WO1986001298A1 - Method for dosing fibrinogen degradation products - Google Patents

Abstract

New method for dosing degradation products (PDF) of fibrinogen and fibrin particularly on plasma. The method which implements a monoclonal anti-PDF antibody in a reaction of the antigene-antibody type is characterized in that a purified monoclonal anti-PDF antibody selected among the group comprising the monoclonal anti-PDF antibodies which A) are elaborated from at least one monomer PDF of the fibrinogen, and B) do not fix the fibrinogen, is contacted with a liquid aqueous medium susceptible of containing at least 1 PDF, the complex formed by said monoclonal antibody and said PDF of said aqueous medium producing a signal which is appropriately amplified. For implementing this method, dosing kits ready for use are provided which comprise as monomer anti-PDF monoclonal antibody particularly an anti-D neo-antibody.

Description

Method for assaying fibrinogen degradation products

The invention relates to a new method for assaying at least one degradation product of fibrinogen and fibrin (abbreviated: PDF) by means of a reaction mechanism of the antigen-antibody type using an anti- Monomeric PDF.

We know that fibrinolysis can have very serious consequences. Indeed, fibrinolyses, whether primary, extremely rare cases, or consecutive to disseminated intravascular coagulation, fairly frequent cases in surgery, obstetrics, resuscitation services, oncology or others, induce severe pathological disorders. It turns out that certain coagulation factors are either destroyed by proteolysis (fibrinogen, factors V, VIII etc.), or consumed (AT III, Protein C, Plasminogen, Factors V, VIII etc ...). This results in a totally unbalanced coagulolytic picture, inducing bleeding disorders which can be very serious. PDFs from fibrinogen (abbreviated

PDFg) or fibrin (abbreviated PDFb) are markers of fibrinolytic status. They are produced by the action of plasmin formed on fibrinogen or fibrin. For all intents and purposes, it is recalled that: in the organism, the degradation of fibrinogen by plasmin gives the fragments X and Y called intermediate and the fragments D and E called terminal; under the action of thrombin fibrinogen transforms into spontaneously crosslinked fibrin; the degradation of the crosslinked fibrin gives fragments or polymer complexes (chains linked together by covalent bonds), in particular the fragment DD (or D dimer) and the complexes DDE and YDE; in terms of antigenic properties PDFg, such as fragment D (other nomenclatures: D monomer, PDF-D or PDFg-D), are different from PDFb, such as the DD fragment and the YDE complex, the differences in antigenic activities result from the presence of specific epitopes on the D fragment and explain in particular the possibilities of distinguish fragment D from fragment DD. All of the above information is taken from the following publications: MARDER et al., "The Struc¬ ture of Fibrinogen Dégradation Products" published in "Progress in Hemostasis and Thrombosis", Vol II, pages 141-174, (1974) ; BUDZYNSKI et al., Biochim. Biophys. Acta, _147, pages 313-323, (1967); BUDZYNSKI et al., Blood 54. (No. 4), pages 794-804, (1979) - where it is stated that the DD (D dimer) fragment originating from fibrin is much more reactive as a marker than fragment D (D monomer) from fibrinogen -; and PLO et al., Proc. Nat. Acad. Sci. USA 70 (n ° 4), pages 1169-1173, (1973) - which describes the production of neo antigens presenting epitopes playing the role of antigenic determinants, hidden in fibrinogen and fibrin and exposed during the- degradation of fibrinogen or fibrin, an epitope being a peptide link of 4 to 6 amino acids approximately -.

Clinically it is important to be able to determine PDF levels in patients' bodily fluids, at least semi-quantitatively.

It is also known that for dosing the PDFs, dosing methods have been used in the serum, the serum being obtained from the plasma by coagulation then centrifugation to collect the supernatant, the plasma being obtained by centrifugation of the blood and collection of the supernatant.

Among the prior techniques are known those which use agglutination of inactivated golden staphylococci, agglutination of a support sensitized by specific polyclonal antibodies or Merskey method which is an inhibition test. These old dosing techniques have a number of drawbacks. In particular, they do not make it possible to distinguish plasma fibrinogen from PDF and therefore imply beforehand a treatment of the plasma to specifically remove the native fibrinogen; this can be done by coagulation under suitable conditions (in the presence of antifibrinolytic agents). The PDFs are then searched for on the serum obtained. These successive operations result in:

- an extension of the dosing time,

- the coagulation of certain early PDFs (in particular the fragment X also designated PDF-X), which causes the elimination of said PDFs with the clot, - a non-negligible proportion of the PDFs formed is capable of being adsorbed on the. fibrin clot,

- if necessary, poor coagulation, leaving residual fibrinogen, which leads to dosing errors.

It is also known that radioimmunoassays have been used in the past to determine PDF levels in serum. However, it should be noted that the use of radioimmunological equipment is not suited to the routine of hospitals and

»Analysis laboratories, having regard to the following:

- the high sensitivity of a radioimmunoassay is unnecessary for this kind of test,

- radioimmunological equipment is heavy and expensive,

- The duration of the radioimmunological assay is too long, when a semi-quantitative assay is sufficient for exploratory research. It is also known that it was recently recommended to use anti-PDF monoclonal antibodies for the study of structured ^é e of fibrinogen, fibrin and their degradation products, on the one hand, and the pathogenic mechanisms involved in the degradation of fibrinogen and fibrin by plasmin and the transformation of fibrinogen into fibrin by thro bine, on the other. Thus, the summary of Chemical Abstracts 100, 119083c (1984) indicates the obtaining, from a PDFb antigen, of a monoclonal antibody specific for a particular epitope of the B _β chain of fibrin; the article by HUI et al., Science ^ 22, 1129-1131, (1983) describes the production of monoclonal antibodies from a synthetic heptapeptide analogous to a portion of one of the protein chains of fibrin , capable of binding to plasma fibrin in the presence of fibrinogen. A technical solution for determining and assaying PDFs, which uses anti-PDFb-DD monoclonal anti¬ bodies produced from the dimeric fragment D of crosslinked fibrin, has been described in the summaries of Chemical Abstracts ^ 9_, 136280t, (1983) and SJ9_, 209051f, (1983) as well as in patent document EP-A-0 122 478 (publication date: October 24, 1984).

According to the invention, a new technical solution for the dosing of PDF in the presence of fibrinogen is recommended which calls for the use of an anti-PDF monoclonal antibody produced from a monomeric PDF, in particular from fragment D fibrinogen.

This new technique, which uses different means from those recommended in CA 99,

136280t and 209051f (1983) and in EP-A-0 122 478 cited above, makes it possible to carry out assays on human plasma effectively. The protocols to be followed and the equipment to be used are simple and inexpensive, in particular according to the operating methods given below. Of plus the new technique according to the invention goes against the teaching of BUDZYNSKI et al., Blood 54 (No. 4), 794-804 (1979) cited above with regard to the ineffectiveness of the fragment D in as a source of labeling, in particular with respect to the DD fragment.

According to one of its aspects, the invention relates to a semi-quantitative dosing process for PDF in body fluids and more specifically plasma.

According to another aspect, the invention relates to a quantitative dosing process for PDF of very high sensitivity in body fluids and more precisely plasma, the detection threshold according to said aspect of the invention being 0.5 ng / ml while it is of the order of 5 to 10 ng / ml according to the technique of EP-A-0 122 478. The method for assaying at least one degradation product (PDF) of fibrinogen and fibrin , according to the invention, by means of a reaction mechanism of the antigen-antibody type using an anti-PDF monoclonal antibody, is characterized in that a purified anti-PDF monoclonal antibody chosen from set including anti-PDF monoclonal antibodies which

(A) are made p ^"artir of at least one monomer PDF of fibrinogen, and (B) do not bind fibrinogen, with an aqueous liquid medium which may contain at least one PDF.

The complex product obtained by the reaction of the monomeric anti-PDF monoclonal antibody with the PDF or PDFs contained in the aqueous liquid medium to be tested constitutes a "signal" which is "amplified" according to a method known per se / _ ~ par example, EIA method (notably EIA-sandwich or EIA-co petition) according to which the complex is coupled with an appropriate enzyme conjugate, a radioimmunological method (RIA), an agglutination method or a luminescence method 7. The presence or absence of PDF in the sample to be tested is thus detected, on the one hand, the quantity of PDF attached to the monoclonalanti-PDF'rnonomer antibody is assayed, on the other p PDF antigens, which are involved in the production of monoclonal antibodies, are the monomeric degradation products of fibrinogen, in particular of human origin.

These are essentially — fragments D and E (and if need be Y) having epitopes playing the role of antigenic determinants, so that the corresponding anti-PDF antibodies fix said PDF without fixing fibrinogen.

Among the antigens which are suitable according to the invention, the D neo and E neo fragments may be mentioned: the epitopes reacting with the corresponding anti-D neo (or anti-E neo) antibodies are hidden or masked inside the fibrinogen molecule intact (or native) but are exposed during the degradation of fibrinogen.

As the PDF fragments substantially include a number of common epitopes, an anti-PDF-D monoclonal antibody is used to recognize the presence of a PDF-D, and can recognize PDF-DD if necessary. or PDF-DDE, and a monoclonal anti-PDF-E antibody recognizes the presence of a PDF-E.

The preparation of the anti-PDF monoclonal antibody which intervenes in the antigen-antibody mechanism according to the assay method of the invention comprises:

1) degradation of fibrinogen by plasmin for 24 h at 37 ° C,

2) the purification of at least one PDF thus obtained,

3 °) the inoculation of said PDF to a mouse, 4 °) the removal of the spleen from the immunized animal,

5 °) the extraction of lymphocytes from the spleen and the fusion of lymphocytes with cells exponentially growing mouse myeloma at a rate of 5 to 20, preferably 10 lymphocytes per myeloma cell,

6) the selection of the secretory hybridomas thus obtained with a view to obtaining clones producing monoclonal antibodies reacting selectively with PDFs but not with fibrinogen.

7) the development of ascites tumors in mice with the selected clones, ⁸ °) ^a recovery of ascitic liquid containing monoclonal antibodies by puncture, 9 °) purification of the antibody, 10 °) the determination of purified monoclonal antibodies on an inert support. In step 9) the purification of the antibodies is carried out by gel filtration according to a method known per se (gelfiltration). So ^{'advantageously} this purification is followed by treatment with pepsin, to remove an element sensitive to the rheumatoid factors present in the plasma, prior to fixing on an inert support of the stadium 10 °).

Schematically, the purified anti-PDF monoclonal antibody comprises a substantially Y-shaped element (see formula I) consisting of three fragments Fa, Fb and Fc. The purpose of digestion with pepsin is to cleave said element in order to remove the Fc fragment which can react in an aspecific manner with Rheumatoid Factor and keep the element in the form of V (see formula II) comprising the fragments Fa and Fb and called ci -after F (ab ') 2. Treatment with pepsin according to the mechanism:

offre l'avantage d'éviter toute interférence avec ledit Facteur Rhumatoide et par suite tout résultat faussement positif lors du dosage des PDF.

offers the advantage of avoiding any interference with said Rheumatoid Factor and consequently any false positive result when dosing PDFs.

The inert support which is involved in fixing the anti-PDF monoclonal antibody is chosen in particular from the group consisting of glass beads, latex particles (in particular polystyrene, polyacrylate, polymetacrylate latex, etc.), red blood cells, clays such as bentonite, carbon black and agarose. Of course, any inert support other than those mentioned above can be used. Advantageously, use will be made of an inert support constituted by particles of polystyrene latex having an average diameter less than or equal to 5 μm and preferably of the order of 0.1 to 1 μm, in particular 0.3 μm. The binding of the anti-PDF monoclonal antibody to the inert support can be carried out by known means. Preferably, one of the following techniques will be used: - direct attachment to the inert support, in particular polystyrene beads,

- fixation by covale bond te after chemical activation of the support or by activation of

The antibody, - fixation by means of a spacer arm, itself activated, or after activation of the antibody,

- fixation on an intermediate layer (protein, chemical or other, etc.) coating the particle or covering the support, by simple adsorption, by chemical or physical activation of the layer or by activation of the antibody. According to a first aspect of the invention, a semi-quantitative assay method is recommended in which the monomeric anti-PDFg monoclonal antibody (preferably anti-E or better anti-D) purified, where appropriate.

5 without Fc fragment sensitive to Rheumatoid Factor, and fixed to an inert support, is brought into contact with an aqueous medium (for example a body fluid such as plasma) to be tested likely to contain PDF, revelation (ie amplification above signal)

10 being carried out by macroscopic agglutination: the agglutination signaling the presence of PDF, and, the absence of agglutination the absence of PDF.

In this assay, an inert support consisting of particles of

15 polystyrene latex having the above-mentioned particle size.

The sensitivity threshold (expressed in fibrinogen equivalent) is here of the order of 1000 to 500 ng / ml. Given this threshold, it is not necessary to use, for example, a distinguishing monoclonal antibody

2o selectively the PDF-D in a mixture which may contain fibrinogen, the D and DD fragments and the other PDFs, it suffices to use an anti-PDF-D monoclonal antibody according to the invention (in particular an anti-Dneo) recognizing the active antigenic determining sites _pc of fragment D which are present on said fragment D and the fragment DD.

According to a second aspect of the invention, a method for the quantitative determination of PDF in a aqueous medium, in particular a body fluid such as

3 plasma. This method comprises contacting the purified anti-PDFg monoclonal antibody, advantageously without Fc fragment, and attached to an inert support, with an aqueous sample to be tested, then reacting the resulting antigenic complex with an immunoconjugate chosen from c polyclonal anti-PDF antibodies, and revelation of the resulting complex according to a known method / _ ^~ immunoenzymatique (EIA), radioimmunologique, lumi¬ nescence, agglutination_7-

Preferably the revelation will be carried out. here by enzyme-linked route, the polyclonal antibody 5 being labeled by an enzyme, peroxidase, and the determination of the PDFs in the test sample being carried out by competition or by the use of a sandwich.

The best mode for implementing the 0 quantitative dosing process includes, for PDF-D,

1) degradation of human fibrinogen by plasmin for 24 h at 37 ° C,

2) purification of at least a PDF-D thus obtained, and - ₅ 3) 1'innoculation said PDF-D mouse,

4) removal of the spleen from the immunized animal,

5) the extraction of lymphocytes from the spleen and the fusion of the lymphocytes with myeloma cells 20 of exponentially growing mice at a rate of 5 to 20, preferably 10 lymphocytes per myeloma cell,

6) the selection of the secretory hybridomas thus obtained with a view to obtaining clones producing monoclonal antibodies reactively selectively with the PDFs but not with fibrinogen,

7) the development of ascites tumors in mice with the selected clones,

8 °) the recovery of ascites liquids containing the monoclonal antibodies by puncture, 3 _θ 9 °) the purification of the monoclonal antibodies thus obtained, then, the elimination of the Fc fragment,

10 °) the fixing of the monoclonal antibodies / _ ^~ F (ab ^l ) 2_7 thus obtained on an inert support,

11 °) bringing said monoclonal antibodies into contact with a test sample (containing .in particular PDF-D, PDF-DD and / or PDF-DDE),

12 °) bringing the resulting antigenic complex into contact with at least one anti-D polyclonal antibody labeled with an enzyme (preferably peroxidase), and 13 °) measuring the enzymatic activity on an appropriate substrate / ^" preferably an orthophenylenediamine substrate (OPD), in the presence of H ₂ 0 for the evaluation of the activity of the peroxidase linked to the antigen-antibody complex 7. Here, a neo D fragment leading to a monoclonal antibody can be chosen as the source of PDF-D anti D-neo which reacts selectively with PDF-D or with PDF-D and PDF-DD, but which does not react with fibrinogen Depending on the selection of anti-neo D monoclonal antibodies the PDF can be determined -D alone or the mixtures of PDF-D and PDF-DD, in combination, where appropriate with PDF-DDE. Everything depends in this selection on the epitope (s) (i) not common between the fragments D and DD, or (ii) common to the said fragments 0 The inert support for fixing monoclonal antibodies can be latex particles having the aforementioned granulo etry, or better can be constituted by tubes or wells to facilitate washing operations here. The anti-D polyclonal immunoconjugate is prepared from a polyclonal antibody of the fragment D, in particular human.

For this a rabbit esr ιrnrnun_.sώ with highly purified -fragment D / _ ~ obtained by prolonged degradation of fibrinogen by plasmin and purification by preparative electrophoresis on PEVIKON block according to MARDER et al. "The purification of fibrinogen degradation products by PEVIKON Block Electrophoresis", Thromb. Diath. Haemorrh. 2_2, 234-239, (1969) _7. The antiserum thus obtained is made specific by exhaustion on a solid support. -. The antibodies are then purified, either by chromatography ion exchange on a DEAE-Trisacryl, DEAE-Sephacel, DEAE-Sepharose exchanger (or any other exchanger type DEAE, QAE, CM or SP). The thus obtained antibodies are then coupled to the -peroxydase selon- the periodate method of Nakane et al ^1. , ^• - "Peroxydase labeled antibody: a new method of con jugation", J. Hystochem.Cytochem.i 22, 1084-1091, (1974). This iπiπunoconjugué - ^' is then lyophilized in the presence of at least one stabilizing agent. According to this best mode of implementation, the sensitivity threshold (expressed in fibrinogen equivalent) is 0.5 ng / ml.

Such a low threshold is particularly interesting in subpathological cases where a low or moderate increase in the content of fragment D has an important diagnostic significance. Such an assay is particularly important for detecting or detecting in particular: infarction and threat syndrome, various cancers, pulmonary embolism, deep venous thromboses; this list is only given for information only; indeed, any modification of the coagulolytic balance resulting in a pre-thrombotic state, will result in an increase in the rate of PDF-D anti-gene structures. It was therefore very important to be able to demonstrate, as is the case according to the invention, an abnormal rise very early, so as to be able to intervene preventively and thus avoid the thrombogenic ace.

Thus according to the invention the above-mentioned semi-quan¬ titative and quantitative assays can be implemented using an anti-PDF monoclonal antibody obtained from one of the easily isolatable PDFs, that is to say PDFg-D and PDFg-E and more precisely D neo and E neo, in particular of human origin.

According to the invention, sets or assay kits comprising the reagents / _ ^{~, that} is to say essentially an anti-PDF-D (or E) antibody adsorbed, are recommended. ^on an inert support, in particular a latex having particles with an average diameter of between 0.1 and

1 μm or even tubes or wells; and thinners_7.

Other advantages and characteristics of the invention will be better understood on reading which will follow from examples of preparation which are in no way limitative but given by way of illustration. EXAMPLE I

Preparation of reagents A. PREPARATION OF PDF

A prolonged degradation of human fibrinogen is carried out (1 g in 50 ml of citrate buffer 50 mg / ml, 0.15 M NaCl at pH 7.00) with human plasmin / _ ^~ 50 CU (Casein Units) _7, for 24 hours at 37 ° C.

The PDF-D and / or PDF-E are then purified by preparative electrophoresis on starch gel ("PEVIKON") according to MARDER et al. "The purification of fibrinogen degradation products by. Pevikon Block Electrophoresis". Thromb. Diath.Haemorrh. , 2, 234-239 (1969); or by ion exchange chromatography according to NILEHN, Thromb. Diath.Haemorrh. ,, 89-100 (1967). B. DEVELOPMENT OF MONOCLONAL ANTIBODIES

The purified PDF-D and PDF-E are used for mouse immunizations (BALB-C) according to standard techniques, see for this purpose "Methods in Immunology". A. Benjamin Inc. Publishers, 159-182, (1970).

After immunizations, the mice are sacrificed and the spleens are removed sterile (2 mice per fusion). The lymphocytes are extracted from the spleen and then the lymphocytes are fused with exponentially growing mouse myeloma cells at the rate of 10 lymphocytes per myeloma cell, in particular according to conventional hybridization protocols. lymphocitaire (see HURRELL JGR: "Monoclonal Hybridoma Antibodies: Techniques and Applications". CRC Press Inc. Edit., 1981 ").

We then proceed to the selection of secretory hybridomas and other screening of the clones producing monoclonal antibodies reacting with PDFs (D or E) but not with fibrinogen (by immunoenzymatic techniques). C. PRODUCTION OF MONOCLONAL ANTIBODIES 0 The selected clones are cultured and then used to develop ascites tumors in BALB-C mice.

The ascites liquids are rich in monoclonal antibodies and are obtained by puncture. ₅ The antibodies are then purified by precipitation with ammonium sulphate at 50% saturation, dialysis against the Tris buffer 0.025 iNaCl 0.035 M pH 8.80, then ion exchange chromatography on DEAE-Trisacryl gel balanced in the same buffer. These purified _Q antibodies (immunoglobulins) are then used for fixing to latex particles or any other solid particle by simple adsorption or by covagent bond. EXAMPLE 2.

25 _-i_- _-_: 20_S ^r _5ii_îPiÊ_ ËS2 £ PÏi2 _-_-! _-_ R __: -____: -.

Take 5 ml of poly¬ styrene latex suspension of 0.3 μm average diameter and add 12.5 mg of immunoglobulins according to Example 1 in 1 ml of 0.1 M glycine buffer - 0.15 M NaCl at pH 8.35.

3 _Q Incubate 4 hours at 37 ° C, then overnight at room temperature (15-25 ° C). Centrifuge for 1 hour at 7000 rpm. , then take up the pellet obtained with 50 ml of 0.1 M glycine buffer, 0.15 M NaCl, bovine albumin 5 mg / ml at pH 8.35. It is resuspended and the reagent c is thus ready for use. EXAMPLE 3

Fixation_par_liaison_covalente We take 5 ml of hydroxylated latex (having free OH functions) and active with metaperiodate 5 0.1 M at a rate of 5 ml. Incubate 30 minutes at room temperature (15-25 ° C) and then dialyse against 3 times 1000 volumes of 0.001 M acetate buffer at pH 4.20.

The dialysate is taken and 10 ml of 1 M ethylenediamine solution at pH 9.5 are added thereto. Incubate 10 'one hour at room temperature and dialysis against

4 times 200 volumes of 0.20 M phosphate buffer containing 0.15 M NaCl at pH 7.6.

The resulting dialysate is taken and 10 ml of 25% glutaraldehyde are added thereto and then incubated overnight at 5 at room temperature. Then dialyzed against

5 times 200 volumes of 0.05 M carbonate buffer containing 0.25 M NaCl at pH 8.3.

The dialysate thus obtained is taken and 12.5 mg of immunoglobulins are added thereto according to Example 1 (in 1 ml 2o of 0.05 M carbonate buffer, 0.25 M NaCl at pH 8.3).

Incubated overnight at room temperature, then centrifuged for 1 hour at 7000 rpm. The pellet is taken up in 50 ml of 0.1 M glycine buffer, 0.15 M NaCl, bovine albumin at 5 mg / ml at pH 8.35. 25 EXAMPLE 4

Fix tion_par_liaison_covalente_ We take 5 ml of latex (having free COOH functions) and couple an ethylene diamine arm using the soluble carbodiimide). For this, 3 _Q 5 mg of EDAC / _ ^~ 3-ethyl- (3-dimethylaminopropyl) -carbo-diimide_7 are added. Incubate for one hour while maintaining the pH at 5.5 and then add 5 ml of 0.1 M ethylene diamine at pH 5.5. The incubation is continued for one hour at ambient temperature, maintaining the pH at 5.5, then it is left overnight at ambient temperature. Dialysis is carried out against 3 times 500 volumes of 0.20 M ^' phosphate buffer containing. 0.15 M aCl at pH 7.6.

The dialysate is taken and 10 ml of 25% glutaraldehyde are added thereto, then incubated overnight at laboratory temperature. Then dialyzed against 5 times 200 volumes of 0.05 M carbonate buffer containing 0.25 M NaCl at pH 8.3.

Take the dialysate and add 12.5 mg of immunoglobulin according to Example 1 (in 1 ml of 0.05 M carbonate _Q buffer, containing 0.25 M of NaCl at pH 8.3).

Incubated overnight at room temperature, then centrifuged for one hour at 7000 rpm. The pellet is taken up in 50 ml of 0.1 M glycine buffer, 0.15 M NaCl, bovine albumin at 5 mg / ml, pH 8.35. , - The reagent is ready for use.

EXAMPLE 5

^D 2 _! - lË _.-! Ê__iz3 _.- H _._: _-__-__: _

To "carry out the assay according to the method of the invention, the procedure is as follows. 0 The blood is taken (9 volumes) and an anticoagulant (1 volume) is added thereto, the anticoagulant being an aqueous mixture of citrate (0.108 M) and excess aprotinin.

The plasma is collected by centrifugation and then the PDFs which it is likely to contain are assayed by bringing the plasma (1 drop) or its dilutions into contact with the reagent (1 drop) according to the invention. The presence of macroscopic agglutination (visible to the naked eye) indicates that PDFs are present, the absence of PDF translating into the absence of agglutination. EXAMPLE 6

_-2Ë! __- 2 S-îfH ^fc i _-___ î__

Monoclonal anti-D neo antibodies are prepared from D neo fibrinogen according to Example 1 above. The purified monoclonal antibodies are subjected to enzymatic digestion with pepsin to discard the Fc fragment.

The antibodies thus obtained are fixed ("coated") on an inert support (wells or tubes). The aqueous sample to be tested is brought into contact with said fixed antibodies, the PDF-D to be assayed (or the PDF-D, PDF-DD and PDF-DDE mixture) are selected selectively compared to the other products contained in the In the sample, the other proteins, in excess, are removed by washing.

The antigenic complex thus formed is then revealed by a polyclonal rabbit antibody specific for fragment D and labeled with peroxidase. The enzymatic activity is demonstrated by the oxidizing action on the orthophenylenediamine substrate (OPD) in the presence of hydrogen peroxide.

The operating procedures are as follows. Reagents

Coating buffer: Carbonate 0.05 M pH 9.60 Dilution buffer: Phosphate 0.05 M NaCl 0.15 M

Tween 20 0.1% BSA 0.5%; pH 7.50

Washing solution: Phosphate 0.01 M NaCl 0.15 M Tween 20 0.1% pH 7.00

Ant ^' i D-neo (monoclonal) coating antibodies according to the present example. used at 2 ug / ml in anti-PDF-D antibody conjugate (polyclonal) buffer conjugated to peroxidase. Use 1/8000 in dilution buffer.

Developer Orthophenylenediamine (OPD) at 0.4 mg / ml in citrate phosphate buffer at pH 5.00 and containing 50 μl per 100 ml of 30% hydrogen peroxide. Stop solution H2 „S04„ 3M Immuno NUNC Type I plates with certificate or Dynatech M129B.

D-Standard Dimer (500 μg / ml solution) After thawing, dilute

1 / 10,000 in dilution buffer

(50 ng / ml solution) and make the following range in dilution buffer: 50 ng / ml 25 ng / ml 10 ng / ml 5 ng / m

2.5rvg / ml 1 ng / ml 0.5 ng / ml 0 ng / m The unconjugate is prepared as follows. A rabbit is immunized with a purified PDF-D / _ ^~ obtained by prolonged degradation of human fibrinogen with plasmin and then purification by electrophoresis according to MARDER et al.,

Thromb. Diath. Haemorrh. 2, 234-239 (1969) supra_7. The resulting antiserum is made specific by exhaustion on a solid support. The anti-D polyclonal antibodies are purified by ion exchange chromatography using a DEAE-Trisacryl exchanger, then coupled to peroxidase according to the aforementioned method of NAKANE et al. The immunoconjugate is lyophilized in the presence of stabilizing agents and is diluted at the time of use.

The calibration is carried out using the DD fragment obtained from a solution with known concentration of fibrinogen, coagulated by thrombin in the presence of Ca, Factor XIII-A and plasmin. A clot forms and is then lysed. The preparation is stabilized with aprotinin. We have a fibrin lysate with known DD concentration. The DD is expressed in initial fibrinogenic equivalent, using concentrations ranging from 0 to 50 ng / ml as indicated above. Plasma_to_test: Normal levels: dilute 1/10 in dilution buffer Pathological rates: dilute 1/50 and 1/200 in dilution buffer. Protocol a) Coating:. Distribute 200 jul of coating solution

(anti-D-neo monoclonal mouse antibody at 2 μg / ml prepared according to example 5) per 5 cuvette.

. Incubate 1 night at 37 ° C.

. wash 5 times in washing solution. b) Antigen:. Distribute 200 µl of standard antigen or diluted plasma to be tested per cuvette. • Incubate for 1 hour at room temperature.

. Wash 5 times in washing solution. c) Conjugated Immuno:

. Distribute 200 μl of diluted immunoconjugate (rabbit polyclonal immunoglobulins, 5 specific to PDF-D and coupled to peroxidase) per cuvette. . Incubate 1 hour at room temperature

(15-25 ⁵ C). . Wash 5 times in washing solution. Do not allow the plate to dry. d) Coloring

. Add the substrate (OPD / H 0.): 200 jul per cuvette. . Incubate 3 minutes (exactly) at ₅ room temperature.

. Stop the reaction with 50 μl of S0.3M H. e) Results:

. After 10 minutes of stabilization, read the optical density at 492 nm. O • Express the results on bilo.garithmic coordinates. COMPARATIVE TESTS

A product according to the invention was compared (product A »anti-D-neo monoclonal antibody without ₅ Fc fragment) specific for the D and DD fragments and prepared according to Example 5, with an anti-PDF-DD monoclonal antibody 20

specific for the D and DD fragments, namely a .. pro _d uit (pro _d uit CP-1, in accordance with the teaching of EP-A-0 122 478) obtained according to the present example 5 from a DD fragment cross-linked fibrin (instead of a D-neo fragment) and free of Fc,

The dosages were carried out:

^~ P2-J £ _iË_δ £ 2 -__-_: J -___ 'according to the methods given in Example 5 above by reaction of the antigenic complex (anti D-neo - PDF to be assayed) with an anti-polyclonal antibody D labeled with peroxidase for development (coloration obtained in 3 minutes)

^~ E2 _ - ._ i2_P £ 2 - yi ---_---- Z ¹ "according to the methods given in Example 5 above by reaction of the complex (anti-DD - PDF to be assayed) with an antibody anti-D monoclonal labeled with peroxidase for development (coloration obtained in 30 minutes)

The results obtained are recorded in Tables I-. And II ^* where the sign + indicates the detection of the substance to be assayed (fibrinogen or FDP), the sign - the absence of detection, and the sign-- a doubt as to the detection of said substance.

These results show that product A according to the invention, which makes it possible to develop a coloration in

3 minutes instead of 30 minutes for CP-1 'has a sensitivity threshold (0.5 ng / ml) lower than that of CP-1 (5-10 ng / ml).

TABLE I Reactivity of product A (according to example 5)

TABLE II Reactivity of product CP-1

(according to the teaching of EP-A-0 122 478)

Claims

1. Method for assaying at least one degradation product (PDF) of fibrinogen and fibrin by means of a reaction mechanism of the antigen-antibody type using an anti-PDF monoclonal antibody, said method being characterized in that that a purified anti-PDF monoclonal antibody chosen from the group comprising monoclonal anti-PDF antibodies which is brought into contact (A) are produced from at least one fibrinogen-containing PDF, and (B) do not bind fibrinogen, with an aqueous liquid medium capable of ^" containing at least one PDF. The complex formed by said monoclonal antibody and said PDF from said aqueous medium constituting a signal which is ^: Emphasizes ^{appropriately.} 2. Method according to claim 1, characterized in that the monomeric anti-PDF means which intervenes in the antigen-antibody reaction mechanism is prepared by ^ar 1) degradation of fibrinogen by plasmin for 24 h at 37 ° C, 2) purification of at least one monomeric PDF thus obtained, 3 °) innoculation of mice, 4) removal of the spleen from the immune animal, 5) extraction of the lymphocytes from the spleen and fusion of the lymphocytes with myeloma cells of exponentially growing mouse at a rate of 5 to 20, preferably 10 lymphocytes per myeloma cell, 6) selection of the secretory hybridomas thus obtained with a view to obtaining clones producing monoclonal antibodies reacting selectively with PDF but not with fibrinogen, 7) development of ascites tumors in mice with the selected clones, 8 °) recovery of the ascites liquids containing the monoclonal antibodies by puncture, 9) purification of the monoclonal anti-PDF monomeric antibodies, 10 °) fixing monoclonal antibodies purified on ^an inert support. 3. Method according to claim 2, characterized in that the inert support involved in fixing the monomeric anti-PDF monoclonal antibody is chosen from the group consisting of glass beads, latex particles, red blood cells, clays, carbon black and agarose. 4. Method according to claim 2, characterized in that the inert support intervening for the fixation of the monomeric anti-PDF monoclonal antibody is chosen from the group consisting of tubes and cups made of glass and plastic. 5. Method according to claim 3, characterized in that the inert support consists of latex particles having an average diameter less than or equal to 5 μm, in particular particles of polystyrene latex having an average diameter of between 0, 1 and 1 µm. 6. Method according to claim 2, characterized in that stage 9 °) comprises the purification of the monoclonal anti-PDF anti-PDF antibodies and the elimination of their Fc fragment sensitive to Rheumatoid Factor by digestion with pepsin. 7. Method according to claim 1, characterized in that (i) the monomeric anti-PDF means which intervenes in the anti-body antigen reaction mechanism is prepared by 1) degradation of fibrinogen by plasmin for 24 h at 37 ° C, 2) purification of at least one monomeric PDF thus obtained, 3 °) innoculation of mice, 4) removal of the spleen from the immunized animal, 5) extraction of lymphocytes from the spleen and fusion of lymphocytes with myleoma cells 10 mice growing exponentially at a rate of 5 to 20, preferably 10 lymphocytes per myeloma cell, 6) selection of the secretory hybridomas thus obtained with a view to obtaining clones producing monoclonal antibodies reacting selectively with the PDFs but not with fibrinogen, 7) development of ascites tumors in mice with the selected clones, 8 °) recovering liquid ascites containing the monoclonal antibodies by puncturing ₂₀ Item 9) Purification of monoclonal antibodies anti-PDF monomer, followed by digestion with pepsin to spread the Fc fragment of said antibody, 10 °) fixation of the purified monoclonal antibodies and without Fc fragment thus obtained, on an inert support, and (ii) in that the antigen-antibody mechanism comprises the following steps: 11 °) bringing said monoclonal antibodies into contact with a test sample, „12 °) bringing the resulting antigenic complex into contact with at least one labeled anti-D polyclonal antibody with an enzyme, 13) measurement of the enzymatic activity on an appropriate substrate. ___. 8. Method according to claim 7, characterized in that the anti-D polyclonal antibody is labeled with peroxidase. 9. Method according to claim 7, characterized in that the measurement of stage 13 °) of the enzymatic activity that, when the polyclonal antibody is labeled with peroxidase, is carried out according to an immunoenzymatic method by means of an orthophenylenediamine substrate in the presence of H _p O. 10. Method according to claim.1, characterized in that the aqueous liquid medium to be tested capable of containing at least one PDF is plasma. 11. Method according to claim 1, characterized in that the monomeric anti-PDF monoclonal antibody is chosen from anti-D and anti-E monoclonal antibodies, in particular anti-D neo and anti-E neo. 12. Method according to claim 1, characterized in that the monomeric anti-PDF monoclonal antibody is a neo-anti-D.