatal autosomal recessive disease affecting Caucasian populations. CF has an incidence in neonates of about 0.05%, indicating a carrier frequency of about 5% of the population. Biological parents of subjects with CF are, by definition, obligatory carriers. Carriers are clinically normal and their detection prior to the birth of an affected child has been precluded by the absence of detectable effects of the gene in single dose.

Methods for detecting CF include DNA sequencing, enzyme immunoassay (Sanguiolo et al, Int. J. Clin. Lab. Res., 25, 142-145, 1995), multiplex DGGE analysis (Costes et al, Hum. Mut. 2, 185-191, 1993), and the use of the polymerase chain reaction (PCR) in conjunction with allele-specifϊc oligonucleotide probes (PCR-ASO). US Patent Application No. 20030008281 (Weston et al.) describes a two tube multiplex ARMS assay for simultaneously detecting 12 of the most prevalent CF mutations in humans, specifically for detecting the CFTR gene mutations 1717-1GGA, G542X, W1282X, N1303K, δF508, 3849+lOkb CDT, 621+1 GDT, R553X, G551D, R117H, R1162X and R334W (Kazazian et al, Hum Mut. 4, 167-177, 1994), as well as the test distinguishing between CF δF508 heterozygotes and homozygotes. The principle of the ARMS test is

that the 3'-end of an ARMS amplification primer confers allele-specificity, and an ARMS product is only generated if the primer is complementary to its target at the 3'- end under the appropriate conditions.

CF is a disease of the exocrine glands, affecting most characteristically the pancreas, respiratory system, and sweat glands. The disease usually begins during infancy and the prognosis for an affected child with CF is a median life expectancy currently estimated to be 30 years.

CF is typified by chronic respiratory infection, pancreatic insufficiency, and susceptibility to heat prostration. It is a major cause of death in children. It is estimated that there are between ten million and twelve million carriers for cystic fibrosis in the United States. Each year, between two thousand and three thousand children are born in the United States who are affected by cystic fibrosis. The cost of therapy for cystic fibrosis patients exceeds US$20,000 per year per patient. Of patients diagnosed in early childhood, fewer than fifty percent reach adulthood.

A serious consequence of CF is an exacerbated clinical condition or exacerbated state. As used herein the term "acute clinical exacerbation", "acute exacerbation", "clinical exacerbation", "exacerbation", or "exacerbated state" in the context of a CF patient shall be understood to mean an exaggeration of a pulmonary symptom of CF.

In most cases, such a clinical exacerbation will be a consequence of a respiratory infection, or increased inflammation. The term "respiratory infection" in this context includes invasion by and/or multiplication and/or colonization of a pathogenic microorganism in one or more components of the respiratory tract, such as, for example, lung, epiglottis, trachea, bronchi, bronchioles, or alveoli. Commonly, such infections result in the inflammation of the respiratory tract.

CF patients are particularly susceptible to respiratory infections from organisms such as, for example, Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenzae, Aspergillus fumigatus, Burkholderia cepacia complex, Stenotrophomonas maltophila, Alcaligenes (Achromobacter) xylosoxidans, B. gladioli, Ralstonia picketti Influenza A virus and Respiratory Syncytial Virus. The most common causes of respiratory infection are the bacteria S. aureus, P. aeruginosa, and H. influenzae.

For example a chronic respiratory infection, particularly an infection of the lung by P. aeruginosa, accounts for almost 90% of the morbidity and mortality in CF. By age 12, about 60-90% of CF patients are infected with P. aeruginosa.

Progressive loss of pulmonary function over many years due to chronic infection with mucoid P. aeruginosa is common in subjects suffering from CF. Smith et al, Cell. 85, 229-236, 1996, reported defective bacterial killing by fluid obtained from airway epithelial cell cultures of CF patients, and suggested that this phenomenon was due to the inhibition of an unidentified antimicrobial factor resulting from increased levels of sodium chloride in the airway epithelial fluid.

Severe chronic pulmonary disease is also associated with cases of CF wherein CFTR expression on the cell surface is reduced, such as, for example, in patients carrying the δF508 mutation. Pier et al. Science. 271, 64-67, 1996 proposed that ingestion and clearance of P. aeruginosa by epithelial cells may protect the lungs against infection, since the specific ingestion and clearance of P. aeruginosa was compromised in a cell line derived from a patient with the δF508 mutation.

US Patent No. 6, 245,735 to Brigham and Womens Hospital disclosed the binding of P. aeruginosa to CFTR via the core portion of the lipopolysaccharide of P. aeruginosa. Also disclosed was a method for upregulating the CFTR in epithelial mucosa to thereby enhance clearance of P. aeruginosa. Such a method comprises contacting mucosal cells expressing the CFTR with the core portion of the lipopolysaccharide of P. aeruginosa.

Patients suffering from CF are extremely susceptible to acute clinical exacerbations, often resulting in a further increase in inflammation and mucus production, thus increasing the risk of bronchiectasis and eventually respiratory failure.

An acute clinical exacerbation is generally assessed using the protocols described in Williams et al Australian Journal of Physiotherapy, 47 _^ 227 — 236, 2001; Dakin et al, Pediatr Pulmonol 34, 436-442, 2001; and/or Rosenfeld et al, J.Pediatr 139 359-365, 2001. In particular, several criteria are assessed, and a patient satisfying four or more of these criteria is considered to have an acute clinical exacerbation. These criteria are as follows: i. Change in sputum production (volume, color, consistency);

ii. New or increased haemoptysis; iii. Increased cough; iv. Increased dyspnoea (shortness of breath); v. Malaise, fatigue or lethargy; vi. Decreased exercise tolerance; vii. Fever; viii. Anorexia or weight loss; ix. Sinus pain/tenderness or change in sinus discharge; x. FVC or FEVi decreased 10% from previous recorded value; xi. Radiographic changes indicative of a pulmonary infection; and xii. Changes in chest sounds.

Alternatively, an acute clinical exacerbation is also diagnosed using by detecting the concentration of C-reactive protein, determining erythrocyte sedimentation rate and/or peripheral neutrophil counts as reviewed in Hϋner et al, Med Bull Istanbul, 32(1), 1999.

Furthermore, several methods have been suggested for the detection of complications of CF, in particular bacterial infection, more specifically P. aeruginosa infection. Such methods include the monitoring of levels of IgG specific to core lipopolysaccharide (US Patent No. 5,179,001), IgG specific to P. aeruginosa (Brett et al, J. Clin. Pathol. 39(10) 1124-1129, 1986), and IgA specific to P. aeruginosa (Brett et al, J. Clin. Pathol. 41(10) 1130-1134, 1988). However, these assays require a significant response by the patient's immune system to a P. aeruginosa infection in order to detect the specific immunoglobulin molecules. Accordingly, such methods are only useful in the monitoring, or prognosis of an established P. aeruginosa infection, rather than being suitable for early detection of infection. Nor do these methods allow for detection of an inflammation-associated exacerbation, rather than an infection-related exacerbation.

Furthermore, other pathogens, such as Staphylococcus aureus and non-typable Haemophilus influenzae, are also commonly isolated from the respiratory tract of CF patients, and are not detected by P. aeruginosa specific assays.

Whilst there has been significant progress in diagnosing CF, the need still exists for further diagnostic and prognostic assays for complications arising in patients suffering from the disease, in particular rapid and reliable methods for determining whether or not a subject suffering from CF is entering an exacerbated condition or state, e.g.,

respiratory infection. Sensitive assays for accurately predicting whether or not a CF subject is developing or suffering from an exacerbated state are highly desirable, as are reliable prognostic indicator for determining whether or not such a subject is responding to treatment for the exacerbated condition.

Summary of invention

In work leading up to the present invention, the inventors identified a defined a class of immune response proteins that are modified or upregulated in cystic fibrosis subjects suffering from an acute clinical exacerbation, i.e., a condition characterized by an inflammatory response and/or a respiratory tract infection and/or pulmonary deterioration. The levels of these proteins were subsequently reduced in CF subjects following successful treatment with antibacterial agents against P. aeruginosa, H. influenzae and/or S. aureus and/or antiinflammatory agents, there being little or no detectable expression in non-CF control subjects. These proteins are: an isoform of α-1 antitrypsin, an isoform of leukocyte elastase inhibitor, an isoform of α-enolase, an isoform of Rho GDP-dissociation inhibitor, an isoform of annexin I, an isoform of annexin III, an isoform of calgranulin C and an isoform of catalase. The present inventors also identified α-1 antitrypsin as being upregulated and/or modified in cystic fibrosis subjects suffering from an acute clinical exacerbation.

The present inventors have also shown that a protein selected from the group consisting of an isoform of α-1 antitrypsin, an isoform of leukocyte elastase inhibitor, an isoform of α-enolase, an isoform of Rho GDP-dissociation inhibitor 2, an isoform of annexin I, an isoform of annexin III, an isoform of calgranulin C and an isoform of catalase has altered mobility or is present at an enhanced level in CF subjects suffering from an acute clinical exacerbation.

The present inventors have also detected an enhanced level of a modified form of α-1 antitrypsin and several neutrophil derived proteins in a sample from a subject suffering from an acute clinical exacerbation. For example, the inventors have shown that annexin I is modified in CF subjects suffering from an acute clinical exacerbation. When CF is exacerbated, such as for example by infection of the epithelial mucosa of the lung, the MW of annexin I is reduced by about 8 kDa, with an associated increase in pi of about 1.2 units, as determined by 2-D gel electrophoresis relative to the mobility of a reference protein. These changes are consistent with cleavage and inactivation of annexin I by a neutrophil elastase enzyme.

The present inventors have also detected an enhanced level of an isoform of α-1 antitrypsin in samples from subjects suffering from an acute clinical exacerbation. The modified form of α-1 antitrypsin has a reduced molecular weight and charge compared to naturally occurring α-1 antitrypsin. These changes are consistent with cleavage and inactivation of αl -antitrypsin by, for example, Pseudomonas aeruginosa elastase enzyme. The present inventors have further shown that CF subjects successfully treated with an antibacterial compound and/or an anti-inflammatory compound following acute clinical exacerbation produce reduced levels of the modified form of αl -antitrypsin. Conversely, in CF patients that do not respond to anti-bacterial treatment and/or anti-inflammatory treatment, the modified αl -antitrypsin remains detectable in patient samples.

Furthermore, the inventors have shown that the amount of a modified form of α-enolase detected correlates significantly with the exacerbated state of a CF subject (i.e. detection of an increased amount of α-enolase indicates that a subject is suffering from a clinical exacerbation).

These findings have provided the means for producing novel diagnostics for the detection of an acute clinical exacerbation in a CF subject, and novel prognostic indicators for the progression of the exacerbated state. Preferably, the markers referred to supra are useful for the early diagnosis of acute clinical exacerbation. It will also be apparent to the skilled person that such prognostic indicators as described herein may be used in conjunction with therapeutic treatments for CF or an acute clinical exacerbation associated therewith.

Accordingly, the present invention provides a method of diagnosing an inflammatory condition in a subject suffering from CF and/or an infection of the respiratory tract in a subject suffering from CF and/or pulmonary deterioration in a subject suffering from CF, said method comprising detecting in a sample from said subject an enhanced level of a neutrophil derived protein and/or α-1 antitrypsin and/or mixtures thereof.

As used herein, the term "inflammatory condition" shall be understood to mean a state of the respiratory tract that is characterized by one or more changes in the physical appearance or functions of a portion of the respiratory tract, such as, for example, dilation of arterioles, capillaries and venules with increased permeability and blood

flow, exudation of fluids (e.g. plasma proteins), leukocytic infiltration, swelling and/or loss of function. For example, an inflammatory condition is caused by injury to the respiratory tract or through infection.

In one example, the inflammatory condition is an acute inflammatory condition.

As used herein, the term "infection" shall be understood to mean invasion and/or- colonization by a microorganism and/or multiplication of a micro-organism, in particular, a bacterium or a virus, in the respiratory tract of a subject. Such an infection may be unapparent or result in local cellular injury. The infection may be localized, subclinical and temporary or alternatively may spread by extension to become an acute or chronic clinical infection. For example, the infection is a respiratory infection.

As used herein the term "respiratory tract" shall be taken to mean a system of cells and organs functioning in respiration. For example, organs, tissues and cells of the respiratory tract include, lungs, nose, nasal passage, paranasal sinuses, nasopharynx, larynx, trachea, bronchi, bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli, pneumocytes (type 1 and type 2), ciliated mucosal epithelium, mucosal epithelium, squamous epithelial cells, mast cells, goblet cells, and intraepithelial dendritic cells.

As used herein the term "pulmonary deterioration" shall be taken to mean that the pulmonary function of a subject is reduced compared to a normal and/or healthy subject. Pulmonary deterioration occurs over time and is usually caused by several acute respiratory tract infections and/or chronic respiratory tract infection and/or chronic respiratory tract inflammation. Symptoms of pulmonary deterioration include fatigue, increased cough, pleuritic chest, increased nasal congestion, fever, polyarthralgias, decreased FEVi, elevation in white blood cell counts and/or weight loss.

As used herein, the term "neutrophil derived protein" shall be understood to mean, any protein that is expressed by a neutrophil, whether that protein is expressed exclusively by a cell of the neutrophil lineage or is expressed in several cell types including a cell of the neutrophil lineage. A neutrophil or cell of the neutrophil lineage is a polymorphonuclear leukocyte that has a plurilobular nucleus in addition to both azurophilic granules and specific granules in the cytoplasm. Generally, a neutrophil or

cell of the neutrophil lineage responds to chemotactic _. signals and/or a proinflammatory cytokine released during an inflammatory response by moving to the site of an injury and/or infection and/or inflammation.

The present invention also provides a method of diagnosing an inflammatory condition in a subject, suffering from CF and/or infection of the respiratory tract in a subject suffering from CF and/or pulmonary deterioration in a subject suffering from CF, said method comprising detecting in a sample from said subject a modified isoform of a neutrophil derived protein and/or α-1 antitrypsin and/or mixtures thereof.

As used herein the term "modified form" shall be understood to mean a protein that differs to the native form of said protein in such a way that is readily detectable using methods known in the art, such as, for example those described herein. Modifications that are detectable by such methods include, for example, proteolytic cleavage, post- translational modification (e.g. phosphorylation or glycosylation), alternative splice forms, and any other modifications result in a change in amino acid composition, molecular weight, charge, and/or protein conformation.

The present invention also provides a method for determining the progression of an inflammatory condition in a subject suffering from CF, infection of the respiratory tract in a subject suffering from CF or pulmonary deterioration in a subject suffering from CF and being administered an amount of a therapeutic compound for the treatment of said inflammatory condition or infection, said method comprising detecting an enhanced level of a neutrophil derived protein and/or α-1 antitrypsin and/or mixtures thereof in a sample from said subject. In accordance with this embodiment, a level of the neutrophil derived protein and/or α-1 antitrypsin and/or mixture thereof that is less than a level of that protein detectable in a subject suffering from an acute clinical exacerbation indicates that the subject is recovering from an exacerbated state.

^■ The present invention also provides a method for determining the progression of an inflammatory condition in a subject suffering from CF and/or infection of the respiratory tract in a subject suffering from CF and/or pulmonary deterioration in a subject suffering from CF wherein said subject is being administered with an amount of a therapeutic compound for the treatment of said inflammatory condition and/or infection and/or pulmonary deterioration, said method comprising detecting a native or unmodified isoform of a neutrophil derived protein and/or α-1 antitrypsin and/or

mixtures thereof in a sample from said subject. In accordance with this embodiment, the detection of said native or unmodified form of the protein indicates that the subject is recovering from an acute exacerbated state. As will be apparent to the skilled artisan, a modified form of the protein may also be present in the sample, such as, for example, if the subject has not fully recovered from the exacerbation.

It is to be understood that the prognostic methods referred to herein are also useful for determining a subject that has not responded to treatment. Accordingly, the present invention additionally provides a method for determining the progression of an inflammatory condition in a subject suffering from CF and/or an infection of the respiratory tract in a subject suffering from CF and/or pulmonary deterioration in a subject suffering from CF wherein said subject is being admiistered a therapeutic compound for the treatment of said inflammatory condition and/or infection and/or pulmonary deterioration, said method comprising detecting an altered level of a neutrophil derived protein and/or α-1 antitrypsin and/or mixtures thereof in a sample from said subject. In accordance with this embodiment of the invention, a level of the protein that is enhanced compared to the level of that protein detectable in a normal or healthy subject indicates that the subject is not responding to said treatment.

The present invention also provides a method for determining determining the progression of an inflammatory condition in a subject suffering from CF and/or an infection of the respiratory tract in a subject suffering from CF and/or pulmonary deterioration in a subject suffering from CF wherein said subject is being admiistered a therapeutic compound for the treatment of said inflammatory condition and/or infection and/or pulmonary deterioration, said method comprising detecting a modified isoform form of a neutrophil derived protein and/or α-1 antitrypsin and/or mixtures thereof in a sample from said subject. In accordance with this aspect of the invention, the detection of a modified form of the protein indicates that the subject is not responding to treatment. .

In accordance with the various aspects of the invention described herein, it is particularly preferred that the neutrophil derived protein that is expressed at an enhanced level is selected from the group consisting of an isoform of leukocyte elastase inhibitor, an isoform of α-enolase, an isoform of Rho GDP-dissociation inhibitor, an isoform of annexin I, an isoform of annexin III, an isoform of calgranulin C and an isoform of catalase. For example, a neutrophil derived protein is an isoform of α-

enolase. Alternatively, a neutrophil derived protein is an isoform of Rho GDP- dissociation inhibitor 2. Alternatively, a neutrophil derived protein is an isoform of annexin I. Alternatively, a neutrophil derived protein is an isoform of Annexin III. Alternatively, a neutrophil derived protein is an isoform of calgranulin C. Alternatively, a neutrophil derived protein is an isoform of catalase. Alternatively, a neutrophil derived protein is an isoform of leukocyte elastase inhibitor.

In one example, a modified form of a neutrophil derived protein is selected from the group consisting of a modified form of annexin I, a modified form of annexin III, a modified form of leukocyte elastase inhibitor, a modified form of α-enolase and a modified form of Rho GDP dissociation inhibitor. For example, a modified neutrophil derived protein is a processed or cleaved annexin I protein. In accordance with this embodiment, the modified annexin I is a processed or cleaved protein, such as, for example, a protein lacking about 20 amino acids to 50 amino acids from the N-terminus of a native annexin I protein, e.g., lacking about 36 amino acids from the N-terminus of a native annexin I protein, such as, for example, a modified annexin I protein consisting essentially of the amino acid sequence set forth in SEQ ID NO: 8 and 11. In another example, a modified neutrophil derived protein is a processed or cleaved leukocyte elastase inhibitor protein. In a further example, a modified neutrophil derived protein is a processed or cleaved annexin III protein. In another example, a modified neutrophil derived protein is a processed or cleaved α-enolase protein. In a further example, a modified neutrophil derived protein is a processed or cleaved Rho GDP dissociation inhibitor protein.

In one example of the diagnostic/prognostic methods described herein, the sample is obtained previously from the subject. Accordingly, the present invention clearly encompasses providing the sample in accordance with such embodiments, the prognostic or diagnostic method is performed ex vivo.

In yet another example, the subject diagnostic/prognostic methods further comprise processing the sample from the subject to produce a derivative or extract that comprises the analyte (e.g., protein).

In another example, a method of the invention as described herein according to any embodiment additionally comprises detecting a modified form of myeloperoxidase and/or detecting an enhanced level of myeloperoxidase in a sample from a subject. In

this respect, detectionof a modified form of myeloperoxidase and/or an increased level of myeloperoxidase is indicative of an inflammatory condition in a subject suffering from CF and/or an infection of the respiratory tract in a subject suffering from CF and/or pulmonary deterioration in a subject suffering from CF, e.g., an acute clinical exacerbation in a subject suffering from CF, or the risk thereof or that a subject is not responding to treatment. A reduced level of myeloperoxidase and/or the detection of a native form of myeloperoxidase is indicative of a reduced risk of developing an inflammatory condition in a subject suffering from CF and/or an infection of the respiratory tract in a subject suffering from CF and/or pulmonary deterioration in a subject suffering from CF, e.g., an acute clinical exacerbation, or that a subject is responding to treatment.

The present invention also provides a method of treatment of a CF subject suffering from an inflammatory condition or infection of the respiratory tract comprising performing a diagnostic method or prognostic method as described herein and administering or recommending administration of a therapeutic composition based on that diagnosis.

The present invention further provides any synthetic or recombinant peptides derived from a neutrophil derived protein and/or α-1 antitrypsin referred to herein, or antibodies thereto, suitable for use or when used in the assays described herein according to any embodiment.

Antibodies or fragments thereof are useful in therapeutic, diagnostic and research applications, including the purification and study of the diagnostic/prognostic proteins, identification of cells expressing the neutrophil derived protein, or for sorting or counting of such cells. Thus, the present invention clearly encompasses the use of an antibody or fragment thereof described herein (e.g., monoclonal antibodies or an antigen-binding fragment thereof) in therapy, including prophylaxis, diagnosis, or prognosis, and the use of such antibodies or fragments for the manufacture of a medicament for use in treatment of an inflammatory condition or infection of the respiratory tract.

Also encompassed by the present invention are methods of identifying ligands of an isoform of a neutrophil derived protein and/or a modified form of a neutrophil derived protein and/or an isoform of α-1 antitrypsin and/or a modified form of α-1 antitrypsin.

Brief description of the figures

Figure 1 is a copy of a photographic representation of a two-dimensional gel separating proteins isolated from sputum derived from a CF patient with an acute clinical exacerbation (spot numbers refer to the proteins identified in Table 3).

Figure 2 shows copies of photographic representations of two-dimensional gels that have been used to separate proteins derived from sputum isolated from CF patients with an acute clinical exacerbation (Exacerbated CF) (i, iv, vii and x), CF patients that have recently received treatment for an acute clinical exacerbation (CF Discharge) (ii, v, viii and xi) and healthy individuals that do not suffer from CF (Control) (iii, vi, ix and xii). Note the presence of an isoform of leukocyte elastase inhibitor, an isoform of α- enolase, annexin III and Rho-GDP-dissociation inhibitor 2 in the protein isolated from CF patients with an acute exacerbation. These proteins are present at a reduced level in CF subjects that do not have an acute clinical exacerbation, and not present in healthy controls.

Figure 3A is a copy of photographic representation of an immunofingerprint of a sputum sample derived from a CF subject suffering from an acute clinical exacerbation. Immunoglobulin isolated from CF subjects was used to capture proteins from a normal, healthy subject, determine those proteins against which CF subjects develop autoantibodies. The spot labeled "lab" represents α-enolase, and the spot labeled "2abc" represents calgranulin C. Spots were detected with rabbit anti-human IgG labeled with horse-radish peroxidase, which was detected by chemiluminescence.

Figure 3 B is a copy of a photographic representation of a 2-dimensional gel that had been used to separate proteins isolated from sputum of a CF subject suffering from an acute clinical exacerbation. Protein was detected with Direct Blue. This gel was overlayed onto the immunofingerprint shown in Figure 3A and the common protein spots in gel digested and identified using MALDI-MS and LCQ. Spots representing calgranulin C and α-enolase are indicated.

Figure 4 shows photographic representations of two-dimensional gels used to separate proteins derived from sputum isolated from an adult CF subject with an acute clinical exacerbation (Exacerbated CF) (i), a CF patients that has received recent treatment for an exacerbated state (CF Discharge) (ii) and a healthy individual that does not suffer from CF (Control) (iii). Note the relative position of proteins representative of α-1 antitrypsin in relation to the protein spot representative of actin. The relative distance between α-1 antitrypsin and actin is reduced in CF patients suffering from an acute clinical exacerbation, when compared to the distance observed in a CF discharge subject or a healthy control. This change in relative position indicates a change in molecular weight and pi of α-1 antitrypsin in a CF patient suffering from an acute clinical exacerbation. Cleaved α-1 antitrypsin is highlighted in the box and uncleaved α- 1 antitrypsin is highlighted by the ellipse.

Figure 5 shows photographic representations of two-dimensional gels used to separate proteins derived from sputum isolated from a child CF subject with an acute clinical exacerbation (CF Child Exacerbated-like) (i), a CF subject (CF Child) (ii) and a healthy individual that does not suffer from CF (Healthy non-CF control) (iii). Note the relative position of proteins representative of α-1 antitrypsin in relation to the protein spot representative of actin. The relative distance between α-1 antitrypsin and actin is reduced in CF patients suffering from an acute clinical exacerbation, when compared to the distance observed in a CF discharge subject or a healthy control. This change in relative position indicates a change in molecular weight and pi of α- 1 antitrypsin in a CF patient suffering from an acute clinical exacerbation. Cleaved α-1 antitrypsin is highlighted in the box and uncleaved α-1 antitrypsin is highlighted by the ellipse.

Figure 6 A is a photographic representation showing cleaved and uncleaved α-1 antitrypsin as detected by two-dimensional gel electrophoresis. Left hand image shows a sample from a healthy adult. Right hand image shows a sample from a healthy child. Uncleaved α-1 antitrypsin is highlighted by the upper ellipse and cleaved alpha- 1 antitrypsin is highlighted by the lower ellipse.

Figure 6B is a graphical representation showing the relative sum of normalized spot volumes for non-cleaved isoforms of α-1 antitrypsin (light bars) and cleaved forms of α-1 antitrypsin (dark bars) as detected in healthy control adults (n=15).

Figure 6C is a graphical representation showing the relative sum of normalized spot volumes for non-cleaved isoforms of α-1 antitrypsin (light bars) and cleaved forms of α-1 antitrypsin (dark bars) as detected in healthy control children (n=3).

, Figure 7 A is a photographic representation showing cleaved and uncleaved α-1 antitrypsin as detected by two dimensional gel electrophoresis. Left hand image shows a sample from a CF adult suffering from an acute clinical exacerbation. Right hand image shows a sample from a CF child with an exacerbation-like pathology. Uncleaved α-1 antitrypsin is highlighted by the upper ellipse and cleaved α-1 antitrypsin is highlighted by the lower ellipse.

Figure 7B is a graphical representation showing the relative sum of normalized spot volumes for non-cleaved isoforms of α-1 antitrypsin (light bars) and cleaved forms of α-1 antitrypsin (dark bars) as detected in healthy control adults (n=48).

Figure 7C is a graphical representation showing the relative sum of normalized spot volumes for non-cleaved isoforms of α-1 antitrypsin (light bars) and cleaved forms of α-1 antitrypsin (dark bars) as detected in healthy control children (n=5). Of the 5 children, 3 are healthy like and 2 are exacerbated like.

Figure 8 is a graphical representation showing the correlation of relative spot volume of cleaved α-1 antitrypsin and disease state. Healthy control adults and healthy control children have very similar cleaved α-1 -antitrypsin values, but adults with CF have significantly higher levels relative to the children with CF.

Figure 9 is a photographic representation showing a two-dimensional gel upon which protein that was isolated using an immunocapture column comprising circulating antibodies isolated from a CF subject has been separated.

Figure 10 is a graphical representation showing results of a two-site ELISA to detect levels of myeloperoxidase in CF subjects suffering from an acute clinical exacerbation (closed bars), CF adult subjects treated for an acute clinical exacerbation (closed bars) and control adult subjects (open bars). Also shown are results attained for CF children (vertical striped) and control children (horizontal stripes). Results are expressed in optical density. Subject IDs correspond to those numbers in Table 1.

Detailed description of the preferred embodiments

An enhanced level of protein markers for an acute clinical exacerbation in a CF subject

As used herein the term "an isoform of leukocyte elastase inhibitor" shall be taken to mean any peptide, polypeptide, or protein comprising an amino acid sequence at least about 80% identical to the amino acid sequence of a leukocyte elastase inhibitor set forth in SEQ ID NO: 1. The term "leukocyte elastase inhibitor" shall also be taken to include a peptide, polypeptide or protein having the known biochemical properties of leukocyte elastase inhibitor. As used herein the term "known biological properties" shall be understood to mean any physico-chemical properties by which a particular peptide, polypeptide, or protein may be characterized, such as, for example molecular weight, post-translational modifications, amino acid composition, or isoelectric point, amongst others.

As used herein the term "isoform" is to be taken in its broadest context to include a native protein or any single form of a protein wherein there are several forms of a protein that differ in some way while maintaining the same function. Protein isoforms may differ at the level of primary sequence, due to alternative splicing, may be a proteolytically cleaved form of a full length or native protein or may be a post- translationally modified form of a protein, such as, for example, a glycoprotein, or a lipidated protein.

For example, the percentage identity to SEQ ID NO: 1 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.

In one example, leukocyte elastase inhibitor is a human leukocyte elastase inhibitor.

For example, an isoform of leukocyte elastase inhibitor has a molecular weight from about 15 kDa to about 21 kDa, more preferably, from about 17 kDa to about 20 kDa and even more preferably from about 18 kDa to about 19 kDa.

In another example, an isoform of leukocyte .elastase inhibitor has a molecular weight from about 39 kDa to about 46 kDa, more preferably from about 40 kDa to about 45 kDa, and most preferably from about 41 kDa to about 44 kDa.

In a further example, an isoform of leukocyte elastase inhibitor has an isoelectric point of about 5.5 to about 7, more preferably about 6 to about 6.75, and most preferably about 6.25 to about 6.

In another example, an isoform of leukocyte elastase inhibitor has a molecular weight of about 18 kDa and/or an isoelectric point of about 6.5. Alternatively, or in addition, an isoform of leukocyte elastase inhibitor has a molecular weight of about 19 kDa and/or an isoelectric point of about 6.6. Alternatively, or in addition, an isoform of leukocyte elastase inhibitor has a molecular weight of about 41 kDa and/or an isoelectric point of about 6.25. Alternatively, or in addition, an isoform of leukocyte elastase inhibitor has a molecular weight of about 44 kDa and/or an isoelectric point of about 6.5.

As used herein the term "an isoform of α-enolase" shall be taken to mean any peptide, polypeptide, or protein comprising an amino acid sequence at least about 80% identical to the amino acid sequence of a α-enolase set forth in SEQ ID NO: 2. The term "α- enolase" shall also be taken to include a peptide, polypeptide or protein having the known biochemical properties of α-enolase.

For example, the percentage identity to SEQ ID NO: 2 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.

For example, α-enolase is a human α-enolase.

In one example, an isoform of α-enolase has a molecular weight of about 20 kDa to about 45 kDa, more preferably from about 25 kDa to about 40 kDa and most preferably from about 28 kDa to about 48 kDa.

In another example, an isoform of α-enolase has a molecular weight from about 10 kDa to about 20 kDa, more preferably from about 12 kDa to about 18 kDa and most preferably about 15 kDa.

In another example, an isoform of α-enolase has an isoelectric point from about 6 to about 7, more preferably from about 6.25 to about 6.8 and most preferably from about

6.45 to about 6.75. Alternatively, or in addition, an isoform of α-enolase has an

isoelectric point from about 7.5 to about 8.5, more preferably from about 7.75 to about 8.25 and most preferably about 8.

For example, an isoform of α-enolase has a molecular weight of about 38 kDa and/or an isoelectric point of about 6.75. Alternatively, or in addition, an isoform of α-enolase has a molecular weight of about 38 kDa and an isoelectric point of about 6.45.

Alternatively, or in addition, an isoform of α-enolase has a molecular weight of about

28 kDa and/or isoelectric point of about 6.45. Alternatvely, or in addition, an isoform of α-enolase has a molecular weight of about 15 kDa and/or an isoelectric point of about 8.0.

As used herein the term "an isoform of Rho GDP-dissociation inhibitor 2" shall be taken to mean any peptide, polypeptide, or protein comprising an amino acid sequence at least about 80% identical to the amino acid sequence of a Rho GDP-dissociation inhibitor 2 set forth in SEQ ID NO: 3 The term "Rho GDP-dissociation inhibitor 2" shall also be taken to include a peptide, polypeptide or protein having the known biochemical properties of Rho GDP-dissociation inhibitor.

For example, the percentage identity to SEQ ID NO: 3 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.

^• In one example, the Rho GDP-dissociation inhibitor 2 is human Rho GDP-dissociation inhibitor.

In one example, an isoform of Rho GDP dissociation inhibitor 2 has a molecular weight from about 10 kDa to about 25 kDa, more preferably from about 12 kDa to about 22 kDa, and most preferably from about 16 kDa to about 19 kDa.

In another example, an isoform of Rho GDP dissociation inhibitor 2 has an isoelectric point from about 5.5 to about 8.0, more preferably from about 6 to about 7.5, and most preferably from about 6.25 to about 7.2.

For example, an isoform of Rho GDP dissociation inhibitor 2 has a molecular weight of about 19 kDa and/or an isoelectric point of about 6.25. Alternatively, or in addition, an

isoform of Rho GDP dissociation inhibitor 2 has a molecular weight of about 16 kDa and/or an isoelectric point of about 7.7.

As used herein the term an "isoform of annexin III" shall be taken to mean any peptide, polypeptide, or protein comprising an amino acid sequence at least about 80% identical to the amino acid sequence of a an isoform of annexin III set forth in SEQ ID NO: 4. The term "an isoform of annexin III" shall also be taken to include a peptide, polypeptide or protein having the known biochemical properties of annexin III.

For example, the percentage identity to SEQ ID ^" NO: 4 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.

In one example, an isoform of annexin III is a human isoform of annexin III.

In one example, an isoform of annexin III has a molecular weight from about 20 kDa to about 30 kDa, more preferably 22 kDa to about 27 kDa, and most preferably of about 24 kDa.

In another example, an isoform of annexin III has an isoelectric point from about 5 to about 8, more preferably from about 5.5 to about 6.5, and most preferably of about 5.8.

For example, an isoform of annexin III has a molecular weight of about 24 kDa and/or an isoelectric point of about 5.8.

As used herein the term an "isoform of calgranulin C" shall be taken to mean any peptide, polypeptide, or protein comprising an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ED NO: 5. The term "calgranulin C" shall also be taken to include a peptide, polypeptide or protein having the known biochemical properties of calgranulin C.

For example, the percentage identity to SEQ ID NO: 5 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.

In one example, calgranulin C is human calgranulin C.

As used herein the term an "isoform of annexin I" shall be taken to mean any peptide, polypeptide, or protein comprising an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 6. The term "annexin I" shall also be taken to include a peptide, polypeptide or protein having the known biochemical properties of annexin I.

For example, the percentage identity to SEQ ID NO: 6 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.

In one example, annexin I is human annexin I.

For example, an isoform of annexin I has a molecular weight from about 25 kDa to about 35 kDa, and most preferably of about 30 kDa.

In another example, an isoform of annexin I has an isoelectric point from about 8 to about 9.5, more preferably from about 8.5 to about 9 and most preferably of about 8.7.

In one example, an isoform of annexin I has a molecular weight of about 30 and/or an isoelectric point of about 8.7.

As used herein the term an "isoform of catalase" shall be taken to mean any peptide, polypeptide, or protein comprising an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 7. The term "catalase" shall also be taken to include a peptide, polypeptide or protein having the known biochemical properties of catalase.

For example, the percentage identity to SEQ ID NO: 7 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.

In one example, catalase is human catalase.

As used herein the term an "isoform of .αl -antitrypsin" shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity

to the amino acid sequence of a αl -antitrypsin set forth in SEQ ED NO: 11. The term "αl -antitrypsin" shall also be taken to include a peptide, polypeptide or protein having the known biochemical properties of αl -antitrypsin. As used herein the term "known biological properties" shall be understood to mean any physico-chemical properties by which a particular peptide, polypeptide, or protein may be characterized, such as, for example molecular weight, post-translational modifications, amino acid composition, or isoelectric point, amongst others.

For example, the percentage identity to SEQ ID NO: 11 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.

In one example, αl -antitrypsin is human αl -antitrypsin.

In determining whether or not two amino acid sequences fall within the defined percentage identity limits supra, those skilled in the art will be aware that it is possible to conduct a side-by-side comparison of the amino acid sequences. In such comparisons or alignments, differences will arise in the positioning of non-identical residues depending upon the algorithm used to perform the alignment. In the present context, references to percentage identities and similarities between two or more amino acid sequences shall be taken to refer to the number of identical and similar residues respectively, between said sequences as determined using any standard algorithm known to those skilled in the art. In particular, amino acid identities and similarities are calculated using software of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America, e.g., using the GAP program of Devereaux et al, Nucl. Acids Res. 12, 387-395, 1984, which utilizes the algorithm of Needleman and Wunsch, J MoI. Biol. 48, 443-453, 1970. Alternatively, the CLUSTAL W algorithm of Thompson et al, Nucl. Acids Res. 22, 4673-4680, 1994, is used to obtain an alignment of multiple sequences, wherein it is necessary or desirable to maximize the number of identical/similar residues and to minimize the number and/or length of sequence gaps in the alignment. Amino acid sequence alignments can also be performed using a variety of other commercially available sequence analysis programs, such as, for example, the BLAST program available at NCBI.

For example, the proteins described hereinabove are used to diagnose or prognose a clinical exacerbation. Preferably, a clinical exacerbation is an acute clinical

exacerbation, wherein an acute exacerbation has a rapid onset (i.e. after initial commencement) and a short but severe course.

As used herein the term "enhanced level of a protein" shall be understood to mean that the amount of the protein detected in a sample derived from a CF subject developing an exacerbated state, or suffering from an acute exacerbation is increased compared to a control sample, e.g., a sample derived from a normal or healthy individual, or a CF subject following successful treatment for an exacerbated state, or a CF subject when they were not suffering from or developing an acute exacerbation.

Assay formats for quantitation of proteins

The amount of a specific protein or. an isoform of a protein or a modified form of a protein in a sample may be determined using any of a variety of techniques known to the skilled artisan such as, for example, a technique selected from the group consisting of, an immunoblot, a Western blot, a dot blot, an enzyme linked immunosorbent assay (ELISA), a fluorescence-linked immunoassay (FLISA), a radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonance energy transfer (FRET), isotope-coded affinity tags (ICAT), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, e.g. LC MS/MS), biosensor technology, evanescent fiber-optics technology or protein chip technology.

For example, the assay used to determine the amount or level of a protein or an isoform of a protein in a sample is a semi-quantitative assay. ^{• '•} •

In another example, the assay used to determine the amount or level of a protein or an isoform of a protein in a sample is a quantitative assay.

As will be apparent to the skilled artisan, such a semiquantitative or quantitative assay may require the use of a reference sample. Suitable reference samples are described herein.

Standard solid-phase ELISA formats or FLISA formats are particularly useful in determining the concentration of a protein from a variety of patient samples.

In one form, such an ELISA or FLISA assay involves immobilizing a sample onto a solid matrix, such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide).

An antibody that specifically binds a neutrophil derived protein or an isoform of α-1 antitrypsin is contacted with the immobilized sample, and forms a direct bond with any of its target protein present in said sample. This antibody is generally labeled with a detectable reporter molecule, such as, for example, a fluorescent label (e.g. FITC or Texas Red), a fluorescent semiconductor nanocrystal (as described in US 6,306,610) in the case of a FLISA or an enzyme (e.g. horseradish peroxidase (HRP)), alkaline phosphatase (AP) or β-galactosidase in the case of an ELISA, or alternatively a second labeled antibody can be used that binds to the first antibody. Following washing to remove any unbound antibody, the label is detected either directly, in the case of a . fluorescent label, or through the addition of a suitable substrate, such as for example hydrogen peroxide, TMB, toluidine, or 5-bromo-4-chloro-3-indol-beta-D- galaotopyranoside (x-gal). Suitable substrates will depend upon the reporter molecule used, and will be apparent to the skilled artisan.

Such FLISA and/or ELISA based systems are particularly suitable for quantification of the amount of a protein in a sample, by calibrating the detection system against known amounts of a protein standard to which the antibody binds, such as for example, an isolated recombinant neutrophil derived protein and/or an isoform of α-1 antitrypsin , or suitable fragment thereof (e.g., a fragment that is capable of binding the antibody used in the FLISA and/or ELISA assay).

In another form, an ELISA or FLISA comprises immobilizing an antibody that specifically binds to a neutrophil derived protein and/or an isoform of α-1 antitrypsin on a solid matrix, such as, for example, a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick or a glass support. A sample is then brought into physical relation with said antibody, and said neutrophil derived protein and/or isoform of α-1 antitrypsin is bound or 'captured'. The bound protein is then detected using a labeled antibody. For example, a neutrophil derived protein and/or an isoform of α-1 antitrypsin captured from a human sample is detected using an anti- neutrophil derived protein antibody and/or an anti-human α-1 antitrypsin antibody that binds to a distinct epitope to the capture antibody. Alternatively, or in addition a third labeled antibody can be used that binds the second (detecting) antibody.

It will be apparent to the skilled person that the assay formats described herein are amenable to high throughput formats, such as, for example automation of screening processes, or a microarray format as described in Mendoza et al, Biotechniques 27(4): 778-788, 1999. Furthermore, variations of the above described assay will be apparent to those skilled in the art, such as, for example, a competitive ELISA.

Alternatively, the presence of an enhanced level of a neutrophil derived protein or modified form thereof and/or an enhanced level of an isoform of α-1 antitrypsin or a modified form thereof is detected using a radioimmunoassay (RIA). The basic principle of this assay is the use of a radiolabeled antibody or antigen to detect antibody-antigen interactions. An antibody that specifically binds to a neutrophil derived protein or an isoform of α-1 antitrypsin is bound to a solid support and a sample brought into direct contact with said antibody. To detect the amount of bound antigen, an isolated and/or recombinant form of the antigen that is radiolabeled is brought into contact with the same antibody. Following washing the amount of bound radioactivity is detected. As any antigen in the sample inhibits binding of the radiolabeled antigen, the amount of radioactivity detected is inversely proportional to the amount of antigen in the sample. Such an assay is quantitated by using a standard curve using increasing, known concentrations of the isolated antigen.

As will be apparent to the skilled artisan, such an assay may be modified to use any reporter molecule, such as, for example, an enzyme or a fluorescent molecule e.g., as described herein, in place of a radioactive label.

Western blotting is also useful for detecting an enhanced level of a protein. In such an assay protein from a sample is separated using sodium doedecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using techniques known in the art and described, for example, in Scopes (Irv, Protein Purification: Principles and Practice, Third Edition, Springer Verlag, 1994). Separated proteins are then transferred to a solid support, such as, for example, a membrane, for example, a PVDF membrane, using methods known in the art, for example, electrotransfer. This membrane is then blocked and probed with a labeled antibody or ligand that specifically binds a neutrophil derived protein and/or an isoform of α-1 antitrypsin. Alternatively, or in addition, a labeled secondary, or even tertiary, antibody or ligand is used to detect the binding of a specific primary antibody.

To determine the amount of a neutrophil derived protein and/or an isoform of α-1 antitrypsin and/or mixtures thereof in a sample, the amount of said protein detected is determined using methods known in the art, such as, for example, densitometry. Following detection of the amount of said protein (i.e. intensity of a strained band or spot), the intensity of a protein band or spot may be normalized against the total amount of protein loaded on a SDS-PAGE gel using methods known in the art. Alternatively, an amount of neutrophil derived protein and/or an isoform of α-1 antitrypsin detected is normalized against the amount of a control/reference protein. The expression of such a control protein should not be affected by the clinical state of a subject from whom a sample is isolated. Such control proteins are known in the art, and include, actin, glyceraldehyde 3 -phosphate dehydrogenase (GAPDH), β2 microglobulin, hydroxy-methylbilane synthase, hypoxanthine phosphoribosyl- transferase 1 (HPRT), ribosomal protein Ll 3c, succinate dehydrogenase complex subunit A and TATA box binding protein (TBP). Antibodies to these proteins are commercially available. Sources of these antibodies will be apparent to the skilled artisan. Following normalization, a relative amount of a neutrophil derived protein or mixtures thereof and/or an isoform of α-1 antitrypsin compared to the control/reference protein is determined. This relative quantity of a protein may be compared to the relative amount of the same protein detected in a reference sample.

The detection of any protein using a method, such as, for example, mass spectrometry, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), protein chip, biosensor technology, evanescent fiber optics, isotope- coded affinity tags (ICAT) or fluorescence resonance energy transfer, is contemplated in the present invention.

High-throughput methods for detecting the presence or absence of a protein are particularly preferred.

In one embodiment, MALDI-TOF is used for the rapid identification of a protein that has been separated by either one- or two-dimensional gel electrophoresis. Accordingly, there is no need to detect the proteins of interest using an antibody or ligand that specifically binds to the protein of interest. Rather, proteins from a sample are separated using gel electrophoresis using methods known in the art and those proteins

at approximately the correct molecular weight and/or isoelectric point are analyzed using MALDI-TOF to determine the presence or absence of a protein of interest.

Alternatively, MALDI or ESI or a combination of approaches is used to determine the concentration of a particular protein in a sample, such as, for example sputum. Such proteins are preferably well characterized previously with regard to parameters such as molecular weight and isoelectric point.

Biosensor devices generally employ an electrode surface in combination with current or impedance measuring elements to be integrated into a device in combination with the assay substrate (such as that described in U.S. Patent No. 5,567,301). An antibody or ligand that specifically binds to a protein of interest is preferably incorporated onto the surface of a biosensor device and a sample isolated from a subject (for example sputum from a CF subject that has been solubilized using the methods described herein) contacted to said device. A change in the detected current or impedance by the biosensor device indicates protein binding to said antibody or ligand. Some forms of biosensors known in the art also rely on surface plasmon resonance to detect protein interactions, whereby a change in the surface plasmon resonance surface of reflection is indicative of a protein binding to a ligand or antibody (see for example, U.S. Patent Nos 5,485,277 and 5,492,840),

Biosensors are of use in high throughput analysis due to the ease of adapting such systems to micro- or nano-scales. Furthermore, such systems are conveniently adapted to incorporate several detection reagents, allowing for multiplexing of diagnostic reagents in a single biosensor unit. This permits the simultaneous detection of several proteins or peptides in a small amount of body fluids.

Evanescent biosensors are also preferred as they do not require the pretreatment of a sample prior to detection of a protein of interest. An evanescent biosensor generally relies upon light of a predetermined wavelength interacting with a fluorescent molecule, such as for example, a fluorescent antibody attached near the probe's surface, to emit fluorescence at a different wavelength upon binding of the diagnostic protein to the antibody or ligand.

Micro- or nano-cantilever biosensors are also preferred as they do not require the use of a detectable label. A cantilever biosensor utilizes a ligand or antibody capable of

specifically detecting the analyte of interest that is bound to the surface of a deflectable arm of a micro- or nano-cantilever. Upon binding of the analyte of interest (e.g. a neutrophil derived protein and/or an isoform of α-1 antitrypsin) the deflectable arm of the cantilever is deflected in a vertical direction (i.e. upwards or downwards). The change in the deflection of the deflectable arm is then detected by any of a variety of methods, such as, for example, atomic force microscopy, a change in oscillation of the deflectable arm or a change in pizoresistivity. Exemplary micro-cantilever sensors are described in USSN 20030010097.

Alternatively, a biosensor that utilizes a lipid membrane is used. Such a biosensor uses a lipid membrane that incorporates a lipid bilayer that comprises an ion channel or ionophore, wherein the lipid bilayer is tethered to a metal electrode (such biosensors are described in AU 623,747, US 5,234,566 and USSN 20030143726). One form of such a biosensor involves two receptors or antibodies that bind to each other being incorporated into a lipid bilayer. One of these receptors/antibodies is bound to an ion channel or ionophore that spans the outer half of the membrane, and this membrane/antibody is also capable of binding to the analyte of interest. The second receptor/antibody is tethered to a membrane molecule (i.e. not the ionophore or ion channel). When the receptors/antibodies are not bound to each other, the ion channel aligns with another half membrane spanning ionophore (i.e. an ionophore that spans the inner half of the membrane) thereby facilitating detectable ion transmission across the membrane. However, when the two receptors/antibodies bind each other, the outer membrane ionophore is displaced thereby disrupting membrane conductivity. The analyte of interest competes with the second receptor/antibody for the binding site on the first receptor/antibody. The presence of the analyte breaks the bond between the two receptors/antibodies and allows the half membrane ionophores to align and provide an ion conductive path.

To produce protein chips, the proteins, peptides, polypeptides, antibodies or ligands that are able to bind specific antibodies or proteins of interest are bound to a solid support, such as, for example glass, polycarbonate, polytetrafluoroethylene, polystyrene, silicon oxide, metal or silicon nitride. This immobilization is either direct

(e.g. by covalent linkage, such as, for example, Schiff s base formation, disulfide linkage, or amide or urea bond formation) or indirect. Methods of generating a protein chip are known in the art and are described in for example U.S. Patent Application No.

20020136821, 20020192654, 20020102617 and U.S. Patent No. 6,391,625. To bind a

protein to a solid support it is often necessary to treat the solid support so as to create chemically reactive groups on the surface, such as, for example, with an. aldehyde- containing silane reagent. Alternatively, an antibody or ligand may be captured on a microfabricated polyacrylamide gel pad and accelerated 'into the gel using microelectrophoresis as described in, Arenkov et al. Anal. Biochem. 275:123-131, 2000.

A protein chip is preferably generated such that several proteins, ligands or antibodies are arrayed on said chip. This format permits the simultaneous screening for the presence of several proteins in a sample.

Alternatively, a protein chip comprises only one protein, ligand or antibody, and is used to screen one or more patient samples for the presence of one polypeptide of interest. Such a chip may also be used to simultaneously screen an array of patient samples for a polypeptide of interest.

For example, a protein sample to be analyzed using, a protein chip is attached to a reporter molecule, such as, for example, a fluorescent molecule, a radioactive molecule, an enzyme, or an antibody that is detectable using methods known in the art. Accordingly, by contacting a protein chip with a labeled sample and subsequent washing to remove any unbound proteins the presence of a bound protein is detected using methods known in the art, such as, for example using a DNA microarray reader.

Alternatively, biomolecular interaction analysis-mass spectrometry (BIA-MS) is used to rapidly detect and characterize a protein present in complex samples at the low- to sub-fmole level (Nelson et al. Electrophoresis 21: 1155- 1163, 2000). One technique useful in the analysis of a protein chip is surface enhanced laser desorption/ionization- time of flight-mass spectrometry (SELDI-TOF-MS) technology to characterize a protein bound to the protein chip. Alternatively, the protein chip is analyzed using ESI as described in U.S. Patent Application 20020139751.

As will be apparent to the skilled artisan, protein chips are particularly amenable to multiplexing of detection reagents. Accordingly, several antibodies or ligands each able to specifically bind a different peptide or protein may be bound to different regions of said protein chip. Analysis of a sample using said chip then permits the detecting of

multiple proteins of interest. Multiplexing of diagnostic and prognostic markers is particularly contemplated in the present invention.

In another example, a sample is analyzed using ICAT, essentially as described' in US Patent Application No. 20020076739. This system relies upon labeling of a protein sample from one source (i.e. a healthy individual) with a reagent and labeling of a protein sample from another source (i.e. a CF subject) with a second reagent that is chemically identical to the first reagent, but differs in mass due to isotope composition. It is preferable that the first and second reagents also comprise, for example, a biotin molecule. Equal concentrations of the two samples are then mixed, and peptides recovered by avidin affinity chromatography. Samples are then analyzed using mass spectrometry. Any difference in peak heights between the heavy and light peptide ions directly correlates with a difference in protein abundance in a sample. The identity of such proteins may then be determined using a method known in the art, such as, for example MALDI-TOF, or ESI.

In another example, an enhanced level of a protein or a modified form of a protein in a sample is detected using two-dimensional gel electrophoresis. In accordance with this embodiment, particulate matter may be removed from the sample prior to electrophoresis, such as, for example, by centrifugation,' filtering, or a combination of centrifugation and filtering. Proteins in the sample are then separated. For example, the proteins may be separated according to their charge using isoelectric focusing an/or according to their molecular weight. Two-dimensional separations allow various isoforms of proteins to be identified, as proteins with similar molecular weight are also separated by their charge. Using image analysis software the presence of a protein of interest may be determined. Furthermore, using mass spectrometry the identity of a protein on the gel may be determined.

As will be apparent from the preceding discussion, it is preferable that a detection system that is antibody or ligand based is used in the method of the present invention. Immunoassay formats are even more preferred.

Any antibody or ligand for use (or when used) in such an assay is encompassed by the instant invention. Methods for the production of such an antibody or ligand are known in the art and described herein.

Antibodies

As used herein the term "antibody" refers to intact monoclonal or polyclonal antibodies, immunoglobulin (IgA, IgD, IgG, IgM and/or IgE) fractions, humanized antibodies, or recombinant single chain antibodies, as well as fragments thereof, such as, for example Fab, F(ab)2, and Fv fragments.

Antibodies referred to herein are obtained from a commercial source, or alternatively, produced by conventional means. Commercial sources will be known to those skilled in the art.

For example, antibodies that bind to α-enolase are described by Lamprecht et al, Clin. Exp. Rheumatol. 16, 513-537, 1998. A rabbit polyclonal antibody against annexin I is available from Zymed Laboratories Inc, South San Francisco, CA, 94080. Polyclonal antibodies against annexin- III are available from Genex Bioscience Inc., Hayward, Ca 94545, USA. A polyclonal antibody against calgranulin C is available from Santa Cruz Biotechnology Inc. Santa Cruz, Ca 95060, USA. A monoclonal antibody against α-1 antitrypsin (human alpha- IAT) designated HYB 191-03-1 is commercially available from BioCore Pty Ltd, Alexandria, New South Wales 2015, Australia.

High titer antibodies are preferred, as these are more useful commercially in kits for diagnostic or therapeutic applications. By "high titer" is meant a titer of at least about l :10 ³ or l:10 ⁴ or 1:10 ⁵ . Methods of determining the titer of an antibody will be apparent to the skilled artisan. For example, the titer of an antibody in purified antiserum may be determined using an ELISA assay to determine the amount of IgG in a sample. Typically an anti-IgG antibody or Protein G is used to bind the IgG. The amount detected in a sample is compared to a control sample of a known amount of purified and/or recombinant IgG to determine the actual amount of IgG. Alternatively, a kit for determining antibody is used, e.g. the Easy TITER kit from Pierce (Rockford,. IL, USA).

Antibodies are preferably prepared by any of a variety of techniques known to those of ordinary skill in the art, and described, for example in, Harlow and Lane (In: Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In one such technique, an immunogen comprising the antigenic polypeptide (e.g. a neutrophil derived protein and/or an isoform of α-1 antitrypsin or an immunogenic fragment thereof) is initially injected into any one of a wide variety of animals (e.g., a mouse, a

rat, a rabbit, a sheep, a dog, a pig, a chicken or a goat). The immunogen is derived from a natural source, produced by recombinant expression means, or artificially generated, such as by chemical synthesis (e.g., BOC chemistry or FMOC chemistry). In this step, the polypeptides or fragments thereof of this invention, may serve as the immunogen without modification. Alternatively, a peptide, polypeptide or protein is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen and optionally a carrier for the protein is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and blood collected from said the animals periodically. Optionally, the immunogen is injected in the presence of an adjuvant, such as, for example Freund's complete or incomplete adjuvant, lysolecithin or dinitrophenol to enhance the immune response to the immunogen. Monoclonal or polyclonal antibodies specific for the polypeptide are then be purified from the blood isolated form an animal by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide of interest are prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol (5:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest) e.g. a neutrophil derived protein and/or an isoform of α-1 antitrypsin or an immunogenic fragment thereof. Such cell lines are produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngenic with the immunized animal. Any of a variety of fusion techniques is employed, for example, the spleen cells and myeloma cells are combined with a nonionic detergent or electrofused and then grown in a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and growth media in which the cells have been grown is tested for the presence of binding activity against the polypeptide (immunogen) of interest. Hybridomas having high reactivity and specificity for the polypeptide (immungen) of interest are preferred.

Monoclonal antibodies are isolated from the supernatants of growing hybridoma colonies using methods such as, for example, affinity purification. In addition, various techniques are employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies are then harvested from the ascites fluid or the blood.

Contaminants are removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.

In one example, an immunogen used in the production of an antibody is one that is sufficiently antigenic to stimulate the production of antibodies, that will bind to the immunogen and is preferably, a high titer antibody. In one embodiment, an immunogen is an entire protein.

In another example, an immunogen consists of a peptide or a fragment of a peptide derived from a neutrophil derived protein and/or an isoform of α-1 antitrypsin. Preferably, an antibody raised to such an immunogen also recognizes the full-length protein from which the immunogen was derived, such as, for example, in its native state or having a native conformation.

Alternatively, or in addition, an antibody raised against a peptide immunogen will recognize the full-length protein from which the immunogen was derived when the protein is denatured. By "denatured" is meant that conformational epitopes of the protein are disrupted under conditions that retain linear B cell epitopes of the protein. As will be known to a skilled artisan linear epitopes and conformational epitopes may overlap.

In one example, a peptide immunogen is determined using the method described by Hopp, Peptide Research, 6: 183-190 1993, wherein a hydrophilic peptide is selected as it is more likely to occur at the surface of the native protein. However, a peptide should not be too highly charged, as this may reduce the efficiency of antibody generation.

In another example, a peptide immunogen is determined using the method described by Palfreyman et al J Immunol. Meth. 75, 383-393, 1984, wherein the amino- and/or

carboxy- terminal amino acids are used to generate a peptide against which specific antibodies are raised.

In yet another example, a peptide immunogen is predicted using an algorithm such as for example that described in Kolaskar and Tongaonkar FEBS Lett. 276(1-2) 112-114, 1990. Such methods are based upon determining the hydrophilicity of regions of a protein, usually 6 amino acids, and determining those hydrophilic regions that are associated with turns in proteins, surface flexibility, or secondary structures, and are unlikely to be modified at the post-translational level, such as, for example by glycosylation. Such regions of a protein are therefore likely to be exposed, that is, at the surface of the three-dimensional structure of the protein. Furthermore, as these regions are not modified, they are likely to remain constant and as such offer a likely site of antibody recognition.

In yet another example, overlapping peptides spanning the entire protein of interest, or a region of said protein are generated by synthetic means, using techniques known in the art. Alternatively, a relatively short protein of low abundance (e.g. calgranulin C) or a portion of a protein that is difficult to purify from a natural source, is produced chemically (e.g. by BOC chemistry or FMOC chemistry).

Synthetic peptides are prepared using known techniques of solid phase, liquid phase, or peptide condensation, or any combination thereof, and can include natural and/or unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (Nα-amino protected Nα-t-butyloxycarbonyl) amino acid resin with the deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield, J. Am. Chem. Soc, §5:2149-2154, 1963, or the base-labile Nα-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids described by Carpino and Han, J Org. Chem., 57:3403-3409, 1972. Both Fmoc and Boc Nα-amino protected amino acids are obtainable from various commercial sources, such as, for example, Fluka, Bachem, Advanced Chemtech, Sigma, Cambridge Research Biochemical, Bachem, or Peninsula Labs.

Synthetic peptides are then optionally screened to determine linear B cell epitopes, using techniques known in the art. In one example, the peptides are screened using an ELISA based assay to determine those peptides against which a CF subject with a clinical exacerbation has raised specific antibodies. Preferred peptides are those

against which a CF subject with a clinical exacerbation has raised specific antibodies, but a CF subject not suffering from an exacerbated state, or a healthy individual has not. Any peptide identified in such a screen is of use in a peptide based diagnostic or prognostic test.

Alternatively, or in addition, such an immunogenic peptide is used to generate a monoclonal or polyclonal antibody using methods known in the art, such as, for example, those described herein. The antibody is then tested to determine its specificity and sensitivity using, for example, an ELISA based assay. An antibody that specifically detects an antigen in a CF subject suffering from an acute exacerbation, but not a healthy CF subject, or a normal or healthy individual is preferred. For example, the antibody is capable of detecting an antigen in a CF subject developing an acute clinical exacerbation, and thus is diagnostic of such a state.

Ligands

As used herein the term "ligand" shall be taken in its broadest context to include any chemical compound, polynucleotide, peptide, protein, lipid, carbohydrate, small molecule, natural product, polymer, etc. that is able to bind selectively and stoichiometrically, whether covalently or not, to . one or more specific sites on a neutrophil derived protein or α-1 antitrypsin. The ligand may bind to its target via any means including hydrophobic interactions, hydrogen bonding, electrostatic interactions, van der Waals interactions, pi stacking, covalent bonding, or magnetic interactions amongst others.

In one example, a ligand is a peptide isolated from a random peptide library. To identify a suitable ligand, a random peptide library is generated and screened as described in U.S. Patent Application No. 5,733,731, 5,591,646 and 5,834,318. Generally, such libraries are generated from short random oligonucleotides that are expressed either in vitro or in vivo and displayed in such a way to facilitate screening of the library to identify a peptide that. is capable of specifically binding to a protein or peptide of interest. Methods of display include, phage display, retroviral display, bacterial surface display, bacterial flagellar display, bacterial spore display, yeast surface display, mammalian surface display, and methods of in vitro display including, mRNA display, ribosome display and covalent display.

A peptide that is capable of binding a protein or peptide of interest is identified by a number of methods known in the art, such as, for example, standard affinity purification methods as described, for example in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994) purification using FACS analysis as described in US Patent No 6,455,63, or purification using biosensor technology as described in Gilligan et al, Anal Chem, 74(9): 2041 - 2047, 2002.

Alternatively, a random peptide is used in a forward 'n'-hybrid assay to determine if said peptide is capable of binding to a protein or peptide of interest. Forward 'n' hybrid methods known in the art, and are described for example, by Vidal and Legrain Nucl Acid Res. 27(4), 919-929 (1999) and references therein, and include yeast two- hybrid, bacterial two-hybrid, mammalian two-hybrid, PoIIII (two) hybrid, the Tribrid system, the ubiquitin based split protein sensor system and the SOS recruitment system. Such methods are incorporated herein by reference

In adapting, a standard forward two-hybrid assay to the present invention, a neutrophil derived protein and/or an isoform of α-1 antitrypsin is expressed as a fusion with a DNA binding domain from, for example, from the yeast GAL4 protein. Methods of constructing expression constructs for the expression of such fusion proteins are known in the art, and are described, for example, in Sambrook et al (In: Molecular Cloning: A laboratory Manual, Cold Spring Harbor, New York, Second Edition, 1989). A second fusion protein is also expressed in the yeast cell, said fusion protein comprising, for example, a random peptide fused to transcriptional activation domain, for example the GAL4 activation domain. These two constructs are then expressed in a yeast cell in which a reporter molecule under the control of a minimal promoter placed in operable connection with a GAL 4 binding site is contained. If the proteins do not interact, a reporter molecule is not expressed. However, if said proteins do interact, said reporter molecule is expressed. Accordingly a protein, polypeptide, peptide that is able to specifically bind a target protein is identified.

A chemical small molecule library is also clearly contemplated for the identification of ligands that specifically bind to a neutrophil derived protein and/or α-1 antitrypsin. Chemical small molecule libraries are available commercially or alternatively may be generated using methods known in the art, such as, for example, those described in U.S. Patent No. 5,463,564.

Antibody based diagnostic methods

The present inventors have also shown that a subject suffering from CF and suffering from an acute clinical exacerbation produce auto-antibodies against a neutrophil derived protein. Accordingly, the present invention also provides a method of diagnosing diagnosing an inflammatory condition in a subject suffering from CF and/or an infection of a respiratory tract of a subject suffering from CF and/or pulmonary deterioration in a subject suffering from CF, said method comprising detecting in a sample from said subject antibodies against a neutrophil derived protein or an immunogenic fragment or epitope thereof and/of α-1 antitrypsin , or an immunogenic fragment or epitope thereof and/or mixtures thereof, wherein the presence of said antibodies in the sample is indicative of said inflammatory condition and/or infection and/or pulmonary deterioration. For example, the method diagnoses an acute clinical exacerbation in a subject suffering from cystic fibrosis.

For example, the present invention provides a method for detecting an inflammatory condition in a subject suffering from CF and/or an infection of a respiratory tract of a subject suffering from CF and/or pulmonary deterioration in a subject suffering from

" CF in a subject, the method comprising contacting a sample derived from the subject with a neutrophil derived protein or an immunogenic fragment or epitope thereof and/or α-1 antitrypsin or an immunogenic fragment or epitope thereof and/or mixtures thereof and detecting the formation of an antigen-antibody complex wherein detection of the complex is indicative of said inflammatory condition and/or infection and/or pulmonary deterioration.

In another example, the diagnostic assays of the invention are useful for determining the progression of an inflammatory condition in a subject suffering from CF and/or an infection of a respiratory tract of a subject suffering from CF and/or pulmonary deterioration in a subject suffering from CF. In accordance with these prognostic applications of the invention, the amount of antibodies against a neutrophil derived protein or an immunogenic fragment or epitope thereof and/or α-1 antitrypsin or an immunogenic fragment or epitope thereof and/or mixtures thereof in a sample from the subject is positively correlated with said inflammatory condition and/or infection and/or pulmonary deterioration. For example, a level of antibodies that is less than the level of the antibodies detectable in a subject suffering from said inflammatory condition and/or infection and/or pulmonary deterioration indicates that the subject is recovering from said inflammatory condition and/or infection and/or pulmonary

deterioration, Similarly, a higher level of antibodies in a sample from the subject compared to a normal and/or healthy individual indicates that the subject has not been rendered free of said inflammatory condition and/or infection and/or pulmonary deterioration.

In a further example, the present invention provides a method for determining the response of a subject suffering from CF and/or an infection of a respiratory tract of a subject suffering from CF and/or pulmonary deterioration in a subject suffering from CF wheren said subject is receiving treatment with a therapeutic compound for said inflammatory condition and/or infection and/or pulmonary deterioration, said method comprising detecting antibodies against a neutrophil derived protein or an immunogenic fragment or epitope thereof and/or α-1 antitrypsin or an immunogenic fragment or epitope thereof and/or mixtures thereof in a sample from said subject, wherein a level of the antibodies that is enhanced compared to the level of the antibodies detectable in a normal or healthy subject indicates that the subject is not responding to said treatment or has not been rendered free said inflammatory condition and/or infection and/or pulmonary deterioration.

In an alternative example, the present invention provides a method for determining the response of a subject suffering from CF and/or an infection of a respiratory tract of a subject suffering from CF and/or pulmonary deterioration in a subject suffering from

CF wheren said subject is receiving treatment with a therapeutic compound for said inflammatory condition and/or infection and/or pulmonary deterioration, said method comprising detecting antibodies against a neutrophil derived protein or an immunogenic fragment or epitope thereof and/or α-1 antitrypsin or an immunogenic fragment or epitope thereof and/or mixtures thereof in a sample from said subject, wherein a level of the antibodies that is lower than the level of the antibodies detectable in a subject suffering from an inflammatory condition and/or an infection of a respiratory tract and/or pulmonary deterioration indicates that the subject is responding to said treatment or has been rendered free of said inflammatory condition and/or infection and/or pulmonary deterioration.

In one example of the diagnostic/prognostic methods described herein, the sample is obtained previously from the- subject. Accordingly, the prognostic or diagnostic method is performed ex vivo.

In yet another example, the subject diagnostic/prognostic methods further comprise processing a sample from the subject to produce a derivative or extract that comprises the analyte (blood, serum, urine or immunoglobulin-containing fraction).

Each of the preveious embodiments directed to detection of an antibody against a neutrophil derived protein or an immunogenic fragment or epitope thereof and/or α-1 antitrypsin or an immunogenic fragment or epitope thereof and/or mixtures thereof shall be taken to apply mutatis mutandis to the detection of an antibody against a modified form of a neutrophil derived protein or an immunogenic fragment or epitope thereof and/or a modified form of α-1 antitrypsin or an immunogenic fragment or epitope thereof and/or mixtures thereof

The methods described hereinabove for the detection of an antibody or α-1 antitrypsin or an immunogenic fragment or epitope thereof and/or mixtures thereof shall be taken to apply mutatis mutandis to the detection of an antibody against myeloperoxidase and/or a modified form thereof. In one example, the method of the invention additionally comprises detecting an antibody against a myeloperoxidase and/or a modified form of myeloperoxidase.

Samples and reference samples

In one example, a sample in which a neutrophil derived protein and/or an isoform of α- 1 antitrypsin and/or mixtures thereof is detected and selected from the group consisting of lung, lymphoid tissue associated with the lung, paranasal sinuses, bronchi, a bronchiole, alveolus, ciliated mucosal epithelia of the respiratory tract, mucosal epithelia of the respiratory tract, squamous epithelial cells of the respiratory tract, a mast cell, a goblet cell, a pneumocyte (type 1 or type 2), broncheoalveolar lavage fluid (BAL), alveolar lining fluid, an intra epithelial dendritic cell, sputum, mucus, saliva, blood, serum, plasma, a PBMC, a buffy coat fraction, a neutrophil and a monocyte. For example, the sample is sputum.

In one example, a sample is obtained previously from a subject.

In one example, the method of the present invention encompasses providing the sample.

In one example, a sample is obtained from a subject by a method selected from the group consisting of surgery or other excision method, aspiration of a body fluid by, for example, hypertonic saline or propylene glycol, broncheoalveolar lavage, bronchoscopy, saliva collection with a glass tube, salivette (Sarstedt AG, Sevelen, Switzerland), Ora-sure (Epitope Technologies Pty Ltd, Melbourne, Victoria, Australia), omni-sal (Saliva Diagnostic Systems, Brooklyn, NY, USA) and blood collection using any method known in the art, such as, for example using a syringe.

In one exemplified form of the invention a sample is sputum, isolated from a lung of a subject using, for example the method described in Gershman, N.H. et al, J Allergy Clin Immunol, 10(4): 322-328, 1999.

Alternatively, a sample is plasma that has been isolated from blood collected from a patient using a method known in the art.

In yet another example, a sample is treated prior to use in a diagnostic or prognostic assay. Accordingly, the methods of the present invention may be performed using a derivative or extract, e.g. a cell extract or solubilized sputum that comprises a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof.

In one example, a sample is treated to lyse a cell in said sample. Such methods include the use of detergents, enzymes, repeatedly freezing and thawing said cells, sonication or vortexing said cells in the presence of glass beads, amongst other methods.

In another example, a sample is treated to denature a protein present in said sample. ^• Methods of denaturing a protein include heating a sample, treatment with 2- mercaptoethanol, or treatment with detergents and other compounds such as, for example, guanidinium or urea.

In yet another example, a sample is treated to concentrate a protein in said sample. Methods of concentrating proteins include precipitation, freeze drying, use of funnel tube gels (TerBush and Novick, Journal of Biomolecular Techniques, 10(3); 1999), ultrafiltration or dialysis.

As will be apparent to the skilled artisan, the diagnostic and prognostic methods provided by the present invention may require a degree of quantification to determine

either, the amount of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof that is diagnostic or prognostic of an inflammatory condition in a subject suffering from CF and/or an infection of the respiratory tract in a subject suffering from CF and/or pulmonary deterioration in a subject suffering from CF, e.g., an acute exacerbation in a CF subject.

Furthermore, certain diagnostic and prognostic methods described herein require the detection of the amount of both an unmodified and/or a modified form of a neutrophil derived protein and/or an isoform of α-1 antitrypsin and/or mixtures thereof that is diagnostic or prognostic of an inflammatory condition in a subject suffering from CF and/or an infection of the respiratory tract in a subject suffering from CF and/or pulmonary deterioration in a subject . suffering from CF, e.g., an acute clinical exacerbation in a CF subject. Such quantification can be determined by the inclusion of appropriate control or reference samples in the assays described herein, wherein said control or reference samples are derived from healthy or normal individuals.

In one example, the level of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof in a sample derived from a subject is compared to the level of the same protein detected in a control or reference sample that was previously derived from the same subject. As will be apparent to a person skilled in the art, this method may be used to continually and/or temporally monitor a subject suffering from CF. In this manner a subject may be monitored for the onset of an inflammatory condition and/or an infection of the respiratoiy tract and/or pulmonary deterioration, e.g., an acute clinical exacerbation, with the goal of commencing treatment for said exacerbation prior to it becoming established and causing damage to the lungs of said subject.

In one example, the control or reference sample comprises, for example, a sample derived from the same subject when that individual was not suffering from an acute clinical exacerbation. In another example, the reference sample comprises, a sample derived from another CF patient that was not suffering from an acute exacerbation at the time of collection of the sample. In yet another example, the reference sample comprises a sample derived from a normal or healthy individual.

As used herein, the term "healthy individual" shall be taken to mean an individual who is not known to suffer from an inflammatory condition and/or an infection of the

respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation. Such a subject preferable is not known to suffer from CF. Methods of determining whether or not a subject suffers from CF are known in the art and/or described herein.

As used herein the term "normal individual" shall be taken to mean an individual having _, a normal amount of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof in a sample and/or a normal amount of a modified form of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof in a sample.

In one example, A reference sample and a test (or patient/subject) sample are both processed, analyzed and/or assayed and data obtained for a reference sample and a test sample are compared. In one embodiment, a reference sample and a test sample are processed, analyzed and/or assayed at the same time. In another embodiment, a reference sample and a test sample are processed, analyzed and/or assayed at a different time.

Alternatively, a reference sample is not included in an assay. Instead, a reference sample is derived from an established data set that has been previously generated. Accordingly, in one example, a control or reference sample comprises data from a sample population study of healthy individuals and/or normal individuals, such as, for example, statistically significant data for the healthy range of the integer being tested. Data derived from processing, analyzing and/or assaying a test sample is then compared to data obtained for the sample population. Accordingly, in one example, a control sample is a data set comprising measurements of a level of a neutrophil derived protein and/or an isoform of α-1 antitrypsin and/or mixtures thereof for a healthy individual or a population of healthy individuals. In another example, a control sample is a data set comprising measurements of a level of a neutrophil derived protein and/or an isoform of α-1 antitrypsin and/or mixtures thereof for a normal individual or a population of normal individuals. In another example, a control sample is a data set comprising measurements of a level of a modified form of a neutrophil derived protein and/or a modified form of α-1 antitrypsin and/or mixtures thereof for a healthy individual or a population of healthy individuals. In another example, a control sample is a data set comprising measurements of a level of a modified form of a neutrophil derived protein and/or a modified form of α-1 antitrypsin and/or mixtures thereof for a normal individual or a population of normal individuals.

Data obtained from a sufficiently large number of reference samples so as to be representative of a population allows the generation of a data set for determining the average level of a particular parameter. Accordingly, the amount of a protein that is diagnostic or prognostic of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation, or the amount of an unmodified and/or modified protein that is diagnostic or prognostic of an acute clinical exacerbation in a CF patient can be determined for any population of individuals, and for any sample derived from said individual, for subsequent comparison to levels of the expression product determined for a sample being assayed. Where such normalized data sets are relied upon, internal controls are preferably included in each assay conducted to control for variation.

Detection of modified proteins as markers of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation in a CF patient

As will be apparent to the skilled artisan the methods described herein relating to the detection of an enhanced level of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof may be modified or used to detect other changes in proteins, for example, to detect the amount of a modified, e.g. cleaved, form of a protein in a sample.

Accordingly, the present invention provides a method of diagnosing an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation in a subject having cystic fibrosis (CF) comprising detecting, in a sample from said subject, a modified form of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof.

In one example a modified form of a neutrophil derived protein and/or an isoform of α- 1 antitrypsin or mixtures thereof is a form that is cleaved in a CF subject suffering from a clinically exacerbated state.

In one example, the present invention comprises detecting a modified level of an isoform of α-1 antitrypsin and/or a modified form of α-1 antitrypsin. Preferably, the assay detects an enhanced level of an isoform of α-1 antitrypsin and/or an enhanced level of a modified form of α-1 antitrypsin to thereby indicate an acute clinical

exacerbation and/or a risk thereof and/or an infection or inflammation. Alternatively, a reduced level of an isoform of α-1 antitrypsin and/or a reduced level of a modified form of α-1 antitrypsin indicates a reduced risk of an acute clinical exacerbation and/or a reduced risk of an infection or a reduced risk of inflammation

In a preferred example a modified form of an isoform of α-1 antitrypsin is a form that is cleaved in a CF patient suffering from a clinically exacerbated state. For example, a modified form of an isoform of α-1 antitrypsin is α-1 antitrypsin that has a decrease in mass of about IkDa to about 1OkDa compared to uncleaved α-1 antitrypsin, e.g., the reduction in Molecular weight is about 3kDa to about 7kDa, e.g., from about 5 kDa to about 7kDa. In addition to a change in mass, a modified isoform of α-1 antitrypsin has a decrease in pi relative to uncleaved α-1 antitrypsin from about 0.01 pi units to about 0.5 pi units, e.g., from about 0.1 pi units to about 0.3 pi units, e.g., from about 0.15 pi units to about 2.5 pi units.

For example, a modified form of α-1 antitrypsin comprises an amino acid sequence set forth in SEQ ID NO: 12 or 13.

Cleavage of α-1 antitrypsin is particularly useful as a diagnostic or prognostic of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation in a patient suffering from CF. For example, a change in molecular weight and pi of an isoform of α 1 -antitrypsin caused by cleavage and/or degradation of said protein is detected with an antibody that specifically binds to the region corresponding to the site of cleavage and/or degradation of an isoform of α-1 antitrypsin. For example, an antibody binds specifically to α-1 antitrypsin that has not been cleaved (i.e. SEQ ID NO: 11), and not a cleaved form of the protein (i.e. SEQ ID NO: 12 or SEQ ID NO: 13). Such an antibody is then useful in determining an amount of uncleaved α-1 antitrypsin in a sample derived from a patient with CF relative to the amount of uncleaved α-1 antitrypsin in a reference sample, e.g., derived from a subject with CF but who does not suffer from a clinical exacerbation, or has recently received treatment for an exacerbated state, or a normal healthy subject. Alternatively, a reference sample may be a sample that was previously derived from the same patient as from which the test was derived. Preferably, the reference sample was derived from the patient prior to the onset of a clinical exacerbation.

Suitable assays for detecting the level of an isoform of α-1 antitrypsin and/or the presence or level of a modified form of α-1 antitrypsin will be apparent to the skilled artisan based on the disclosure herein. For example, the presence or amount of an isoform of α-1 antitrypsin or a modified form of α-1 antitrypsin is detected with an immunoassay known in the art and/or as described herein.

In one example, a modified form of a neutrophil derived protein is annexin I that has a reduction in mass of about 3kDa to about 15kDa, e.g., about 5 kDa to about 10 kDa, e.g., from about 7 kDa to about 9 kDa, e.g., about 8 kDa. Alternatively, or in addition to a change in mass, it is preferable that there is also an increase in the pi of annexin I from about 1 pi units to about 3 pi units, e.g., from about 1.5 pi units to about 2.75 pi units, e.g., from about 1.75 pi units to about 2.5 pi units, e.g., about 2.2 pi units.

In one example, a change in molecular weight and pi is caused by cleavage of annexin I.

Cleavage of annexin I is useful as a diagnostic or prognostic of an acute clinical exacerbation in a patient suffering from CF.

In one example, a change in molecular weight and pi of a neutrophil derived protein and/or an isoform of α-1 antitrypsin caused by modification, cleavage and/or degradation of said protein is detected with an antibody or ligand that specifically binds to the region corresponding to the site of cleavage and/or degradation of a neutrophil derived protein and/or an isoform of α-1 antitrypsin. Accordingly, this antibody or ligand only binds to an unmodified form of a neutrophil derived protein and/or an isoform of α-1 antitrypsin.

In another example, an antibody or ligand binds specifically to a neutrophil derived protein and/or an isoform of α-1 antitrypsin that has not been modified (e.g. uncleaved annexin I, SEQ ID NO: 6 or α-1 antitrypsin, SEQ ID NO: 11), and not a cleaved form of the protein (e.g. cleaved annexin I, SEQ ID NO: 8 or SEQ ED NO: 9 or cleaved α-1 antitrypsin, SEQ ID NO: 12 and SEQ ID NO: 13). Such an antibody is then useful in determining a level of a unmodified neutrophil derived protein and/or an isoform of α-1 antitrypsin in a sample derived from a subject with CF relative to a level of an uncleaved neutrophil derived protein and/or an isoform of α-1 antitrypsin in a reference sample.

In one example, a decreased level of an unmodified neutrophil derived protein and/or an isoform of α-1 antitrypsin is detected in a reference sample compared to a level detected in a test sample. This level may be either an absolute level or a relative level. This indicates that the subject from whom the test sample is isolated is suffering from or is likely to develop and/or will develop an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In another example, an antibody or ligand specifically binds to a modified form of a neutrophil derived protein and/or an isoform of α-1 antitrypsin. Such an antibody may, for example, bind to a specific protein conformation that is induced by said modification.

In one example, a modified form of a neutrophil derived protein and/or an isoform of α- 1 antitrypsin or mixtures thereof is detected in a sample isolated from a CF subject, but said modified protein is not detected in a reference sample, indicating that the CF subject is suffering from or will develop an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In another example, a modified form of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof is detected in a reference sample, however, an increased level of said protein is detected in a test sample isolated from a CF ^' subject, indicating that the CF subject is suffering from or is likely to or is at risk of developing an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In another example, an antibody or ligand specifically binds to both a modified form of a protein and an unmodified form of a protein, such as for example an N-terminal fragment of a cleaved annexin I (SEQ ID NO: 8). Accordingly, by separating proteins, polypeptides and peptides, for example, according to their molecular weight using, for example sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferring said protein from the SDS-PAGE gel to a membrane, for example a PVDF membrane using methods known in the art, and described, for example, in Harlow and Lane {In: Antibodies: A Laboratory Manual,

Cold Spring Harbor Laboratory, 1988), a modified form of neutrophil derived protein and/or an isoform of α-1 antitrypsin may be detected using methods known in the art.

Alternatively, should the difference in molecular weight of a fragment of a neutrophil derived protein and/or an isoform of α-1 antitrypsin and an unmodified form of a neutrophil derived protein and/or an isoform of α-1 antitrypsin be sufficient, size exclusion chromatography or filtration is useful for isolating the modified form, and the amount of said form determined using methods known in the art and/or described herein.

In one example, a modified form of a neutrophil derived protein and/or an isoform of α- 1 antitrypsin is not detected in a reference sample, but is detected in a sample isolated from a CF subject, indicating that said subject is suffering from or is likely to or is at risk of developing an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In another example, a modified form of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof is detected in a reference sample, however, it is detected at significantly greater levels in a sample isolated from a CF subject indicating that the CF subject is suffering from or is likely to or is at risk of developing an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

Monitoring the progression of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation in a CF subject

The present invention provides a method for determining the progression of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation in a subject suffering from CF and being administered an amount of a therapeutic compound for the treatment of said infection, said method comprising detecting an enhanced level of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof, wherein a reduced level of said protein or loss of expression of said protein indicates that a subject is recovering from an exacerbated state. ^■

In one example, a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof is detected in a sample isolated from a CF subject at a level that is less than the level of that protein detectable in a subject suffering from an acute clinical exacerbation indicating that the CF subject is recovering from said exacerbation.

Accordingly, this example of the invention provides a prognostic indicator of the response of a CF subject suffering from an acute clinical exacerbation to a treatment with a therapeutic compound for the treatment of said exacerbation. Using this prognostic method the effectiveness of a therapeutic compound in the treatment of an acute clinical exacerbation is monitored and the therapy being used may be modified to ensure more rapid recovery of a subject suffering from CF.

In one example, a detection method as described herein is used to detect a level of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof in a sample isolated from a CF subject that is receiving treatment for an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In one example, failure to detect an enhanced level of said neutrophil derived protein and/or an isoform of α-1 antitrypsin indicates that said subject is likely to recover or has recovered from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In another example, the' level of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof detected in said subject sample is compared to the level of the same protein detected in a reference sample.

In one example, an enhanced level of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof in a test sample indicates that the subject from whom the test sample was derived has recovered, or is recovering, from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In another example, the level of a protein detected in a test sample is compared to an amount of a protein detected in a reference sample derived from a normal or healthy individual. Using methods known in the art and/or described herein, a level of a

neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof is determined in both a test sample and said reference sample. Detection of a level of said protein in a test sample equivalent, or not significantly different to a level of the same protein detected in a reference sample, indicates that the subject from whom the test sample was derived has recovered or is recovering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In yet another example, a prognostic indicator of the present invention is used to characterize the ongoing treatment of a CF subject with an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation. For example, several samples are isolated from a CF subject suffering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation, each sample being collected at a different point in time during treatment. For example, a sample is isolated from a CF subject with an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation each day following the commencement of administration of a therapeutic compound for the treatment of said exacerbation. The level of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof is then determined in each of these samples. Results of such an assay are then compared to previous results and/or those obtained from a reference sample. Such an assay monitors the level of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof in a subject during treatment, and as such provides a prognostic indication of the effectiveness of treatment for an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

Such an assay may also be used to determine whether or not a subject is responding to treatment for an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation. As such, the present invention also provides a method for determining the progression of an acute clinical exacerbation in a subject suffering from CF and being administered an amount of a therapeutic compound for the treatment of said inflammatory condition and/or infection and/or pulmonary deterioration and/or acute clinical exacerbation, said method comprising detecting an enhanced level of a neutrophil derived protein and/or an isoform of α- 1 antitrypsin or mixtures thereof, wherein detection of an enhanced

level of a protein indicates that a subject is not recovering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In one example, a level of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof is detected and compared to an amount of the same protein in a reference sample, e.g., as described herein.

In one example, a reference sample is derived from the same subject as that from which the test sample is derived. Preferably, the reference sample is derived prior to the test sample, e.g., the reference sample is derived during the inflammatory condition and/or infection of the respiratory tract and/or pulmonary deterioration and/or acute clinical exacerbation. Accordingly, such an assay determines the ongoing status or recovery of a CF subject suffering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

An amount of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof detected in a test sample that is equivalent, or not significantly different to such a reference sample indicates that the subject from whom the test samples was derived is not recovering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In another example, a reference sample is derived from one or more normal or healthy individuals or a data set comprising measurements from such subjects or a population thereof. Detection of a level of a neutrophil derived protein and/or an isoform of α-1 antitrypsin in a sample derived from a subject that is significantly greater than that detected in a reference sample indicates that the subject is not recovering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

Indication that a CF subject is not recovering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation suggests that the treatment used for the control of an acute clinical exacerbation in a CF patient may require modification. For example, the patient may be infected with a multiple-drug resistant bacteria requiring treatment with an antibiotic such as, for example, Tobramycin™, Meropenum ¹ or vancomycin .

As will be apparent to the skilled artisan, the methods described herein are also useful to produce and prognostic assay wherein a modified protein is detected. Accordingly, the present invention also provides a method for determining the progression of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation in a subject suffering from CF and being administered with an amount of a therapeutic compound for the treatment of said exacerbation, said method comprising detecting a native or unmodified isoform of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof, wherein the detection of said native or unmodified form of the protein indicates that the subject is recovering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In one example, a level of a native or unmodified protein is detected in a subject using methods known in the art and described herein.

In one example, the level of an unmodified or a native form of a protein; a modified form of a protein; or both an unmodified and modified form of a protein is determined in a subject suffering from CF and currently undergoing treatment for an acute clinical exacerbation. The level of protein detected is then compared to a reference sample, e.g., as described herein.

In one example, a reference sample is derived previously from the same subject as a test sample, preferably while said subject was suffering from an inflammatory condition andVor an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation. Detection of significantly greater levels of unmodified protein and/or less modified protein in a test sample compared to said reference sample indicates that said subject is recovering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In another example, a reference sample is a sample derived from one or more CF subjects suffering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation. A result indicating that the test sample contains significantly greater levels of unmodified protein and/or less modified protein that a subject is recovering from an inflammatory

condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In another example, a reference sample is a sample derived from one or more normal and/or healthy individuals or a suitable data set. Detection of a level of an unmodified form of a neutrophil derived protein and/or an isoform of α-1 antitrypsin and/or a modified form of said protein where that level does not significantly vary from the reference sample indicates that a subject from whom the test sample was derived is recovering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

Such an assay is also used to determine those subjects that are not recovering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation. Accordingly, the present invention also provides a method for determining the response of a CF subject having . an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation to treatment with a therapeutic compound for the treatment of said inflammatory condition and/or infection and/or pulmonary deterioration and/or acute clinical exacerbation, said method comprising detecting a native or unmodified isoform of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof, wherein the. detection of a modified form of the protein indicates that the subject is not responding to treatment.

In one example, a reference sample is derived from the same subject as that from which the test sample is derived. Preferably, the reference sample is derived prior to the test sample, e.g., the reference sample is derived during the same clinical exacerbation event.

In another example, a reference sample is derived from one or more subjects with CF that suffer from an acute clinical exacerbation.

In accordance with either of the two previous paragraphs, detection, in a test sample, of a level of a modified protein and/or an unmodified protein that is not significantly different to the reference sample suggests that a subject from whom the test sample was derived is not recovering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

A result that shows significantly greater levels of a protein in a test sample than a reference sample suggests that said subject is entering at risk of entering or suffering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In another example, a reference sample is derived from one or more normal and/or healthy individuals. Detection, in a test sample of an enhanced level of a modified form of a neutrophil derived protein and/or an isoform of α-1 antitrypsin or mixtures thereof when compared to the level detected in a reference sample indicates that a subject is not recovering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

In a preferred example, a diagnostic method described herein is used in the early diagnosis of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation. In this manner a subject suffering from CF may be treated with antibacterial compounds before an infection becomes established. This reduces the overall effect of anclinical exacerbation by reducing the inflammation associated with such an infection, thereby reducing the risk of submucosal gland hypertrophy and excessive mucus secretion in the respiratory tract. In the long term, the reduction in the number and severity of cases of clinical exacerbation suffered by a CF patient reduces the risk of progressive bronchiectasis and respiratory failure.

As used herein the term "early diagnosis" shall be understood to mean that an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation is diagnosed before bacteria associated with an exacerbation can be cultured from a sample derived from the respiratory tract of a CF subject.

Preferably, an acute clinical exacerbation or infection is diagnosed within about 5 to 10 days after exposure to an infectious organism, more preferably about 3 to 6 days after exposure to an infectious organism, even more preferably about 2 to 3 days after exposure to an infectious organism, and most preferably about 1 day after exposure to an infectious organism.

Following the diagnosis of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation an assay that determines if such a state is induced by a respiratory infection may be performed. Furthermore, it is important that the organism causing such an infection be identified. Methods of identification of a bacterial species causing an infection are known in the art and include, but are not limited to phage typing, multilocus enzyme electrophoresis (MLEE)(as described in, for example, Selander et al, Appl. Environ. Microbiol. 51(5) 873-874, 1986), analysis of microorganism toxin production, serology and antibiograms such as described in US Patent Number 4, 421, 349, ribotyping (or the identification of the molecular weight of rRNA genes), sequencing of 16s rRNA genes, the semi-automated Random Amplified Polymorphic DNA (RAPD) techniques described in US Patent Number 6,395,475 (Florida State University) or determining the number (of base pairs) of variable ribosomal DNA sequences as described in US Patent Number 5, 753, 467.

Accordingly, following identification of an infectious agent, a subject suffering from an acute exacerbation is treated with an anti-bacterial agent specific to the pathogen causing said infection, thereby increasing the probability that a CF subject will rapidly recover from the respiratory tract infection. Furthermore by diagnosing a respiratory tract infection early and effectively treating said infection, there is a reduced risk of damage to the respiratory tract of said CF subject.

A related exampleof the present invention relates to the detection of inflammation in a CF subject, wherein a diagnostic or prognostic method of the present invention suggests the onset of a clinically exacerbated state, however no infectious agents are detected in the respiratory system of said CF patient. Tests for inflammation in a CF subject are known in the art, such as, for example, detecting the levels of lactoferrin in the serum of a CF subject (as described in Eichler et al, Eur. Respir. J. 14(5), 1145- 1149, 1999), or measuring the level of carbon monoxide or nitric oxide exhaled by a CF subject (Kharitonov and Barnes Biomarkers, 7(1): 1-32, 2002).

Accordingly, the methods of the present invention allow the early diagnosis of an acute clinical exacerbation in a CF subject, allowing treatment of a bacterial infection and/or inflammation to commence at an early stage, preferably to inhibit the onset of said exacerbated state-.

Multiplex assays

As will be apparent to those skilled in the art a diagnostic or prognostic assay may measure an enhanced level of one protein selected from the group consisting of an isoform of α-1 antitrypsin, an isoform of leukocyte elastase inhibitor, an isoform of α- enolase, an isoform of Rho GDP-dissociation inhibitor, an isoform of annexin I, an isoform of annexin III, an isoform of calgranulin C and an isoform of catalase. Alternatively, a prognostic or diagnostic assay may measure an enhanced level of any two or more proteins selected from the group consisting of an isoform of α-1 antitrypsin, an isoform of leukocyte elastase inhibitor, an isoform of α-enolase, an isoform of Rho GDP-dissociation inhibitor, an isoform of annexin I, an isoform of annexin III, an isoform of calgranulin C and an isoform of catalase. Such multiplexed assays are useful in increasing the specificity and accuracy of a prognostic or diagnostic assay.

As used herein the term "multiplex", shall be understood not only to mean the detection of two or more diagnostic or prognostic markers in a single sample concurrent, but also to encompass consecutive detection of two or more diagnostic or prognostic markers in a single sample, concurrent detection of two or more diagnostic or prognostic markers in distinct but matched samples, and consecutive detection of two or more diagnostic or prognostic markers in distinct but matched samples. As used herein the term "matched samples" shall be understood to mean two or more samples derived from the same initial sample, or two or more samples isolated at substantially or approximately the same point in time from the same subject.

In the case of analysis of two samples isolated at substantially or approximately the same point in time, these samples may be the same type of sample, e.g. sputum, or different samples, e.g. sputum and plasma.

Accordingly, a multiplexed assay may be an assay that detects the amount or modification of more than one neutrophil derived protein and/or isoform of α-1 antitrypsin in the same reaction. As will be apparent to the skilled artisan, if such an assay is antibody or ligand based, both of these antibodies should bind and/or detect the relevant neutrophil derived protein and/or isoform of α-1 antitrypsin under substantially the same conditions.

Alternatively, or in addition, a multiplexed assay comprises first detecting the amount or modification of one or more neutrophil derived protein(s) and/or isoform(s) of α-1 antitrypsin, followed by the detection of the amount or modification another one or more neutrophil derived protein(s) and/or isoform(s) of α-1 antitrypsin in the same sample. Accordingly, based on the result of the first step in this process it may be unnecessary to proceed to the second step, e.g. if the first assay indicates that a patient is not suffering from an acute clinical exacerbation.

A multiplexed assay also comprises of the detection of the amount or modification of one or more neutrophil derived protein(s) and/or isoform(s) of α-1 antitrypsin in separate reactions. Such an assay comprises of each of these proteins being detected in a separate reaction or one or more of these proteins may be detected in one reaction and one or more proteins in another reaction. Again such an assay detects all of these proteins simultaneously, that is all reactions including all samples proceed at the same time. Alternatively, these reactions are consecutive, with each reaction proceeding following either the commencement or completion of another assay. Accordingly, based on the result of the first step, in this process it may be unnecessary to proceed to the second step, e.g. if the first assay indicates that a patient is not suffering from an acute clinical exacerbation.

In another example, a prognostic or diagnostic assay detects a modified form of a protein selected from the group consisting of an isoform of α-1 antitrypsin, an isoform of leukocyte elastase inhibitor, an isoform of α-enolase, an isoform of Rho GDP- dissociation inhibitor, an isoform of annexin I, an isoform of annexin III, an isoform of calgranulin C and an isoform of catalase. Alternatively, a prognostic or diagnostic assay detects a modified form of two or more proteins selected from the group consisting of an isoform of α-1 antitrypsin, an isoform of leukocyte elastase inhibitor, an isoform of α-enolase, an isoform of Rho GDP-dissociation inhibitor, an isoform of annexin I, an isoform of annexin III, an isoform of calgranulin C and an isoform of catalase.

In yet another example, an assay of the present invention is used to detect an enhanced level of one or more proteins selected from the group consisting of an isoform of α-1 antitrypsin, an isoform of leukocyte elastase inhibitor, an isoform of α-enolase, an isoform of Rho GDP-dissociation inhibitor, an isoform of annexin I, an isoform of annexin III, an isoform of calgranulin C and an isoform of catalase and one or more

modified proteins selected from the group consisting of an isoform of α-1 antitrypsin, an isoform of leukocyte elastase inhibitor, an isoform of α-enolase, an isoform of Rho GDP-dissociation inhibitor, an isoform of annexin I, an isoform of annexin III, an isoform of calgranulin C and an isoform of catalase.

In another example, a method of the present invention as described herein according to any embodiment is performed in combination with an assay to detect a modified level of an isoform of myeloperoxidase and/or a modified form of myeloperoxidase. For example, the assay detects an enhanced level of an isoform of myeloperoxidase thereby indicating an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation. Alternatively, a reduced level of an isoform of myeloperoxidase indicates a reduced risk of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation.

As used herein, the term "myeloperoxidase" shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence of a myeloperoxidase set forth in SEQ ID NO: 15. Preferably, the degree of sequence identity is at least about 85% or 95% or 95% or 98% or 99%.

Suitable assays for detecting the level of an isoform of myeloperoxidase will be apparent to the skilled artisan based on the disclosure herein. For example, the presence or amount of an isoform of myeloperoxidase is detected with an immunoassay known in the art and/or as described herein.

In yet another example, an assay of the present invention as described in any embodiment additionally comprises detecting the level of an isoform of α- 1 antitrypsin and the level of an isoform of myeloperoxidase. In another example, an assay of the present invention as described in any embodiment additionally comprises detecting the level of a modified form of α-1 antitrypsin and the level of an isoform of myeloperoxidase.

In yet another example, a diagnostic or prognostic assay of the present invention is, for example, multiplexed, with an assay that monitors mucin proteins. ^'

Mucin proteins are elevated in CF patients and that the glycosylation of these proteins is different in subjects suffering from CF compared to healthy controls (Jiang et al, Am J. Physiol 273 913-920, 1997). Furthermore, the glycosylation of mucins has been shown to be modified in response to the inflammatory state of the patient, for example, as the respiratory system becomes more inflamed in a CF patient, mucins become highly sulfated and highly sialylated.

Accordingly, a diagnostic or prognostic assay that determines the glycosylation patterns of mucins in a CF patient may be used as a general indicator of the degree of inflammation and/or infection in the respiratory tract or even the intestinal tract. Through multiplexing a diagnostic or prognostic assay of the present invention with an assay that determines the glycosylation pattern of mucin, for example, an antibody or ligand that binds to a particular sugar moiety, the general systemic state of a patient is determined.

As used herein, the term "general systemic state" shall be understood to mean that a multiplexed assay indicates the general pulmonary health of a CF patient. Accordingly, such an assay determines the presence of an infection of the respiratory tract of a CF patient, the level of inflammation of the respiratory tract of a CF patient and the degree of damage to the respiratory tract of said patient. Accordingly, such a diagnostic assay permits the skilled artisan to continually monitor a CF patient thereby facilitating the correct treatment of said patient for inflammation or infection of the respiratory system.

Chronic or repeated episodes of acute inflammation and infection damage the lungs of CF patients resulting in bronchiectasis and eventually respiratory failure requiring a lung transplant. Accordingly, a diagnostic or prognostic method that can effectively monitor both inflammation and infections and allow the early detection and treatment of these complications will result in a reduction of the damage caused to the respiratory tract of a CF patient.

In another example, a diagnostic or prognostic assay of the present invention is multiplexed with another assay or marker that is diagnostic or prognostic of an acute clinical exacerbation of CF.

In one example, a diagnostic or prognostic assay or marker of the present invention is multiplexed with another protein that is diagnostic of an acute clinical exacerbation of a

CF subject, or neutrophil activation, or infection or inflammation. Such markers include, for example, defensin 1, defensin 2 or defensin 3, nitrotyrosine, lactoferrin, or elastase 1 or 2, amongst others. The detection of such a marker may be assayed by any method known in the art and/or described herein. ^'

In one example, an additional assay is the measurement of forced expiration volume in one second (FEVi). , Such an assay will be known to those skilled in the art. FEVi is

. measured by, for example a spirometer. Other such measurement that may be of use in a multiplexed assay include, peak expiratory flow. (PEF), vital capacity (VC) which is the maximum volume of air that can be inhaled or exhaled, forced expiratory flow at

50% of FEVi (FEF ₅₀ %), and forced expiratory time. Methods of measuring such are known in the art.

In yet another example, an assay or marker of the present invention is multiplexed with an .assay that detects the presence of a bacterial infection, such as a P. aeruginosa infection. Such assays include the detection of IgG in a CF subject that is specific to the core lipopolysaccharide .of P. aeruginosa (US Patent No. 5,179,001), or IgA specific to P. aeruginosa cells (as described by Brett et al, J. Clin. Pathol. 41(10),

1130-1134, 1988), or an antibody specific to sodium alginate exoploysaccharide of P. aeruginosa (as described in Bryan et al, J. Clin. Microbiol. 18(2), 276-282, 1983).

Alternatively, this assay detects a type-Ill secretory protein of P. aeruginosa (as described by Roy-Burman et al, J. Infect. Dis. 183(12), 1767-1774, 2001).

Diagnostic assay kits The present invention also provides an antibody or fragment thereof, ligand or synthetic or recombinant peptide that is generated for use and/or used in a diagnostic or prognostic assay as described herein. Methods of isolating such an antibody, fragment, peptide or ligand are known in the art and/or described herein.

The present invention also provides for the use of any novel or previously undescribed antibody, ligand or synthetic or recombinant peptide in other therapeutic or diagnostic applications or for research. Such applications include the purification and study of the diagnostic/prognostic proteins, identification of cells expressing said proteins, and sorting or counting cells. Accordingly, the present invention encompasses the use of a novel antibody or fragment thereof, ligand or synthetic or recombinant peptide in therapy, including, prophylaxis, diagnosis, prognosis, or the use of such agents in the

manufacture of a medicament for use in treatment of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation in a CF patient.

The present invention also provides a kit comprising an antibody or fragment thereof or a ligand that is capable of specifically binding to a neutrophil derived protein and/or an isoform of α-1 antitrypsin for the diagnosis or prognosis of an inflammatory condition or an infection of the respiratory tract or pulmonary deterioration in a subject suffering from CF, e.g., for the diagnosis of an acute clinical exacerbation in a subject suffering from CF.

In one example, an antibody or a ligand specifically binds to a neutrophil derived protein and/or an isoform of α-1 antitrypsin and facilitates detection and estimation of the amount of said protein.

In another example, an antibody or a ligand specifically binds an isoform of a neutrophil derived protein and/or an isoform of α-1 antitrypsin and facilitates detection and estimation of the amount of said protein.

In a further example, an antibody or a ligand specifically binds a neutrophil derived protein or an isoform of a neutrophil derived protein selected from the group consisting of an isoform of leukocyte elastase inhibitor, an isoform of α-enolase, an isoform of Rho GDP-dissociation inhibitor, an isoform of annexin I, an isoform of annexin III, an isoform of calgranulin C and an isoform of catalase. Alternatively, an antibody specifically binds an isoform of α-1 antitrypsin.

In yet another example, an antibody, or ligand specifically binds a modified form of a neutrophil derived protein and/or α-1 antitrypsin. Such an antibody permits the skilled artisan to detect the binding of the antibody to said modified protein, and diagnose or prognose an acute clinical exacerbation in a CF subject.

In a further example, an antibody or a ligand specifically binds a modified form of a neutrophil derived protein selected from the group consisting of an isoform of leukocyte elastase inhibitor, an isoform of α-enolase, an isoform of Rho GDP- dissociation inhibitor, an isoform of annexin I, an isoform of annexin III, an isoform of

calgranulin C and an isoform of catalase. Alternatively, an antibody specifically binds a modified form of α-1 antitrypsin.

In another example, an antibody, or ligand specifically binds to an unmodified form of a protein. Such an antibody is of particular use in diagnosis or prognosis of an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinieal exacerbation in a CF subject wherein said exacerbation induces a reduction in the amount of the unmodified form of the protein.

In another example, an antibody, or ligand binds to both a modified form and an unmodified form of a protein. Such an antibody enables the skilled artisan to determine the relative concentration of each of these forms of a protein and diagnose or prognose a disease.

Optionally, the kit further comprises means for the detection of the binding of an antibody, fragment thereof or a ligand to a neutrophil derived protein and/or an isoform of α-1 antitrypsin. Such means include a reporter molecule such as, for example, an enzyme (such as horseradish peroxidase or alkaline phosphatase), a substrate, a cofactor, an inhibitor, a dye, a radionucleotide, a luminescent group, a fluorescent group, biotin or a colloidal particle, such as colloidal gold or selenium. Preferably such a reporter molecule is linked to the antibody or ligand.

In another example, a kit according to any embodiment of the present invention comprises an antibody or fragment thereof or a ligand that is capable of specifically binding to an isoform of α-1 antitrypsin for the diagnosis or prognosis of an acute clinical exacerbation of CF.

In another example, a kit according to any embodiment of the present invention comprises an antibody or fragment thereof or a ligand that is capable of specifically binding to a modified form of α-1 antitrypsin for the diagnosis of prognosis of an acute clinical exacerbation of CF.

In another example, a kit according to any embodiment of the present invention comprises an antibody or fragment thereof or a ligand that is capable, of specifically binding to an isoform of myeloperoxidase for the diagnosis of prognosis of an acute clinical exacerbation of CF.

The present invention also provides a kit comprising a neutrophil derived protein or an immunogenic fragment or epitope thereof, and/or α-1 antitrypsin or an immunogenic fragment or epitope thereof and/or mixtures thereof. Such a kit is suitable for detecting an antibody against a neutrophil derived protein and/or α-1 antitrypsin and/or mixtures thereof in a sample from a subject, e.g., in a method described herein according to any embodiment.

In yet another example, a kit additionally comprises a reference sample, for example, a protein sample derived from a sample isolated from one or more CF subjects suffering from a clinical exacerbation. Alternatively, a reference sample comprises a sample isolated from one or more CF subjects that do not suffer from a clinical exacerbation, or one or more normal healthy individuals. Such a reference sample is optionally included in a diagnostic or prognostic assay. Results obtained from a sample are compared to results obtained from a reference sample facilitating a diagnosis or prognosis of the clinical status of the patient from whom the sample was isolated.

In another example, a reference sample comprises a peptide that is detected or bound by an antibody or a ligand. Preferably, the peptide is of known concentration. Such a peptide is of particular use as a standard. Various known concentrations of such a peptide are detected using a prognostic or diagnostic assay described herein. Accordingly, these results may be used to determine concentration of a neutrophil derived protein and/or an isoform of α-1 antitrypsin in a sample derived from a subject, facilitating a diagnosis or prognosis of the clinical state of said subject.

In a related example, the peptide is the peptide against which an antibody was raised. Such a peptide is of particular use in control samples in an assay. In such samples saturating amounts of the peptide is added to a sample in addition to an antibody that binds a neutrophil derived protein and/or an isoform of α-1 antitrypsin. Accordingly, this will block the binding of said antibody. Such a sample acts as a negative control, in which the specific binding of said antibody is determined.

In yet another example, a kit optionally comprises means for sample preparation. Preferably such means are means of solubilizing sputum, such as, for example, a detergent (e.g. tributyl phosphine, C7BZO, dextran sulfate, or Polyoxyethylenesorbitan monolaurate).

Optionally, the kit is packaged with instructions for use, e.g., in a method as described herein according to any embodiment.

The present invention also provides. a method of treatment of a CF subject suffering from an inflammatory condition and/or an infection of the respiratory tract and/or pulmonary deterioration and/or an acute clinical exacerbation, said method comprising performing a diagnostic or prognostic method described herein and recommending treatment or treating said subject with a therapeutic compound for said inflammatory condition and/or infection and/or pulmonary deterioration and/or acute clinical exacerbation.

In another example, the present invention provides a method of treatment of a CF subject suffering from an acute clinical exacerbation comprises performing a diagnostic or prognostic method described herein according to any embodiment; identifying the source of said exacerbation using a method known in the art; and recommending treatment or treating said subject with a suitable therapeutic compound and, optionally, monitoring the effectiveness of said treatment using a method described herein according to any embodiment.

The present invention is further described with reference to the following non-limiting examples.

Example 1 Sample groups for sputum analysis.

Sputum and blood samples were isolated from 20 healthy individuals and 20 CF patients suffering from an acute clinical exacerbation, in addition to 13 CF patients that had been treated for an acute clinical exacerbation and discharged from hospital. The clinical data regarding the subjects from which the samples were isolated are shown in Table 1.

Table 1. Summary of clinical data associated with all recruited adult subjects.

BMI, body mass index; F, female; FEV ₁ , forced expiratory volume in 1 second; FSV, fat soluble vitamins; HM, herbal medicine; M, male; N/A, not applicable; neb, nebulised; NS, natural supplement; PERT, pancreatic enzyme replacement therapy; SaIb, salbutamol.

Sputum was also isolated from 5 healthy control children (aged 8 to 14 years) and 7 CF children. Clinical data relating to the children are set forth in Table 2.

Table 2. Summar of clinical data associated with all recruited child sub ects

Example 2 Preparation of Sputum Samples for Analysis

Sputum was aspirated from a patient's lungs, using the method described in Gershman, N. H. et alJ. Allergy Clin. Immunol. 10(4), 322-328 (1999).

Sputum isolated from CF patients is known to have increased viscosity due to the hypersecretion of mucins in addition to increased amounts of extracellular DNA and actin filaments from bacteria (for example Pseudomonas aeruginosa) and human inflammatory cells (e.g. necrosing neutrophils). Accordingly, prior to any analysis of sputum from CF patient it is necessary to reduce this viscosity, or solubilize the sputum sample. Conditions used for sputum solubilization are set forth in Table 3.

Sputum samples are then solubilized prior to analysis, essentially as described by, Out et al, Monaldi. Arch. Chest Dis. 56(6): 493-499, 2001.

Solubilization was compared to that observed in the vehicle control, and was assessed on the reduction in visco-elasticity and increased solubilization of particulate matter.

Example 3 2-dimensional gel electrophoresis analysis of sputum samples

Six hundred micrograms of protein prepared in Example 2 was used in the analysis of sputum proteins in CF patients. Sputum proteins (<10OkDa) from 20 healthy control subjects, 20 CF patients with an acute clinical exacerbation and 13 hospital discharged CF patients were analyzed by 2-DE over the pH ranges 4-7 and 6-11.

Prior to analysis, 600μg of sputum protein was made up to a final volume of 210μl in a 7M urea/2M thiourea/5mM tris/2% CHAPS sample buffer. The sample was then ultrasonicated for 30 seconds, before being reduced and alkylated with tributyl phosphine and acrylamide, respectively.

Prior to rehydration of IPG strips, samples were ultrasonicated for 2 minutes and then centrifuged at 21000 x g for 5 minutes. The supernatant was collected and lOμl of Orange G (Sigma) added as an indicator dye.

First Dimension

Dry 11cm IPG strips (Amersham-Pharmacia Biotech., Uppsala, Sweden) were rehydrated for 8 hours with 210μl of protein sample. Rehydrated strips were focused on a Protean IEF Cell (Bio-Rad, Hercules, CA) or Proteome System's IsoElectrIQ electrophoresis equipment (Proteome Systems Ltd, Sydney Australia) for 120 kVhr at a

maximum . of 10 kV. Focused strips were then equilibrated in urea/SDS/Tris-HCl buffer.

Second Dimension Equilibrated strips were inserted into loading wells of 6-15% (w/v) tris-acetate SDS- PAGE pre-cast prototype 10cm x 15cm GelChips (Proteome Systems, Sydney Australia) or 14% homogenous SDS-PAGE gels. Electrophoresis was performed at 5OmA per gel for 1.5 hours, or until the tracking dye reached the bottom of the gel. Proteins were stained using SpyroRuby (Molecular Probes) or Direct Blue 71. Gel images were scanned using an Alphalmager System (Alpha Innotech Corp.)

Example Gel Images are shown in Figures 1, 2 and 3.

Example 4 Image analysis of 2-DE gels from sputum samples

The 2-DE gels described in Example 3 were run in at least triplicate and proteins visualized by SPYRO Ruby Staining. Fluorescent images were then collected using an Alphalmager System. Image analysis was then performed using the image analysis software ImagepIQ (available from Proteome Systems Limited, North Ryde, Sydney, Australia).

The best resolved gel from each replicate was then chosen as a representative image for analysis.

The following strategy was then used to analyze sputum images:

1. Spot detection coupled with segmentation (boundary detection) to determine the integrated intensity and integrated area of spots;

2. Editing images to sharpen the accuracy of spot data, i.e. eliminating artefacts; 3. Choosing anchor points to synchronize or warp each of the gel images to maximize the efficiency of gel overlaying;

4. Choosing a reference image for matching;

5. Matching of gel images;

6. Normalization of spot volumes to correct for differences in protein load and staining per gel and image acquisition parameters; and

7. Generation of result reports that contain information on the matches and spot data. These reports can be queried to find, for example, all the spots that are unique to a particular group of gels

Default parameters were used for spot area thresholds and intensity thresholds. The spot volumes were normalized using the formula: normalized volume — (individual spot volume)/(total volume), wherein total volume = (the sum of the volumes for all of the spots detected on the gel).

Gel images were compared between patient samples electrophoresed between the pH range of 4-7 or the pH range of 6-11. A minimum of 3 anchor points were used for registration, and orientation of gels.

The complete matching algorithm of ImagepIQ was used, where all gels uploaded as reference gels were compared to all gels uploaded as target gels. After normalization, the results report was queried to identify protein spots common to all gel images identified in a particular sample group.

The major differences observed between healthy control patients and CF patients with an infection were protein trains in the sputum of healthy controls that were not observed in CF patients with an infection, and an increase in expression of lower molecular weight proteins and protein fragments in sputum from CF patients with an acute clinical exacerbation. It was interesting that the gel images from CF patients that had been discharged from hospital more closely resembled that images of healthy controls.

Clinical criteria were used to define 'well' CF children as a second control group to exacerbated CF adults. Despite all the CF children having FEVi values similar to healthy controls (Table 2), subjects 64 and 69 presented sputum profiles clearly indicating signs of inflammation as observed in exacerbated CF adults.

Interestingly, CF child subject 64 was clinically diagnosed as having a flare of allergic bronchopulmonary aspergillosis 96 days after sputum collection and analysis. An elevation of total IgE associated with a drop in lung function, new infiltrates and acute respiratory signs were found at the time of the flare. Child CF subject 69 was clinically diagnosed 49 days later with a flare of Staphylococcus aureus infection. The remaining

5 CF children analyzed did not exhibit any signs of ensuing exacerbation. This supports the proteomic profile data as predictive for exacerbation onset. Children with a 2-DE profile similar to an adult subject suffering from an acute clinical exacerbation are referred to as having an "exacerbated-like" condition.

Example 5 Identification of proteins that are differentially expressed between CF and healthy control sputum

A number of proteins were found to be consistently expressed in the majority, and in some cases all, of the CF patients with an acute clinical exacerbation, but not in healthy control subjects. These proteins were then identified by mass spectrometry in multiple CF patients.

Prior to mass spectrometry it was necessary to prepare protein samples by in-gel tryptic digestion Protein gel pieces were excised, destained, digested and desalted using an Xcise™, an excision/liquid handling robot (Proteome Systems, Sydney, Australia and Shimadzu-Biotech, Kyoto, Japan) in association with the Montage In-GeI Digestion Kit (developed by Proteome Systems and distributed by Millipore, Billerica, Ma, 01821, USA). Prior to spot cutting, the 2-D gel was incubated in water to maintain a constant size and prevent drying. Subsequently, the 2-D gel was placed on the Xcise, a digital image was captured and the spots to be cut were selected. After automated spot excision, gel pieces were subjected to automated liquid handling and in-gel digestion. Briefly, each spot was destained with 100 μl of 50% (v/v) acetonitrile in 50 mM ammonium bicarbonate. The gel pieces were dried by adding 100% acetonitrile, the acetonitrile was removed after 5 seconds and the gels were dried completely by evaporating the residual acetonitrile at 37°C. Proteolytic digestion was performed by rehydrating the dried gel pieces with 30 μl of 20 mM ammonium bicarbonate (pH 7.8) containing 5 pg/mL modified porcine trypsin and incubated at 3O ⁰ C overnight.

Ten μl of the tryptic peptide mixture was removed to a clean microtitre plate in the event that additional analysis by Liquid Chromatography (LC) - Electrospray Ionisation (ESI) MS was required. ^' . ^■

Automated desalting and concentration of tryptic peptides prior to MALDI-TOF MS was performed using Cl 8 ZipTip (Millipore, Bedford, MA). Adsorbed peptides were eluted from the tips onto a 384-position MALDI-TOF sample target plate (Kratos, Manchester, UK) using 2 μl of 2 mg/ml α-cyano-4-hydroxycinnamic acid in 90% (v/v) acetonitrile and 0.085% (v/v) TFA.

Digests were analyzed using an Axima-CFR MALDI-TOF mass spectrometer (Kratos, Manchester, UK) in positive ion reflectron mode. A nitrogen laser with a wavelength of 337 ran was used to irradiate the sample. The spectra were acquired in automatic mode in the mass range 600 Da to 4000 Da applying a 64-point raster to each sample spot. Only spectra passing certain criteria were saved. All spectra underwent an internal two point calibration using an autodigested trypsin peak mass, m/z 842.51 Da and spiked adenocorticotropic hormone (ACTH) peptide, m/z 2465.117 Da. Software designed by Proteome Systems, as contained in the web-based proteomic data management system BioinformatlQ ^m (Proteome Systems), was used to extract isotopic peaks from MS spectra.

Protein identification was performed by matching the monoisotopic masses of the tryptic peptides (i.e. the peptide mass fingerprint) with the theoretical masses from protein databases (Hnman-Pseudomonas aeruginosa-Staphylococcus aureus) using IonlQ ² database search software (Proteome System Limited, North Ryde, Sydney, Australia). Querying was done against the non-redundant SwissProt (Release 40) and TrEMBL (Release 20) databases (June 2002 version), and protein identities were ranked through a modification of the MOWSE scoring system. Propionamide-cysteine (cys-PAM) and oxidized methionine modifications were taken into account and a mass tolerance of 100 ppm was allowed.

Miscleavage sites were only considered after an initial search without miscleavages had been performed. The following criteria were used to evaluate the search results: the MOWSE score, the number and intensity of peptides matching the candidate protein, the coverage of the candidate protein's sequence by the matching peptides and the gel location.

In addition, or alternatively, proteins were analyzed using LC-ESI-MS. Tryptic digest solutions of proteins (10 μl) were analyzed by nanofiow LC/MS using an LCQ Deca

Ion Trap mass spectrometer (ThermoFinnigan, San Jose, CA) equipped with a Surveyor

LC system composed of an autosampler and pump. Peptides were separated using a PepFinder kit (Thermo-Finnigan) coupled to a Cl 8 PicoFrit column (New Objective). Gradient elution from water containing 0.1% (v/v) formic acid (mobile phase A) to 90% .(v/v) acetonitrile containing 0.1% (v/v) formic acid (mobile phase B) was performed over a 30-minute period. The mass spectrometer was set up to acquire three scan events - one full scan (range from 400 to 2000 amu) followed by two data dependant MS/MS scans.

Proteins were identified using TurboSequest (Thermo-Finnigan) software making use of a combined Huτnan-Pseudomonas aeruginosa-Staphylococcus aureus database created from SwissProt and TrEMBL protein entries (non-redundant, June 2002 version). Peptides were identified from MS/MS spectra in which more than half of the experimental fragment ions matched theoretical ion values, and gave cross-correlation

(a raw correlation score of the top candidate peptide), delta correlation (difference in correlation between the top two candidate peptides) and preliminary score (raw score used to rank candidate peptides) values greater than 2.2, 0.2, and 400, respectively.

Those proteins that represented potential diagnostic or prognostic markers of an acute clinical exacerbation are shown in Table 4.

Semi-quantitative differential display analysis was performed to establish the amounts of the proteins in Table 4 in a CF patient with an acute clinical exacerbation, and following treatment for said infection. Normalized spot volumes were estimated using

ImagepIQ and compared between pre- and post-treatment results.

Table 4 Proteins dia nostic of an acute clinical exacerbation in a CF atient

^a A Molecular Weight as defined herein is determined by 2-dimensional gel electrophoresis or SDS/Page shall be understood to include the stated molecular weight ±1 kDa. ^b An isoelectric point (pi) is determined by 2-dimensional gel electrophoresis or isoelectric focusing shall be understood to include the stated pi ± 0.25 units. Spot number relates to the corresponding spot number in Figure 1.

Example 6

Analysis of proteins that are modified in CF patients with an acute clinical exacerbation.

In addition to differential expression of proteins between the sample groups, some proteins were modified in response to an acute clinical exacerbation in CF patients. In

particular it was found that and annexin I, annexin III, leukocyte elastase inhibitor, α- enolase and Rho-GDP dissociation inhibitor 2 showed a change in molecular weight and pi in CF patients with an acute clinical exacerbation, when compared to healthy control subjects and hospital discharged CF patients.

MALDI-TOF and ESI LC MS/MS analysis of annexin I indicates that the protein is being cleaved in CF subjects with an acute clinical exacerbation. It has previously been suggested that annexin I is inactivated by leukocyte elastase by cleavage at the Val36- Ser37 peptide bond, to generate the fragments shown in SEQ ID NO: 8 and SEQ ID NO: 9. Such a cleavage event results in a mass loss of approximately 8 kDa and a change in charge of approximately 1.2 pi units, concurring with the changes observed in CF patients.

Example 7 LC-MS/MS Analysis of unfractionated sputum

Volumes of sputum equivalent to 50 μg of protein from CYFB 1-8 (healthy control), CYFBI-20 (exacerbated CF) and CYFBI-20B . (discharged CF subject, partially recovered) were dried, the pellets resuspended in 50 μl of 4 M Urea/0.4 M ammonium bicarbonate and proteins reduced by the addition of 0.5 μl of 1.0 M DTT. After incubation at room temperature for 2 hours, the samples were alkylated by the addition- of 2.5 μl of 1.0 M iodoacetamide and incubated for an additional 3 hours. The samples were diluted with 62.5 μl of water after which 62.5 μl of trypsin (40 ~Lg/ml) was added and digestion was performed overnight at 37 ⁰ C. Twenty μl of each sample was introduced via the pepfmder nanoflow LC-MS configuration into a ThermoFinnigan LCQ-Deca ion trap mass spectrometer. The LC gradient was developed over 94 min from 0.1% (v/v) formic acid to 0.1% (v/v) formic acid/54% (v/v) acetonitrile. A scan sequence was performed so that each full scan mass spectrum (400-2000 amu) was ^■ followed by two consecutive MS/MS scans of the most intense peaks in each scan. Intense ions found more than twice within a minute were excluded for further fragmentation by MS/MS within the following 3 minutes. Proteins were identified using the TurboSequest (ThermoFinnigan) software, using a combined Human- Pseudomonas aeruginosa-Staphylococcus aureus database created from the SwissProt and TrEMBL databases as above. Matches were considered when more than one peptide from the same protein was found within any of the samples, with a Sequest scoring of more than 2.2. The amount of individual proteins present in the samples was

obtained from the peak intensity of the most abundant peptide identified within the protein.

Using this approach the proteins of Table 4 are detected. Furthermore it is shown that the amount of each of the proteins or isoforms detected are in a similar ratio between the sample groups as those detected in Example 5.

Example 8

Immuno-profϊling of CF sputum samples

As CF subjects suffer from cyclic inflammation that develops into a chronic inflammation that in turn manifests itself as recurrent phases of exacerbation and recovery, the immune system of CF subjects is frequently induced. This leads to the generation of auto-antibodies against cytoplasmic proteins/enzymes from cells of the CF subject.

Accordingly, using such auto-antibodies in immuno-profϊling experiments is a useful process for identifying proteins that are involved in acute clinical exacerbations.

Sputum samples were prepared essentially as described herein-above, and immunoprofiling performed essentially as described in Ballot et ah, Clin Chem. 49:634-643 2003. Essentially, proteins isolated from sputum samples from CF subjects or normal, healthy individuals, were separated using 1 -dimensional and/or two- dimensional gel electrophoresis, the proteins transferred to a membrane and probed with plasma from a CF subject suffering from an acute clinical exacerbation. Bound antibodies were then detected with an anti-human antibody labeled with horseradish peroxidase and binding detected with chemiluminescence.

Immuno-matching was employed to identify antigenic targets commonly expressed in exacerbated CF subjects. Interestingly, many proteins that were detected in immunoprofiling experiments were found in the majority of patient samples tested.

Protein spots detected using immuno-profiling methods were then identified using mass spectrometry (essentially as described in Example 5). The protein identification data on the immuno-reactive antigens was determined by overlaying the immuno-profiles

19

onto duplicate two-dimensional gel electrophoresis protein gel upon which proteins are visualized via Coomassie staining or Sypro Ruby staining and as such can be subsequently identified by mass spectrometry. Using this approach α-enolase (spot 1) and Calgranulin C (spot 2) were identified (as shown in Figures 3A and B).

Example 9 _;

Detection of antibodies that bind enolase in a CF subject

6.1 Biological samples Clinical CF samples were collected and crude plasma isolated using a capture column. The crude plasma used from the capture column were combined from four exacerbated CF adults in the age group 22- to 37-years old. Predicted FEVi values were between 22-65 % and the subjects have had 2-4 exacerbations in the last 12 months. Microbiological testing was performed on collected sputum samples. All adult CF subjects used in current study had profuse P. aeruginosa growth in the lungs. In addition, one CF adult also had pulmonary S. aureus infection.

Saline-induced sputum was collected from healthy control- and CF subjects and subsequently liquefied. Sputum used for 2DE arrays was acetone precipitated and proteins were resuspended in the CHAPS containing ProteomlQ Resuspension Reagent (Proteome Systems, Woburn, US). The samples were cleaned up on 100 kDa and 5 kDa spin columns (Millipore, Billerica, MA).

Sputum used for immuno-capturing: Sputum samples were pooled from two exacerbated CF subjects of 22 and 31- years old (total of 16mL). They had predicted

FEVi values of 14 % and 51 % and had been treated for 1-2 exacerbations in the last 12 months. Microbiological testing showed profuse P. aeruginosa in the lungs of both patients. In addition, one of the two patients also contained profuse S. aureus. The pooled sputum were incubated with 30 mM IAA to inactivate residual DTT used in the liquifϊcation protocol and IgG depleted by using Protein G coupled Sepharose beads as recommended by manufacturer (Amersham Pharmacia (Uppsala, Sweden);

6.2 Preparation of an immunocapture column

An immuno-capture column was generated from a total of 5 mL pooled plasma from five exacerbated CF patients (total protein concentration of -40 mg/mL). IgG was bound to Protein G sepharose by incubating the pooled plasma with 10 mL 50% slurry

of Protein G sepharose. The matrix was washed in 10 mM PBS pH 7.4 and bound IgG was irreversibly immobilized utilizing DSS. The generated column is referred to as the capture column.

6.3 Capture of immunogenic protein from CF samples

The capture column was incubated overnight with the captured plasma from CF subjects (as described in Example 6.1) at 4 ⁰ C at constant rotation and beads were subsequently harvested by centrifugation. The flow-through was collected and saved for subsequent incubation ^' steps (the protein extract was passed over the capture column three times in each capture). The harvested beads were washed 3 times in 10 mM PBS pH 7.4 and captured proteins were eluted with 5OmM glycine pH 2.7. The column was extensively washed with first 50 mM glycine pH 2.7 then 10 mM PBS pH 7.2 prior subsequent incubation steps.

Eluted proteins were alcohol precipitated and subsequently resolubilized in Cellular and Organelle Membrane solubilizing reagent from the ProteoPrep Universal Extraction kit (Sigma, St. Louis, MO). Following the instruction in the ProteoPrep kit the solubilized proteins were reduced and alkylated with a final concentration of 5 mM tri-n-butylphophine and 10 mM acrylamide, respectively.

6.4 Two-dimensional gel electrophoresis

Eleven centimeter pH 3-10 or pH 4-7 PGs were purchased from Amersham (Uppsala, Sweden). Isoelectric focusing was conducted as per manufacturer's instructions using an IsoElectrIQ ² unit from Proteome Systems (Wobura, MA). Second dimension 6-15% or 14% homogenous Tris-Acetate Gelchip gels were run as recommended by manufacturer (Proteome Systems, Woburn, MA). Arrayed proteins were visualized by silver-staining (Shevchenko et ah, Mass spectrometric sequencing of proteins silver- stained polyacrylamide gels. 68, 850-858. 1996) or transferred to PVDF-P membranes (Millipore, Billerica, MA) by using semi-wet membrane-blotting cassettes accompanying the IsoElectrIQ ² unit from Proteome Systems (Woburn, US).

6.5 MS analysis

Proteins spots of interest were excised and washed twice in 100 mM NH ₄ HCO ₃ : 50% acetonitrile (ACN) pH 8.2 and dehydrated at 50degC for 30 minutes. Proteins were digested as described by Katayama et al (Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight

mass spectrometry) and digested for 3 hours at 37degC. Tryptic peptides was extracted by sonication and purified as described by Kussmann et al. Peptides were eluted with ~1.5μl MALDI matrix solution (70% ACN, 0.1% TFA, 1.5mg/ml alpha-cyano-4- hydroxycinnamic acid (Sigma, St. Louis, MO). Peptide mass fingerprints (PMF) were generated by matrix-assisted laser desorption/ionization-time-of-flight- mass spectrometry (MALDI-TOF-MS) using an Axima CFR (Rratos, Manchester, UK) or an ABI MALDI MS/MS (AME Bioscience, London, UK).

6.6 Results As shown in Figure 3, 14 proteins were isolated from the sputum of CF subjects. Of these human α enolase (enolase 1) was identified as spot 12 using mass spectrometry.

Example 10

Antibodies that detect an unmodified form of Annexin I

Annexin I is cleaved in response to an acute clinical exacerbations. Accordingly, a peptide spanning the predicted site of cleavage of this protein is generated. The sequence of this peptide is:

GIy GIy Pro GIy Ser Ala VaI Ser Pro Tyr Pro Thr Phe Asn Pro (SEQ ID NO: 10)

Peptide antigens and peptide probes are synthesized essentially using the methods described in Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer- Verlag, Heidelberg and Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg.

Peptides are purified using HPLC and purity assessed by amino acid analysis.

Female BalB/c mice are immunized with a purified form of the peptide (SEQ ID NO: 10). Initially mice are sensitized by intraperitoneal injection of Hunter's Titermax adjuvant (CytRx Corp., Norcross, GA,). Three boosts of the peptide are administered at 2, 5.5 and 6.5 months post initial sensitization. The first of these boosts is a subcutaneous injection while the remaining is administered by intraperitoneal injection. The final boost is administered 3 days prior to fusion.

The splenocytes of one of the immunized BALB/c mice are fused to X63-Ag8.653 mouse myeloma cells using PEG 1500. Following exposure to the PEG 1500 cells are incubated at 37 ⁰ C for 1 hour in heat inactivated fetal bovine serum. Fused cells are then transferred to RPMI 1640 medium and incubated overnight at 37 ⁰ C with 10% CO ₂ . The following day cells are plated using RPMI 1640 media that has been supplemented with macrophage culture supernatants. ,

Two weeks after fusion, hybridoma cells are screened for antibody production by solid phase ELISA assay. Standard microtitre plates are coated with isolated annexin 1 (essentially as described' by Weng et al, Protein ScL 2(3), 448-458, 1993) in a carbonate based buffer. Plates are then blocked with BSA, washed and the test samples (i.e. supernatant from the fused cells) added, in addition to control samples, (ie supernatant from an unfused cell). Antigen-antibody binding is detected by incubating the plates with goat-anti-mouse HRP conjugate (Jackson ImmunoResearch Laboratories) and then using ABTS peroxidase substrate system (Vector Laboratories, Burlingame, Ca 94010, USA). Absorbance is read on an automatic plate reader at a wavelength tof 405 nm.

Any colonies that are identified as positive by this screen continue to be grown and screened for several further weeks. Stable colonies are then isolated and stored at -8O ⁰ C.

Positive stable hybridomas are then cloned by growing in culture for a short period of time and diluting the cells to a final concentration of 0.1 cells/well of a 96 well tissue culture plate. These clones are then screened using the previously described annexin I assay. This procedure is then repeated in order to ensure the purity of the clone.

Four different dilutions, 5 cells/well, 2 cells/well, 1 cell/well, 0.5 cells/well of the primary clone are prepared in 96-wells microtiter plates to start the secondary cloning. Cells are diluted in BVIDM tissue culture media containing the following additives: 20% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml of penicillin, 100 μg/ml of streptomycin, 1% GMS-S, 0.075% NaHCO ₃ . To determine clones that secrete anti-annexin I antibody, supernatants from individual wells of the 0.2 cells/well microtiter plate are withdrawn after two weeks of growth and tested for the presence of anti-annexin I antibody by ELISA assay as described above.

All clones are then adapted and expanded in RPMI media containing the following additives: 10% FBS, 2 mM L-glutamine, 100 units/ml of penicillin, 100 μg/ml of streptomycin, 1% GMS-S, 0.075% NaHCO ₃ , and 0.013 mg/ml of oxalaacetic acid. A specific antibody is purified by Protein A affinity chromatography from the supernatant of cell culture.

Example 11 Assessment of specificity of anti-annexin I antibody

Purified annexin I is combined with P. aeruginosa (Calbiochem, San Diego, Ca 92121, USA) and incubated at 37 ⁰ C. Following incubation samples are ethanol precipitated, supernatant fluid removed and the samples freeze-dried. Samples are then reconstituted and adsorbed onto a microtitre plate. Purified annexin I is adsorbed onto another microtitre plate.

Cell culture supernatant from the hybridomas generated in Example 10 are then screened to determine those that produce an antibody that is able to specifically recognise the uncleaved form of annexin I.

Microtitre plates are blocked with BSA, washed and then the test samples (ie supernatant from a hybridoma) is added. Additionally, control samples are used (i.e. supernatant from an unfused cell). Antigen-antibody binding is detected by incubating the plates with goat-anti-mouse HRP conjugate (Jackson ImmunoResearch Laboratories) and then using ABTS peroxidase substrate system (Vector Laboratories, Burlingame, Ca 94010, USA). Absorbance is read on an automatic plate reader at a wavelength tof 405 nm.

An antibody that is able to specifically recognise the uncleaved form of annexin I is of particular use in a diagnostic and/or prognostic assay.

Example 12

An immunoassay to diagnose or prognose an acute clinical exacerbation in a CF

. patient.

The antibody of Example 11 is adsorbed to a microtitre plate at a concentration appropriate for detecting the presence of annexin I in a biological sample. Samples are

adsorbed. for 1 hour at room temperature or overnight at 4 ⁰ C. Plates are then washed with tris-buffered saline. Plates are blocked with BSA in TBS and then washed with TBS.

A positive control, i.e. isolated annexin I, is included in the assay at various concentrations. Additionally one or more biological samples, ie sputum samples, isolated from a normal and/or healthy individual is included, to determine the amount of uncleaved annexin I that is observed in a control individual. These samples are each added to a well of the microtitre plate.

A diluted biological sample isolated from a CF subject is added to the microtitre plate.

Samples are then incubated for approximately 1 hour before being washed with TBS containing 0.01 % Tween 20 (Sigma Aldrich).

A dilution (in TBS) of a secondary, goat-anti-human HRP conjugated antibody • (Jackson ImmunoResearch Laboratories) is then added to each of the wells of the microtitre plate. Samples are again washed with TBS and Tween, before binding of the secondary antibody is measured using ABTS peroxidase substrate system (Vector Laboratories, Burlingame, Ca 94010, USA). Absorbance is read on an automatic plate reader at a wavelength of 405 nm.

The absorbance detected in the control (normal and/or healthy individual) sample/s are then compared to the samples of known concentration in order to determine the approximate concentration of annexin I in these samples. The absorbance detected in the test sample/s is also compared to this standard in order to determine the amount of

' protein in this sample.

These results are then used to determine the clinical status of the CF subject. If there is less uncleaved annexin I detected in the sample from CF subject than a sample from a normal healthy individual, this indicates that the CF subject is suffering from, or will soon develop an acute clinical exacerbation.

Example 13 An antibody to recognise an isoform of calgranulin C

A set of peptides spanning the calgranulin C polypeptide (SEQ ID NO: 5) are generated using methods known in the art, such as, for example as described n Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer- Verlag, Heidelberg and Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg. These peptides are approximately 15 amino acids long, and are generated using a 6 amino acid moving window.

Each of these peptides is then used to generate a monoclonal antibody essentially as described in Example 10. The cell supernatant of the fused cells is screened in a solid phase ELISA assay, however, in this case recombinant human calgranulin C is used.

Recombinant human calgranulin C is produced and purified essentially as described in Gottsch and Liu Curr. Eye Res. 17(9), 870-874, 1998. This protein is then adsorbed to a microtitre plate. The protein is incubated (in TBS) in each of the wells of the microtitre plate either for 3-4 hours at room temperature or overnight at 4 ⁰ C. Each well is then washed three times with TBS, and then blocked with TBS and 5% skim milk powder for a period of 1 hour. Samples (i.e. monoclonal antibodies) are then added and incubated at room temperature for 1 hour before all wells are washed with TBS and 0.1% skim milk powder and 0.01% Tween 20.

A dilution (in TBS) of a secondary, goat-anti-mouse HRP conjugated antibody (Jackson ImmunoResearch Laboratories) is then added to each of the wells of the microtitre plate and incubated for approximately 1 hour. Samples are again washed three times with TBS and Tween, before binding of the secondary antibody is measured using ABTS peroxidase substrate system (Vector Laboratories, Burlingame, Ca 94010, USA). Absorbance is read on an automatic plate reader at a wavelength tof 405 nm.

Those cells that show antibodies that are able to bind calgranulin C are selected for cloning, and the clones again selected by this method.

The titer of the antibodies produced using this method are determined using the Easy Titer kit available from Pierce (Rockford, II, USA). This kit utilises beads that specifically bind mouse antibodies, and following binding of such an antibody these

beads aggregate and no longer absorb light to the same degree. Accordingly, the amount of an antibody in the supernatant of a hybridoma may be assessed by comparing the OD measurement obtained from this sample to the amount detected in a standard, such as for example mouse IgG.

Example 14

An ELISA to detect the presence of an enhanced level of calgranulin C in a CF patient suffering from an acute clinical exacerbation

The antibody of Example 13 is adsorbed to a microtitre plate at a concentration appropriate for detecting the presence of calgranulin C. Samples are adsorbed for 1 hour at room temperature or overnight at 4 ⁰ C. Plates are then washed with tris buffered saline. Plates are blocked with BSA in TBS and then washed with TBS.

As a positive control purified recombinant calgranulin C is included in the assay at various concentrations, i.e. as a standard curve. Additionally, one or more biological samples, i.e. sputum samples, isolated from a normal healthy individual is also included, in order to determine the amount of calgranulin C that is observed in a control individual. These samples are added to a well of the microtitre plate.

A diluted biological sample isolated from one or more CF patients is added to the microtitre plate.

Samples are then incubated for approximately 1 hour before being washed with TBS containing 0.01% Tween 20 (Sigma Aldrich).

A dilution (in TBS) of a secondary, goat-anti-human HRP conjugated antibody (Jackson ImmunoResearch Laboratories) is then added to each of the wells of the microtitre plate. Samples are again washed with TBS and Tween, before binding of the secondary antibody is measured using ABTS peroxidase substrate system (Vector Laboratories, Burlingame, Ca 94010, USA). Absorbance is read on an automatic plate reader at a wavelength tof 405 nm.

The absorbance detected in the control (normal healthy individual) sample/s are then compared to the samples of known concentration in order to determine the approximate concentration of calgranulin C in these samples. As calgranulin C is not normally

observed unless a subject is suffering from an acute clinical exacerbation or is suffering from a respiratory infection, it is expected that there will be no detectable levels of calgranulin C in the normal healthy control. The absorbance detected in the test sample/s is also compared to the standard in order to determine the amount of protein in this sample.

These results are then used to determine the clinical status of the CF saubject. If there is more clagranulin C detected in the CF subject than a normal healthy individual, this suggests that the CF subject is suffering from, or will soon develop, an acute clinical exacerbation.

Example 15 A multiplexed assay for detection of an acute clinical exacerbation in a CF subject

The antibodies of example 11 and example 13 are adsofbed to a microtitre plate, each to a separate well, at a concentration appropriate for detecting the presence of annexin I and calgranulin C respectively. Plates are then blocked with BSA in TBS and then washed with TBS.

A fraction of a biological sample is then added to each of these wells such that each of these wells is effectively exposed to the same biological sample. Samples are incubated for 1 hour at room temperature or overnight at 4 ⁰ C. Plates are then washed with tris-buffered saline.

As a positive control purified recombinant Annexin I and/or calgranulin C (as determined by the antibody used) is included in the assay at various concentrations, ie as a standard curve. Additionally, one or more biological samples, ie sputum samples, isolated from a normal and/or healthy individual is also included, in order to determine the amount of annexin I and calgranulin C that is observed in a control individual. These samples are added to a well of the microtitre plate.

A diluted biological sample isolated from a CF patients is added to the microtitre plate.

Samples are then incubated for approximately 1 hour before being washed with TBS containing 0.01 % Tween 20 (Sigma Aldrich).

A dilution (in TBS) of a secondary, goat-anti-human HRP conjugated antibody (Jackson ImmunoResearch Laboratories) is then added to each of the wells of the microtitre plate. Samples are again washed with TBS and Tween, before binding of the secondary antibody is measured using ABTS peroxidase substrate system (Vector Laboratories, Burlingame, Ca 94010, USA). Absorbance is read on an automatic plate reader at a wavelength tof 405 nm.

The absorbance detected in the control (normal healthy individual) sample/s is/are then compared to the samples of known concentration in order to determine the approximate concentration of unmodified annexin I and calgranulin C in these samples. As annexin I is not cleaved unless a subject is , suffering from an acute clinical exacerbation, a higher concentration of uncleaved annexin I will be detected in a normal and/or healthy subject than that detected in a CF subject suffering from an acute clinical exacerbation. Furthermore, as calgranulin C is not normally observed unless a subject is suffering from an acute clinical exacerbation or is suffering from a respiratory infection, there will be reduced detectable levels of calgranulin C in the normal healthy control. The absorbance detected in the test sample/s is also compared to the standard in order to determine the amount of protein in this sample.

These results are then used to determine the clinical status of the CF saubject. If there is less uncleaved annexin I and/or more calgranulin C detected in the CF subject than a normal healthy individual, this suggests that the CF subject is suffering from, or will soon enter, an acute clinical exacerbation. The provision of multiple markers of an acute clinical exacerbation increases the accuracy of this assay, as it is preferred (although not essential) that an enhanced amount of uncleaved annexin I and more calgranulin C be detected in a biological sample derived from a CF subject that is developing an acute exacerbation in order to produce an accurate diagnosis.

EXAMPLE 16 Detection of modified levels of α-1 antitrypsin and modified forms thereof in subjects suffering from an acute clinical exacerbation

Using methods essentially as described in Examples 1-5 the proteins described in Table 5 were identified as markers of an acute clinical exacerbation.

Semi-quantitative differential display analysis was performed to establish the amounts of the proteins in Table 5 in a CF patient with an acute clinical exacerbation, and following treatment for said infection. * Normalised spot volumes were estimated using ImagepIQ and compared between pre- and post-treatment results.

^a A Molecular Weight as defined herein is determined by 2-dimensional gel electrophoresis or SDS/Page shall be understood to include the stated molecular weight

±1 kDa. ^b An isoelectric point (pi) is determined by 2-dimensional gel electrophoresis or isoelectric focussing shall be understood to include the stated pi ± 0.25 units.

Spot number relates to the corresponding spot number in Figure 1.

Example 16

Analysis of proteins that are modified in CF patients with an acute clinical exacerbation.

In addition to differential expression of proteins between the sample groups, it also appeared that some proteins were modified in response to an acute clinical exacerbation in CF patients. In particular it was found that α-1 antitrypsin showed a change in molecular weight and pi in CF patients with an acute clinical exacerbation, when compared to healthy control subjects and hospital discharged CF patients.

Figure 4 shows the position to several proteins representing different isoforms of α-1 antitrypsin relative to actin. Note the apparent change in both molecular weight (of about 5kDa) and pi (of about 0.2pI units) in a CF patient suffering from an acute clinical exacerbation. Such a change iindicates s suggestive of post-translational

modification of the α-1 antitrypsin protein. Figure 5 shows a similar result in CF children suffering from an exacerbated type state.

Glycoproteomic analysis of the α-1 antitrypsin protein suggest that changes in the mass and charge of the protein are not due to changes in the sugars that are normally found attached to this protein.

MALDI-TOF and ESI LC MS/MS analysis of α-1 antitrypsin suggest indicate that the protein is being cleaved in CF subjects with an acute clinical exacerbation. For example, It has previously been suggested that α-1 antitrypsin is inactivated by P. aeruginsoa by cleavedage at the Pro-357-Met-358 peptide bond, to generate the fragments shown in SEQ ID NO: 12 and SEQ ID NO: 13. Such a cleavage event results in a mass loss of approximately 6.5kDa and a change in charge of approximately 0.2 pi units, concurring with the changes observed in CF patients. Furthermore, the apparent change in mass and charge of α-1 antitrypsin is not observed in CF patients that have been treated for an acute clinical exacerbation and recovered from said infection (as shown in Figure 4). However, samples isolated from those patients that have been treated and do not recover from an acute clinical exacerbation contain a modified α-1 antitrypsin. Accordingly, the monitoring of the modification of α-1 antitrypsin is particularly useful not only in diagnosing an acute clinical exacerbation in CF patients but also monitoring the effectiveness of treatment for said exacerbation.

EXAMPLE 17

Association between, cleavage of alpha- 1 antitrypsin and disease state

17.1 Determining Detecting the amount ofalpha-1 antitrypsin in a subject Relative levels of both cleaved and uncleaved α-1 antitrypsin was determined in samples from in 18 CF adult subjects (CYFB 1-4, -6, -10, -11, -12, -15, -16, -20, -35, - 37, -38, -39, -40, -41, -45, -46, -47 and -48), 15 healthy control adults (CYFB 1-7, -8, - 9, -13, -17, -18, -19, -22, -23, -24, -25, -26, -27, -30, -36), 5 CF children (CYFB2-61, - 63, -64, -65 and -69) and 3 healthy control children (CYFB2-70, -73, -79).

Using the method described in Example 5 to estimate spot volume, we have estimated the sum total for cleaved or non-cleaved α-1 antitrypsin of the previously described

subjects using both 6% and 15% two-dimensional gels (For example as shown in Figure 4).

The healthy adult controls and the healthy control children were found to have significantly greater quantities of the non-cleaved α-1 -antitrypsin relative to the cleaved form (Figures 6 and 7). In contrast, the α-1 -antitrypsin expression pattern in exacerbated CF adults and CF children who had 'exacerbated-like' 2-D profiles of their sputum, shows the cleaved form of the protein dominates relative to the non-cleaved form.

17.2 Statistical analysis of results

Statistical analysis was performed to evaluate the differences between the healthy and CF subjects (both adults and children). In addition, the interaction of disease status (i.e. CF versus healthy control) with subject group (i.e. adult versus children) was also explored using statistical methods.

The sum of normalised spot volumes for the non-cleaved and cleaved forms of α-1 - antitrypsin were analysed for a total of 41 subjects: 23 with CF (18 adults and 5 children) and 18 healthy controls (15 adults and 3 children).

Where it could be ascertained by spot detection that a protein train was not present, protein train volume was recorded as zero. Spot volumes were log transformed before analysis, following the addition of a small positive constant (10) to prevent problems with the logarithm of zero. Spot volumes were analysed for the non-cleaved and cleaved variants of the α-1 -antitrypsin. In addition, the log ratio of non-cleaved to cleaved forms was analysed. Data were analysed using a two-way analysis of variance, with effects for. disease status (control or CF), subject group (adult or child) and the disease status by subject group interaction (the difference in disease status effect between adults and children).

Table 6 shows the analysis of variance results for the non-cleaved, cleaved and the ratio of the non-cleaved: cleaved forms of α-1 -antitrypsin. In each of these instances, the levels of the α-1 -antitrypsin (non-cleaved, cleaved, ratio of non-cleaved: cleaved) are significantly different between the CF and healthy control subjects including both adults and children. In addition, the variance analysis of the cleaved form and the ratio of non-cleaved: cleaved forms of α-1 -antitrypsin shows that their corresponding levels

are statistically significant between the adults and the children (age effect). The effect of age (i.e. subject group) and disease status is also statistically significant and the interaction plot in Figure 8 illustrates the significant effect of age and disease status for the cleaved form of α- 1 -antitrypsin.

The statistical data presented in Table 6 show that measurements of non-cleaved, cleaved, ratio of non-cleaved: cleaved of α-1 -antitrypsin can be are useful ford to defminge a patient's disease status (i.e. CF or healthy control). In addition, measurements of the cleaved and the ratio of non-cleaved: cleaved α-1 -antitrypsin can distinguish between adult and children samples and whether or not the sample was CF adult, CF child, or a healthy control (adult or child).

Table 6. Analysis of variance for log of non-cleaved, cleaved and the log ratio of non- cleaved: cleaved α-1 -antitrypsin normalized spot volumes in CF and healthy control adults and children.

Df Sum Sq Mean Sq F value Pr(>F)

Age 1 1.042 1.042 0.536 0.469

Non-cleaved form of Disease Status 1 45.893 45.893 23.604 0 α-1-antitrypsin

Age:Disease Status 1 0.006 0.006 0.003 0.957

Residuals 37 71,939 1.944

Df Sum Sq Mean Sq F value Pr(>F)

Age 1 15.181 15.181 10.559 0.002

Cleaved form of α-

Disease Status 1 154.826 154.826 107.692 0

JL-antitrypsin

Age:Disease Status 1 15.554 15.554 10.818 0.002

Residuals 37 53.194 1.438

Df Sum Sq Mean Sq F value Pr(>F)

Ratio of Non-

Age 1 24.179 24.179 8.518 0.006 cleaved: Cleaved

Disease Status . 1 369.306 369.306 130.104 0 antitrypsin Age:Disease Status 1 16.152 16.152 5.69 0.022

Residuals 37 105.026 2.839

Statistically significant p values less than 0.05 are highlighted in bold. Note that age refers to adult or child status.

From these analyses, it is clear that α-1 -antitrypsin is a marker for CF lung exacerbation. Measuring the levels of the cleaved form as well as the ratio of the non- cleaved: cleaved levels allows significant conclusions to be made regarding the health status of the patient's lung.

EXAMPLE 18 Association between the ratio of normal to cleaved alpha- 1 antitrypsin and disease state

18.1 Samples

Relative levels of both cleaved and uncleaved α-1 antitrypsin was determined from spot volumes essentially as described in Example 5 for fourteen adults with CF (eight of whom suffer from an exacerbation); six healthy controls; seven children suffering from CF; and four healthy control children.

FEVi was also determined for subjects as a measure of lung function.

18.2 Statistical methods

Data for α-1 antitrypsin levels and ratios were analysed using a linear model with terms for disease status (CF or control) and age (adult or child).

Analyses for cleaved α-1 antitrypsin levels or ratio of normal to cleaved α-1 antitrypsin were performed using log transformed data to stabilize variances and to give a symmetric distribution of the residuals. A small constant (0.2) was added to all values- prior to taking the log transformation. FEVi values were not log transformed prior to analysis.

The relationship between cleaved α-1 antitrypsin levels and lung function or the ratio of normal to cleaved α-1 antitrypsin and lung function was tested by studying the reduction in sums of squares obtained when the marker is added to a model containing the age effect.

18.3 Results

Results of analysis are shown in Tables 7 and 8. In particular, Table 7 shows the log cleaved α-1 antitrypsin level, log ratio of normal to cleaved α-1 antitrypsin, F ratios and p values for the hypothesis of no disease differences in marker concentration for adults or for children.

Both cleaved α-1 antitrypsin levels and ratio of normal to cleaved α-1 antitrypsin are statistically different between disease groups as is FEVi.

Table 8 shows the relationship of the level of cleaved α-1 antitrypsin or the ratio of normal to cleaved α-1 antitrypsin with FEV ₁ . Both markers are statistically significantly associated with FEV ₁ , with the log of the ratio of normal to cleaved α-1 antitrypsin showing the strongest correlation.

Table 7: Mean lo transformed marker concentration or ratio b disease rou .

CO c

CO "Cleaved" is the log of the spot volume for cleaved form of α-1 antitrypsin; and "ratio" is the log ratio of normal to cleaved form of α-1 antitrypsin. m

CO

I m 5 Table 8; F tests for the relationship between α-1 antitrypsin cleavage and FEVi m F value Pr(F)

73 c Log α-1 antitrypsin 7.45 0.013 m Log ratio of normal to cleaved 74 O.001

IO α-1 31. antitrypsin

*J O

C

EXAMPLE 19

Detection of modified levels of myeloperoxidase in subjects suffering from an acute clinical exacerbation

Using methods essentially as described in Examples 1-5 the proteins described in Table 95 were identified as markers of an acute clinical exacerbation.

Semi-quantitative differential display analysis was performed to establish the amounts of the proteins in Table 9 in a CF patient with an acute clinical exacerbation, and following treatment for said infection. Normalised spot volumes were estimated using ImagepIQ and compared between pre- and post-treatment results.

Table 9 Proteins dia nostic of an acute clinical exacerbation in a CF atient

^a A Molecular Weight as defined herein is determined by 2-dimensional gel electrophoresis or SDS/Page shall be understood to include the stated molecular weight

±1 kDa. ^b An isoelectric point (pi) is determined by 2-dimensional gel electrophoresis or isoelectric focussing shall be understood to include the stated pi ± 0.25 units.

Spot number relates to the corresponding spot number in Figure 1.

Example 20 Detection of auto-antibodies that bind myeloperoxidase in a CF subject

20.1 Biological samples Clinical CF samples were collected and crude plasma isolated using a capture column. The crude plasma used from the capture column were combined from four exacerbated CF adults in the age group 22- to 37-years old. Predicted FEVi values were between 22-65 % and the subjects have had 2-4 exacerbations in the last 12 months. Microbiological testing was performed on collected sputum samples. All adult CF subjects used in current study had profuse P. aeruginosa growth in the lungs. In addition, one CF adult also had pulmonary S. aureus infection.

Saline-induced sputum was collected from healthy control- and CF subjects, and subsequently liquefied. Sputum used for 2DE arrays was acetone precipitated and proteins were resuspended in the CHAPS containing ProteomlQ Resuspension Reagent (Proteome Systems, Woburn, US). The samples were cleaned up on 100 kDa and 5 kDa spin columns (Millipore, Billerica, MA).

Sputum used for immuno-capturing: Sputum samples were pooled from two exacerbated CF subjects of 22 and 31 years old (total of 16mL). They had predicted

FEVi values of 14 % and 51 % and had been treated for 1-2 exacerbations in the last 12 months. Microbiological testing showed profuse P. aeruginosa in the lungs of both patients. In addition, one of the two patients also contained profuse S. aureus. The pooled sputum were incubated with 30 mM IAA to inactivate residual DTT used in the liquification protocol and IgG depleted by using Protein G coupled Sepharose beads as recommended by manufacturer (Amersham Pharmacia (Uppsala, Sweden).

20.2 Preparation of an immunocapture column

An immuno-capture column was generated from a total of 5 mL pooled plasma from five exacerbated CF patients (total protein concentration of ~40 mg/niL). IgG was bound to Protein G sepharose by incubating the pooled plasma with 10 mL 50% slurry of Protein G sepharose. The matrix were washed in 10 mM PBS pH 7.4 and bound IgG was irreversibly immobilised utilizing DSS. The generated column is referred to as the capture column.

17.3 Capture of immunogenic protein from CF samples

The capture column was incubated overnight with the captured plasma from CF subjects (as described in Example 20.1) at 4 ⁰ C at constant rotation and beads were subsequently harvested by centrifugation. The flow-through was collected and saved for subsequent incubation steps (the protein extract was passed over the capture column three times in each capture). The harvested beads were washed 3 times in 10 mM PBS pH 7.4 and captured proteins we.re eluted with 5OmM glycine pH 2.7. The column was extensively washed with first 50 mM glycine pH 2.7 then 10 mM PBS pH 7.2 prior subsequent incubation steps.

Eluted proteins were alcohol precipitated and subsequently resolubilised in Cellular and Organelle Membrane solubilizing reagent from the ProteoPrep Universal Extraction kit

(Sigma, St. Louis, MO). Following the instruction in the ProteoPrep kit the solubilized proteins were reduced and alkylated with a final concentration of 5 mM tri-n- butylphophine and 1O mM acrylamide, respectively.

20.4 Two-dimensional gel electrophoresis

Eleven centimetre pH 3-10 or pH 4-7 IPGs were purchased from Amersham (Uppsala, Sweden). Isoelectric focusing was conducted as per manufacturer's instructions using an IsoElectrIQ ² unit from Proteome Systems (Woburn, MA). Second dimension 6-15% or 14% homogenous Tris-Acetate Gelchip gels were run as recommended by manufacturer (Proteome Systems, Woburn, MA). Arrayed proteins were visualised by silver-staining (Shevchenko et ah, Mass spectrometric sequencing of proteins silver- stained polyacrylamide gels. 68, 850-858. 1996) or transferred to PVDF-P membranes (Millipore, Billerica, MA) by using semi-wet membrane-blotting cassettes accompanying the IsoElectrIQ ² unit from Proteome Systems (Woburn, US).

20.5 MS analysis

Proteins spots of interest were excised and washed twice in 100 mM NH ₄ HCO ₃ : 50% acetonitrile (ACN) pH 8.2 and dehydrated at 50degC for 30 minutes. Proteins were digested as described by Katayama et al (Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and digested for 3 hours at 37degC. Tryptic peptides was extracted by sonication and purified as described by Kussmann et al. Peptides were eluted with ~1.5μl MALDI matrix solution (70% ACN, 0.1% TFA, 1.5mg/ml alpha-cyano-4- hydroxycinnamic acid (Sigma, St. Louis, MO). Peptide mass fingerprints (PMF) were

generated by matrix-assisted laser desorption/ionisation-time-of-flight- mass spectrometry (MALDI-TOF-MS) using an Axima CFR (Rratos, Manchester, UK) or an ABI MALDI MS/MS (AME Bioscience, London, UK).

20.6 Results

As shown in Figure 9, 14 proteins were isolated from the sputum of CF subjects. Of these human myeloperoxidase was identified as spot 10.

EXAMPLE 20 A two-site ELISA to determine the amount of myeloperoxidase in sputum from a subject

Sputum was isolated from subjects and solubilized essentially as described in Examples 1 and 2. Excess dithiόthreitol remaining in liquefied sputum was first quenched with 30 mM iodoacetamide for 2 hr. Myeloperoxidase levels were measured in liquefied sputum, diluted 1/50 in blocking buffer (1% (w/v) casein, 0.1% (v/v) tween-20, 0.1% (w/v) sodium azide in PBS, pH 7.4), by coating the plate overnight with 50 μl of an anti-human myeloperoxidase monoclonal antibody, clone 2C7, (Serotec, Oxford, UK) diluted 1/2000 in phosphate buffered saline (PBS), pH 7.4. Non-specific binding sites were blocked using 400 μl/well blocking buffer. Rabbit anti-human myeloperoxidase polyclonal antibody (Chemicon International Inc., Temecula, CA), diluted 1/500 in PBS, followed by addition of 100 μl/well anti-rabbit horseradish peroxidase conjugated antibody, diluted 1/5000 was used for detection. Colour was developed by the addition of TMB substrate for 30 min. The reaction was stopped by the addition of 80 μl of 0.5 M sulfuric acid. All reactions were conducted in volumes of 50 μl per well for 1 hr at room temperature, unless specified otherwise. The plates were washed three times with PBS between all antibody/conjugate incubation steps. Differential absorbance (450 nm - 620 nm) was measured using a PowerWavex 340 plate reader (Bio-Tek Instruments Inc., Winooski, VT). .

As shown in Figure 10 CF samples derived from ^" subjects that were suffering from an acute clinical exacerbation had considerably higher levels of myeloperoxidase detected than control subjects. Furthermore, the level myeloperoxidase in some subjects were reduced following treatment for an acute clinical exacerbation (see, for example, the level of myeloperoxidase detected in the column labelled 11 versus HB and 44 versus 44B).

With regard to the results attained for the CF children, those subjects with a 2-DE gel profile similar to adult subjects suffering from an acute clinical exacerbation (subjects 64 and 69) were shown to have increased levels of myeloperoxidase in their sputum. Those subjects that had 2-DE profiles similar to healthy adult CF subjects (61, 63, 65, 71 and 76) had similar levels of myeloperoxidase in their sputum as observed in the sputum of healthy control children.