FR2640143A1 - POLYMERIZED ANTIBODIES, DIRECTED AGAINST IMMUNOGLOBULINS - THEIR USE IN DIAGNOSTIC TESTS - Google Patents

Abstract

Antibodies directed against immunoglobulins capable of producing an immunological reaction with specific antibodies present in a biological sample; said antibodies are characterized in that they are polymerized. The invention also relates to the use of these antibodies in tests for detecting the presence of specific antibodies in a biological sample, in particular in the case of a parasite-induced or virus-induced infection. These polymerized antibodies are used in conjunction with particulate antibodies of a contrasting colour, or vice versa; this improves the sensitivity of agglutination tests because the aggregates are of a larger size.

Description

POLYMERIZED ANTIBODIES. LEADS AGAINST IMMUNOGLO
BULINES - THEIR USE IN DIAGNOSTIC TESTS.

 Antibodies circulating against several parasite antigens are produced for the duration of an infection. Such antibodies can be detected in whole blood, plasma or serum by direct or specific agglutination tests.

 These specific tests which include the use of specific antigens allow the detection of specific agglutinins. Their usefulness is however limited by a lack of sensitivity. Thus, certain infections are not detectable if the antigen used in the test is expressed only in a late phase of the infection or if this antigen is not sufficiently immunogenic.

 In addition, some parasites do not have the gene encoding the antigen used can escape detection. The lack of sensitivity can also be attributed to so-called "incomplete and blocking antibodies which, when alone, do not clump together.

 The use of anti-immunoglobulin antibodies of the Ig type (anti-Ig antibodies) as a Coomb reagent, considerably improves the sensitivity. However, under these conditions, reading the test results is made more difficult because of the small size of the aggregates formed after the immunological reaction or of a prozone phenomenon well known in immunology which manifests itself in the presence of low dilutions of serum. (for example dilutions of the order of 1/2 or 1/10). In this case, when the antibodies to be assayed are present in high concentration, the agglutination with the anti-Ig is inhibited and the signal for the presence of the antibodies sought disappears.

The implementation of an infection detection test due to a parasite requires additional manipulations such as prior dilutions of the sample to be tested.

 The inventors have demonstrated that an appropriate quantity of anti-Ig antibodies in polymerized form added to an antigen, causes an increase in sensitivity during an immunological detection test involving agglutination reactions, and the reduction, otherwise the abolition of the prozone phenomenon. In addition, no additional manipulation is required prior to the implementation of the reactions, such as for example prior dilutions of the sample to be assayed.

 The present invention relates to polymerized Ig-antibodies. The invention also relates to the use of these antibodies as a reagent in immunological tests for the detection, for example of parasites or viruses.

 The present invention also relates to rapid tests of antibodies determined for example due to infectious diseases in humans or animals through the use of polymerized anti-Ig antibodies.

This application includes all kinds of tests which allow the detection of aggregates of particles (consisting of multicomplexes of the anti-Ig type, antibody sought, specific antigen of said antibody sought) of sufficient size to be visible to the eye. given the conditions for revealing these aggregates.

 The invention makes use of the capacity preserved by antibodies-Ig in polymerized form to non-selectively fix other antibodies and the immunological complexes containing them, among which the complexes formed between specific antibodies contained in a biological sample and corresponding antigens.

 Biological samples can be made up of biological fluids such as whole blood, serum, or plasma.

 Polymerized anti-Ig antibodies of the invention are further characterized by a rate of polymerization sufficient to allow the formation of aggregates visible to the naked eye, when these are formed during the contacting of a biological sample containing antibodies.

 The polymerized anti-Ig antibodies of the invention are further defined by their ability to bind non-specifically to the constant parts of specific determined antibodies present in a biological sample, these determined antibodies being themselves recognized by a specific antigen.

 Particularly within the scope of the invention are polymerized anti-Ig antibodies composed of oligomers comprising at least 2 motifs, in particular 2 to 25 anti-Ig antibodies covalently coupled together. It is remarkable that the polymers thus obtained retain the capacities of the antibodies in their unpolymerized form to form non-specific complexes with other antibodies among which the determined or chosen antibodies possibly present in the test sample, these antibodies being themselves same immunologically recognized by a specific, labeled antigen.

 The polymerization rate of the polymerized anti-Ig antibodies does not exceed that which would cause the insolubilization of the polymer. This polymerization rate is however sufficient so that, in the presence of distinct antibodies, the polymerized anti-Ig antibodies are capable of producing, by agglutination with other antibodies, aggregates visible with the naked eye, if necessary after concentration of the medium. in which the operation is carried out.

 The polymerized antibodies are capable of binding to the constant part of the antibodies present in the medium, for example following an infection, to form the aforesaid aggregates, those of these aggregates in which the sought antibodies intervene then being themselves recognized. by homologous antigens labeled, for example by staining, normally capable of selectively forming with the desired antibody a complex of the antigen-antibody type; whereby those of the aggregates formed which include labeled antigens can be easily identified by the marker, among the aggregates involving the polymerized anti-Ig and other antibodies, heterologous towards the labeled antigen.

 The invention also relates to a diagnostic test for the presence of determined antibodies in a biological sample comprising the following steps - bringing the biological sample into contact with a labeled cell specific for the antibody sought in the presence of anti - Polymerized Ig as defined above, - the detection of those of anti-Ig aggregates which also carry the labeled antigen.

 It should be noted that the Ig immunoglobulins used for the production of anti-Ig antibodies must come from the same mammalian host as that providing the biological medium, in particular serum or plasma., In which the antibody sought is to be assayed. If the host is human, the anti-Ig antibodies are obtained by immunization of a non-human animal with immunoglobulins G obtained from a human serum.

 It is easily conceivable that the preferred polymerization rate for anti-Ig antibodies is to be chosen according to the nature of the biological samples and of the antibodies sought. This rate of polymerization must be sufficient for the aggregates to be capable of forming and to individually retain a maximum rate of the antibodies sought. This rate will not exceed that which could cause too massive agglutination in the presence of other antibodies.

 In general, we will place ourselves in conditions allowing the production of multiple agglutinates.

 Due to the specific affinity of the antigen used for detection with the antibody sought, we are further led to make the following observations depending on whether the biological sample contained or did not contain the antibody sought - case where the sample contained the antibody sought selective attraction of the labeled antigen towards the complexes formed between them by the anti-Ig antibodies and the antibody sought: hence the individualization and consequently visualization of the particles retaining the marker, in particular when it is colored, - case of that absence of the antibody sought: absence of individualization and consequently of visualization of particular aggregates among others.

 According to an advantageous embodiment of the diagnostic method carried out on a biological sample in solution optionally containing the antibodies to be assayed, this is brought into contact with the labeled antigens and the polymerized anti-Ig antibodies and. concentrates the medium until it causes insolubilization of the aggregates formed. Advantageously small quantities of the medium are deposited in spots on a support, in particular a paper and one causes the evaporation. In the case where the marker is colored, the presence of the antibody sought is manifested by coloring points at the level of the aggregates then individualizable (concentration of the coloring at several points in the sample) On the contrary, a uniform coloring of the The sample shows that the antibodies sought are absent.

 The determined antibodies sought may be antibodies formed as a result of infection by a parasite or by a virus. By way of example, mention may be made of the trypanosome, the vector of brucellosis, the HIV virus.

Detection of the aggregates formed can be carried out by means of a step of detecting the coloration of these aggregates, as soon as the specific antigen incorporated in the medium has been labeled. This marking can be obtained with a dye such as blue
Coomassie or Bengal rose or even with a fluorescent agent. Enzyme markers can also be used. The revelation of the aggregates is in this case carried out by means of an enzyme activity indicator.

 Radioactive labeling of antigens can also be performed. In this case, the step of detecting aggregates is preceded by a step of physical separation of the non-aggregated complexes and of the sought-after aggregated complexes.

 The above separation can take place by centrifugation or by filtration.

 The invention further relates to a kit for diagnosing the presence of determined antibodies comprising - polymerized antibodies, - specific labeled antigens.

 Advantageously, this diagnostic kit can be used to detect specific antibodies due to infection by a parasite or a virus. In this case the antigens of the kit are specific for this parasite or this virus.

 The antigens in this kit can be labeled as defined above.

 The invention also relates to a process for the preparation of polymerized anti-Ig antibodies, comprising the polymerization of anti-Ig antibodies in the presence of a polymerization agent, in particular under the standard conditions used for protein coupling reactions.

 Suitable polymerization agents are those usually used to polymerize or couple proteins. It is possible, for example, to react the antibodies in solution with ethyl chloroformate, glutaraldehyde or any polyfunctional reagent for coupling between active functions carried respectively by the proteins represented by these anti-g antibodies. Such active functions consist, for example, of amine, carboxyl or both. By adjusting the relative proportions of the antibodies and the polyfunctional reagent, the rate of polymerization can be adjusted in a known manner.

 The invention also relates, as new reagents, to compositions containing both the polymerized anti-Ig antibody and the labeled antigen in relative proportions preferably adjusted as a function of the possible concentrations of the antibodies sought in the samples to be tested. Appropriate proportions must in each case be established -experimentally, by using variable proportions of the two constituents and by reacting them with samples containing standard concentrations of the antibody immunologically homologous to the antigen, in particular at concentrations often encountered in clinically suspect subjects.

EXAMPLE 1: Aalutination test on a Trvpanozoma evansi card.

 In chronic human trypanosomiasis, research into the mechanism of antigenic variation and the immune response has led to the development of a diagnosis based on a simple card agglutination test.

 The principle of such a test is based on the detection of circulating antibodies directed against several antigens of the infectious parasite by using a type of variable antigen of T aambiense appearing early during the infection.

When this test is used under the conditions known for the diagnosis of camel trypanosomiasis (5) and infection with buffalo T. evansi (2), a low sensitivity is observed. Modifications have been made to this test in the context of the invention so that the sensitivity is brought to a higher level by the addition to the antigen of a polymerized anti-Ig antibody. This new test is original in that it is based on the use of a variable antigen expressed early during an infection, namely the antigen (VAT) RoTat 1.2 (English abbreviation of Rode Trypanozoma type antigen) of T evansi which is present in all parasites
T evansi, in combination with a polymerized anti-Ig antibody.

 This test detects the agglutination of antibodies that have appeared during the first two weeks of infection.

- Preparation of anti-I polymerized anticorPs.

 The polymerized anti-Ig antibodies will sometimes be designated below by the term "anti-Ig adsorbent".

Antisera specific for buffalo, camel and pig immunoglobulins were obtained by immunization of rabbits with specific immunoglobulins above, of each of the above animal species and respectively obtained from the sera of these animals and isolated on protein A by complexation with a protein A immobilized on an appropriate support, in particular that known under the brand
SEPHAROSIS 4B. The technique used is that described in (3).

 The anti-immunoglobulins obtained were then polymerized with ethyl chloroformate and stored at 4C in a phosphate buffer (PBS) containing 1% bovine serum albumin with 0.1% azides as described in (4).

- Execution of the test.

 This test is carried out as follows - 45p liters of serum diluted in phosphate buffer at pH 7.4 and 45p liters of the fixed antigen RoTat 1.2 colored with a blue dye and containing 10% of polymerized anti-Ig antibodies are deposited on the reaction surface -constituted by an agglutination card covered with a white plastic material (Bruster) which can be substituted by photographic paper RC. The suspension is spread out and subjected to reaction for 10 minutes in a rotary evaporator.

 The results are indicated as follows +++ (very strongly positive), ++ (strongly positive), - + (positive), +/- (weakly positive) - (negative).

The highest dilution of serum giving a positive result (+) is considered to be the limit of detection - SDecificity of the test
The test is significant for the following dilutions of the serum with or without the use of polymerized anti-immunoglobulin antibodies - buffalo (water buffalo):> 1/4 - cattle: 1/2 - pig: 1/2 - camel: 1/2
This test is in very good correlation with parasitaemia and it is complementary to the Elisa tests using antigens (see Table I) based on monoclonal antibodies produced against T aambiense or T evansi.

- Test sensitivity
The sensitivity of the test was demonstrated by titration of 43 antisera produced in rabbits in cattle and buffaloes experimentally infected with Tevansi (see Figure 1) and the titration of sera obtained from 4 infected camels and two camels uninfected, from 139 samples collected from pigs, cattle and buffaloes from different regions of Thailand.

 The addition of polymerized anti-Ig antibodies to the antigen led to a 10 to 100-fold increase in sensitivity. The test detected antibodies circulating against parasites in whole blood, serum or plasma during the first two weeks of infection (1), in the absence of prozone phenomena constantly observed when using unpolymerized anti-Ig antibodies in place of the polymerized antibodies. On the contrary, the use of anti-Ig antibodies leads to reliable results, even at low dilutions of serum (2).

TABLE I: Comparison between 4 tests for diagnosis
from T evansi in buffalo.

Number Parasitic- antibodies tested by: Ag-ELISA of crumbed animals CATT CFT 1 + 320 40 0.087 2 +++ 160 40 0.193 3 ++ 320 160 0.098 5 + 640 80 0.855 7 - 640 80 0.078 9 +++ 320 80 0.262 12 + 160 160 0.198 13 - - 120 0.034 23 - 640 20 0.757 24 +++ 320 20 0.836
Control .- 4 2 0.020 (D.0.)
CATT = Test for the search for Trypanosome by agglutination on a card.

CFT = Fixation test on the complement.

D.O = Optical Density: values greater than or equal to 0.050 are considered to be positive.

EXAMPLE 2 - Card Alutination Test For sleeping sickness in humans.

 The test includes the use of a conventional Testryp (R) CATT antigen and RoTat 1.2 antigen as well as the use of a polymerized anti-Ig antibody (antibodies directed against human G immunoglobulins).

 The preparation of the anti-Ig adsorbent and the execution of the test are carried out as described in Example 1. The sensitivity of the test was demonstrated after titration of known sera from African patients infected with T aambiense and T rodesiense and after titration of control sera obtained from healthy European people.

 The addition of an anti-Ig adsorbent led to a 10 to 100-fold increase in sensitivity.

EXAMPLE 3 - Brucellosis test.

This test is based on the use of an antigen in acid buffer, colored with Bengal pink and
Supplemented with polymerized anti-human Ig antibodies.

 This. test is carried out on disposable agglutination cards and these specific reactions take place in an interval of 5 minutes. The sensitivity of the test was demonstrated by the titration of sera from European patients with antibodies blocking brucellosis as well as with sera obtained from healthy Europeans. The addition of an anti-Ig adsorbent to the conventional antigen stained with Bengal pink made it possible to obtain a 2-8 fold increase in sensitivity.

EXAMPLE 4.

- A4alutination test for HIV.

 A solid support is suitably colored and consisting of particles of a polymerized substance such as a plastic or another organic polymer. The particles can also consist of insoluble bile proteins which do not cross-react with the polymerized antibodies. These protein particles are prepared by controlled coupling.

 The HIV surface protein is then coupled to this support via covalent bonds. A suspension of particles coated with antigens is prepared to carry out the agglutination test. Antibodies to IgM or IgC are detected using specific anti-IgM or anti-IgG polymerized adsorbents. The test is carried out as in Example 1.

CONCLUSION
Agglutination tests using polymerized antibodies meeting the definitions of the invention have made it possible to detect circulating antibodies directed against parasites or viruses, from whole blood, plasma or serum. Such tests are very quick (10mn), reliable, very sensitive and can be adapted for screening in large numbers. Consequently, the treatment of the infectious diseases described in the examples can be started immediately after a positive diagnosis,
BIBLIOGRAPHY (1) - Bajyana Songa E., et al, Ann. Soc. Belgian Med too. 1987, 67, 51-57.

(2) - Bajyana Songa E., et al, Ann. Soc. Belgian Med.

too., 1987, 67, 137-148.

(3) - Goding J.W. Journ. Imm. Meth., 1976, 13, 215-226.

(4) - Van der Loo W., et al, European Journal of
Immunology, 1977, 7 (1), 15-22.

(5) - Zweygarth E. et al, Ann. Soc. Belgian Med. too much.

1984, 64, 309-313.

Claims (9)

1 / - Antibodies directed against immunoglobulins (anti-Ig antibodies) in the polymerized state, soluble capable in particular of attaching in a non-specific manner to the constant parts of antibodies produced during an infection due to a virus or to a parasite such as the trypanosome, the vector of brucellosis, the HIV virus.  2 / - Anti-Ig antibodies according to claim 1 composed of oligomers comprising at least 2 motifs, in particular 2 to 25 anti-Ig antibodies covalently coupled. 3 / A method of in vitro diagnosis of the presence of determined antibodies in a biological mammal sample comprising the following steps - bringing the biological sample into contact with a labeled antigen specific for the antibody sought, in the presence of antibodies Anti-polymerized Ig according to any one of claims 1 or é, these anti-Ig antibodies being formed against Ig supplied by a mammal of the same species as that from which the biological medium tested. - the detection of those of the anti-Ig aggregates which also carry the labeled antigen.  4 / In vitro diagnostic method according to claim 3, characterized in that the medium obtained after contacting the sample to be assayed with the anti-Ig antibodies polymerized according to any one of claims 1 or 2 and with the antigens marked, is concentrated until it causes insolubilization of the aggregates formed. 5 / A method of in vitro diagnosis according to any one of claims 3 or 4, characterized in that small amounts of the medium obtained after contacting the sample to be assayed with the anti-Ig antibodies polymerized according to any one claims 1 or 2 with the labeled antigens, are deposited in spots on a support and subjected to evaporation prior to the implementation of the detection step. 6 / A method of in vitro diagnosis according to any one of claims 3 to 5, characterized in that the detection of the aggregates formed is carried out either through a step of detecting the coloration of these aggregates, after labeling of the specific antigen incorporated into the medium thanks to a dye, such as Coomassie blue or pink. Bengal or with a fluorescent agent, either through a step of detecting an enzymatic marker when the specific antigen incorporated in the medium is labeled with an enzymatic marker. 7 / Kit for diagnosing the presence of determined antibodies comprising - polymerized antibodies according to any one of claims 1 to 2, - labeled antigens specific for the determined antibodies sought.  8 / A method of preparing anti-Ig antibodies according to any one of claims 1 to 2, comprising the polymerization of anti-Ig antibodies in the presence of a polymerization agent usually used to polymerize or couple proteins, such as ethyl chloroformate, glutaraldehyde or any polyfunctional reagent for coupling between active functions carried respectively by the proteins represented by these anti-Ig antibodies. 9 / Reagent for the diagnosis of the presence of antibodies determined in a biological sample consisting of a composition containing both the anti-Ig antibody polymerized according to any one of claims 1 to 2 and a labeled antigen specific for antibody sought, in relative proportions preferably adjusted as a function of the possible concentrations of antibody sought in the sample tested.