FR2573533A1 - Method for the detection or titration of immunoglobulins directed towards a particular antigen and device for carrying out this method - Google Patents

Abstract

Method for the detection or titration of immunoglobulins, directed towards a particular antigen, in a liquid medium liable to contain them, wherein the aforementioned liquid medium is brought into contact, according to standard sero-immunological techniques, with a solid support to which the aforementioned antigen is bound, and wherein the system obtained is then brought into contact with a solution containing an antibody directed towards an isotypic antigenic determinant of the aforementioned immunoglobulins to be detected or titrated. The aforementioned antibody is a monoclonal antibody, and the system obtained is then brought into contact with a solution containing a second antibody labelled with a tracer agent, this second antibody being directed towards an isotypic antigenic determinant of the first antibody, and thereafter the presence, where appropriate, of the aforementioned tracer agent is determined qualitatively and/or quantitatively on the system obtained; and device for carrying out this method.

Description

 The subject of the present invention is a method for detecting or titrating immunoglobulins directed against a given antigen, in a liquid medium capable of containing them, as well as a device intended for carrying out this method.

 The method of the invention is in particular applicable to the detection of immunoglobulins (or antibodies) present in the serum.

 Serological methods are mainly used to detect or titrate antibodies present in human body fluids and also in body fluids and samples obtained in the laboratory for experimental purposes. These methods are based on the antigen-antibody reaction which has given rise to the development of various techniques such as radio-immunological techniques, immunofluorescence techniques and immuno-enzymatic techniques.

 It is known that the search for immunoglobulins constitutes an important element in establishing the diagnosis of certain infections, in particular of bacterial or viral origin.

 In addition, knowledge of the classes of immunoglobulins involved often makes it possible to determine the degree of progression of the disease.

 One of the drawbacks of recent sero-immunological methods, such as the ELISA method, used for the detection or titration of immunoglobulins, is that they often require the use of purified antigens; see for example J. LUKA et al., J. Imm. Meth., 67, 145-156 (1984).

 However, obtaining purified antigens is generally a long and costly operation, in particular when it is a question of obtaining relatively large quantities of antigens.

 In the case of viral infections, additional difficulties arise from the fact that viruses generally contain or release several kinds of antigens, the characterization of which is often useful for determining the stage of the disease. The preparation of purified antigens is complicated.

 The subject of the present invention is a method for detecting antibodies which has in particular the advantage of not requiring the use of purified antigens.

 It is known that, according to conventional sero-immunological techniques, the antibodies directed against a particular antigen can be detected by fixing this purified antigen on a solid adsorbent support, by bringing the support obtained into contact with the biological fluid capable of containing the desired immunoglobulins, then by bringing the solid system obtained into contact with a solution containing antibodies labeled anti-immunoglobulins. We then look for the possible presence of the marker on the support, this presence signifying that the biological fluid studied contained indeed antibodies directed against the antigen.

 We have now discovered that it is possible to search for immunoglobulins, without the need to use purified antigens, provided that the single labeled antibody is replaced by a system of two antibodies, at least one of which is a monoclonal antibody, as will be explained below.

 The present invention relates to a method of detecting or titrating immunoglobulins directed against a particular antigen, in a liquid medium capable of containing them, in which said liquid medium is brought into contact, according to the usual sero-immunological techniques, with a solid support on which said antigen is fixed, and in which the system obtained is then brought into contact with a solution containing an antibody directed against an isotypic antigenic determinant of said immunoglobulins to be detected or titrated, characterized in that said antibody is an antibody monoclonal and that the system obtained is then brought into contact with a solution containing a second antibody labeled with a tracer agent, said second antibody being directed against an isogenic antigenic determinant of the first antibody, then that is determined qualitatively and / or quantitatively the possible presence of said tracer agent on the system obtained.

 As indicated above, the method of the invention can be carried out with unpurified antigens. For example, in the case of viral antigens, the supernatant obtained after lysis of the cells originating from a culture of cells in which the virus has been multiplied may be used as the preparation of the antigens.

The lysis of the cells is either due to the action of the infectious agent within the cell, or caused by any known means (sonication, grinding, etc.)
The antigen preparation can be fixed on the solid support by any known means, for example by adsorption, by covalent bond or not, in particular by chemical coupling with a bifunctional coupling agent, by immunochemical affinity, etc.

 The solid support can be produced with any solid, biological or synthetic material, endowed with adsorbent properties or capable of fixing a coupling agent. These materials are known and described in the literature. Among the solid materials capable of fixing, for example, antigens by adsorption, mention will be made in particular of polystyrene, polypropylene, latex, etc. Among the materials which can be used to fix antigens by covalence using a coupling agent , examples include dextran, cellulose, their amino derivatives, etc.

 The support can be presented, for example, in the form of discs, cubes, rods or plates, in particular microtiter plates.

 The first antibody used in the process of the invention is a monoclonal antibody directed against an isotypic antigenic determinant (specific to the species) of the individual in whom the presence of immunoglobulins is sought. They will preferably be antibodies directed against an antigenic site of the Fc fragment of the immunoglobulins of the species in question.

 In other words, the first antibody is an antibody originating from an animal species different from that of the individual in which the presence or absence of immunoglobulins directed against the particular antigen used is sought.

 It is known that anti-immunoglobulin antibodies can be obtained by immunizing, with immunoglobulins originating from individuals of a first species, animals of another species, and by selecting, for example by means of immunosorbing techniques , the immunoglobulins obtained specific for a particular antigenic determinant.

 However, as the first antibody used, in the process of the invention, monoclonal antibodies which can be obtained, for example, according to the hybridoma technique by immunization of mice with human immunoglobulins3 then fusion of the lymphocytes of the immunized mice with lymphocytes myeloma. Cell clones are then selected which produce antibodies specifically directed against an isotypic antigenic determinant of human immunoglobulins.

 Of course, monoclonal antibodies obtained by any other method can also be used.

 The fluid in which the presence of antibodies is sought is for example a bodily fluid, such as a blood serum, a laboratory sample, etc.

 The second antibody used in the method of the invention is also an antibody directed against an isogenic antigenic determinant of the first antibody. Preferably, this second antibody is also a monoclonal antibody.

 For example, if the first antibody is a mouse monoclonal antibody, the second antibody could be an anti-rabbit antibody (mouse immunoglobulin), in particular an antibody directed against an antigenic site of the Fc fragment of the mouse antibodies.

 The second antibody is an antibody modified by labeling with a tracer agent according to conventional techniques for preparing the labeled antibodies.

 For example, the tracer is a radioactive tracer obtained by isotopic exchange with a radioactive isotope such as iodine 125. Labeling of antibodies with a radioactive tracer is known per se.

 The labeled antibody can also be a fluorescent conjugate antibody obtained by coupling of antibodies with a fluorochrome (for example derived from fluorescein or rhodamine) according to known methods.

 The coupling of antibodies with an enzymatic tracer is also known per se. The enzyme is for example a peroxidase, or an alkaline phosphatase.

 The enzymatic activity possibly present on the reagent, after carrying out a test, can be determined according to known methods with an appropriate substrate allowing for example a revelation by colorimetry, by fluorescence, by potentiometry, etc.

 Of course, the various stages of the process of the invention (incubation, washing) are carried out in a liquid medium which does not dissociate the antigen / antibody complexes. Buffer solutions commonly used for seroimmunological reactions of this type are used for these various operations.

 The invention also relates to a device intended for the implementation of the method defined above, characterized in that it comprises one or more units of said solid support on which said antigen is optionally already fixed, containers containing solutions of said first and second antibodies, dilution, incubation and washing buffers, and, optionally, a container containing a solution of said antigen, as well as a manual, the whole being gathered in the same packaging with several compartments constituting an analysis box.

 The invention will now be described in more detail with more specific reference to the detection of antibodies directed against viral antigens3 and in particular antigens of Herpes virus. The case of the Epstein-Barr virus will be described more particularly. It should be noted that it is only to facilitate understanding of the presentation and to illustrate concretely the advantage of the process that the research for antibodies directed against the antigens of the Epstein-Barr virus has been described more particularly.

Specialists will understand that the process described is in no way restricted to the search for these particular antibodies, and the rest of the description should in no way be considered as limiting in this regard.

 The Epstein-Barr virus, discovered in 1964, is a lymphotropic herpes virus affecting all populations of the globe, although the age of primary infection varies greatly from one human group to another. The pathological manifestations due to the Epstein-Barr virus are of four types. In western countries, it is mainly infectious mononucleosis, which affects adolescents from groups of high socioeconomic level.

Certain infectious mononucleoses are accompanied by malignant polyclonal proliferation which can lead to death, in particular in the immuno-depressed. In Equatorial Africa, the same virus is responsible for childhood cancer of the face, called Burkitt Lymphoma. But it is in Southeast Asia and the Mediterranean region that the tumor pathology linked to the Epstein-Barr virus is the most important. It is indeed cancer of the nasopharynx which is one of the most frequent cancers in these regions.

 It is very important to be able to detect, in a simple and inexpensive manner, the antibodies directed against the Epstein-Barr virus. Indeed, thanks to the detection of a specific antibody directed against this virus, we can not only detect this cancer early at the very beginning, when it is perfectly curable, but also we can characterize a pre-tumor state to prevent progression to a cancerous state.

Knowledge of the Epstein-Barr virus was reviewed in the book "The Epstein-Barr Virus" Ed. By MA Epstein and BG
Achong, Springer-Verlag, Berlin, Heidelberg, New-York (1979).

 The Epstein-Barr virus can be multiplied in the laboratory in vitro, by obtaining lines of B lymphocytes obtained either in certain cases of cancers associated with this virus, or even from normal individuals.

Within these lymphocytes, the virus is generally latent, that is to say that it does not multiply but that one can detect its presence either by certain immunofluorescence techniques detecting the presence of viral antigens within cells, or again by molecular biology methods detecting the presence of viral DNA.

In practice, to obtain the antigens of the Epstein-Barr virus, the virus can be multiplied on the following known cell lines
P3HR1, Raji, etc ... We induce the replication of the virus using the usual techniques, in particular by addition of viral latency preactivators (croton oil, TPA, sodium butyrate, etc ...).

 It is recalled that TPA is an abbreviated notation for 12-0-tetradecanoylphorbol acetate.

 We know that the examination of lymphocytes infected with the Epstein-Barr virus has made it possible to highlight various antigenic specificities, notably the early antigens or EA antigens (EA-R: early antigen - restricted; EA-D: early antigen - diffused) , the nuclear antigen (EBNA = Epstein-Barr virus nuclear antigen) and a viral capsid antigen (VCA = Viral capsid antigen); see the work "The Epstein-Barr Virus already cited, pages 39-52; see also" The Herpes Viruses ", Ed. by B. Roizman.

Plenum Press Publ. New York, N.Y. (1982), Vol. 1, in particular chapter 2, entitled "Epidemiology of Epstein-Barr Virus ans As soc iated Diseases in Man" by Guy de THE.

 The known methods of detection and titration of antibodies directed against the antigens of this Epstein-Barr virus use indirect immunofluorescence on smears of cells expressing one or other, or several, viral antigens. In practice, we have spreads of cells prepared so that they only express certain viral antigens. After fixation, serum to be tested is deposited on the fixed cells. After incubation, washing and then depositing antibodies labeled with fluorescein and directed against human immunoglobulins.

 It is thus possible to detect the antibody molecules of the serum to be tested which have possibly fixed on the viral antigens present.

 This technique, described by Henle G. and Henle W., J. Bacteriol., 91, 1248-1256 (1966), is very long and does not make it possible to set up large-scale screening campaigns in order to detect very early on the nasopharyngeal cancer mentioned above. It was therefore important to be able to have a rapid, specific and very inexpensive test, which could be used very widely in populations at risk for this cancer, which are relatively poor populations and who cannot use a long technique. , expensive and impractical.

 The method of the invention makes it possible in a simple, effective and specific way, to detect for example the antibodies of the IgA class directed against the EA antigen in 97% of patients suffering from cancer of the nasopharynx.

By way of comparison, the immunofluorescence techniques hitherto used in industrialized countries detected only 20 to 30% of such antibodies in these patients; see from THE et al., Nasopharyngeal
Carcinoma: Etiology and control, IARC publication, pp. 471-481 (1978).

 The comparison of this technique and the classical ELISA technique showed that the sera negative for this antigen according to the conventional technique (from THE et al., Article cited) are indeed negative by this new technique, which proves the specificity of the proposed test.

Another immunoenzymatic technique used in
People's Republic of China described by ZENG et al., Intervirology, Vol.13, pp. 162-168 (1980) gives a positivity proposal of up to 60%.

 The technique of the present invention, compared to these two previous techniques, makes it possible to increase this proportion up to 97%.

 The advantages of the new technique are: the simplicity, the specificity and above all the very low cost of this test compared to the immunofluorescence tests and to the ELISA test used until now and most often using antigens and antibodies highly purified.

 The following examples illustrate the invention without, however, limiting it.

EXAMPLE 1
1. Preparation of the EA antigen
The BURKITT P3HR-1 lymphoma cell line (reference
HINUMA et al., J. Virol.1,1045, 1967) is cultured at 37 "C in RPMI medium with 20% fetal calf serum at an initial cell concentration
5 of 5x105 cells per ml; add croton oil, up to a concentration of 500ng / ml (or TPA 20ng / ml), sodium n-butyrate (4mM)
6 and 0.25 x 10- g / ml of cytosine arabinoside (macaw C). After 48 hours of culture in this medium, the cells are collected, washed twice in PBS buffer and stored at -200 C. The frozen cell pellets are resuspended in 1 ml of an aqueous solution containing 150 mM NaCl, 20 mM buffer Tris (pH 7.5), 1 mM EDTA and 0.5 mM phenyl methyl sulfonyl fluoride (PMSF) per 108 cells. After sonication repeated eight times for 10 seconds each in crushed ice, centrifugation is carried out at 18,000 rpm for 45 minutes.

The supernatant is then used as an antigen and the protein concentration is measured. The antigen is stored at -70 C.

2. Preparation of the VCA antigen
The procedure is as above, but without the use of the cytosine arabinoside.

3. Preparation of the EBNA antigen
The BURKITT lymphoma cell line called Raji (reference: PULVERTAFT, RV, Lancet, 10.94.1964) is used with 20% fetal calf serum. We operate from frozen cells.

4. Other reagents
- Monoclonal antibodies
Monoclonal antibodies are used against human Ig A, G or M, prepared in mice according to the hybridoma technique.

- Labeled antibodies
Rabbit antibodies against mouse TgGs and labeled with horseradish peroxidase (dilution 1: 30000).

5. Preparation of ELISA plates
100 microliters of an antigen suspension, containing 10 micrograms of the antigen suspension at pH 9.6 in a 0.05M sodium carbonate buffer, are added to each well of a polystyrene microtiter plate. These plates are incubated overnight at 40C.

They are then washed with a 0.05M sodium carbonate buffer at pH 9.6 containing 0.1% bovine serum albumin, then dried for 20 minutes at room temperature. The unbound sites are saturated with a 0.05M carbonate buffer (pH 9.6) added with 1% of serum bovine albumin. After 3 hours in a humid chamber at room temperature, the plates are washed twice with PBS buffer containing 0.05% Tween 20, then dried at room temperature. The plates can then be kept at + 40C until they are used.

6. Antibody detection and titration technique
We operate as follows
- 100 microliters of the test serum diluted 1/40 are added in duplicate to each well and incubated for one hour at room temperature. The plates are then washed five times in washing buffer as above.

 - 100 microliters of mouse monoclonal antibodies directed against human Ig of the class IgA, IgM or IgG, diluted 1/500 in the same washing buffer, are then added to each well. Again, the plates are incubated at room temperature for one hour. The plates are then washed five times in the washing buffer.

 - 100 microliters of rabbit serum containing antibodies directed against mouse IgG and labeled with peroxidase, diluted 1: 30000 with the washing buffer, are added to each well and incubated at room temperature for one hour in a humid room. The plates are again washed five times with the buffer.

 - the substrate for the enzymatic reaction is then added. For this, 100 microliters of a solution containing 40 mg of orthophenylene diamine (OPD) and 30 microliters of H202 at 30% in 100 ml of a phosphate / citric acid buffer (pH 5.0) are added to each well. , 2 M Na2HPO4 and 0.1M citric acid. After 20 minutes at room temperature, the reaction is stopped by adding 50 micrograms of a 4M solution of H2 SO4. The colored reaction is read with a spectrometer (Titertek Multiskan) at 492nm.

The results are given by the P / N ratio, P being the optical density of the sample minus the optical density of the control and N being the optical density of the negative reference serum minus the optical density of the PBS buffer.

 If the P / N ratio is greater than 2, the serum is considered positive, and negative if it is less than 2. The higher this ratio, the higher the corresponding antibody titer in the serum.

Results
Antigen titration
To determine the optimal concentration of antigen for the test
ELISA, microtiter plates are coated with serial dilutions of cell extracts containing 0.09 micrograms to 100 micrograms per well while the positive reference serum is diluted 1:40 to 1: 25-60. The P / N values of a positive reference serum are calculated corresponding to the different concentrations of the antigen and serum dilutions. Similar dc P / N values (2.4-3.0) and the same antibody titer are obtained when the concentration of the antigen used varies from 6.25 micrograms to 100 micrograms per well. Consequently, it is possible to use 10 micrograms of antigen per well in routine assays.

 Titration of anti-human IgA monoclonal antibodies.

To determine the optimal dilution of anti monoclonal antibodies
Human IgA for the ELISA test, serial dilutions ranging from 1: 125 to 1: 4000 of anti-IgA antibodies are tested. This study showed that the values of
P / N for sera from patients with nasopharyngeal cancer decreases with dilution of the anti-IgA antibody. The greatest dilution of anti-IgA antibodies giving positive results (P / N> to 2) was never less than 1: 500 for the sera studied. It is therefore this dilution of 1: 500 of the anti-IgA antibody which can be used for the routine tests.

Specificity and sensitivity of the ELISA test
Two known positive sera and one serum recognized as negative were tested with serial dilutions with an optimal concentration of EA antigens at a rate of 10 micrograms per well. The curves representing the optical density and the P / N values of positive and negative serum are very clearly different.

 Twenty-one known negative sera and twenty-one known positive sera were then tested using both the ELISA test and the enzyme-linked immunosorbent assay. These tests have shown that there is a good correlation between the antibody titers detected by the ELISA test and by the immunoenzymatic test. The mean geometric titer of the positive sera detected by ELISA was 1: 710, ie 8 times higher than the mean geometric titer (1: 88.8) obtained with the enzyme immunoassay.

Reproducibility of the ELISA test
To determine the reproducibility of the ELISA test, 10 sera from patients with nasopharyngeal cancer and 8 sera from healthy individuals were tested in two separate experiments. The results of these two series of experiments are very similar, the variation in the P / N value for each serum being most often between 0.1 and 0.4.

 Detection of anti-EA IgA antibodies in patients with nasopharyngeal cancer, in patients with other cancers and in healthy individuals, by the ELISA test and by the enzyme immunoassay.

 91 sera from patients with nasopharyngeal cancer, 59 sera from patients with other cancers and 90 sera from healthy individuals were tested both by the test according to the invention and by the immunoenzymatic test. It was found that the percentages of anti-HA antibodies detected by the enzyme immunoassay were 60%, OZ and 0Z respectively, while with the ELISA test, the percentages were 97Z, 3.4Z and 2.2% respectively .

 Thus, not only the results of detection with the test of the invention are markedly improved, but in addition, obtaining certain positive results in individuals at a pre-cancerous stage makes it possible to envisage the detection of the disease. at a stage where it is easily curable.

Claims (13)

 1. Method for detecting or titrating immunoglobulins directed against a particular antigen, in a liquid medium capable of containing them, in which said liquid medium is brought into contact, according to the usual sero-immunological techniques, with a solid support on which is fixed said antigen, and in which the system obtained is then brought into contact with a solution containing an antibody directed against an isogenic antigenic determinant of said immunoglobulins to be detected or to be titrated, characterized in that said antibody is a monoclonal antibody and that the the system obtained is then brought into contact with a solution containing a second antibody labeled with a tracer agent, said second antibody being directed against an isogenic antigenic determinant of the first antibody, and then the possible presence is determined qualitatively and / or quantitatively of said tracer agent on the system obtained.  2. Method according to claim 1, characterized in that the antigen is an unpurified antigen.  3. Method according to claim 2, characterized in that said antigen is a viral antigen.  4. Method according to claim 3, characterized in that said antigen is a herpes virus antigen.  5. Method according to claim 4, characterized in that the said virus is the Epstein-Barr virus.  6. Method according to claim 5, characterized in that the said antigen is an EA antigen, the Atlantis VCA or the EBNA antigen.  7. Method according to any one of the preceding claims, characterized in that the said immunoglobulins are IgG, IgM, or IgA.  8. Method according to any one of the preceding claims, characterized in that the said second antibody is a monoclonal antibody.  9. Method according to any one of the preceding claims, characterized in that the said tracer agent is an enzymatic, radioactive or fluorescent tracer.  10. Method according to any one of the preceding claims, characterized in that the said solid support is a material capable of fixing an antigen, either by adsorption or by means of a coupling agent.  11. Method according to claim 10, characterized in that said support is in the form of discs, cubes, rods, balls or plates, including microtiter plates.  12. Process according to any one of the preceding claims, characterized in that the said liquid medium is a blood serum.  13. A device intended for implementing the method as defined in any one of the preceding claims, characterized in that it comprises one or more units of said solid support on which said antigen is optionally already fixed, containers containing solutions of said first and second antibodies, dilution, incubation and washing buffers, and optionally a container containing a solution of said antigen, as well as instructions for use, the whole being combined in the same package with several compartments constituting an analysis box.