FR2571968A1 - Purified lymphadenopathy and acquired immune deficiency syndrome virus and envelope antigens of this virus, method for obtaining this virus and these envelope antigens of this virus, uses of this virus or of these antigens in the preparation of immunogenic compositions or for the diagnosis of the abovementioned pathologies - Google Patents

Abstract

The invention relates to a purified virus of the LAV type containing an envelope antigen having a molecular weight of the order of 110,000, and more especially to this antigen itself. This antigen may be detected by labelling the virus with a high concentration of [<35>S]cysteine. The invention relates still more especially to a method for the diagnosis of the presence of antibodies against the aetiological agent of Aids in the serum of patients suffering from Aids or LAS or of asymptomatic carriers of the virus. Lastly, it relates to immunogenic compositions containing the antigen of the type in question as an immunogenic principle.

Description

Ourified virus of lvmohadenooathies and acquired dimmuno-depression syndrome and envelope antigens of this virus, process for obtaining this virus and these envelope antigens of this virus. aDDli-cations of this virus or of these antigens in the Drenaration of immunogenic comnositions or Dour the diagnosis of the aforesaid affections.

The present invention relates to a virus purified from lymphadenopathies (hereinafter referred to by the abbreviation SLA) and from the acquired immune depression syndrome (hereinafter referred to as AIDS), to a method of obtaining these antigens from the envelopes of these viruses, for their applications in the preparation of immunogenic compositions or for the diagnosis of the aforesaid affections.

A retrovirus exhibiting characterizations of the etiologic agent of AIDS has been identified. It was first described in an article by F.

Barré-Sinoussi et al, Science, 220, 868 (1983).

This retrovirus has the following characteristics. It is T-lymphotropic, its preferred target consists of Leu 3 cells (or T4 lymphocytes); it has reverse transcriptase activity requiring the presence of Mg2 + ions and exhibits a strong affinity for poly (adenylate-oligodeoxy-thymidylate) [poly (A) oligo (dT) 12-18]; it has a density of 1.16-1.17 in a sucrose gradient, an average diameter of 139 nanometers and a core having an average diameter of 41 nanometers, the lysates of this virus are immunologically recognized, these lysates contain a p25 protein recognized by the same sera but which is not immunologically recognized by the p24 protein of the HTLVI and II viruses.

Retroviruses of this type (sometimes designated by the generic abbreviation LAV) have been deposited in the National Collection of Cultures of Microorganisms of the INS-
TITUT PASTEUR of Paris, under the numbers 1-232. I-240 and 1-241.

Morphologically and immunologically similar strains of virus have been isolated in other laboratories; we will mention for the record the strain of retrovirus called HTLV-III (RC Gallo et al., Science, 224, 500 (1984) and MG Sarngadharan et al. Science 224, 506 (1984) and that called ARV, isolated by M. Jay Levy et al., Science, 225, 840-842 (1984). Reference is also made to the European patent application filed September 14, 1984, under priority of UK application No. 83 24800 filed September 15, 1983 for which relates to a more detailed description of LAV retroviruses or the like and of the applications to which extracts of these viruses give rise.

Only the internal antigens (core) of the virus could be recognized. after lysis of the virus, by sera from patients affected by AIDS or ALS. A p41 protein has been described in the above articles on HTLV3 as a possible component of the virus envelope. But formal proof that it was an envelope protein has not been provided.

Methods for obtaining an LAV virus or a related virus have also been described. Reference may in particular be made to the article already mentioned by F. Barré-Sinoussi et al., As regards the preparation of the virus in cultures of T lymphocytes originating either from the blood or from the umbilical cord, or also from cells. bone marrow from healthy adult donors This process comprises in particular the following essential steps e - viral infection of these T lymphocytes, after activation by a mitogenic lectin, with a viral suspension obtained from a crude supernatant of virus-producing lymphocytes ( initially obtained from a patient infected with AIDS or ALS), - culture of the infected cells with TCGF, in the presence of anti-α-interferon sheep serum, - purification of the virus produced (production generally begins between ninth and fifteenth days following infection and lasts 10 to 15 days) by precipitation of the virus in polyethylene glycol to carry out a prior concentration of the virus, then centrifugation of the virus. preparation in a 20-60% sucrose gradient or in an isotonic metrizanide gradient (sold under the trademark NYCODENZ by NYEGAARD, Oslo), the virus being recovered with the appropriate density band (1.16-1.17 in the case of the sucrose gradient or 1,10-1,11 in a NYCODENZ gradient).

LAV virus can also be produced from continuous T-type lines, such as the CEM line, or from lvmphoblastoid B cell lines, such as obtained by transforming B cells from a healthy donor with D virus. 'Epstein-Barr.

The lines obtained are then capable of continuously producing a virus (LAVIB) which possesses the essential antigenic and morphological lines of the LAV viruses (except that it is collected in a band of density sometimes slightly higher than in the case of above, especially 1.18) in sucrose. The final purification of the virus can also be carried out in a gradient of
NYCODENZ. For general techniques for producing viruses of the 8-LAV type, reference may also be made to the French patent application filed on May 9, 1984 under number 84 07151.

The invention relates to a novel variety of purified retrovirus, related to those which have been defined above, but which differs from them (or which is characterized) by the fact that these viruses contain one or more antigens, exhibiting the characteristics. of a glycoprotein in the tests which are described below, these antigens possibly being revealed by labeling the virus with labeled cysteine, in particular 35-S-cysteine, in a sufficiently high concentration for this purpose in the medium of culture of the virus, in particular at a rate of 200 microcuries per ml of medium, devoid of unmarked cysteine. These antigens are selectively recognized by sera from patients affected by AIDS or ALS or by sera from asymptomatic carriers of the virus.

A preferred antigen conforming to the preceding definition, as it can be obtained from a lysate of this virus (or by gentle stripping of the envelopes of the virus), has a molecular weight of the order of 110,000 daltons, this molecular weight which can be appreciated in a system of comparative migration distances of said antigen and of proteins of known molecular weight (MW) respectively, in particular of the following proteins (marketed by AMERSHAM) and of - lysozyme- [14 C] -methylated (MW: 14,300) .

- carbon dioxide - [14C] -methylated (MW: 30,000).

- ovalbumin - [14C] -methylated (MW: 46,000).

- [14C] -methyl bovine serum albumin (MW: 69,000).

- phosphorylase b - [14C] -methylated (MW: 92,500), - myosin - [14C] -methylated (MW: 200,000).

The compared migration was carried out on a polyacrylamide gel at 12.5%, then under a voltage of 35 V for 18 hours.

The invention also relates to the antigens themselves, in particular that of molecular weight of approximately 110,000, which furthermore have the capacity to be recognized by sera from patients affected by AIDS or ALS or by sera from persons who have been treated. exposed to LAV viruses or the like. These antigens also have the characteristic of forming complexes with concanavalin 'A, said complex being able to be dissociated in the presence of O-methyl- -D-mannopyranoside.

The antigens according to the invention can also bind to other pectins, for example those known under the designation “LENTYL-LECTINE”. The preferred antigen according to the invention, of molecular weight 110,000, is also sensitive to the action of endoglycosidases. This action is manifested by the production from the antigen of molecular weight 110,000 of a protein having a molecular weight of the order of 90,000, the latter being able to be separated for example by an immunoprecipitation or by separations by putting the differences in molecular weights (differentiated migrations on gel) come into play.

The preferred antigens of the invention consist of glycoproteins.

The invention also relates to a process for producing the viruses in accordance with the invention, this process being essentially distinguished from those which were recalled above at the level of the final purification operation.

In particular, the purification step of the process according to the invention is no longer carried out in gradients, but involves carrying out differential centrifugations carried out directly on the supernatants of the culture media of the producing cells. These centrifugation operations include in particular a first centrifugation at an angular speed of centrifugation, in particular 10,000 revolutions per minute, allowing the elimination of the non-viral constituents, more particularly of the cellular constituents, then a second centrifugation at a higher angular speed, in particular at 45,000 revolutions per minute, to obtain sedimentation of the virus itself. In preferred embodiments, the first centrifugation, at 10,000 rpm, is maintained for 10 minutes and the second, at 45,000 rpm, for 20 minutes. These are of course only indicative values, it being understood that it remains within the scope of the specialist to modify the centrifugation conditions, since he takes care to ensure the separation of the cellu-lar constituents and viral constituents.

This modification of the purification process leads to obtaining viral preparations from which the mentioned antigen can then be isolated. However, this has not been obtained to date from virus preparations prepurified by previous methods. In any event, the viruses finally obtained according to the process of the present invention are distinguished from the preceding viral preparations, in that they are recognized by sera from patients or from people who have been exposed to the LAV virus or to strains. morphologically and antigenically similar.

The antigen according to the invention can itself be obtained from these latter viruses, by lysis (or other appropriate treatment) of the latter in the presence of any suitable detergent and by recovery and separation of the released antigens. Advantageously, the lysis of the viruses is carried out in the presence of aprotinin or any other agent capable of inhibiting the action of proteases. The separation of the antigens according to the invention can then be carried out by any method known per se; for example one can proceed with a separation of the proteins by bringing into play their respective different migrations in a determined gel, the desired protein then being isolated from the zone of the gel in which it should normally be found after an electrophoresis operation under conditions well determined, having regard to its molecular weight.

The antigen according to the invention can however be separated from the lysate of the aforementioned viruses, thanks to their affinity for lectins, in particular concanavalin A or lentyllectin. The lectin used is preferably immobilized on a solid support, such as the crosslinked polymer based on agarose, and marketed under the trademark SEPHAROSE.

After bringing the lysate into contact with an appropriate buffer, the retained antigen can be eluted in any suitable manner, in particular by using a solution of O-methyl-a-D-mannopyranoside.

Further purification of these antigens can be carried out by immunoprecipitation with sera from patients known to possess active antibodies against said protein, with concentrated preparations of antibodies (polyclonal antibodies) or even with monoclonal antibodies, more particularly oriented. against the antigen according to the invention, in particular that having the molecular weight of 110,000, hereinafter referred to under the abbreviation gel 10.

Additional characteristics of the invention will become apparent in the course of the following description of the isolation of a virus in accordance with the invention and of an envelope antigen of this virus. Reference will be made to FIG. 1, -which is derived from a photographic reproduction of gel bands which were used to perform electrophoresis of extracts of lysates of T lymphocytes, respectively infected and uninfected (control) by a suspension of LAV.

T lymphocytes from a healthy donor and infected with LAV1, 'under the conditions described by F.

Barré-Sinoussi et al., Or CEM cells from a patient with leukemia and also C-vitro infected with LAV1, were maintained in culture in a
Medium containing 200 microcuries of S-cysteine and free of unlabeled cysteine. The infected lymphocytes are maintained in culture in a non-denaturing medium to prevent degradation of the desired antigen. The supernatant of the culture medium is then subjected to a first centrifugation at 10,000 revolutions per minute for 10 minutes. to remove the non-viral constituents, produced at 45,000 revolutions per minute for 20 minutes, to produce sedimentation of the virus. The virus pellet is then lysed using detergent, in the presence of aprotinin (5 Z), in particular in the conditions described in the article by F. Barré-Sinoussi and Cool.

The same operations are repeated on lymphocytes from a healthy donor as a control.

The different lysates are then immunoprecipitated with sera from patients affected by AIDS or ALS, as well as with sera from healthy donors or those affected by other diseases. The media were then subjected to electrophoresis in an SDS-polyacrylamide gel,
The results are shown in fig. 2. The gel bands numbered 1 to 6 were obtained from
35 from preparations labeled with S-cysteine. The bands numbered 7-10 correspond to the results observed on preparations of infected or uninfected lymphocytes, labeled with 35S-methionine. Finally, the M band corresponds to the migration distances of the reference proteins identified above, the molecular weights of which have been recalled in the right part of the figure.

To the left of the figure are referenced the viral proteins which have been identified.

It is noted that columns 7 to 9 show the p25 protein specific for LAV. labeled with 35Smethionine, the same protein being absent from columns 8 and 10 corresponding to an observation made on preparations obtained from healthy lymphocytes.

Columns 3 and 5 correspond to the results which were observed on preparations obtained from lymphocytes infected and labeled with 35S-cysteine. We find both the p25 and p18 proteins characteristic of the LAV nucleotide proteins and the gap110 glycoprotein, also specific for LAV. We also see appear in the various preparations, although less clearly, images corresponding to a p41 protein ( molecular weight of the order of 41,000) and not specific for the LAV virus, It is also observed in witnesses,
The virus according to the invention and the antigen according to the invention can be either precipitated by lectins, in particular concanavalin A, or attached to a column of
SEPHAROSE-concanavalin A, This fixing can in particular be carried out by bringing the lysate of the aforesaid virus into contact in a buffer allowing the following composition
Tris 10 mM
0.15 M NaCl
1 mM CaCl2
MgCl2 1 mM
Detergent marketed under the brand TRITON 1 Z
pH 7.4
Once the fixation is completed, the SEPHA is washed
ROSE-concanavalin A with a buffer of the same composition, except for the TRITON content which is lowered to 0.17 ..

Elution is then carried out with a 0.2 M solution,
O-methyl-aD-mannopyranoside, within the wash buffer.

The protein can be further concentrated by immunoprecipitation with the antibodies contained in sera from patients affected by AIDS or with polyclonal antibodies obtained from a serum obtained from an animal previously immunized against the virus according to the invention or the invention. aforesaid glycoprotein. The protein can then be recovered by dissociation of the complex with a solution having an adequate content of ionic salt.

Preferably, the aforementioned antibody preparations have been immobilized beforehand in a manner known per se on an insoluble support of the SEPHAROSE B type.

It is also possible to have recourse to monoclonal antibodies secreted by hybridomas prepared beforehand against gel 10. These monoclonal antibodies, as well as the hybridomas which produce them, also form part of the invention.

The conditions under which the monoclonal antibodies according to the invention can be prepared are described below.

Immunization of mice
Groups of 6-8 week old Balb / c mice were used. One group received the virus comprising the aforementioned glycoprotein, another the gap110 glycoprotein. The immunization protocol, identical for the 4 mice, consisted of the injection, intraperitoneally three times, then once intravenously, of 10 μg of the antigen preparation in the presence of complete Freund's adjuvant on day O. incomplete Freund's adjuvant on day 14 and no adjuvant on days 28 and 42.

Hybrid fusion and culture
The 6.53 non-secreting variant of P3 X 63 myeloma
Ag8, resistant to azaguanine, itself from the MOPC-21 line, was used. The fusion with the splenocytes of immunized mice was carried out in the presence of polyethylene glycol 4.000 according to the technique of FASEKAS DE
ST-GROTH and SCHEIDEGGER on day 45 (8). The selection of the hybrids in RPMI 16-40 “HAT” medium was carried out according to the same culture technique in 24-well plates (Costar).

The hybridomas producing antibodies of adequate specificity were then cloned in 96-well plates. in the presence of a feeder layer of syngeneic thymocytes. The producer clones thus selected were then expanded in 24-well plates, still in the presence of thymocytes. When confluence appeared in one of the wells, the clone was injected intraperitoneally into a mouse
BALB / c having received an injection of Pristane 8 days previously and / or maintained in liquid culture.

Demonstration of anti-LAV antibodies
Five different techniques made it possible to characterize the clones producing antibodies of interesting specificity. As a first step, the hybrids producing antibodies were determined by an ELISA test revealing the mouse immunoglobulins in the supernatants. From this first selection, we looked for the supernatants which presented antibodies directed against viral constituents using an ELISA test revealing anti-LAV antibodies (9), or by immunofluorescence on human virus-producing cells. Finally, the supernatants were analyzed by radioimmunoprecipitation of virus labeled with 35S-cysteine and by the technique of Western-blot on viral preparation (10) which made it possible to determine the speciflcitos of these anti-LAV antibodies;
RESULTS
The cells obtained from the different fusions were cultured in 648 wells. Microscopic examination showed that most of these wells contained a single hybrid clone capable of growing in selective "HAT" medium. More than 50% of them produced antibodies giving rise to a positive response to the antivirus ELISA examination. The most representative fusions were tested by the Western-blot technique and several of them were undergone. -cloned, taking into account their specificity, their reactivity in antivirus ELISA and their behavior in culture. We more particularly retained the hybrids which produced antibodies which recognized the glycoprotein of molecular weight corresponding to the viral glycoprotein gap110. All the subclonings gave rise to antibody producing clones which, after expression, were injected into syngeneic mice. Analysis of the specificities of the antibodies present in the various ascites fluids confirmed the specificity of the antibodies of said ascites with respect to gpl 10.

The monoclonal antibodies obtained can themselves be used to purify proteins containing an antigenic site also contained in gap110. The invention therefore also relates to this purification process as such. This method is advantageously applied to virus lysates or T lymphocyte lysates or any other cells producing LAV or the like, as long as before lysis. care will have been taken to avoid the uncontrolled separation of gap110 (it being understood that the method can also be applied to any solution containing gpi10 or a protein. polypeptide or glycoprotein comprising an antigenic site of envelope protein, recognized by the monoclonal antibody, whatever the nature of this solution). For the implementation of this method, the monoclonal antibodies are advantageously immobilized on a solid support, preferably suitable for affinity chromatography operations. For example, these monoclonal antibodies are fixed on a three-dimensional crosslinking agarose network, marketed under the trademark SEPHAROSE by the Swedish company PHARMACIA A.G, for example by the cyanogen bromide method.

The invention therefore relates more particularly to a process for separating these antigens characterized by the operations consisting in passing the medium capable of containing them in contact with an affinity column carrying the aforesaid monoclonal antibodies, in order to selectively bind said polypeptides, proteins. or glycoproteins and then recovering them by dissociation of the antigen-antibody complex by means of an appropriate buffer, in particular a solution of adequate ionic strength, for example a salt, preferably ammonium acetate (which does not leaves no residue when the lyophilization of the preparation is then carried out), It is also possible to use an acidified solution to pH 2-4 or to a glycine buffer at the same pH.

These antigens are themselves capable of being used as reagents, or even as in vitro diagnostic agents, for the detection of anti-LAV antibodies. It goes without saying that the invention also relates to polypeptide fractions which may have lower molecular weights, since they would carry antigenic sites capable of being recognized by the same monoclonal antibodies. It will be clear to the specialist that From the moment when the monoclonal antibodies according to the invention are available, it is possible to envisage the isolation, from the above-mentioned antigens, of smaller peptide sequences containing the same antigenic sites, for example by using to techniques known per se of cutting the initial polypeptide by enzymes capable of cutting larger polypeptides at specific sites By way of example of such proteins, mention may be made of the StaDhvlococcus aureus V8 enzyme, alpha-chvmotrypsin , the “mouse submaxillary gland protease” marketed by the company
BOEHRINGER, the collagenase of Vibrio alinolvticus chemovar iophaus, which specifically recognizes said peptides Gly-Pro and Gly-Ala, etc.

It is also possible to search for polypeptides or fragments of antigens of the envelopes of the virus, a cloning of the fragments excised from a cDNA constructed from the genomes of the various varieties of LAV and similar viruses.

Fig. 2 is representative of restriction maps of some of these cDNAs, comprising a total of 9.1 to 9.2 kb. We will focus more particularly on the polypeptides encoded by cDNA fragments located in the region extending between the KpnI site (position 6100) with respect to the restriction maps of FIG. 2 and the site
BglII (position 9150). The presence of a site characteristic of an envelope antigen of the LAV virus or the like in the polypeptide capable of being expressed (in an appropriate cellular host previously transformed by such a fragment or by a vector containing this fragment) can be detected. by any appropriate immunochemical method,
The antigens of the genus in question can also be used to separate antibodies having the above-mentioned characteristics from a polyclonal mixture of antibodies. In this case, the polypeptides of the genus in question will in turn be immobilized on an affinity chromatography support, for example of the genus indicated above. The separation process will therefore comprise the step of passing a solution containing the polyclonal antibodies into contact with the immobilized polypeptides, and the step of recovering the retained antibodies by means of a solution or a buffer similar to that. or the one that was considered above.

Finally, the invention relates to immunogenic compositions characterized by the association of an antigen in accordance with the invention, as an immunogenic principle, in particular at a rate of 10 to 500, for example 50 to 100 micrograms / kg, with a physiologically acceptable excipient allowing its administration to a living host, more particularly man, with a view to conferring on it immunity with regard to said antigens, including viruses
LAV or whole analogues, These antigens constitute active principles whose immunogenicity can be used whenever protection ia vivo against LAV viruses or viruses related to them is sought.

The invention also relates to a method using the antigens according to the invention for the detection of the presence of anti-LAV antibodies, in particular in blood samples from humans or animals, with a view to carrying out the detection. AIDS or ALS.

Finally, the invention relates to an in vitro diagnostic method involving an envelope antigen in accordance with the present invention for the detection of anti-antibodies.
LAV in the serum of patients affected by the disease or of persons immune to the virus. It relates more particularly to a kit or "kit" comprising this antigen.

The diagnostic method comprises in particular - the deposition of determined quantities of an antigen in accordance with the present invention in the wells of a microtiter plate - the introduction into these wells of increasing dilutions of the serum to be diagnosed - the incubation of the microplate - careful washing of the microplate - introduction into the wells of the microplate of labeled antibodies specific for blood immunoglobulins, the labeling being carried out by an enzyme chosen from those capable of hydrolyzing a substrate such that the latter then undergoes a modification of its radiation absorption, at least in a determined wavelength band and - the detection, preferably in a comparative manner with respect to a control, of the extent of the hydrolysis of the substrate in as a measure of the potential risks or the actual presence of the disease.

Of course, quantitative titrations of antibodies can be carried out on the sera studied.

Preferred methods involve immunoenzymatic or immunofluorescent assays. in particular in accordance with the ELISA technique. The assays can be immunofluorescence assays or direct or indirect enzyme-linked immunosorbent assays.

Claims (15)

1 - Retrovirus exhibiting the characteristics of the etiological agent of AIDS. and more particularly LAV retroviruses, characterized in that it comprises one or more antigens exhibiting the characteristics of a glycoprotein and which can be revealed by labeling the virus using a high concentration of 35S-cysteine . in particular at a rate of 200 microcuries per milliliter of medium, and this in the absence of unlabeled cysteine, these antigens also being recognized selectively by sera from patients affected by AIDS or ALS, or even by sera of asymptomatic carriers of the virus. 2 - Retrovirus according to claim 1, characterized in that it comprises an envelope antigen having a molecular weight of the order of 110,000 daltons. 3 - Antigen having a molecular weight of approximately 110,000 or antigen of lower molecular weight derived from the above, which has the capacity to be recognized by sera of patients affected by AIDS or ALS or by sera of asymptomatic carriers of the virus. 4 - Antigen according to claim 3, which also has the characteristic of forming complexes with concanavalin A, said complex being capable of being dissociated in the presence of O-methyl-a-D-mannopyranoside 5 - Antigen according to claim 3 or claim 4 characterized in that it can also bind to other lectins. 6 - Antigen according to any one of claims 3 to 5, which has a molecular weight of the order of 110,000 daltons and which is also sensitive to the action of endoglycosidases. 7 - Process for obtaining a retrovirus according to claim 1 or claim 2, comprising the infection of T lymphocyte lines or B lymphoblastoid cell lines, the culture of these infected cells and the purification of the virus obtained, characterized in that the final virus purification step comprises carrying out differential centrifugations carried out directly on the supernatants of the culture media of the producing cells and recovering the final virus, Which is labelable with S-cysteine used in high concentrations and in the absence of unlabeled cysteine. 8 - Process for obtaining an antigen according to any one of claims 3 to 6, comprising the isolation of this antigen from the purified virus obtained at the end of claim 7, in particular by lysis of this virus or stripping of the envelopes of this virus, and the separation of the antigens which have the capacity to be recognized by sera from patients affected by AIDS or ALS, more particularly from that which has a molecular weight of the order of 110. ode. 9 - Process according to claim 8, characterized in that the separation of the antigen comprises contacting the medium containing the antigen, as obtained at the end of the process defined in claim 8, with an affine lectin , such as concanavalin A or lentyl-lectin, this lectin preferably being immobilized on a solid support, then elution of the aforesaid antigen, in particular using a solution of O-methyla-D-mannopyranoside. 10 - Method according to claim 8, characterized in that said purification is carried out by immunoprecipitation with the sera of patients known to possess active antibodies against said antigen or else with monoclonal antibodies produced by hybridomas previously formed and secreting said monoclonal antibodies, these more particularly recognizing the antigen according to any one of claims 3 to 6. 11 - Monoclonal antibody characterized by its ability to specifically recognize the antigen according to claim 6. 12 - The hybridomas secreting the monoclonal antibody according to claim 11. 13 - Immunogenic composition characterized by the association of an antigen according to any one of claims 3 to 6, with a physiologically acceptable excipient, allowing its administration to a living host, more particularly 1 man. 14 - Immunogenic composition according to claim 13, characterized in that it is dosed with antigen so as to allow the administration of a dose of 10 to 500, in particular 50 to 100 micrograms per kilogram. 15 - A diagnostic method comprising - the deposit of determined quantities of an antigen according to any one of claims 3 to 6 in the wells of a microtiter plate - the introduction into these wells of increasing dilutions of the serum to be diagnosed - incubation of the microplate - careful washing of the microplate - introduction into the wells of the microplate of labeled antibodies specific for blood immunoglobulins, the labeling being carried out by an enzyme chosen from those capable of hydrolyzing a substrate such that the latter then undergoes a modification of its radiation absorption, at least in a determined wavelength band and - the detection, preferably in a comparative manner with respect to a control, of the importance of hydrolysis of the substrate as a measure of potential risks or the actual presence of disease.