METHOD OF DETECTION OF PROTEOLYSIS PRODUCTS IN PLASMA AND A DIAGNOSTIC SYSTEM FOR ITS APPLICATION

Abstract

The present invention relates to the fields of medicine and immunology. In particular, this invention relates to immunological diagnostic methods that use the whole-length molecule of plasminogen or its peptide fragments as universal detectors of proteolysis products having a C-terminal lysine. The method of this immunological diagnostic is to identify the human diseases associated with increased activity of proteolytic enzymes. The inventions also relate to a diagnostic test system comprised of a detectorthe full-length molecule and its presented peptide fragments. The technical result is comprised of achieving the required degree of dissociation of the antigen-antibody complex in a sample from the subject, as well as changing the conformation of proteins using an incubation buffer containing organic solvents in the disclosed ratios, that can significantly increase the sensitivity of the method for determining the concentration of proteolytic fragments with a C-terminal lysine binding with plasminogen or fragments thereof.

Claims

1. A method for identifying a subject having an increased blood plasma concentration of proteolytic products having a C-terminal lysine, comprising the following steps: providing a blood plasma sample obtained from a subject; contacting the blood plasma sample or a component thereof with plasminogen or a fragment thereof in the presence of a buffer composition, wherein said plasminogen or fragment thereof consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and wherein said buffer composition comprises a compound selected from the group consisting of dimethylsulfoxide, dimethylformamide, methanol, ethanol, propanol, propanol-2, acetone, acetonitrile, chloroform, ethylene glycol, N-methylpropanamide and combinations thereof; and detecting complexes comprising the plasminogen or the fragment thereof bound to the proteolytic product having a C-terminal lysine; and/or determining the level of proteolytic products having a C-terminal lysine in the sample; wherein an increased amount of complexes and/or an increased level of proteolytic products having a C-terminal lysine in the sample relative to a control sample is indicative of said subject having an increased blood plasma concentration of proteolytic products having a C-terminal lysine.

2. The method of claim 1, where in an increase of the level of proteolytic products having a C-terminal lysine exceeding the level in the control sample by more than 30% is taken as an indicator of the presence of a pathological process in a subject.

3. The method of claim 1 or 2, which comprises the use of an enzyme linked immunosorbent assay (ELISA).

4. The method of claim 1, wherein said contacting comprises contacting the blood plasma sample with a solid support, where in said plasminogen or fragment thereof is immobilized on the surface of the solid support.

5. The method of claim 1, wherein said contacting comprises (i) contacting the blood plasma sample with a solid support, wherein antibodies specific to the products of proteolysis having a C-terminal lysine are immobilized on the surface of the solid support, (ii) allowing the products of proteolysis having a C-terminal lysine present in the sample to bind to the antibodies, (iii) removing unbound components from the solid support, and (iv) contacting said plasminogen or fragment thereof with the solid support, wherein said plasminogen or fragment thereof is optionally labeled with a detectable label.

6. A test system for identifying a subject having an increased concentration of proteolytic products having a C-terminal lysine, comprising a detection system for these products, comprising (i) a plasminogen molecule or a fragment thereof, wherein said plasminogen molecule or fragment consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, (ii) a buffer composition comprising a compound selected from the group consisting of dimethylsulfoxide, dimethylformamide, methanol, ethanol, propanol, propanol-2, acetone, acetonitrile, chloroform, ethylene glycol, N-methylpropanamide and combinations thereof.

7. The test system of claim 6, which comprises the use of an enzyme linked immunosorbent assay (ELISA).

8. The test system of claim 6, further comprising a solid support with said full-length plasminogen molecule or the fragment thereof being immobilized on its surface.

9. The test system of claim 6, further comprising a solid support with antibodies specific to the products of proteolysis having a C-terminal lysine immobilized on its surface.

10. The use of a test system according to claim 1 for identifying a subject with a risk of developing a pathological process.

11. The method of claim 1, wherein the subject is a human subject.

12. The method of claim 1, wherein the proteolytic products with C-terminal lysines to be detected are proteolytic fragments of immunoglobulin.

13. The method of claim 12, wherein the proteolytic fragments of immunoglobulin are fragments of IgG and/or fragments of IgA.

14. The method of claim 2, wherein the pathological process is a cancer or an autoimmune disease.

15. The method of claim 14, wherein the cancer is prostate cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1. The primary structure of human plasminogen.

[0047] 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.sup.77) resulting in the conversion of Glu-plasminogen to Lys.sup.78-plasminogen or Glu-Plasmin to Lys.sup.78-Plasmin; (c) the activation of plasminogen to plasmin by the cleavage of the Arg.sup.561-Val.sup.562 peptide bond. Unfilled arrows indicate the 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.sup.603, Asp.sup.741, and Ser.sup.741, is also indicated by anasterisk (*). Disulfide bonds are depicted by heavy lines. A filled diamond (.diamond-solid.) indicates a phosphorylation site.

[0048] The 20 amino acids and the abbreviations used are as follows:

TABLE-US-00003 alanine-ala-A;arginine-arg-R;asparagine-asn-N; asparticacid-asp-D;cysteine-cys-C;glutamine- gln-Q;glutamicacid-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; trytophan-trp-W;tyrosine-tyr-Y;valine-val-V.

EMBODIMENTS

[0049] 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.

[0050] The method for the preparation of the heavy chain (Glu-H) Glu1-Arg561 and light chain (L) Val562-Asn791 of human plasminogen:

[0051] The basic method consists of the activation of plasminogen to plasmin, followed by the reduction of SS 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.

[0052] 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.sub.4).sub.2SO.sub.4 (0.31 g/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.

[0053] 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 KIU/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.

[0054] Third step: The reduction of SS-bonds between the heavy and light chains of plasmin. Mercaptoethanol was added to the plasmin solution to a final concentration of 0.25 mM 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.

[0055] 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/mil 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 K1-5S 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.

[0056] 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.

[0057] The purification of Lys-plasminogen (Lys78-Asn791) and its heavy chain (Lys-H Lys78-Arg561) was performed by the same method, but without aprotinin.

[0058] 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.

[0059] The isolation of kringles K1-4, 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 (10 mg/ml) was activated with urokinase (600 ME/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 KIU/ml aprotinin, and applied to a column of Lys-Sepharose 4B equilibrated with the same buffer. After a washing step, the adsorbed K1-4, 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 K1-4, 5 was assessed by 12% SDS-polyacrylamide gel electrophoresis. [0060] a. The isolation of kringle domains K1-4 (Tyr80-Ala440), K1-3 (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-75 column was performed to separate low and high molecular weight proteins. Protein fractions of the second peak containing K1-3, K1-4, 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 K1-3, K1-4 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 K1-4 and K4-5 were eluted with the buffer and fragment K1-3 was eluted with a solution of 0.25 M KCl in the same buffer. The purified fragment K1-3 was dialyzed against water and lyophilized. Fragments K1-4 and K4-5 were separated by gel filtration on Sephadex G-75. [0061] b. Kringles K5 (Ser449 (or Pro452)-Phe546), K1-3 (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 KCl in buffer containing 20 mM NaOAc, pH 5.0. We used KCl 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 K1-3 (Tyr80-Val354) were obtained using a similar scheme, but with a different gradient. [0062] c. 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, Elcna 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). [0063] d. Kringles K1-4 (Lys78-Pro446) and K-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 K1-4 (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.

[0064] We developed two types of diagnostic systems, direct and inverse.

[0065] 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.

[0066] The ligand was diluted in 0.1M carbonate-bicarbonate buffer, pH 9.6, at a maximum concentration of 5 g/ml for molecules with a molecular weight greater than 25 kDa, and 10 g/ml for molecules of mole 1PBS (phosphate buffered saline):0.14M NaCl; 0.003MKCl; 0.005M Na2HPO4; 0.002M KH2PO4

[0067] Preparation of 1 liter of 10PBS: NaCl80 g; KCl2 g; Na.sub.2HPO.sub.418 g; KH.sub.2PO.sub.42 g

[0068] Substrate buffer (pH 4.3): 31 mM citric acid, 0.05 N NaOH, H.sub.2O.sub.2 3 mM

[0069] TMB solution: 5 mM 3,3, 5,5-tetramethylbenzidine in 70% DMSO

[0070] Chromogenic substrate solution: 4 parts of substrate buffer mixed with 1 part TMB solution.

[0071] 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.

[0072] 100 L of the ligand solution were added to each well of a microwell 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.

[0073] To increase the sensitivity and specificity of the method, a number of components were used in the incubation buffer, as presented in Table 2.

[0074] 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 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 or Mab 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.sub.2SO.sub.4). Photometry was performed on an UNIPLAN photometer (Pikon, Russia) at a wavelength of 450 nm.

[0075] 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 antibodies 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% Tween-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.

[0076] 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 1 mg/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.

[0077] 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

[0078] Blood samples were drawn from the patients' median cubital veins, using EDTA vacutainer tubes. The samples were then centrifuged for IS min. Plasma was dispensed out into 100 L aliquots and stored at 40C.

[0079] 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.

[0080] 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.sub.2SO.sub.4). Photometry was performed on an UNIPLAN photometer (Pikon, Russia) at a wavelength of 450 nm.

[0081] 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.

[0082] 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.sub.2SO.sub.4). Photometry was performed on an UNIPLAN photometer (Pikon, Russia) at a wavelength of 450 nm.

[0083] 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.

[0084] 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.

[0085] 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 50 mg/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. 100 L 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

[0086] No difference was observed between the control sample and the sample from a patient 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

[0087] Identification of the Binding of IgG and IgA with a C-Terminal Lysine in Prostate Cancer Using Direct ELISA.

[0088] 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.

[0089] 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.

[0090] Results:

[0091] Using the following sequences as the ligand: SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO6, SEQ ID NO9, SEQ ID NO10, SEQ ID NO11, SEQ ID NO12, SEQ ID NO13, 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.

[0092] Using the following sequences as the ligand: SEQ ID NO 7, SEQ ID NO8, SEQ ID NO14, SEQ ID NO15, SEQ ID NO16, SEQ ID NO17, SEQ ID NO18, SEQ ID NO19, 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 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.

Example 2

[0093] Identification of the Binding of IgG and IgA with a C-Terminal Lysine in Prostate Cancer Using Inverse ELISA.

[0094] 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.

[0095] 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.

[0096] Results:

[0097] Using the following biotinylated sequences as a detector: SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO6, SEQ ID NO9, SEQ ID NO10, SEQ ID NO11, SEQ ID NO12, SEQ ID NO13 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.

[0098] Using the following biotinylated sequences as a detector: SEQ ID NO7, SEQ ID NO8, SEQ ID NO14, SEQ ID NO15, SEQ ID NO16, SEQ ID NO17, SEQ ID NO18, SEQ ID NO19, SEQ ID NO020, 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.