TITLE METHODS OF DIAGNOSIS OF INFLAMMATORY BOWEL

DISEASE

FIELD OF THE INVENTION The present invention relates to diagnosis of bowel disease. More particularly, this invention relates to detection of human antibodies which bind bacterial markers as indicators of inflammatory bowel disease, or a susceptibility thereto.

BACKGROUND OF THE INVENTION

Inflammatory bowel disease (IBD) comprises two main forms of chronic intestinal inflammation, Crohn's disease (CD) and ulcerative colitis (UC). Indeterminate colitis (IC) describes another form of IBD when a discrimination between CD and UC can not be made with certainty. Characterization of each has been empirically defined by their respective clinical, pathological, endoscopic and radiological profiles (Podolsky, 2002). CD is a chronic transmural inflammatory process which can affect any segment of the gastrointestinal tract. However, whilst the course of the disease is variable, most sufferers tend to progress toward relapsing and chronic disease. In contrast, intestinal inflammation in UC is primarily confined to the colon. Key features of UC include diffuse superficial mucosal inflammation and the concurrent production of a complex array of inflammatory mediators (Xavier and Podolsky, 2007).

Current methods for diagnosing an individual as having CD or UC, while highly specific, are relatively costly, requiring labor-intensive immunofluorescence assays and careful analysis of staining patterns. Further, IBD is currently primarily diagnosed via colonoscopy, which is an expensive and complex procedure requiring anesthesia. In addition to causing discomfort to the patient, it can typically be a lengthy amount of time before results can be obtained. Often, it takes months from the time the physician orders the test until the results are obtained and presented to the patient.

At present, the aetiology of IBD remains unresolved although it is thought to be an immunologically mediated disease in a genetically susceptible host (Bossuyt, 2006). Furthermore, current research indicates that chronic gut inflammation results from an aberrant immune response and loss of tolerance to

the innate intestinal flora (Hugot et al, 2001; Kucharzik et ah, 2006). Several autoantibodies have been implicated in IBD (Saxon et al., 1990; Rump et al., 1990; Nassberger et al., 1992; Klebl et al., 2005) with atypical perinuclear anti- neutrophil cytoplasmic antibodies (pANCAs) and anti-Saccharomyces cerevisiae antibodies (ASCAs) being the most extensively examined. Various studies have revealed heterogeneous and conflicting results with regards to pANCAs and ASCAs potential for diagnosing and discriminating between CD and UC (Mayet et al., 1992; Yang et al., 1996; Walmsley et al., 1991; Peeters et al., 2001). Moreover, a study by Peeters et al. (2001) which evaluated the diagnostic accuracy of pANCA and ASCA for IBD demonstrated that although the specificity of these serological markers were moderately high (50-60%), the low sensitivity (44%) reduces their efficacies as diagnostic tests. Thus there are currently no biomarkers with the ability to successfully classify disease or predict severity and course available.

SUMMARY OF THE INVENTION

The difficulty in diagnosing IBD and distinguishing between Crohn's disease (CD), ulcerative colitis (UC) and indeterminate colitis (IC) hampers early and effective treatment of these chronic diseases. Thus, there is a need for non- invasive, rapid and sensitive testing methods for distinguishing between different forms of IBD to prescribe the most favorable treatment procedure.

The present invention arises from the surprising discovery of one or more bacterial proteins, or antibodies thereto, that are indicative of IBD in a mammal, or a susceptibility thereto. A preferred advantage of the invention is the ability to discriminate between CD, UC, IC and unaffected individuals in the diagnosis of IBD.

In one broad form, the invention relates to one or more bacterial proteins, fragments or derivatives thereof, or antibodies thereto, that are indicative of, or associated with, IBD in a mammal, or a susceptibility thereto. In a first aspect, the invention provides a method of diagnosis of IBD in a mammal, including the step of determining the presence or absence of one or more bacterial proteins, or one or more antibodies thereto in a sample obtained, or obtainable from said mammal, which are indicative of IBD in said mammal, or a susceptibility thereto.

Preferably, the sample is serum.

In a second aspect, the invention provides a kit for diagnosis of IBD in a mammal, comprising one or more isolated bacterial proteins, and/or antibodies thereto, which are indicative of IBD in a mammal, or a susceptibility thereto, and one or more detection reagents.

In a third aspect, the invention comprises an antibody which binds one or more isolated bacterial proteins, which are indicative of IBD in a mammal, or a susceptibility thereto.

Preferably, according to the aforementioned aspects, the bacterial proteins may be derived from, obtained or obtainable from a bacterium selected from the group consisting of Gram negative (G ^" ) or Gram positive (G ⁺ ) bacteria.

More preferably, the bacterial proteins are derived, obtained or obtainable from bacteria of a genus selected from the group consisting of Staphylococcus, Bacteroides, Klebsiella, and Enterococcus. Even more preferably, the bacterial proteins are derived, obtained or obtainable from bacteria of a species selected from the group consisting of S. aureus, E. faecalis, B. vulgatus, and K. pneumoniae.

Preferably, according to the aforementioned aspects the mammal is a human. Forms of IBD include, but are not limited to, CD, UC and IC.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Typical Western blot profiles of Klebsiella pneumoniae (K. pneumoniae), Enterococcus faecalis (E. faecalis), Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) protein extracts probed by sera from (a) CD, (b) UC, (c) IC, (d) unaffected relative (UR), (e) disease control (DC) ₃ and (f) healthy control (HC) patients. Arrows indicate the differential band for the various IBD groups i.e. CD = ~17.5 kDa S. aureus protein; UC = -16 kDa S. aureus protein; IC = ~32 kDa and ~62 kDa K. pneumoniae proteins. The arrow in

(d) indicates that the differential band for the UR group is a -36 kDa E. faecalis protein. Exposure time after detection was 5 minutes.

Figure 2. Typical Western blot profiles of Bacteroides fragilis (B. fragilis), Bacteroides vuϊgatus (B. vulgatus), Clostridium difficile (C. difficile), and Saccharomyces cervisiae (S. cerevisiae) protein extracts probed by sera from (a) CD, (b) UC, (c) IC, (d) UR, (e) DC, and (f) HC patients. The arrow in (a) indicates that the differential band for the CD group is a ~47 kDa B. vulgatus protein.

Figure 3. Typical Western blot profiles of Klebsiella pneumoniae (K pneumoniae), Enterococcus faecalis (E. faecalis), Escherichia coli (E. coli) and

Staphylococcus aureus (S. aureus) protein extracts probed by sera from HC patients. The arrow indicates that the differential band for the HC group is a ~48 kDa E. faecalis protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

To enable accurate diagnosis of inflammatory bowel disease (IBD), the present inventors analyzed the relationship between different forms of inflammatory bowel disease and the presence of human serum antibodies which bind or otherwise recognize specific bacterial proteins, and thereby indicate whether or not an individual suffers from, has had or is susceptible to, IBD.

As used herein, the term "inflammatory bowel disease" (IBD) is a collective term referring to CD, UC and IC. Thus, an individual having either CD,

UC, or IC is defined herein as having IBD. The term "IBD" distinguishes CD, UC and IC from all other disorders, syndromes or abnormalities of the gastroenterological tract, including irritable bowel syndrome (IBS).

Throughout the specification, the term "indeterminate colitis" is synonymous with '7C" and will be employed when a distinction between UC and CD can not be made with certainty.

For the purposes of this specification, the term "sample" as used herein, means any extract, specimen or fraction derived, obtainable or obtained from an organism. A sample may be, for example, whole blood, serum, plasma, saliva or other bodily fluid or tissue, a cell extract, lysate or fraction.

In one embodiment, the sample may be a "test sample" obtained or obtainable from a mammal, such as a human.

Preferably, according to this embodiment, the test sample is selected from the group consisting of blood, serum and plasma.

More preferably, the test sample is serum.

In another embodiment, the sample may be a bacterial protein sample. In one broad form, the present invention arises from the surprising discovery of the presence, or absence of one or more isolated bacterial proteins, or antibodies to said one or more proteins, that are indicative of IBD in a mammal, or a susceptibility thereto.

As used herein, the term "susceptibility" broadly encompasses any positive or negative predisposition or preponderence to IBD. This can include an increased likelihood of, or predisposition to, displaying at least one or more symptoms of IBD and can also include decreased or reduced likelihood of displaying at least one or more symptoms of IBD. This latter example is predicated, in part, on the discovery that the presence of one or more antibodies to one or more E. faecalis proteins in patient samples correlates with an absence of

IBD symptoms, which may suggest that patients with these antibodies have reduced susceptibility to IBD.

As described herein, the term "mammal" includes and encompasses humans, domestic and farm animals, such as dogs, horses, cats, sheep, pigs, cows, etc.

Preferably, the mammal is a human.

For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.

By "protein" is meant an amino acid polymer comprising D- or L-amino acids and/or natural or non-natural amino acids as are well understood in the art. A "peptide" is a protein having no more than fifty (50) amino acids.

A "polypeptide" is a protein having fifty (50) or more amino acids.

Preferably, in the context of bacterial proteins, the protein is a native protein. Such bacterial proteins may be purified, partially purified or enriched as is well known in the art.

For example, bacterial proteins may be purified or partially purified from a whole bacterial cell extract or lysate by any suitable procedure known to those of skill in the art inclusive of electrophoresis, centrifugation, dialysis, and protein chromatography although without limitation thereto. Also in the context of bacterial proteins, the invention contemplates fragments and/or derivatives of said proteins.

Fragments may constitute as few as 6 contiguous amino acids, or may be longer, such as comprising 10, 12, 15, 20, 30, 50 or 100 or more amino acids of a protein. In other embodiments, fragments may constitute at least 5%, 10%, 15%,

20%, 40%, 50%, 60%, 70%, 80% or 90-95% of a bacterial protein.

Preferably, bacterial protein fragments are antigenic fragments which are bound or recognized by a human serum antibody that is indicative of IBD, or a susceptibility thereto. Derivatives of bacterial proteins include proteins that have been chemically-modified and/or conjugated with molecules such as radioisotopes, biotin, avidin, a fluorophore, a chromogen, an enzyme (e.g. horseradish peroxidase, alkaline phosphatase), and the like.

Preferably, derivative bacterial proteins are conjugated with one or more molecules that assist detection in a diagnostic method or kit of the invention.

For the purposes of this invention, the term "antibody" means an immunoglobulin molecule, which may be polyclonal, monoclonal, recombinant and/or naturally-occurring (e.g. obtained from a test sample such as serum). More specifically, the invention provides antibodies which bind, recognize and/or have been raised against bacterial proteins described herein.

Also contemplated are antibody fragments such as Fc fragments, Fab and Fab'2 fragments, fragments comprising VH and/or VL CDRS, diabodies and ScFv fragments. Preferred antibody fragments are antigen-binding fragments.

Antibodies may be made in suitable production animal such as a mouse, rat, rabbit, sheep, chicken or goat.

For example, purified or partially purified bacterial proteins, or mixtures thereof, may be administered to a production animal for polyclonal or monoclonal antibody production.

Monoclonal antibodies may be produced by standard methods such as described in Current Protocols in Immunology, Antibodies: A Laboratory Manual

(1988). Such methods generally involve obtaining antibody-producing cells, such as spleen cells, from an animal immunized as described above, and immortalizing said cell, such as by fusion with an immortalized fusion partner cell.

Monoclonal antibodies or fragments thereof, may also be produced by recombinant means. Such recombinant methods are well known in the art and a variety of commercial sources are available for production of recombinant antibodies. As is well understood in the art, antibodies may be conjugated with labels selected from a group including an enzyme, a fluorophore, a chemiluminescent molecule, biotin, radioisotope or other label.

Examples of suitable enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzyme label may be used alone or in combination with a second enzyme in solution or with a suitable chromogenic or chemiluminescent substrate.

Examples of chromogens include diaminobanzidine (DAB), permanent red, 3-ethylbenzthiazoline sulfonic acid (ABTS), 5-bromo-4-chloro-3-indoryl phosphate (BCIP), nitro blue tetrazolium (NBT), 3,3',5,5'-tetramethyl benzidine (TNB) and 4-chloro-l-naphthol (4-CN) , although without limitation thereto.

A non-limiting example of a chemiluminescent substrate is Luminol™, which is oxidized in the presence of horseradish peroxidase and hydrogen peroxide to form an excited state product (3-aminophthalate). Fluorophores may be fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), allophycocyanin (APC), Texas Red (TR), Cy5 or R-Phycoerythrin (RPE), although without limitation thereto.

Radioisotope labels may include ¹²⁵ 1, ¹³¹ 1, ⁵¹ Cr and ⁹⁹ Tc, although without limitation thereto. Other antibody labels that may be useful include colloidal gold particles and digoxigenin.

Suitably, although not exclusively, an antibody may be used as a positive control in methods and/or kits of the invention.

Another aspect of the invention provides a kit for diagnosis of IBD in a mammal, comprising one or more bacterial proteins, or antibodies thereto, which are indicative of IBD in a mammal, or a susceptibility thereto, and one or more detection reagents. Suitably, the kit will be employed for determining the presence or absence of an antibody present in a test sample.

For example, the kit may comprise purified or partially purified bacterial proteins, fragments or derivatives thereof as hereinbefore described, and/or one or more antibodies or antibody fragments as hereinbefore described, and optionally one or more detection reagents (e.g. a labeled secondary antibody, such as conjugated with biotin, an enzyme, a fluorophore or a chromogen) to facilitate detection of one or more antibodies in a test sample.

The terms "detect", "detected" "detecting" or "detection" are used in the broadest sense to include both qualitative and quantitative measurements of a target molecule. In one aspect, the detecting method as described herein is used to identify the mere presence of the antibody of interest in a biological sample. In another aspect, the method is used to test whether the antibody of interest in a sample is present at a detectable level. In yet another aspect, the method can be used to quantify the amount of the antibody of interest in a sample and further to compare the antibody levels from different samples.

Bacterial proteins detected according to the invention are typically obtainable from bacteria that are capable of colonizing, inhabiting, existing in or otherwise residing in a mammal. In some embodiments, the bacteria may be capable of colonizing, inhabiting, existing in or otherwise residing in a mammalian gastrointestinal tract. In particular, non-limiting embodiments, the bacteria may be commensal bacteria which, by way of example only, are gut- associated.

In one embodiment, such bacteria may be gram negative (G ^' ) or gram positive (G ⁺ ). Suitably, although not exclusively, Gram negative bacteria include

Bacteroides and Klebsiella.

Suitably, although not exclusively, Gram positive bacteria include Staphylococcus and Enterococcus.

In another embodiment, such bacteria may be aerobic or anaerobic.

Suitably, although not exclusively, the bacteria are selected from the group consisting of Staphylococcus aureus (S. aureus), Enter ococcus faecalis (E. faecalis), Bacteroides vulgatus (B. vulgatus), and Klebsiella pneumoniae (K pneumoniae). In one embodiment, the presence of one or more antibodies directed against one or more proteins from S. aureus in a sample obtained from a mammal determines whether said mammal has, has had, or is susceptible to, CD.

In one particular embodiment, said one or more antibodies are directed against a S. aureus protein of approximate molecular weight 17.5 kDa. Typically, according to this embodiment, a human has or has had CD, or has an increased susceptibility to CD.

In another particular embodiment, typically although not exclusively, a human that does not have antibodies directed against a S. aureus protein of 16 kDa, has or has had CD, or has an increased susceptibility to CD. In another embodiment, the presence of one or more antibodies directed against one or more proteins from B. vulgatus in a sample obtained from a mammal determines whether said mammal has, has had, or is susceptible to, CD.

In one particular embodiment, said one or more antibodies are directed against a B. vulgatus protein of approximate molecular weight 47 kDa. Typically, according to this embodiment, a human has or has had CD, or has an increased susceptibility to CD.

In yet another embodiment, the presence of one or more antibodies directed against one or more proteins from E. faecalis in a sample obtained from a mammal determines whether a mammal has, has had, or is susceptible to, CD. In one particular embodiment, typically, although not exclusively, a human that does not have antibodies directed against E. faecalis proteins of approximate molecular weights of 36 kDa and/or 48 kDa, has or has had CD, or has an increased susceptibility to CD.

In one embodiment, the presence of one or more antibodies directed against one or more proteins from S. aureus in a sample obtained from a mammal determines whether said mammal has, has had or is susceptible to, UC.

In one particular embodiment, said one or more antibodies are directed against a S. aureus protein of approximate molecular weight 16 kDa. Typically,

according to this embodiment, a human has or has had UC, or has an increased susceptibility to UC.

In another particular embodiment, typically although not exclusively, a human that does not have antibodies directed against a S. aureus protein of approximate molecular weight 17.5 kDa, has or has had UC, or has an increased susceptibility to UC.

In another embodiment, the presence of one or more antibodies directed against one or more proteins from B. vulgatus in a sample obtained from a mammal determines whether said mammal has, has had or is susceptible to, UC. In one particular embodiment, typically although not exclusively, a human that does not have antibodies directed against a B. vulgatus protein of approximate molecular weight 47 kDa, has or has had UC, or has an increased susceptibility to UC.

In yet another embodiment, the presence of one or more antibodies directed against one or more proteins from E. faecalis in a sample obtained from a mammal determines whether said mammal has, has had, or is susceptible to, UC.

In one particular embodiment, typically, although not exclusively, a human that does not have antibodies directed against E. faecalis proteins of approximate molecular weights of 36 kDa and/or 48kDa, has or has had UC, or has an increased susceptibility to UC.

In one embodiment, the presence of one or more antibodies directed against one or more proteins from K. pneumoniae in a sample obtained from a mammal determines whether said mammal has, has had or is susceptible to, IC. In one particular embodiment, said one or more antibodies are directed against K. pneumoniae proteins of approximate molecular weights of 32 kDa and 62 kDa. Typically, according to this embodiment, a human has or has had IC, or has an increased susceptibility to IC.

In another embodiment, the presence of one or more antibodies directed against one or more proteins from S. aureus in a sample obtained from a mammal determines whether said mammal has, has had or is susceptible to, IC.

In one particular embodiment, typically although not exclusively, a human that does not have antibodies directed against S. aureus proteins of approximate

molecular weights of 16 kDa and/or 17.5 kDa, has or has had IC, or has an increased susceptibility to IC.

In yet another embodiment, the presence of one or more antibodies directed against one or more proteins from B. vulgatiis in a sample obtained from a mammal determines whether said mammal has, has had or is susceptible to, IC.

In one particular embodiment, typically although not exclusively, a human that does not have antibodies directed against a B. vulgatus proteins of approximate molecular weight 47 kDa, has or has had IC, or has an increased susceptibility to IC. In yet another embodiment, the presence of one or more antibodies directed against one or more proteins from E. faecalis in a sample obtained from a mammal determines whether a mammal has, has had or is susceptible to, IC.

In one particular embodiment, typically although not exclusively, a human that does not have antibodies directed against E. faecalis proteins of approximate molecular weights of 36 kDa and/or 48 kDa, has or has had IC, or has an increased susceptibility to IC.

In one embodiment, the presence of one or more antibodies directed against one or more proteins from E. faecalis in a sample obtained from a mammal determines whether said mammal has a reduced susceptibility to IBD ₅ is resistant to IBD, unaffected by IBD or otherwise protected from IBD.

In one particular embodiment, said one or more antibodies are directed against E. faecalis proteins of approximate molecular weights of 36 kDa and/or

48 kDa. Typically, according to this embodiment, a human has a reduced susceptibility to IBD, is resistant to IBD, unaffected by IBD or otherwise protected from IBD.

In another embodiment, the presence of one or more antibodies directed against one or more proteins from S. aureus in a sample obtained from a mammal determines whether said mammal has a reduced susceptibility to IBD, is resistant to IBD, unaffected by IBD or otherwise protected from IBD. In one particular embodiment, typically although not exclusively, a human that does not have antibodies directed against S. aureus proteins of approximate molecular weights 16 kDa and 17.5 kDa has a reduced susceptibility to IBD, is resistant to IBD, unaffected by IBD or otherwise protected from IBD.

In yet another embodiment, the presence of one or more antibodies directed against one or more proteins from B. vulgatus in a sample obtained from a mammal determines whether said mammal has a reduced susceptibility to IBD, is resistant to IBD, unaffected by IBD or otherwise protected from IBD. In one particular embodiment, typically although not exclusively, a human that does not have antibodies directed against a B. vulgatus proteins of approximate molecular weight 47 kDa has a reduced susceptibility to IBD, is resistant to IBD, unaffected by IBD or otherwise protected from IBD.

In yet another embodiment, the presence of one or more antibodies directed against one or more proteins from K. pneumoniae in a sample obtained from a mammal determines whether said mammal has a reduced susceptibility to IBD, is resistant to IBD, unaffected by IBD or otherwise protected from IBD.

In one particular embodiment, typically although not exclusively, a human that does not have antibodies directed against K. pneumoniae proteins of approximate molecular weights of 32 kDa and 62 kDa has a reduced susceptibility to IBD, is resistant to IBD, unaffected by IBD or otherwise protected from IBD.

Furthermore, this aspect of the invention, in combination with other tests, provides a method for improving the diagnosis, and prognosis of IBD. Preferably, although not exclusively, said other tests include ASCA and pANCA tests.

With regard to diagnostic methods and kits, Figures 1, 2 and 3 provide non-limiting examples of detecting the presence of specific antibodies in patient sera and comparisons with sera from unaffected controls using immunoblotting techniques.

A non-limiting exemplary method provides an immunoblot that is coated with cell wall and cytoplasmic membrane bacterial extracts (e.g. from K. pneumoniae, E. faecalis, E. coli, B. vulgatus and S. aureus). A serum sample is collected from a human. The serum is applied to the immunoblot. Antibodies present in the sample will bind to the bacterial proteins. The immunoblot is washed with a wash buffer. Then, a rabbit antibody that has been engineered to bind to the invariant region of mammalian antibodies is applied to the immunoblot. The rabbit antibody includes therein a detecting molecule, e.g., HRP, which will change colour in the presence of a developing solution (HRP-

conjugated rabbit anti-mammalian antibody). The HRP-conjugated rabbit anti- mammalian antibody that binds to the immunoblot will reveal the presence of specific antibodies against bacterial proteins in the sample.

While the examples in Figures 1, 2 and 3 detect serum antibody using immunoblots, a person skilled in the art will be familiar with other methods of detecting antibody presence/levels. For example, ELISA, immunohistochemical staining, flow cytometry, phage display technology, immunoassays, including competitive and non-competitive immunoassays (e.g. immunochromatographic assays such as "Rapid assays", CEIA and liposome immunoassays), radioimmunoassays, and protein arrays may be used to detect antibody presence/levels.

One preferred method, particularly suitable for large scale and/or high throughput diagnostic tests, is an ELISA-based method.

A non-limiting example of an ELISA method includes binding of one or more bacterial proteins, fragments or derivatives by an antibody, if present in a test sample, wherein detection is facilitated by a labeled secondary antibody, as hereinbefore described.

It will be appreciated from the foregoing that the method, kit and antibodies of the invention provide improved diagnosis of IBD and, preferably, discrimination between UC, IC and CD. Furthermore, such diagnosis may assist in prescribing the correct treatment and/or drug regime for an individual that has, has had, or is susceptible to IBD and/or may assist in determining the responsiveness of an individual to a treatment and/or drug regime aimed at treating IBD. So that the invention may be fully understood and put into practical effect, the skilled reader is directed to the following non-limiting detailed Example.

EXAMPLE Determination of patient antibody status The following example shows specific analyses of the presence of specific antibodies in patient sera and comparisons with sera from unaffected controls using immunoblotting techniques.

Bacterial propagation and protein extraction

K. pneumoniae, E. faecalis, E. coli, S. aureus, B. vulgatus, B. β'agilis, S. cerevisiae and C. difficile were grown overnight on 5% Horse Blood Agar at 37°C. Cells from the eighth bacteria were harvested by centrifugation at 900Og at 4°C for 10 minutes. Cells were washed once in sodium phosphate buffer (1OmM, pH 7.0) and resuspended in sodium phosphate buffer (1OmM, pH 7.0) and lysozyme (lOmg/mL) (Sigma Aldrich) was added plus ethylenediamine- tetraacetic acid (EDTA) (4mM). The cell suspension was incubated at 37°C for 1.5 hours with intermittent mixing. Cells were disrupted by sonication (Soniprep 150, 20μ per 15 seconds). Bacterial cell walls were collected by centrifugation at 4000g at 4 ⁰ C for 10 minutes. Cytoplasmic membranes were collected by ultra centrifugation at 100 00Og at 4°C for 30 minutes. To each cell wall and cytoplasmic membrane fraction, ImL of 2X SDS buffer (0.09M Tris-HCl pH 6.8, 20% glycerol, 2% SDS, 0.1M dithioerythritol and 0.02% bromophenol blue) was added. All bacterial protein samples were denatured at 100 ⁰ C for 10 minutes and then stored at -20°C for later use.

Patients and sera: Immunoblotting was performed on IBD patients and healthy control volunteers. Samples remained coded until the analyses were completed.

Identification of immunoreactive bacterial proteins by SDS-PAGE and immunoblotting

To identify potential diagnostic bacterial proteins, patient sera from CD, UC, IC and unaffected controls were probed against standardised K. pneumoniae, E. faecalis, E. coli, B. vulgatus, S. aureus, B. fragilis, S. cerevisiae and C. difficile cell proteins. Briefly, 10 μg of each protein extract was loaded onto 12.5% polyacrylamide SDS-PAGE gels and electrophoresed at 110 volts for 80 minutes at room temperature. Proteins were transferred at 4 ⁰ C, 100 volts for 1 hour to Hybond C Extra nitrocellulose membranes (Amersham Biosciences) using 3- (cyclohexylamino)-l -propane sulphonic acid (CAPS) buffer (1OmM CAPS ₅ 10% methanol, pH 11.0) and the membranes blocked (5% skim milk powder in PBS containing 0.1% Tween 20 (SM-PBS-T)) for 1 hour at room temperature with rocking. Patient samples were diluted 1 : 1000 in SM-PBS-T and incubated with the membranes at room temperature for 1 hour with shaking. After washing with

PBS-T (PBS, 0.1% Tween 20), membranes were incubated with the secondary antibody, conjugated rabbit anti-human horseradish peroxidase (HRP) IgG (Roche) diluted 1:4000 in SM-PBS-T at room temperature for 1 hour with shaking. Membranes were washed 4X for 15 minutes in PBS-T then detected via chemiluminescence (Pierce). The presence of unique bands in the diseased groups when compared to unaffected-relative controls (UR), disease controls (DC), and healthy controls (HC) were identified and subsequently categorised as differential.

Figure 1 shows the typical Western blot profiles when sera from CD, UC, IC, unaffected-relative controls (UR), disease controls (DC) and healthy controls (HC) were used to probe K. pneumoniae, E. faecalis, E. coli and S. aureus cell wall and cytoplasmic membrane protein extracts. Four proteins, ~16 kDa and ~17.5 kDa from S. aureus and -32 kDa and —62 kDa from K. pneumoniae were shown to be differential between diseased groups when probed by patient sera (Table 1). Moreover, antibodies to one — 36 kDa E. faecalis protein was found exclusively in control patient sera. Unique to the UC group was the -16 kDa S. aureus protein detected in 100% of patients. The -17.5 kDa S. aureus protein was immunoreactive in all but one (93%) of the CD serum samples that were screened. In contrast, only a single UC patient (7%) demonstrated antibodies to this particular (-17.5 kDa) S. aureus protein. Interestingly, the differential K. pneumoniae proteins (~32 kDa and -62 kDa) were shown to be immunoreactive in CD, UC and IC patient sera. However, only those patients with IC simultaneously demonstrated antibodies (100%) to both the -32 kDa and -62 kDa K. pneumoniae proteins. Importantly, neither S. aureus protein was detected in IC or control patient sera. Furthermore, unaffected sera were not reactive against either differential K. pneumoniae protein. Conversely, only unaffected control sera were immunoreactive (100%) to the -36 kDa E. faecalis protein.

Figure 2 shows the typical Western blot profiles when sera from CD, UC, IC ₅ unaffected-relative controls (UR), disease controls (DC), and healthy controls (HC) were used to probe B. fragilis, B. vulgatus, C. difficile and S. cerevisiae cell wall and cytoplasmic membrane protein extracts. Antibodies to a -47 kDa protein from B. vulgatus were exclusively found in all (100%) of the CD serum samples that were screened (n-36). In contrast, the -47 kDa B. vulgatus protein was not detected (0%) when probed with UC, IC or control sera (i.e. UR, DC, and

HC) ₅ which suggests that antibodies to this particular ~47 B. vulgatus protein are specific for patients affected by CD (Table 1).

Figure 3 shows the typical Western blot profiles when sera from healthy controls (HC) were used to probe K. pneumoniae, E. faecalis, E. coli and S. aureus cell wall and cytoplasmic membrane protein extracts. Antibodies to a 48 kDa E. faecalis protein were found in the majority (73%) of the healthy control

(HC) samples (Table 1).

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

AU computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

Table 1. The percentage prevalence and estimated molecular weights of the seven differential bacterial markers.

Table 2. Diagnostic efficacy of the novel markers for the diagnosis and discrimination of the various forms of IBD and unaffected individuals. (Sens. = sensitivity, Spec. = specificity, PPV = positive predictive value, NPV = negative predictive value, SE = standard error, + = both antigens must be present).

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