DESCRIPTION FIELD OF INVENTION

The present invention relates to the fields of medicine and immunology. In particular, this invention relates to immunological diagnostic methods that utilize the full-length molecule of plasminogen or its peptide fragments which may be used as universal detectors of proteolytic products having a G-terminal lysine in an immunological diagnostic to identify the human diseases associated with increased activity of protcoivtic enzymes. The group of inventions also relates to a diagnostic test system utilizing as a detector the full-length molecule and its presented peptide fragments.

Terminology

Technical and scientific terms used in the description have the same meaning and value that are commonly used in the relevant areas of science and technology.

The term "antigen" as used herein refers to a protein or fragments thereof, capable of binding antibodies.

The term "detector" refers to a peptide sequence capable of binding to the C-tcrminal lysine of proteins formed alter proteolysis.

The term "kringle" refers to a protein domain having a structure . stabilized by three disulfide bonds.

The term "domain" refers to a part, of a protein characterized by certain structural or functional properties.

The term "analysis" refers to methods of identifying of the molecular compounds, comprising the steps of: (a) the interaction with the antigen within a biological sample under suitable conditions to form an antigen-antibody complex: and (b) the detection of these complexes.

The term "marker" refers to particular molecular compounds of a specific structure, the presence of which in human tissue samples is associated with a aspecific range of diseases.

The term "epitope" herein refers to a region of a protein molecule which is capable of interacting with the antibody. The term "ligand" herein refers to a protein molecule which is capable of forming a non- covalent bond with another protein molecule.

The term "diagnostic test" is the detection of a diagnostic determinant using a specific laboratory method, the analytical parameters of which remain constant.

BACKGROUND

There is presently unclear need for the development of new markers for the diagnosis of diseases associated with elevated levels of proteolytic cleavage of proteins in the body, particularly those involved in the early stages of tumorigenesis. Tumor development is accompanied by a high level of proteolytic activity (Al-Majid S., Waters H. The biological mechanisms of cancer-related skeletal muscle wasting: the role of progressive resistance exercise // Biol. Res. Nurs. 2008. Vol. 10, JN° 1. pp. 7-20). Indeed, the level of proteolytic activity is currently considered one of the factors of carcinogenesis (Bashir T., Pagano M. Aberrant ubiquitin-mediated proteolysis of cell cycle regulatory proteins and oncogenesis // Adv. Cancer. Res. 2003.Vol. 88.pp. 101-144). Several types of proteases are involved in the process of carcinogenesis by enhancing the proliferation, invasion, and metastasis of tumor cells (Chilingirov AD proteolysis inhibitor effect on some bacterial pathogens, and for inflammatory processes // Pat. Fiziol.And experimental. Therapy. 1997. N° 3. C. 37-39; Soreide K. Proteinase- activated receptor 2 (PAR-2) in gastrointestinal and pancreatic pathophysiology, inflammation and neoplasia // Scand. J. Gastroenterol. 2008. Vol. 43, N° 8. pp. 902-909). Of these,serine proteases are reported to most significantly contribute to the process of carcinogenesis (Zorio E., Gilabert-Estelles J., Espana F., Ramon LA, Cosin R., Estelles A. Fibrinolysis: the key to new pathogenetic mechanisms / / Curr. Med. Chem. 2008. Vol. 15, N° 9,pp. 923-929).

Serine proteases usually cleave peptide bonds between positively charged amino acids lysine and arginine, as well as the esters and amides of these amino acids (Fersht E. The structure and mechanism of action of enzymes. Ed. Kurganova BI Moscow, "Mir" 1980. 432 pp...). To date, some authors have shown that the products of proteolytic activity can serve as a universal marker, the detection of which is associated with various autoimmune and oncogenic processes. For example, the high content of the products of proteolytic cleavage of immunoglobulins can be used as a marker of autoimmune disease or cancer (Robert Jordan et al, patent US08501907). In that work, the authors proposed a method for the detection of proteolytic cleavage of immunoglobulins using polyclonal and monoclonal antibodies. Other authors presented data on the specific proteolysis of immunoglobulins by plasmin (Peter S.Harpel et al The J. of biological chemistry Vol. 264, No. 1 , Issue of January 5, pp. 616-624 (1989)). Following cleavage, the immunoglobulin molecule was shown to specifically interact with plasminogen due to the presence of a C-terminal lysine.

Plasmin, a trypsin-like serine protease, is usually generated by the activation of plasminogen by streptokinase, urokinase or tissue plasminogen activator (tPA). It is well known that plasminogen exhibits fibrinolytic activity and can block clot formation by binding to the C- terminal lysine residues of fibrin and the proteolysis of fibrin fibers.. Both plasminogen and plasmin bind to fibrin through kringle regions, each of which is a triple loop region formed by disulfide bonds. Kringles Kl , K2, K3, K4, and K5 have a strong affinity for lysine.

The participation of the C-terminal lysine in protein binding to plasminogen has beendemonstrated by Marco Candela et al., (Binding of Human Plasminogen to Bifidobacterium, Journal of bacteriology, Aug. 2007, p. 5929-5936). In this study, proteins which bind to plasminogen were treated with carboxypeptidase B, which specifically cleaves only C-terminal lysine and arginine. After this treatment, the proteins lost their ability to bind to plasminogen, indicating that C- terminal lysine participation is essential for the binding to plasminogen and its fragments.

The plasminogen/plasmin system not only takes an active part in the process of fibrinolysis, but has also been shown to be closely associated with angiogenesis and carcinogenesis. Interestingly, some products of plasminogen degradation may be more active than the intact plasminogen molecule in the processes of angiogenesis and carcinogenesis. (YG Klys, et all, Proteolytic plasminogen derivatives in the development of malignancies, Oncology, V 12, N°1 , 2010). The following variants of fragments of plasminogen in the plasma have been described: l -3; K2-3; Kl -4; Kl -4,5; and Kl -5 (Perri S, Martineau D, Francois M, et al. Plasminogen kringle 5 blocks tumor progression by antiangiogenic and proinflammatory pathways. Mol Cancer Ther 2007; 6: pp. 441-449). It has previously been shown that all kringle domains are actively involved in angiogenesis and carcinogenesis. The activity of the first four kringles (Kl -4) is the best-studied - they play a role in angiogenesis. For example, this sequence of kringle domains is found in angiostatin (Francis J. Castellino, Victoria A. Ploplis, Structure and function of the plasminogen / plasmin system, ThrombHaemost 2005; 93: pp. 647-54; C. Boccaccio and Paolo M. Comoglio Cancer Res 2005; 65 (19): pp. 8579-82; Rijken DC, Lijnen HR. New insights into the molecular mechanisms of the fibrinolytic system. J ThrombHaemost 2009; 7: pp. 4- 13). The activation of plasminogen and some of the other serine proteases leads to an increase in the amount of C-terminal lysine containing protein proteolysis products during carcinogenesis. Since the intact plasminogen moleculeas well as its fragments has lysine binding sites, they can bind the degradation products with a C-terminal lysine generated by serine proteases and be used as detectors of the process of carcinogenesis and other pathological processes. This detector has universal properties compared with other proposed methods of detecting degradation products, which require using monoclonal antibodies specific for each product of proteolysis.

The detection of the products of proteolysis in plasma in both human and animal samples can be performed using enzyme-linked immunosorbent assay (ELISA), where the plasminogen molecule or its fragments are used as a detector. The ELISA was first developed in 1971 and currently, an extensive range of types and modifications of ELISA are used. The basic principles of ELISA, regardless of modifications, are as follows:

1. At the first stage of the reaction, antigens or antibodies are adsorbed onto a solid phase. The reagents or compounds not bound to the solid phase are easily removed by washing.

2. Test samples and controls are incubated in the coated wells - thus, immune complexes can be formed on the surface of the solid phase. Unbound components are removed by washing.

3. Antibody-enzyme or antigen-enzyme conjugates, which bind a complementary site on the antigen (or antibody) on the solid phase are then added. Their binding is detected via a colorimetric reaction after the adding of the substrate for the conjugated enzyme. This reaction can be stopped and optical density can be measured.

The levels of the immunoglobulin and other proteins after proteolysis are determined using an indirect ELISA. The wells are coated by antibodies to the desired protein (antigen) and incubated with the serum (plasma) samples or other biological material from the patient (cerebrospinal fluid, saliva, etc.). Specific antigens bound to antibodies at the solid phase are detected using a second antibody-enzyme conjugate to another epitope of the antigen. Depending on the purpose of the assay, different antigens are used, either with antibodies universal for all isotypes or specific to certain classes and subclasses of immunoglobulins. The main advantage of this method is in the versatility of the conjugate. This reaction is also methodologically simple. The main stages of an indirect ELISA for the determination of specific antigens (or antibodies) in the sample are as follows:

1. The antigen, or the ligand (antibody) is adsorbed onto a solid phase, and then washed free of unbound components.

2. The free binding sites are blocked. The wash step is repeated.

3. The samples are added to the wells, incubated and then the wells are washed to remove unbound components. Samples serving as positive and negative controls are incubated in parallel wells.

4. The antibody-enzyme or antigen-enzyme conjugate is added at a working dilution, incubated and the unbound components are washed away.

5. The colorimetric substrate is added. The color reaction is stopped by adding a stop solution.

6. The optical density is measure on a reader.

Under optimum conditions, this method has both a high specificity and a high sensitivity. It can detect nanogram quantities of antigen (or antibody) in serum (or plasma). However, existing methods of immunoassay detection of antigen have a limitation associated with the fact that some antigen in the sample can be present in a complex with other proteins. This complex does not bind to the solid phase, that masks the true concentration of the antigen in the sample. At sufficiently high concentrations of the complex, false negatives may result. To determine the true concentration of antigens, the dissociation of this complex is required. In a previous study of the binding properties of the antigen-antibody complex, it was demonstrated that the use of different organic solvents can increase the sensitivity of the reaction (Mohd. Rehan, HinaYounus, Int. J. of Biol. Macromolecules, Effect of organic solvents on the conformation ant interaction of catalase and anticatalase antibodies).

We have developed a method and a test system with an increased sensitivity of detection of proteolytic products. In the claimed invention, both full-length plasminogen molecules as well as plasminogen fragments of a defined structure are used as antigens and detectors . The claimed invention furthermore uses organic components to detect C-terminal lysine containing proteolysis fragments of immunoglobulin and other protein, which significantly increases the sensitivity of the diagnostic test system. DISCLOSURE OF THE INVENTION

Increased level of immunoglobulins and other proteins having a C-terminal lysine residue after proteolysis and that can bind to plasminogen or fragments thereof, can serve as diagnostic markers of diseases associated with elevated levels of proteolytic fragments of immunoglobulin and other proteins.

The inventors hypothesized that serine proteases are activated in the area of chronic inflammation associated with tumor progression, which leads to the accumulation of proteolitic products with C-terminal lysines. These proteolytic products enter circulation and their amounts can be determined by immunoanalysis. Experiments performed by the inventors have shown that proteolytic products with a C-terminal lysine can be detected using plasminogen or fragments thereof. Thus, the elevated levels of proteolytic products with a C-terminal lysine in blood are a marker of disease associated with elevated levels of proteolytic products having a C- terminal lysine, and the measurement of the levels of these proteins in blood can be used as a diagnostic indicator of a developing disease process.

To increase the sensitivity of detection of the concentration of proteolytic fragments with a C-terminal lysine residue, the inventors developed novel buffer solutions which include a number of organic solvents (dimethylsulfoxide, dimetlformamid, methanol, ethanol, propanol, propanol-2, acetone, acetonitrile, chloroform, ethylene glycol, N-methylpropanamide) to decrease the hydrophobic interactions between molecules. To date, there are no reports in the published literature of using the proposed organic solvents in incubation buffers to determine the level of proteolytic fragments with a C-terminal lysine.

The inventors describe a new method of detection of the concentration of proteolytic fragments having a C-terminal lysine in blood, which is a diagnostic test system for identifying subjects with a high concentration of proteolytic fragments with a C-terminal lysine that are capable of binding to plasminogen or fragments thereof. This diagnostic test system is comprisedof an incubation buffer prepared with organic solvents, the antigen or the detector - a full length plasminogen or its fragments containing at least one sequense of SEQ ID NO: 1-20, and the control sample (C). The composition of the incubation buffer includes at least one component selected from the following group of solvents: dimethylsulfoxide, dimetlformamid, methanol, ethanol, propanol, propanol-2, acetone, acetonitrile, chloroform, ethylene glycol, N- methylpropanamide. The fragment of plasminogen in the claimed test system is selected from the list in Table 1. The control sample used in the test system is a sample from a healthy subject.

Plasminogen and/or the fragments thereof may be immobilized on a solid support which may also be included in the test system. The antibodies to the products of proteolysis with a C- terminal lysine can also be immobilized on the solid support and, in turn, bind plasminogen or fragments thereof containing at least one sequence of SEQ ID NO: 1-20. To assess the concentration of proteolytic fragments with a C-terminal lysine in blood using the claimed test system, the fragment of plasminogen is selected from the list in Table 1. The identification of proteolytic fragments having a C-terminal lysine is performed via an immune reaction, followed by a subsequent colorimetric, fluorescence, or conductivity methods of detection. Thus, the concentration of proteolytic fragments with a C-terminal lysine can be determined using any known methods of detection: colorimetric, fluorescent or conductometric. A concentration of proteolytic fragments with a C-terminal lysine in the test sample which is 30% higher compared to the control sample is taken to indicate the presence of a pathological process in the subject.

The claimed test system can be used to identify subjects with risk of having of cancer or autoimmune diseases. To this end, the method allows the use of the claimed diagnostic test system for identifying subjects with increased levels of proteolytic fragments with a C-terminal lysine that are capable of binding to plasminogen or fragments thereof. To detect elevated levels of proteolytic fragments with a C-terminal lysine, a sample from a subject is compared to a control sample from a healthy donor.In the claimed diagnostic test system, the authors show a technical result in the achievement of the required degree of dissociation of the antigen-antibody complex in the subject sample, as well as in changing the conformation of proteins using the incubation buffer containing organic solvents in the disclosed ratios, which allows a significant enhancement of the sensitivity of detection of concentration of proteolytic fragments with a C- terminal lysine that are capable of binding to plasminogen or fragments thereof.

Ligands and detectors for the immunoassay

Plasminogen is a single-chain glycoprotein present in plasma at a concentration of about 2 □M (Wohl et al., Thromb. Res. 27:523-535, 1982; Kang et al., Trends Cardiovasc. Med. 90:92-102, 1999). Plasminogen contains 791 amino acid residues and 24 disulfide bonds. The protein consists of a single polypeptide chain, with an N-terminal glutamine and a C- terminal asparagine. It contains 2%-3% carbohydrates, which are localized within the heavy chain. Oligosaccharides are attached to Asp288 and Tre345. Plasminogen is a precursor of plasmin, which is formed by the cleavage of plasminogen between Arg-561 and Val-562 by tissue plasminogen activator (tPA) or urokinase-type plasminogen activator. In the process of activation of plasminogen, a bond between Arg560 and Val561 is cleaved; light and heavy chain are formed, connected by disulfide bonds. The light chain (Val561 - Asn790) has an active protease site, including the amino acid sequence of SerHisAsp. The plasmin heavy chain(Lys78 - Arg560) has five triple disulfide-linked loops known as kringle regions - or kringle domains - which are compact globular structures with a hydrophobic core. These structures are involved in the process of protein interaction in blood clotting. Both plasminogen and plasmin bind to fibrin through amino-terminal kringle regions, each of which is a triple loop region formed and fixed by disulfide bonds. ringles of the heavy chain consist of Kl, K2, K3, K4, and K5. Kringles 1-4 have domains which have a strong affinity for lysine, ε-aminocaproic acid, parabens, and other co-carbon amino acids having antifibrinolytic properties. Lysine binding sites (LBS) play an important role in the interaction between plasmin (plasminogen) and fibrin, as well as plasmin and its inhibitor - a2-AP (antiplasmin). Any fragment of plasminogen containing a kringle, regardless of whether it is a product of natural cleavage in vivo or a result of cleavage of plasminogen in vitro (e.g., by enzymatic action) can be used to detect proteolytic fragments with a C-terminal lysine. To assess the concentration of proteolytic fragments with a C-terminal lysine in diseases associated with the accumulation of such fragments, either plasminogen fragments or the full-length plasminogen molecule can be used as detectors. Specifically, a full- length plasminogen molecule, its heavy chain alone, as well as any of its fragments containing a kringle domain may be used asligands, immobilized on a solid phase, to determine the concentration of proteolytic fragments with a C-terminal lysine in blood samples.

These ligands and detectors can be either native Glu-plasminogen or its proteolytic derivatives may be prepared using genetic engineering techniques, by synthesis of a recombinant peptide in bacterial or eukaryotic expression systems. The recombinant ligands and detectors correspond to the amino acid sequence of human plasminogen. Specifically, the ligand and the detector for the disclosed invention are the full-length plasminogen and its fragments presented in Table 1.

The inventors were the first to describe and confirm that plasminogen or the fragments thereof may be used as ligands and detectors to determine the concentration of proteolytic fragments having a C-terminal lysine in a sample of human blood plasma by enzyme immunoassay and the result of this assay can be used as an important diagnostic sign to detect the presence of a pathological process in the subject.

The authors were the first to have applied a method of processing of plasma samples using dimethylsulfoxide, dimetlformamid, methanol, ethanol, propanol, propanol-2, acetone, acetonitrile, chloroform, ethylene glycol, N-methylpropanamide in an incubation buffer composition, that allows to increase the sensitivity of the method, compared to the methods, where the incubation buffer solution does not include the proposed components.

Despite the fact that the proposed method of detection is based on the use of specific well-defined polypeptides and buffer solution components, it should be clear to any qualified specialist in this scientific field that other proteins and peptides having the same composition of amino acid sequences and any combination of the proposed components in an incubation buffer identical to those in the disclosed invention can be used in an analogous fashion.

It should be clear to all qualified persons in this scientific field that the antigens and detectors in the present invention may be replaced by any amino acid having an polypeptide homology of more than 80% with the proposed polypeptides, since the replacement of amino acids that does not alter the structure of the kringle domains will not be an obstacle to the detection of proteolytic fragments having a C-terminal lysine.

Table 1 describes the various polypeptides -the derivatives of plasminogen that can be used for the enzyme immunoassay reaction with samples of human blood plasma for identification of proteolytic fragments with a C-terminal lysine.

Table 2 shows the different organic components in the buffer used for the preliminary treatment of plasma samples. The composition of the incubation buffer of can include anywhere from 5-25% of the organic components shown in Table 2 in 0.15 M Tris-HCl pH 8.8.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure l .The primary structure of human plasminogen.

Filled arrows indicate the cleavage sites for: (a) the release of the signal peptide, which is required for the production of the mature form of the protein (Glu-plasminogen); (b) the release of the activation peptide (Glu'-Lys ⁷⁷ ) resulting in the conversion of Glu'- plasminogen to Lys ⁷⁸ - plasminogen or Glu'-Plasmin to Lys ⁷⁸ - Plasmin; (c) the activation of plasminogen to plasmin by the cleavage of the Arg ⁵⁶¹ -Val ⁵⁶² peptide bond. Unfilled arrows indicatethe introns in the gene sequence. Triangles identify the N-linked oligosaccharide site at sequence position 289 and the O-linked glycan at position 346. The catalytic triad, His ⁶⁰³ , Asp ⁶⁴⁶ , and Ser ⁷⁴¹ , is also indicated by anasterisk(*). Disulfide bonds are depicted by heavy lines. A filled diamond (+) indicates a phosphorylation site.

The 20 amino acids and the abbreviations used are as follows:

alanine— ala— A; arginine— arg— R; asparagine— asn— ; aspartic acid— asp— D; cysteine— cys— C; glutamine— gin— Q; glutamic acid— glu— E; glycine— gly— G; histidine— his— H; isoleucine— ile— I; leucine— leu— L; lysine— lys— K; methionine— met— M; phenylalanine— phe— F; proline— pro— P; serine— ser— S; threonine— thr— T; tryptophan— ti — W;

tyrosine— tyr— Y; valine— val— V.

Figures 2. A schematic of the formation of heavy chain (Kl-5 +30 r) and light chain (micro- plasmin) from Glu-plasminogen.

EMBODIMENTS

The plasminogen molecule and fragments thereof, disclosed herein (Table. 1) were obtained either as recombinant proteins or purified from plasma, and used as antigens and detectors to create the immunoassay for the detection of the concentration of proteolytic fragments with a C-terminal lysine in blood samples of patients with various pathologies, including cancer.

Isolation of ligands for the immunoassay.

The method for the preparation of the heavy chain (Glu-H) Glul-Arg561 and light chain (L) Val562-Asn791 of human plasminogen:

The basic method consists of the activation of plasminogen to plasmin, followed by the reduction of S-S bonds between heavy and light chains in conditions that exclude autolysis, and then isolating the fragments using affinity chromatography on Lys-Sepharose 4B. Urokinase cleaves the Arg561 -Val562 bond in plasminogen. The resulting plasmin cuts the 77-78 bond and cleaves off the N-terminal peptide (1-77) . Mercaptoethanol reduces the two bonds between Cys558-Cys566 and Cys548-Cys666 which link the heavy and light chains.

First step:Glu-plasminogen was isolated from frozen human donor plasma by affinity

chromatography on Lys-Sepharose 4B at 4° C, pH 8.0. Blood plasma was thawed in the presence of aprotinin, centrifuged for 30 min at 4° C and diluted 2-fold in 0.02 M phosphate buffer, pH 8,0, containing 20 KIU / ml aprotinin. Prepared plasma was then applied onto a Lys- Sepharose 4B column, equilibrated with 0.1 M K-phosphate buffer, pH 8.0, containing 20 KIU / ml aprotinin. The column was washed to remove unbound protein with 0.3 M phosphate buffer, pH 8,0, containing 20 KIU / ml aprotinin, overnight to an absorbance at A280 = 0.05-0.01. Glu-plasminogen was eluted with a solution of 0.2 M 6-aminocaproic acid in 0.1 M K- phosphate buffer, pH 8.0, containing 20 KIU / ml aprotinin. Fractions containing protein were pooled and subjected to further purification by precipitation (NH ₄ ) ₂ SO ₄ (0.3 lg /ml protein solution). The precipitate was stored at 4° C for 18-24 hours and then separated by

centrifugation and dissolved in 0.05 M Tris-HCl buffer, pH 8.0 to a concentration 1.5 -2.0 mg / ml. The purified Glu-plasminogen was then dialyzed at 4° C against water (pH 8.0) and lyophilized.

Second step: Urokinase was added to a final concentration of 600 IU / ml to a solution of Glu- plasminogen (5 mg/ml) in 0.05 M Tris-HCl buffer, pH 8.8, containing 0.02 M L-lysine, 0.15 M NaCl, 20% glycerol, and 6000 IU / ml aprotinin, and incubated for 4 h at 37° C. The progression of conversion of Glu-plasminogen to plasmin was monitored by the hydrolysis of the specific substrate S-2251 (HD-Val-Leu-Lys p-nitroanilide, Sigma, USA) by plasmin in samples from the reaction, with complete conversion identified by observation of the maximum conversion rate for the substrate.

Third step:The reduction of S-S-bonds between the heavy and light chains of plasmin.

Mercaptoethanol was added to the plasmin solution to a final concentration of 0.25mM and incubated under nitrogen in the dark for 20 minutes at room temperature. The resulting free SH- groups were blocked by adding a freshly prepared solution of iodoacetic acid in 0.1 M Na- phosphate buffer, pH 8.0 (to a final concentration of 0.315 M) and incubated for 20 min.

Fourth step: The separation of the heavy and light chains of plasmin by column chromatography on Lys-Sepharose 4B. The reaction mixture was diluted to a concentration of 1 mg/ml of protein with 0.1 M Na-phosphate buffer, pH 8.0, containing 20 KIU / ml aprotinin and applied to a Lys-Sepharose 4B column equilibrated with the same buffer. Chromatography was performed at 25° C. The heavy chain of plasmin, containing kringles Kl-5 and 30 amino acid residues of the connecting peptide, was adsorbed onto the sorbent, whereas the light chain was washed away with equilibration buffer. Heavy chain (MR ~ 56-57 kDa) was eluted with a 0.2 M solution of 6- aminocaproic acid in 0.1 M Na-phosphate buffer, pH 8.0. The pooled fractions were dialyzed against water (pH ~ 8.0) and lyophilized.

The purity and molecular weight of the protein were assessed by 12% SDS- polyacrylamide gel electrophoresis. The absence of amidase activity (for S-2251) before and after incubation with urokinase confirmed that the solution of the heavy chain did not contain trace concentrations of miniplasminogen, which may go undetected by electrophoresis. The purification of Lys-plasminogen (Lys78-Asn791) and its heavy chain (Lys-H Lys78- Arg561) was performed by the same method, but without aprotinin. Miniplasminogen, which consists of K5 and plasmin light chain (Val442-Asn791),was obtained by incubation of Lys-plasminogen (Lys78-Asn791) with elastase followed by gel filtration on G-75 Sephadex.

The isolation of kringles Kl-4, 5 (Lys78-Arg530) was performed according to the method described by Cao and colleagues (Cao R.,Wu H.L., Veitonmaki N., Linden P., Farnedo J., Shi C.Y., and Cao Y. 1999. Proc. Natl. Acad. Sci. USA,.96, 5728-5733) with some modifications. Glu-plasminogen (lOmg/ml) was activated with urokinase (600ME/ml) in 0.05 M phosphate buffer, pH 9.0, containing 0.02 M L-lysine and 0.1 M NaCl, at 37° C. Complete conversion of plasminogen to plasmin was monitored by the increase in the amidase activity of the solution to the maximum activity value. An equal volume of 0.2 M glycerol buffer, pH 12.0 was added to a solution of plasmin, to a final pH of 10.5, and incubated for 18 hours at 25°C. The reaction mixture was diluted 5-fold with buffer containing 0.1 M phosphate buffer, pH 8.0, and 40 IU / ml aprotinin, and applied to a column of Lys-Sepharose 4B equilibrated with the same buffer. After a washing step, the adsorbed K 1-4,5 was eluted from the column with 0.2 M solution of 6-aminocaproic acid in 0.1 M phosphate buffer, pH 8.0, and 40 KIU / ml aprotinin, dialyzed against water, and lyophilized. The purity of the obtained Kl-4,5 was assessed by 12% SDS-polyacrylamide gel electrophoresis.

The isolation of kringle domains Kl-4 (Tyr80-Ala440),Kl-3 (Tyr80-Val338), and K4-5 (Val355 - Phe546) was performed using elastase treatment of Glu-plasminogen by the method described in the work of Cao and colleagues(Cao Y., Ji R. W., Davidson D., Schaller J., Marti D., Sohndel S., McCanse S. G., O 'Reilly M. S., Llinas M., and Folkman J. (1996) J. Biol. Chem., 271, 29461-29467). Glu-plasminogen was incubated with elastase at a ratio of 50:1 in a buffer containing 0.05 M Tris-HCl, pH 8.5, 0.5 M NaCl, and 200 KIU aprotinin, for 5 hours at room temperature. The reaction was stopped by adding PMFS to a concentration of 1 mM for 40-50 min. Gel-filtration on a Sephadex G-75column was performed to separate low and high molecular weight proteins. Protein fractions of the second peak containing Kl-3, Kl-4, K4-5 and miniplasminogen were applied to Lys-Sepharose 4B affinity column equilibrated with buffer containing 0.05 M Tris-HCl, pH 8.5 and 0.15 M NaCl. After the removal of

miniplasminogen which was not adsorbed onto the Lys-Sepharose4B in the flow-through fraction, the adsorbed fragments Kl-3, Kl-4 and K4-5 were eluted with a solution of 0.2 M 6- aminocaproic acid in the same buffer, dialyzed against a buffer containing 0.02 M Tris-HCl, pH 8.0, and applied to a column of heparin-agarose equilibrated with the same buffer. Unbound fragments Kl-4 and K4-5 were eluted with the buffer and fragment Kl-3 was eluted with a solution of 0.25 M KC1 in the same buffer. The purified fragment Kl-3 was dialyzed against water and lyophilized. Fragments Kl-4 and K4-5 were separated by gel filtration on Sephadex G-75.

Kringles K5 (Ser449 (or Pro452) - Phe546), Kl-3 (Tyr80-Val338), and K-4 (Val335- Ala440) were prepared according to the 1997 report from Cao and colleagues (Cao, Y., Chen, A., An, S. S. A., Ji, R. W., Davidson, D., and Llinas, M. (1997) J. Biol. Chem. 272, 22924- 22928). The method involves the digestion of Lys-plasminogen (Lys78-Asn791) by

elastase. After processing, the elastase mixture was applied to a column of Mono-S (Bio-Rad) equilibrated with buffer containing 20 mM NaOAc, pH 5.0. Fragments of plasminogen were eluted by a gradient of up to 1 M KC1 in buffer containing 20 mM NaOAc, pH 5.0. We used KC1 gradients of 0-20%, 20-50%, 50-70% and 70-100%. The K-5 fragment eluted at 50%. Fragments containing kringle domain K-4 (Val335-Ala440) and kringle domains Kl-3 (Tyr80- Val354) were obtained using a similar scheme, but with a different gradient.

The method for the isolation of kringle K5 (Val442-Arg561) involves digesting miniplasminogen(Val442-Asn791 Containing K5 within its heavy chain with elastase, followed by digestion of the resulting fragment by pepsin and then using gel filtration and ion exchange chromatography, as described by Thewes and colleagues (Theresa Thewes, Vasudevan Ramesh, Elena L. Simplaceanu and Miguel Llinfis, Isolation, purification and I H-NMR characterization of a kringle 5 domain fragment from human plasminogen (Biochimica et BiophysicaActa 912 (1987), 254-269).

Kringles Kl-4 (Lys78-Pro446) and Kl-4 (Lys78-Lys468) were prepared according to the method described by Patterson and Sang ( Patterson, B. C. and Sang, Q. A. (1997)

J.Biol.Chem. 272, 28823-28825 ), using metalloproteinases. Kringle Kl-4(Asn60-Pro447) was prepared according to the method reported by Lijnen and colleagues (Lijnen, H. R., Ugwu, F., Bini, A., and Collen, D. (1998) Biochemistry 37, 4699-4702), using metalloproteinases.

The production of a diagnostic test system for ELISA to assay proteolytic fragments with a C-terminal lysine.

We developed two types of diagnostic systems, direct and inverse.

When preparing a direct diagnostic system, full-length plasminogen or fragments thereof containing at least one kringle domain are used as the ligands for coating the solid phase. The various ligands used in ELISA are listed in Table 1. Their primary amino acid sequences are in the sequence listing. The ligand was diluted in 0.1M carbonate-bicarbonate buffer, pH 9.6, at a maximum concentration of 5 [ j g/ml for molecules with a molecular weight greater than 25 kDa, and 10

□ g/ml for molecules of molecular weight less than 25 kDa.

I X PBS (phosphate buffered saline):0.14M NaCl; 0.003MKC1; 0.005M Na2HP04; 0.002M H2PO4

Preparation of 1 liter of 10X PBS:NaCl - 80g; KC1 - 2g; Na ₂ HPO ₄ - 18g; KH ₂ PO ₄ - 2g Substrate buffer (pH 4.3): 31mM citric acid, 0.05 N NaOH, H ₂ O ₂ 3mM

TMB solution: 5 mM 3,3 ', 5,5'-tetramethylbenzidine in 70% DMSO

Chromogenic substrate solution: 4 parts of substrate buffer mixed with 1 part TMB solution.

To create the immunoassay kit, the immobilization of the ligand was preliminarily performed on a solid phase. Various types of carriers for immobilization of a ligand can be used, including cellulose acetate, glass beads or other particles that can adsorb proteins, as well as immunological plates or plastic strips.

100 L of the ligand solution were added to each well of a microtiter plate (Costar).The solution was incubated for 14-16 hours at 4°C in a humidified chamber. The contents of the wells were removed by shaking out and the plate was then washed twice with a solution containing PBS with 0.05% Tweeen-20 at 200□L / well to remove unbound ligand. A blocking solution consisting of 200 □L of a 1 % solution of bovine serum albumin (BSA) in PBS was added to wells and incubated for 1.5-2 hours at room temperature. After incubation, the blocking solution was removed, the plate dried overnight at room temperature and then used in further applications.

To increase the sensitivity and specificity of the method, a number of components were used in the incubation buffer, as presented in Table 2.

The test and control plasma samples were diluted 100-fold with incubation buffer containing one of the components listed in Table 2, incubated for 1 hour at 37° C and then diluted with diluent buffer (0.1 M Tris-HCl c 0.05% Tween-20, pH 8.0) at al : 10 dilution. 100

□ L of the solution were added to the appropriate wells and incubated for 1 hour at 37°C. After incubation, the solution was aspirated, the plate was washed 4 times with wash solution (PBS with 0.05% Tween-20). A working concentration of the conjugate solution diluted in PBS with 0.5% BSA (Mab Fc IgG-peroxidase orMab Fc IgA-peroxidase conjugates were used for determination of IgG and IgA levels,respectively) was added to the appropriate wells at 100 □L / well, and incubated for 1 hour at 37°C. Unbound components were removed by washing the plate 4 times with washing solution. 100 □L of the chromogenic substrate-solution were then added to all the wells and incubated for 15 minutes at 37° C. The reaction was stopped by the addition of 100 □L of stop solution (2M H ₂ SO ₄ ). Photometry was performed on an "UNIPLAN" photometer (Pikon, Russia) at a wavelength of 450 nm.

To manufacture the inverse diagnostic system, a labeled full-length plasminogen molecule or fragments thereof, containing at least one kringle domain listed in Table 1 was used as the detector in an ELISA of proteolytic fragments with a C-terminal lysine. The primary amino acid sequences of these peptides are in the sequence listing. To create the inverse diagnostic system (or kit), the first step involved the immobilization of mouse monoclonal antiodies to human immunoglobulins or other proteins onto a solid substrate (solid phase). Several types of immobilization substrates for monoclonal antibodies can be used, such as cellulose acetate, glass beads or other particles that can adsorb proteins, immunological plates or plastic. 100 □L of the monoclonal antibody(10 □g/ml) solution was added to each well of a microtiter plate (Costar). The solution was incubated for 14-16 hours at 4°C in a humidified chamber. The contents of the wells were removed by shaking out and the plate was then washed twice with a solution containing PBS with 0.05% Tweeen-20 at 200□L/well to remove unbound ligand. A blocking solution consisting of 200 □L of a 1% solution of bovine serum albumin (BSA) in PBS was added to wells and incubated for 1.5-2 hours at room temperature. After incubation, the blocking solution was removed, the plate dried overnight at room temperature and then used in further applications.

The full-length plasminogen or fragments thereof were subjected to a biotinylation procedure. 10 mg of a biotinylation reagent, biotinamidohexanoic acid N-hydroxysuccinimide ester (Sigma, B-2643), were dissolved in 0.5 ml of dimethylformamide. Plasminogen or its fragments were dissolved in 0.1 M phosphate buffer, pH 7.4, at a concentration of lmg/ml. 5 □l of the biotinylation reagent in dimethylformamide were added to 1 ml of this solution and incubated for 1 hour at room temperature on a shaker. Aprotinin solution was added to a final concentration of 20 IU/ml, the resulting peptide solution was transferred to a dialysis bag (4000 Da) and left overnight to dialyze against 0.01 M phosphate buffer with 20 IU/ml aprotinin, at 4°C. The resulting solution was diluted 2-foldin glycerol and frozen.

To demonstrate the involvement of C-terminal lysines in the protein binding to plasminogen, following incubation with incubation buffer, the plasma samples were diluted to a final dilution of 1 :1000 and incubated with carboxypeptidase B (Sigma- Aldrich) at 50 □g/ml in PBS. After incubation with carboxypeptidase B, the enzymatic reaction was stopped the addition of 1,10-Phenathroline (Sigma-Aldrich) in methanol (180 mg/ml). 100□L of the sample were used for testing in an ELISA assay to determine the concentration of immunoglobulins or other proteins with a C-terminal lysine after proteolysis.

An immunoassay method for the detection of proteolytic fragments with a C- terminal lysine

Blood samples were drawn from the patients' median cubital veins, using EDTA vacutainer tubes. The samples were then centrifuged for 15 min. Plasma was dispensed out into 100□L aliquotes and stored at -40 ^° C.

The control group consisted of plasma samples taken from 5 healthy donors. Each donor sample tested negative for hepatitis A, B, C and HIV viruses, as well as tuberculosis and syphilis.

Titers of proteolytic fragments of immunoglobulins IgG and IgA with C-terminal lysines in control samples were measured using the direct and inverse immunoassay according to the described procedure. The dilution of control samples was selected so that the optical density did not exceed 0.2.For the direct immunoassay, the final dilution of the sample (1 :1000) was empirically established, and this dilution was then used for all samples. The full-length plasminogen molecule, as well as its fragments, were used as the ligands. For increased accuracy of measurement, each sample was tested in duplicate. The optical density of the control sample was determined by taking the mean optical density of the pooled samples from 5 healthy controls. The test and control plasma samples were diluted 100-fold with incubation buffer containing one of the components listed in Table 2, incubated for 1 hour at 37°C, and then diluted with diluent buffer (0.1 M Tris-HCl c 0.05% Tween-20, pH 8.0) at a 1 : 10 dilution.100 □L of each sample were added to the appropriate wells and incubated for 1 hour at 37°C. After incubation, the solution in the wells was removed and the plate was washed 4 times with wash solution (PBS with 0.05% Tween-20). Conjugate solution in PBS with 0.5% BSA (Mab Fc IgG-peroxidase and Mab Fc IgA-peroxidase conjugates were used for determination of IgG and IgA concentrations, respectively) was added to appropriate wells at 100 □L/well, and incubated for 1 hour at 37°C. Unbound components were removed by washing the plate 4 times with washing solution. 100 □L of the chromogenic substrate-solution were then added to all the wells and incubated for 15 minutes at 37°C. The reaction was stopped by adding 100□L of stop solution (2M H ₂ SO ₄ ). Photometry was performed on an "UNIPLAN" photometer (Pikon, Russia) at a wavelength of 450 nm. For the inverse immunoassay, the final dilution of the sample (1 : 1000) was empirically established and used for all samples. A full-length biotinilated molecule of plasminogen as well as its biotinilated fragments were used as detectors. For accuracy of measurement, each sample was tested in duplicate. The optical density of the control sample was determined by obtaining the mean optical density of the pooled samples from five healthy controls. 96-well plates with adsorbed mouse monoclonal antibodies to IgG and IgA (DIATECH-M, Russia) were used.

The test and control plasma samples were diluted 100-fold with incubation buffer containing one of the components listed in Table 2, incubated for 1 hour at 37°C, and then diluted with diluent buffer (0.1 M Tris-HCl c, 0.05% Tween-20, pH 8.0) at a 1 :10 dilution. 100 □L of each sample were then added to the appropriate wells and incubated for 1 hour at 37°C. After incubation, the solution in the wells was removed and the plate was washed 4 times with wash solution (PBS with 0.05% Tween-20). A working dilution of plasminogen conjugated with biotin or biotinilated fragments thereof, in PBS with 0.5% BSA, were added to appropriate wells at 100 □L/well and incubated for 1 hour at 37° C. Unbound components were removed by washing the plate 4 times using washing solution. 100 □L of streptavidin-peroxidase conjugate solution were then added to all wells and incubated for 30 minutes at 37°C. Unbound components were removed by washing the plate 4 times with washing solution. 100 □L of the chromogenic substrate-solution were then added to all the wells and incubated for 15 minutes at 37° C. The reaction was stopped by adding 100 □L of stop solution (2M H ₂ SO ₄ ). Photometry was performed on an "UNIPLAN" photometer (Pikon, Russia) at a wavelength of 450 ran.

Direct and inverse ELISA of control samples was performed using each individual ligand and biotinylated detector. For comparison, five samples from the healthy control group were taken as controls; these were chosen so that the optical density of each one differed from the group mean by no more than 5%. These 5 samples were pooled and the resulting sample was used as the control sample (C),and was taken to indicate the normal concentration level of immunoglobulins with a C-terminal lysine. The samples with an optical density exceeding that of the control samples by more than 30% were considered positive. This cutoff range avoids false positives.

To evaluate the effectiveness of using various fragments of plasminogen and various organic solvents in the incubation buffer (listed in Table 2), plasma samples of patients with various forms of cancer and autoimmune diseases were used in an ELISA for immunoglobulin fragments with a C-terminal lysine. The examples provided below show the results of using various ligands and detectors in the test system designed to identify high titer of proteolytic fragments of immunoglobulin and other proteins with a C-terminal lysine.

To prove the involvement of C-terminal lysines in the binding to plasminogen or its fragments, plasma samples, after incubation with incubation buffer, were diluted up to a final dilution of 1 : 1000 and incubated with carboxypeptidase B at a final concentration of 50mg/ml in PBS (Sigma-Aldrich). After an 1 hour incubation with carboxypeptidase B, the enzymatic reaction was stopped with 1,10-Phenathroline (Sigma-Aldrich) diluted in methanol to a final concentration of 1.8 mg/ml. 100DL of the sample were used for testing in ELISA to detect the concentration of proteolytic fragments of immunoglobulins and other proteins containing a C- terminal lysine.

EXAMPLES

No difference was observed between the control sample and the sample from apatient in an ELISA for immunoglobulin fragments with a C-terminal lysine when using an incubation buffer without the proposed components listed in Table 2 (dimethylsulfoxide, dimetlformamid, methanol, ethanol, propanol, isopropanol, acetone, acetonitrile, chloroform, ethylene glycol, N- methylpropanamide) at a final dilution of 1 : 1000. By contrast, when using an incubation buffer with the proposed components listed in Table 2, a clear difference was observed in all cases between the control and the test samples at a final dilution of 1 : 1000. Notably, after incubation of the samples at a final dilution of 1 : 1000 with carboxypeptidase B, the difference between the control samples and the patient sample disappeared.

EXAMPLE 1

Identification of the binding of IgG and IgA with a C-terminal lysine in prostate cancer using direct ELISA.

Diagnoses of patients with prostate cancer were established on the basis of the following parameters: clinical examination and confirmation by prostate biopsy. The group consisted of 5 patients with prostate cancer.

ELISA of samples from prostate cancer patients and a control sample was performed according to the method described above. The samples where the optical density exceeded the control by more than 30% were considered positive.

Results: Using the following sequences as the ligand: SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N06, SEQ ID N09, SEQ ID NO10, SEQ ID NOl 1, SEQ ID N012, SEQ ID NO 13, in an ELISA, only 2 out of 5 samples from prostate cancer patients were positive for IgG and IgA when using an incubation buffer without the proposed components listed in Table 2. The dilution of samples in this case was 1 : 100. By contrast, when using an incubation buffer with the proposed components listed in Table 2, the number of positive samples from prostate cancer patients rose to 5 out of 5 positive for IgG and IgA. The final dilution here was 1 : 1000. After incubation of the samples at a final dilution of 1 : 1000 with carboxypeptidase B, the difference between the cancer patient samples and the control sample disappeared.

Using the following sequences as the ligand: SEQ ID NO 7, SEQ ID N08, SEQ ID N014, SEQ ID NOl 5, SEQ ID N016, SEQ ID N017, SEQ ID N018, SEQ ID N019, SEQ ID NO20 in an ELISA, only 2 out of 5 samples from prostate cancer patients were positive for IgG and IgA when using an incubation buffer without the proposed components listed in Table 2. The dilution of samples in this case was 1 : 100. By contrast, when using an incubation buffer with the proposed components listed in Table 2, the number of positive samples from prostate cancer patients rose to 4 out of5 positive for IgG and IgA. The final dilution here was 1 : 1000. After incubation of the samples at a final dilution of 1 : 1000 with carboxypeptidase B, the difference between the cancer samples and the control sample disappeared.

EXAMPLE 2.

Identification of the binding of IgG and IgA with a C-terminal lysine in prostate cancer using inverse ELISA.

Diagnoses of patients with prostate cancer were established on the basis of the following parameters: clinical examination and confirmation by prostate biopsy. The group consisted of 5 patients with prostate cancer.

ELISA of samples from prostate cancer patients and a control sample was performed according to the method described above. The samples where the optical density exceeded that of the control sample by more than 30% were considered positive.

Results:

Using the following biotinylated sequences as a detector: SEQ ID NOl, SEQ ID N02, SEQ ID N03, SEQ ID N04, SEQ ID N06, SEQ ID N09, SEQ ID NO10, SEQ ID NOl 1, SEQ ID N012, SEQ ID NOl 3 in an ELISA, only 2 out of 5 samples from prostate cancer patients were positive for IgG and IgA using an incubation buffer without the proposed components listed in Table 2. The dilution of samples in this case was 1 : 100. By contrast, when using an incubation buffer with the proposed components listed in Table 2, the number of positive samples from prostate cancer patients rose to 5 out of 5 positive for IgG and IgA. The final dilution here was 1 : 1000. After incubation of the samples at a final dilution of 1 : 1000 with carboxypeptidase B, the difference between the cancer samples and the control sample disappeared.

Using the following biotinylated sequences as a detector: SEQ ID N07, SEQ ID N08, SEQ ID N014, SEQ ID N015, SEQ ID N016, SEQ ID N017, SEQ ID N018, SEQ ID N019, SEQ ID NO20. in an ELISA, only 2 out of 5 samples from prostate cancer patients were positive for IgG and IgA using an incubation buffer without the proposed components listed in Table 2. The dilution of samples in this case was 1 : 100. By contrast, when using an incubation buffer with the proposed components listed in Table 2, the number of positive samples from prostate cancer patients rose to 4 out of 5 positive for IgG and IgA. The final dilution here was 1 : 1000. After incubation of the samples at a final dilution of 1 : 1000 with carboxypeptidase B, the difference between the cancer patient samples and the control sample disappeared.

Table 1. Fragments of plasminogen having kringle structures.

Table 2: Solvent of buffer for incubation