FR2863616A1 - New antibodies specific for protein S not cleaved by factor Xa, useful for determining uncleaved protein S in blood, particularly for diagnosis of coagulation disorders - Google Patents

Abstract

Antibody (Ab) that recognizes specifically plasma protein S (I) that has not been cleaved by factor Xa. Independent claims are also included for the following: (1) fragments of Ab that contain the antigen recognition site; (2) reagent for determining uncleaved (I) comprising Ab or its fragments, either as immunoconjugates or in a form suitable for performing immunoassays; (3) kit for determining uncleaved (I) comprising: (a) Ab, its fragments or the reagent of (2); and (b) antibodies (Ab1), or their fragments, that recognize free protein S; (4) method for determining uncleaved (I) in blood plasma; (5) method for preparing monoclonal antibodies that recognize uncleaved (I) specifically; (6) method for determining the anticoagulant potential in a patient; and (7) peptides C26C and Y29C as new compounds. Cys-Leu-Arg-Ser-Phe-Gln-Thr-Gly-Leu-Phe-Thr-Ala-Ala-Arg-Gln-Ser-Thr-Asn-Ala-Tyr-Pro-Asp-Leu-Arg-Ser-Cys (C26C) Cys-Leu-Arg-Ser-Phe-Gln-Thr-Gly-Leu-Phe-Thr-Ala-Ala-Arg-Gln-Ser-Thr-Asn-Ala-Tyr-Pro-Asp-Leu-Arg-Ser-Cys-Val-Leu-Tyr (Y29C).

Description

ASSAY OF FREE S PROTEIN NOT CLIVE BY FACTOR Xa. THE USE OF

  THIS ASSAY FOR EVALUATION OF COAGULATION POTENTIAL.

  The invention relates to the assay of free S protein not cleaved by factor Xa, involved in the physiology of coagulation and in particular in the sequence of enzymatic reactions leading to the coagulation of blood. The invention particularly relates to the use of this assay for assessing the coagulation potential in a patient.

  The subject of the invention is, in particular, polyclonal or monoclonal antibodies, usable for carrying out the above assay, these antibodies recognizing the S protein uncleaved by factor Xa.

  Coagulation is a complex phenomenon comprising a cascade of in vivo reactions, involving various molecules, comprising constituents of a protein nature as well as ions such as calcium ion but also cells, in particular those of the vascular wall, as well as platelets.

  Protein components of the coagulation cascade include plasma proteins that include coagulation factors and coagulation inhibitors, and tissue protein, also called tissue factor, which is closely associated with phospholipids in cell membranes.

  Coagulation factors are activated during the coagulation process and are mostly synthesized especially in hepatocytes. Among these factors, which will be mentioned in the context of the present invention include factor Xa.

  Other plasma proteins, physiological inhibitors of coagulation, include a large number of proteins among which, belonging to a particular family, the protein called protein C, or more exactly the system of protein C, which actually comprises two plasma proteins vitamins K-dependent proteins C and S as well as a membrane protein, thrombomodulin. Protein C is a zymogen of a double-stranded serine protease.

  Protein S, belonging to the same family is a single-stranded protein that carries a Gla domain followed by four EGF domains and then an extended carboxy-terminal region. After being synthesized in hepatocytes, protein C and protein S are secreted into the plasma after carboxylation of Glu residues in the amino-terminal position.

  Protein S contains about 7% carbohydrates and is at a plasma concentration of about 25 mg / l.

  Activated C and S proteins play an essential role in coagulation disorders, as evidenced by thromboembolic diseases in patients with a deficiency of one of these two proteins (Comp PC, et al. : Recurrent venous thromboembolism in patients with partial deficiency of protein SN Engl J. Med 311, 1525-1528, 1984; Schwartz H., et al., Familial protein S deficiency is asociated with recurrent thrombosis, Blood 64, 1297-1300, 1984; Koster et al., Protein C deficiency in a controlled series of unselected outpatients: a brief but clear risk factor for venous thrombosis (Leiden thrombophilia study), Blood 85, 2756-2761, 1995, Gladson CL et al, The frequency of type I heterozygous protein S and protein C deficiency in 141 unrelated young patients with venous thrombosis, Thromb Haemost, 59, 18-22, 1988, Pabinger I., et al, Hereditary deficiency of antithrombin III, protein C and protein S: prevalence in patients wi history of venous thrombosis and criteria for rational patient screening. Blood Coag. Fibrinol., 3, 547-553, 1992; Pabinger I, et al: The risk of thromboembolism in asymptomatic patients with protein C and protein deficiency: a prospective cohort study. Thromb. Haemost., 71, 441-445, 1994; Girolami A., et al., Severe arterial cerebral thrombosis in patients with protein S deficiency (moderately reduced total and markedly reduced free protein S): a family study. Thromb. Haemost. 61, 144-147, 1989; Aliaart, C., et al, Hereditary protein deficiency in young adults with arterial occlusive disease. Thromb. Haemost. 64, 206-210, 1990). When protein C is activated by thrombin linked to thrombomodulin, the latter constituting an endothelial cell membrane receptor is capable, in the presence of the protein S acting as a cofactor, to inactivate the coagulation factors Va and VIIIa. which results in a limitation of the generation of thrombin.

  Protein S has been characterized and a cDNA sequence encoding this protein has been obtained from isolated mRNA (Lundwall et al., Isolation and sequence of the cDNA for human protein S, regulator of blood coagulation, Proc. Natl Acad Sci 83: 6716-6720, 1986) The obtained cDNA contains 3344 bp and encodes a polypeptide extending from the amino acid at position -15 belonging to the Leader sequence to the amino acid 635. In addition, this cDNA contains a 3 'non-coding region of 1148 bp. Two potential polyadenylation sequences were identified in this noncoding region, one in position 3274 and the other in position 3309. The genomic DNA encoding the human S (PS) protein is transcribed into a 4 kb majority mRNA. , representing approximately 0.01% of total hepatic mRNAs (Hoskins et al., Cloning and characterization of human cDNA encoding a protein S precursor, Proc Natl Acad Sci 84: 349-353, 1987).

  Protein S is therefore a glycoprotein, which can be purified from human plasma, characterized for the first time by Di Scipio et al (Comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S. Biochemistry 16: 698-706, 1977). The single-stranded protein S has a molecular weight of about 75 kDa and a plasma concentration of 350 nM (25 mg / l) in normal plasma.

  On SDS polyacrylamide gel (SDS-PAGE) and under reducing conditions, the plasma protein S migrates as a doublet suggesting that a fraction of this protein circulates in double stranded form (Dahlbâck, B. Purification of human vitamin K-depenent protein S and its limited proteolysis by thrombin Biochem J. 209: 837-846, 1983b).

  Further observation shows that protein S, synthesized in several cell types including liver cells, endothelial cells, Leydig cells, and platelets, is first obtained in the form of a precursor of 676 amino acids, which is then cleaved to give a mature protein of 635 amino acids and a pre-propeptide of 41 amino acids. Once cleaved from its signal peptide, the S protein undergoes post-translational modifications before being secreted into the circulation. These modifications include N-glycosylation at residues Asn458, Asn468 and Asn489 as well as gamma-carboxylation of the 11 N-terminal Glu residues to γ-carboxy-glutamic acids (Gla) and 13-hydroxylation. The mature protein thus obtained comprises various domains, represented in FIG. 1, among which will be mentioned for the purposes of the present application, the thrombin sensitive loop (BST) domain encoded by exon IV of the PRO-SI gene. (Ploos van Amstel et al A mutation in the protein S pseudogene is linked to protein S deficiency in a thrombophilic family, Thromb Haemost, 62, 897-901, 1989) of the protein S. The thrombin sensitive loop, coded by exon IV of the PRO-SI gene is constituted by amino acids 46 to 74 of the protein and comprises two cysteine residues (in positions 47 and 72) linked together by a disulfide bridge. The secondary structure and the three-dimensional structure were studied for the BST loop and a molecular modeling of BST was performed (Villoutreix et al, A model for the Gla-TSR-EGF-1 region of the anticoagulant cofactor protein S: from biostructural pathology to species-specific cofactor activity J. Computed Aided Mol Des., 11: 293-304, 1997a).

  It has also been shown that protein S circulates in the plasma in four forms: on the one hand the free or bound forms of a complement system protein, C-4b-BP, on the other hand the intact forms or cleaved in the plasma. Cleavage of the S protein was examined, showing that it can be performed differently in vivo and in vitro. At the BST loop, thrombin (or Factor Ila) cleavage is observed in vitro at the potential sites marked by amino acids 49 and 70 and, moreover, factor Xa cleavage at an Arg60 site, the latter requiring the presence of calcium and phospholipids. In vivo observations show that cleaved protein S would result at least in part from factor Xa cleavage while cleavage by thrombin can not occur. However, the role of other enzymes in the cleavage observed in vivo can not be ruled out.

  The bound S protein (in cleaved or intact form) is linked to a complement protein, the C4b-binding protein (or C4b-BP) which is a high molecular weight protein (530 to 570 kDa) identified by Dahlbäck et al 1983 (Degradation of human complement component C4b in the presence of C4b-binding protein-protein S complex, Biochem J. 209: 857-863). The interaction of protein S with C4b-BP would involve different regions of a so-called sex hormone binding globulin (SHBG) domain encoded by exons IX to XV of the PROS1 gene of protein S. The four forms of protein S are shown in Figure 2, it being understood that the cleavage illustrated is that which would involve the factor Xa of the cascade of coagulation. The four plasma forms of protein S are distributed in the following proportions: 62% of bound S protein, of which 53% of bound, uncleaved S protein, and 9% of bound and cleaved S protein, and moreover 38% of protein S free, including 6% free S protein, cleaved (inactive) and 32% free protein, uncleaved (active). These values were determined by Griffin et al (Reevaluation of total, free and bound protein S and C4b-binding protein levels in plasma anticoagulated with citrate or hirudin, Blood 79: 3203-3211, 1992).

  Given the activity of free and intact (uncleaved) protein S, when it is a cofactor of protein C in anticoagulant activity, and the abolition of this anticoagulant function after cleavage of protein S by the factor Xa generated during the activation of coagulation, it seemed interesting to consider the detection and specific assay of the free protein S uncleaved by factor Xa. Thus, the subject of the invention is the means for carrying out this assay, which means would advantageously make it possible to evaluate the anticoagulant potential of patients suffering from coagulation disorders. In particular, the means for detecting and assaying the free and intact S protein may be used in the context of performing a diagnosis of coagulation disorders in a biological sample, in particular plasma, taken from a patient.

  The present patent application therefore relates to means for the assay of free S protein not cleaved by factor Xa, as well as the use of these means for the production of detection and assay kits, as well as in methods detecting and assaying the free factor S uncleaved protein S, in particular for use in diagnostic protocols.

  The free S protein not cleaved by factor Xa will also be referred to hereinafter as the uncleaved free protein S text, in particular to take into account the possible intervention of other factors in the cleavage in vivo.

  The means defined to allow the assay of the uncleaved free protein S are, first and foremost, antibodies having an antigen specificity for the protein S which is not cleaved by factor Xa.

  A second category of antibodies is used to perform the assay of free S protein not cleaved by factor Xa: they are antibodies with a capacity to recognize free S protein.

  For carrying out the invention, the antibodies defined above capable of recognizing the free plasma protein S (first category of antibodies) and, moreover, the antibodies capable of recognizing the factor S non-cleaved protein (second category of antibodies). antibodies), are further characterized by the fact that the epitopes recognized by each of these two categories of antibodies are different on the protein S. The recognition specificity of the antibodies recognizing the protein S which is not cleaved by the factor Xa can in in addition to being characterized by the fact that these antibodies recognize neither the cleaved free plasma protein S nor the bound forms of the cleaved plasma S protein.

  In the context of the invention, the antibodies are therefore used for their specificity of recognition with respect to one or the other of the above antigens.

  The subject of the invention is antibodies having an antigenic specificity for the factor Xa-cleaved S protein, as such, obtained specifically against this protein, that is to say against an epitope which does not find itself recognized by antibodies prepared against free S protein.

  The specificity of the recognition can be evaluated by an immunological competition test between the different forms of the protein S, it being understood that the antibodies of the invention react in such a test significantly more strongly with the protein S not cleaved by the factor Xa with the other forms of this protein S. A plastic support, for example a micro-ELISA plate, is brought into contact overnight with uncleaved purified protein S and with factor Xa cleaved protein S to achieve the coating of the plate. The different antibodies to be tested are then deposited on the plate. Depending on their specificity, the antibodies are fixed on the coating or not. The bound antibodies are revealed with the aid of the polyclonal anti-antibody antibodies to be tested and they are coupled to the enzyme, for example peroxidase, alkaline phosphatase or acethylcholinesterase. The bound enzyme is visualized by its activity on the specific substrate for example ortho-phenylenediamine (O.P.D.) or tetramethylbenzidine (TMB) for peroxidase. The antibody of the invention results from staining with factor Xa uncleaved protein S and does not produce a reaction against factor Xa-cleaved protein S.

  In a particular embodiment of the invention, the antibodies are monoclonal antibodies.

  According to a particular embodiment of the invention, the antibodies are characterized in that they recognize the peptide CLRSFQTGLFTAARQSTNAYPDLRSC (C26C) or the peptide CLRSFQTGLFTAARQSTNAYPDLRSCVLY (peptide Y29C).

  The presence of the Y residue facilitates coupling of the peptide to the carrier proteins (such as KLH) used for immunization.

  Monoclonal antibodies according to the invention can be prepared by various methods including immunizing an animal, in particular a mammal, to trigger a reaction of its immune system against the administered antigen consisting of the purified protein S, not cleaved or for example by one of the peptides identified above. From the antibody-producing cells of the immunized animal, cell fusion is performed with cells, in particular immortalized cells, to obtain hybridomas enabling the production of the antibodies by cell lines in culture.

  Other techniques than that of the hybridoma production according to Kohler & Milstein (Continuous culture of fused cells secreting antibody of predefined specificity, Nature 1975, 256: 495-497) are also available to produce antibodies, such as that of the hybridomas obtained. with human B cells (Cote et al., 1983, PNAS 80: 20262030) or the EBV hybridoma technique (Cole et al., 1985, Alan R. Liss, Inc., pp. 77-96).

  In the context of the present invention, in particular hybridoma-secreted IgM antibodies prepared with splenocytes from immunized mice fused with myeloma cells have been obtained.

  The invention also relates to antibodies whose antigen specificity is that specified above, which belong to other isotypes and in particular IgG. If necessary, the IgG can be derived from IgM according to known techniques including switch class.

  A preferred monoclonal antibody according to the invention is the S2P11B6 antibody, secreted by the hybridoma deposited with ATCC under n PTA-5425 on August 27, 2003.

  A protocol for the preparation of this S2P11B6 antibody is described in the following experimental section, including the purification of the antibody.

  The antibodies according to the invention may also be obtained by other techniques, such as expression in host cells transformed with DNA sequences encoding the desired specificity of the antibodies.

  Anti-uncleaved protein S antibodies according to the invention can also be polyclonal, for example prepared in rabbits or hens.

  The invention also relates to fragments of the antibodies defined in the context of the invention, in particular functional fragments, having preserved the antigenic specificity of the antibodies of the invention, for example Fab, Fab ', F (ab') fragments. 2 or subfragments thereof, such as fragments containing all or part of the CDR domains responsible for the antigenic specificity, sufficient to specifically bind the uncleaved PS antigen by factor Xa.

  Antibody fragments may be prepared by enzymatic digestion, for example, with pepsin or papain with mercaptoethanol, or by expression of their coding sequences in cellular hosts or by chemical synthesis.

  The invention also relates to antibodies or chimeric antibody fragments obtained from previous ones, the properties of which have for example been improved for use in an immunoassay.

  The subject of the invention is also a reagent for the determination of the free plasma protein S which is not cleaved by factor Xa, comprising antibodies corresponding to the definition given above, or their functional fragments defined above, said antibodies or their fragments being prepared for use in immunological assays such as ELISA (immunoconjugate antibody) tests, immunochromatographic detection, detection by aggregation of latex specific 2863616 antibody-specific latex specimens, or microfluidic detection or by tests carried out with microbeads coated with the antibodies.

  For example, antibodies or their functional fragments may be conjugated with peroxidase or with alkaline phosphatase, or colloidal gold.

  A protocol for carrying out the immunoconjugate is described in the experimental part.

  A reagent thus constituted is preferably such that the concentration of immunoconjugates in solution makes it possible to avoid the risks of aggregation and is, for example, less than or equal to 1 mg / ml.

  The present invention also relates to a kit for assaying free plasma protein S not cleaved by factor Xa, comprising: antibodies having an antigen specificity for protein S not cleaved by factor Xa as defined above, or fragments thereof functional, or a reagent as defined above, antibodies recognizing the free protein S or their fragments recognizing the free protein S.

  Antibodies recognizing free S protein are used especially when the kit allows the immunoassay, for example by assay based on aggregation of latex microparticles or by ELISA. The antibodies, or their functional fragments can in this case be used to sensitize a plastic support or latex particles. The antibodies or their functional fragments may also be coupled with an enzyme, for example peroxidase, alkaline phosphatase or acetylcholinesterase. Such antibodies are, for example, those produced by the company STAGO and marketed under the names 15C4 and 34G2 (marketed in the ASSERACHROM TOTAL PROTEIN S and ASSERACHROM FREE PROTEINS kits of DIAGNOSTICA STAGO). Both monoclonal antibodies recognize the free protein S not bound to C4b-BP, ie the functional protein S as a cofactor of activated protein C. The 15C4 antibody is IgG1 kappa and the 34G2 antibody is IgG2b kappa.

  The kit for assaying the free factor Xa non-cleaved protein S may further contain a reagent for assay calibration, and / or a reagent for establishing assay quality control.

  The assay calibration reagent may be a freeze-dried human plasma containing a known amount of uncleaved free S protein close to 100% after reconstitution and dilution (relative to a secondary standard of International Standard 93/590 of 1995).

  The assay quality control reagent may be lyophilized human plasma containing a known amount of uncleaved free S protein. This rate is determined relative to a secondary standard of International Standard 93/590 of 1995.

  Advantageously, the assay kit thus defined further comprises a dilution buffer for the immunoconjugate, for example an EDTA buffer.

  The assay kit also contains, as appropriate, instructions for assaying the uncleaved free S protein.

  The subject of the invention is also the use of antibodies as defined above, or their functional fragments for the assay of free S protein not cleaved by factor Xa, in a biological sample, preferably a sample of blood plasma.

  This assay includes the performance of an immunoassay, for example a radioimmunoassay (RIA) type assay or an ELISA type assay.

  The invention also relates to a method for assaying free S protein, not cleaved by factor Xa in a blood plasma sample, characterized in that an ELISA test is carried out by means of, on the one hand, specific antibodies for the above-defined factor Xa non-cleaved S protein or functional fragments of these antibodies, preferably used in the form of an immunoconjugate, or by means of a reagent defined above, and secondly of antibodies recognizing the free S protein or antibody fragments recognizing the free S protein, used for coating the ELISA plates.

  The invention also relates to a method for the preparation of monoclonal antibodies specifically recognizing the factor Xa-cleaved non-cleaved protein S comprising: - immunization of an animal selected by mammals with an antigen selected from the protein S , uncleaved purified, a peptide having the sequence CLRSFQTGLFTAARQSTNAYPDLRSC or the sequence CLRSFQTGLFTAARQSTNAYPDLRSCVLY or one of these peptides coupled to a carrier molecule, the antigen being advantageously associated with an adjuvant of the immunization, the splenocyte fusion of the immunized animal with immortalized cells, for example myeloma cells compatible for fusion, - the selection of hybridomas produced by the fused cells, for their ability to secrete anti-bodies capable of recognizing the factor S uncleaved protein and / or the peptides used for the immunization stage and for their ability to not recognize the pro F-cleaved S-protein, - the recovery of the selected antibodies.

  Such a method allows the production of monoclonal antibodies either in the form of ascites or in vitro. If necessary, the antibodies thus produced are purified by known techniques. An antibody purification protocol is described in particular in the experimental section which follows. These antibodies belong for example to the class of IgM.

  The invention furthermore relates to a method for detecting the anticoagulant potential of a patient comprising the assay of the free factor Xa non-cleaved protein S in a sample of plasma taken from the patient, using antibodies defined above. , their functional fragments, or a reagent defined above. The detection carried out in the context of the invention is for example advantageous in the context of the detection of thromboses, in particular venous thromboses, or pulmonary embolism, or more generally of a thromboembilic risk which is linked to a protein deficiency. S. This method is such that it advantageously comprises for the detection, carrying out an immunological test, for example ELISA.

  The use of the antibodies according to the invention thus makes it possible to detect and quantify the protein S, which is not cleaved by factor Xa, in a biological sample and in particular in a sample of blood plasma.

  Other means and advantages of the invention will appear in the figures or in the examples which follow.

  Figure 1: Correlation between the organization of the PROS 1 gene and the structural domains of the PS.

  Figure 2: Mean plasma concentration of the different molecular forms of protein S based on the values determined by Griffin et al (1992) and considering the level of PS cleaved at 15%.

  Figure 3 (a and b): Recognition of cleaved and uncleaved PS by two assay systems.

  Figure 4: Inhibition of the assay of free PS uncleaved by Xa by C4b-BP.

  Figure 5: Comparison of two assays of uncleaved plasma PS: 15C4 or 34G2 in coating and S2P11 C6 in conjugate.

  1. PREPARATION OF SPECIFIC MONOCLONAL ANTIBODIES BY CELLULAR FUSION 1.1. Antigens for Immunization of Mice The purified whole protein S and the peptide sequences located around the factor Xa S-protein cleavage site were selected as antigens for immunization.

  Firstly, 3 peptides were synthesized: Peptide G13Y: GLFTAARQSTNAY Peptide C26C: CLRSFQTGLFTAARQSTNAYPDLRSC Peptide Y29C: CLRSFQTGLFTAARQSTNAYPDLRSCVLY The G13Y peptide was coupled to the carrier protein, KLH. The C26C peptide, being quite long (26 amino acids), was used as.

  In a second step, the peptide Y29C was synthesized. This peptide contains the peptide C26C and 3 amino acids of the physiological sequence more, in order to allow the coupling to the KLH. It has been used alone or coupled to KLH via either BDM or bromoacetyl.

  1.2. Immunization of Mice Two immunization strategies were applied: Short-term immunization (2-4 weeks), mainly with ImmunEasy adjuvant (QUIAGEN).

  This recently developed adjuvant contains oligonucleotides with unmethylated cytosine / guanidine residues. The immune system in mammals recognizes these sequences, which are found in bacterial DNA. The answer is very fast and strong. Only 2 or 3 injections in two or three weeks are enough. In addition, it is less toxic than conventional adjuvants.

  Long-term immunization (5-6 months minimum) with classic Freund's adjuvants: complete and incomplete.

  For both strategies, the last immunization is performed 4 days before the merger.

  1.3 Preparation of Free Factor Xa Cleaved Protein Free F factor Xa cleaved protein S was prepared to test the antigenic specificity of the antibodies produced.

  To prepare the cleaved S protein, the purified or recombinant S protein (300 nM final concentration) was proteolyzed by purified factor Xa (final concentration 6 nM) in the presence of calcium and phospholipid surfaces (Long GL, Lu D., Xie RL, Kalafatis M .: Human protein S cleavage and inactivation by coagulation factor Xa J. Biol Chem: 273, 11521-11526, 1998). For the enzymatic cleavage reaction, different phospholipid surfaces may be used, for example synthetic phospholipids such as phosphatidylserine and phospatidylcholine or platelet substitutes such as cephalin or lecitin.

  Factor Xa-cleaved S protein preparations were sufficient to test the specificity of the different antibodies produced during cell fusions.

  1.4 Cell Fusions Cell fusions occurred between NS1 (Flow Lab) murine myeloma cells and immunized mouse splenocytes (BALBIC). After selection of the hybridomas in HAT medium, the culture supernatants were tested on the micro-ELISA plates sensitized with one of the following antigens: The purified S protein uncleaved by factor Xa The purified S protein cleaved by factor Xa corresponding synthetic peptides coupled or not to KLH - KLH, in case of immunization with peptides coupled to KLH. In the supernatants selected, the determination of the isotype was performed.

  Ten cellular fusions were performed: nine after short-term immunization and one after long-term immunization.

  The results of these ten mergers are shown in Table 1.

  The fusions carried out made it possible to obtain some hybridomas secreting antibodies of the desired specificity and having a greater or lesser reactivity. (These hybridomas were obtained after immunization with the purified uncleaved free protein S, C26C peptide and Y29C not coupled to KLH and peptide Y29C coupled to KLH).

  Selection of specific hybridomas was performed by the culture supernatant reactivity test against cleaved S protein and factor Xa uncleaved protein S. Both forms of the S protein are coated on a micro ELISA plate for 1 night. After washing with the washing solution, the culture supernatants to be tested diluted 1/5 in dilution buffer are incubated for 2 hours with the antigens fixed on the micro-ELISA plate. After the incubation, the wells are washed and the sheep antibody-anti-mouse antibody-peroxidase conjugate is deposited. During the incubation, this conjugate binds to the antibodies bound to the corresponding antigens. The revelation is achieved by adding the peroxidase substrate, TMB, after the new wash. The reaction is stopped by the addition of 1 M sulfuric acid. The optical density (OD) is measured at 450 nm. For the supernatants retained, the OD with the factor Xa non-cleaved S protein varied from 0.9 to 3.7 and with the factor Xa cleaved S protein it was in the range of 0.1 to 0.3 .

  o All the hybridomas obtained, specific for protein S, not cleaved by factor Xa, are IgM. To use these pentamers, very high molecular weight (about 900 000 daltons) it is necessary to prevent the possible aggregation of the molecules.

  A hybridoma (S2P11B6) secreting IgM and having a high reactivity (Do> 3.0) with the Xa-cleaved S protein and very low (0.1) with the factor Xa cleaved S protein was retained to study the feasibility of assaying the free F factor Xa uncleaved protein S.

Table 1

  Cell Fusions: PROTEIN S NOT CLASSIFIED BY FACTOR Xa Date Immunogen Adjuvant Number Hybridomas Isotype Reactivity OD at 492 Additional data, Injectio Retained for nm comments ns tests number, name PS n.v. PS c.

  24/04/2001 C26C-KLH ImmunEasy 3 None 12/06/2001 Purified Ps ImmunEasy 3 3: P4E10 IgG2a 0.9 3.2 PS c> PS n.c.

  P8G8 IgM 1.1 0.2 Recognition G13Y, Q12C and P1 0B9 IgM 0.8 0.1 C26C, r. weak, PS nc> PS c.

  03/07/2001 Q12C-KLH Incomplete Freund 3 3: P8G4 IgG, 3.6 3.7 PS c = PS n.c.

  ImmunEasy 1 P13E2 IgG1 3.7 3.7 PS c = PS n.c.

  P11F7 IgG, 0 0 Recognition Q12 PS only 09/10/2001 Purified PS Incomplete Freund 5 No ImmunEasy 2 20/11/2001 G13Y-KLH ImmunEasy 3 7 2IgG2a 0 0 No recognition of PS 2 IgM Recognition G13Y 1IgG3 1 IgG1 / IgM 1 IgG2a / IgM 18/12/2001 Purified PS ImmunEasy 5 None 12/02/2002 C26C ImmunEasy 4 12 7 IgM 0 - Recognition C26C 4 IgM> 1.0> 0.2 r average, PS nc> PS c 1 M: S2P11B> 3.0> 0.1 r. strong, PS nc.> PS c.

  16/04/2002 Y29C-bromo-KLH ImmunEasy 1: P24E3 IgM> 3.0 0.2 r. strong, PS nc. <PS c.

r. weak, PS nc. <PS c.

  28/05/2002 Y29C6BDB-KLH ImmunEasy 4 2: 8G3 IgM 1.4 0.4 r. weak, PS nc. <PS c.

  + Y29C 14G2 IgM 0.6 0.2 r. weak, PS nc. <PS c.

  08/10/2002 Y29C Complete Freund 1 4 1 IgM 6G8 3.6 3.5 PS n. = PS c.

  Incomplete Freund 4 1 IgM 8F2 1.7 0.3 r.moyenne, PS n.c.> PS c.

  2 IgM and 1.9 0.6 r. average, PS n.> PS c.

  IgG2a *: 0.4 0.1 r. weak, PS nc. <PS c. 4G9 1F2

  R = reactivity IV PS c. = S cleaved protein Xa PS nc = S protein uncleaved by factor Xa Co 0) * Difficulty of separating IgM and IgG2a W 0) 0) 2. Feasibility of the dosaqe of free S protein uncleaved by factor Xa 2.1 Production of S2P11B6 Antibodies S2P11B6 antibodies were produced: * In ascites Anti-factor Xa monoclonal anti-factor Xa monoclonal antibodies were prepared by the usual ascitic induction technique in Balb c. For this, mice, previously sensitized by intraperitoneal injection of pristane, are injected intraperitoneally 1 month later with 10 'hybridoma cells per mouse. When ascitic tumors develop, ascites fluids are punctured. The monoclonal antibodies are precipitated by ammonium sulfate prior to purification.

  In vitro on Cell Line In parallel, the hybridomas are cultured in vitro in the culture chamber based on membrane technology: Cell Line (Integra Biosciences SARL, Cergy, France). Cells and FCS are injected into the cell compartment and RPMI 1640 medium into the other compartment. Secreted monoclonal antibodies are regularly removed from the cell compartment.

  The amount of antibody produced by these two methods was not evaluated in the absence of a commercial test to quantify IgM in the unpurified medium.

  After IgM purification, the concentration can be determined by measurement of OD at 280 nm.

  2.2 S2P11B6 Antibody Purification Feasibility Two types of gels were tested to purify the S2P11B6 antibody: * Thiosorb M (Bioprocessing Inc., Princeton, USA) * and HiTraplgM (small column ready for use).

  Purification of S2P11B6 ascites on Thiosorb M did not give good results. Purified antibodies have lost their reactivity. However, the prior treatment of this ascites by ammonium sulphate does not influence this reactivity. It is possible that the ethylene glycol used for elution is not suitable for this antibody.

  S2P11B6 ascites purification on HiTrap IgM was not possible due to clogging.

  On the other hand, the purification of the Cell Line supernatants on this gel did not pose any problem. Five purifications were successfully performed (Table 3).

Table 3

  Yield of purifications of supernatant S2P11B6 on HiTrap IgM cell harvest Volume deposited Quantity Quantity Line on ml column of purified antibody purified antibodies mg / l ml supernatant (mg / ml) 1 7.8 11.02 1.4 2 6 , 2 9,36 1,5 3 14 20,8 1,5 4 9 71,0 7,9 7,7 11 1,4 Sufficient quantities of the antibody S2P11B6 were obtained. The highest yield of production and purification probably coincides with the maximum cell density in the Cell Line.

  Feasibility of Manufacturing the S2P11B6 Conjugate The S2P11B6 antibody recognizes the factor S uncleaved protein S.

  Protein S circulates in plasma in two free forms and bound to C4b-BP. The cleavage of this protein by factor Xa concerns both the free form and the complexed form. The antibody of the invention recognizes both of these forms. In order to assay the free F factor Xa-free protein S, therefore the anticoagulant activity of protein S as the cofactor of activated protein C, it is necessary to have the free protein S specific antibody. The antibodies of Diagnostica Stago 15C4 and 3402 are quite suitable for this purpose.

  For the determination of the free S protein in ELISA, it is preferable to carry out the coating with the antibody specific for the free S protein. In addition, IgMs can pose a cleavage problem in F (ab ') 2. On the other hand, the coating of whole IgM molecules increases the risk of nonspecific reactions.

  For this reason, the S2P11B6 antibody was chosen as the conjugate. Currently, there is no standard protocol for making the conjugate between IgM and peroxidase. It has therefore been necessary to develop a method for the S2P11B6 antibody.

  Before coupling, the IgM antibodies are dialyzed in carbonate buffer at pH 9.6. Peroxidase (Roche) is activated by m-periodate and dialyzed in acetate buffer at pH 4.2. Dialyzed antibodies and activated peroxidase are incubated for 2 hours at room temperature. The residual active groups are blocked by the sodium borohydride solution (Nakane, J. Histochem, Cytochem .: 22, 1084-1091, 1974). The S2P11B6 peroxidase conjugate is precipitated from saturated ammonium sulfate and centrifuged for 15 min at 4000 rpm at room temperature. The pellet containing the conjugate is taken up in 0.2 M phosphate buffer containing 0.015 M NaCl and 0.02 M MgCl 2 at pH and dialyzed in the same buffer. S2P11B6 antibodies aggregate rapidly at concentrations above 1 mg / ml and cool (2-8 C). The protocol giving the satisfactory results therefore includes the use of this antibody at a concentration of less than 1 mg / ml and the ambient temperature during all the manipulations. In addition, the recovery of the pellet by distilled water led to the aggregation of the conjugate. On the other hand, the suspension of the pellet in the dialysis buffer containing MgCl 2 and NaCl made it possible to avoid aggregation.

  2.4. Assay of Free S-protein Assay Uncleaved by Factor Xa Principle The coating of a microELISA plate with one of two antibodies AntiagoS Stago Free Anti-Protein: 15C4 or 34G2 assures the specificity of the assay for protein S free. The above antibodies are coated at 10 μg / ml in 50 mM carbonate buffer at pH 9.6 overnight.

  The S2P11B6 antibody used for the conjugate will bind only to the free F factor Xa uncleaved protein S.

  Figure 3 presents a comparison of two assays 15C41S2P11B6 * and 34G2 / S2P11B6 * with respect to the recognition of purified S protein cleaved and uncleaved by factor Xa.

  The specificity of two dosages is good. It seems that it is a little better for the 15C4 coating antibody.

  Figure 4 shows a comparison for these two assays with respect to free protein S not complexed with C4b-BP.

  The tests confirm that the added C4b-BP inhibits the S protein assay. The more a normal C4b-BP plasma is overloaded, the less the S protein is dosed. The two assays give parallel inhibition curves.

  The following test focused on the assay of free factor Xa non-cleaved S protein in the pool of normal plasmas (Figure 5). The antibody 34 G2 coating gives the signal a little stronger. Finally, the 15C4 antibody, currently used as a coating in the free ASSERACHROM PS (ASSERACHROM FREE PROTEINS), was selected for coating.

  3 ASSAY OF FREE S PROTEIN AND FREE CLONIC FREE S PROTEIN By way of example, the table below shows the results of the comparative assay of free S protein and intact free S protein in normal subjects (15). women and 15 men), 11 subjects with disseminated intravascular coagulation (DIC), 18 subjects with hepatocellular insufficiency (IHC) and 20 pregnant women.

  Disseminated intravascular coagulation is an anatomoclinic syndrome characterized by abnormal diffusion of the coagulation process which should normally remain localized. A CIVD is typically accompanied by biological abnormalities that associate a decrease in the plasma concentration of several coagulation factors, in particular fibrinogen, factors II, V and VII as well as platelets, and an increase in the circulating rate of degradation of fibrin (D-dimers). This complex biological syndrome is called consumption coagulopathy. It can also be encountered during localized intravascular coagulation.

  In normal subjects, the level of free S-protein cleaved is not decreased relative to the level of protein S. In the other groups studied, the concentration of cleaved protein S is lower than the concentration of protein S This is due to its cleavage by factor Xa under these physiopathological conditions.

  In view of these results, it appears that the determination of the free protein F uncleaved by factor Xa could be a better reflection of the activity than the free protein S and could thus advantageously replace the assay of the free protein S.

  Rates of free S protein (PS) and free uncleaved S protein Sample Free PS% Free PS Uncleaved% Women 89.0 19.1 89.7 19.

1 N = 15 Men 96.1 17.4 102.4 22.8 N = 15 CIVD 67.1 16.6 59.3 13.7 N = 11 IHC 52.8 13.9 42.4 15.6 N = 18 Pregnant women N = 49.9 16. 2 45.5 16.3

Claims (22)

  1. Antibodies characterized by specifically recognizing the factor Xa-uncleaved plasma S protein.   2. Antibody according to claim 1, characterized in that they are monoclonal.   3. Antibody according to any one of claims 1 or 2, characterized in that they recognize the peptide CLRSFQTGLFTAARQSTNAYPDLRSC (C26C) or peptide CLRSFQTGLFTAARQSTNAYPDLRSCVLY (Y29C).   4. Antibody according to any one of claims 1 to 3, characterized in that they do not recognize the factor Xa cleaved plasma protein S.   5. monoclonal antibody according to any one of claims 2 to 4, characterized in that it is IgM.   6. monoclonal antibody according to claim 5, characterized in that it is the S2P11B6 antibody secreted by the hybridoma deposited with ATCC under n PTA-5425.   An antibody fragment according to any one of claims 1 to 6, comprising the antigenic recognition site of said antibody.   A reagent for the assay of the factor Xa-cleaved, non-cleaved plasma S protein comprising antibodies according to any one of claims 1 to 6 or an antibody fragment according to claim 7 in the form of immunoconjugates.   9. Reagent according to claim 8, characterized in that the antibodies are conjugated with peroxidase.   A reagent for the assay of free factor Xa-cleaved, free plasma protein S, comprising antibodies according to any one of claims 1 to 6 or an antibody fragment according to claim 7 in a presentation allowing the production of an immunological assay.   A kit for assaying Factor Xa uncleaved free plasma protein S comprising: antibodies according to any one of claims 1 to 6, or an antibody fragment according to claim 7, or a reagent according to any of claims 8 to 10, antibodies recognizing free S protein or fragments thereof recognizing free S protein.   12. Use of antibodies according to any one of claims 1 to 6, antibody fragments according to claim 7, or a reagent according to any one of claims 8 to 10, for the determination of the free protein S no cleaved by factor Xa, in a biological sample, preferably a sample of blood plasma.   13. A method for assaying the free protein S uncleaved by factor Xa in a sample of blood plasma, characterized in that an ELISA test is carried out by means of, on the one hand, antibodies according to any one of the claims 1 to 6 used in the form of an immunoconjugate or antibody fragments according to claim 7, or by means of a reagent according to any one of claims 8 to 10, and secondly of antibodies recognizing free protein S or their fragments recognizing the free protein S, used for coating the ELISA plates.   A process for the preparation of monoclonal antibodies specifically recognizing the Factor Xa uncleaved plasmid S protein comprising: immunizing an animal selected by the mammals with an antigen selected from purified, uncleaved protein S, a peptide having the sequence CLRSFQTGLFTAARQSTNAYPDLRSC (C26C) or the peptide CLRSFQTGLFTAARQSTNAYPDLRSCVLY (Y29C) or one of these peptides coupled to a carrier molecule, the antigen being associated with an adjuvant, the splenocyte fusion of the immunized animal with immortalized cells, by example of the myeloma cells, compatible for the fusion, the selection of hybridomas produced by the fused cells, for their ability to secrete antibodies capable of recognizing the protein S not cleaved by factor Xa and / or the peptides used for the step immunization and for their ability to not recognize factor Xa-cleaved protein S, recovery of selected antibodies.   15. The method of claim 14, characterized in that the monoclonal antibodies are produced in ascites.   16. The method of claim 14, characterized in that the monoclonal antibodies are produced in vitro.   17. The method according to any one of claims 14 to 16, comprising a step of purifying the selected antibodies.   18. A method for detecting the anticoagulant potential in a patient comprising assaying the factor Xa non-cleaved S protein in a plasma sample taken from the patient, using antibodies according to any one of claims 1 to 6 or a reagent according to any one of claims 7 to 9.   19. The method of claim 18, characterized in that the detection comprises carrying out an immunological test.   20. The method of claim 18 or claim 19, characterized in that one looks for a deficit of free protein S uncleaved by factor Xa.   21. Peptide characterized in that its amino acid sequence is as follows CLRSFQTGLFTAARQSTNAYPDLRSC (C26C) or CLRSFQTGLFTAARQSTNAYPDLRSCVLY (Y29C).   22. Use of a peptide according to claim 21, optionally associated with a carrier protein, for the preparation of antibodies.