EP0309496A1

EP0309496A1 - SOLUBLE FORMS OF LOW AFFINITY Fc GAMMA RECEPTORS, PROCESS FOR THEIR IDENTIFICATION AND DOSAGE, A CORRESPONDING DOSAGE KIT, AND APPLICATIONS

Info

Publication number: EP0309496A1
Application number: EP88902150A
Authority: EP
Inventors: Jay Unkeless; Claude Jacquillat; David Khayat
Original assignee: Universite Pierre et Marie Curie Paris 6
Current assignee: Universite Pierre et Marie Curie Paris 6
Priority date: 1987-02-24
Filing date: 1988-02-23
Publication date: 1989-04-05
Also published as: US5219728A; FR2611913B1; FR2611913A1; WO1988006733A1

Abstract

Ces récepteurs sont caractérisés par le fait qu'ils consistent en le produit obtenu par chromatographie d'affinités sur colonne couplée à des anticorps 3 G8 ou à des lectines ou à des anticorps polyclonaux anti-récepteur FcR, d'un fluide biologique d'origine humaine, puis par filtration sur gel, le spectre dudit produit, en électrophorèse sur gel d'acrylamide en condition réductrice, comportant une bande majeure correspondant à une masse moléculaire comprise entre 72000 et 76000 daltons, et une pluralité de bandes mineures. Suivant sa forme purifiée, le récepteur consiste en une glycoprotéine de masse moléculaire 72000-76000 daltons, qui est reconnue en ELISA et en Western Blotting par l'anticorps monoclonal anti-Leu 11b. Ces récepteurs sont applicables au diagnostic et au suivi de la thérapeutique de maladies mettant en jeu les récepteurs Fc, (maladies infectieuses, maladies auto-immunes, rejets de greffes, cancers et myélomes et SIDA), ainsi qu'à l'étude des polymorphismes humains.These receptors are characterized by the fact that they consist of the product obtained by affinity chromatography on a column coupled to 3 G8 antibodies or to lectins or to polyclonal anti-FcR receptor antibodies, of a biological fluid of origin. human, then by gel filtration, the spectrum of said product, by electrophoresis on acrylamide gel under reducing condition, comprising a major band corresponding to a molecular mass of between 72,000 and 76,000 daltons, and a plurality of minor bands. Depending on its purified form, the receptor consists of a glycoprotein with a molecular mass of 72,000-76,000 daltons, which is recognized in ELISA and in Western blotting by the anti-Leu 11b monoclonal antibody. These receptors are applicable to the diagnosis and monitoring of the therapy of diseases involving Fc receptors (infectious diseases, autoimmune diseases, graft rejection, cancers and myelomas and AIDS), as well as to the study of polymorphisms. humans.

Description

The present invention relates to the soluble forms of the low-affinity receptors for the Fc fragment of IgGs; the present invention also relates to a method for identifying and assaying soluble forms of low affinity gamma Fc receptors, in particular human soluble Fc receptors; it also relates to a set of reagents necessary for the implementation of this process; finally, it relates to the applications of this method for the diagnosis of pathologies in which the above-mentioned receptors are involved, such as infectious diseases, autoimmune diseases, graft rejection, diseases of human complexes, cancers, myelomas , and acquired immunodeficiency syndrome (AIDS), for the therapeutic monitoring of the evolution of these pathologies, as well as for the study of human polymorphisms.

On the surface of many cells, especially cells of the immune system (macrophages, B lymphocytes, polymorphonuclear cells, NK cells etc.), receptors exist which allow a relationship between antibodies or immunoglobulins and these cells of immunity. These receptors have an affinity for the Fc fragment of antibodies (FcR). This Fc fragment corresponds to the part not provided with antibody activity of the immunoglobulins. The importance of these receptors is considerable, because it is through them that foreign antigens (viruses, tumor cells, etc.) can be recognized and the initiation of a cytotoxic reaction by T lymphocytes in the humoral with secretion of specific antibodies by B cells; phagocytosis, that is to say the extraction of tissues and blood from particles recognized as foreign by the cells of the reticuloendothelial system (monocytes, macrophages); cellular cooperation between the different cells of immunity; transplacental transfer of antibodies from the mother to the fetus; and the clearance of immune complexes, the pathogenic role of which is known in many affections.

The existence of secretion of these receptors for the Fc fragment (FcR) by cultures of mouse T cells, activated by a specific activator, has been demonstrated. These Fc receptor molecules, secreted in vitro, under very specific conditions of activation and culture (in particular in a medium devoid of any serum causing suffering of the cells in culture) still have the capacity to bind, by their Fc fragment, the immunoglobulins and they have therefore been called "immunoglobulin binding-factor" (IBF). The existence of this type of IBF has been demonstrated for three classes of immunoglobulins IgG, IgA, IgE, namely the factors IgGBF, IgABF and IgEBF.

It has also been demonstrated that these IBF molecules are capable, when in contact with cultured B lymphocytes, or even myeloma cells in vitro, of completely inhibiting the activation of these cells and, thereby even, the secretion of immunoglobulins by these B cells or myeloma. They are therefore molecules secreted by cells belonging to the immune system under artificial conditions of culture in vitro, but nevertheless endowed with a functional capacity to bind the antibodies and to bring about a significant suppression of the activation of the B lymphocyte system. (lymphokine). The existence of an Fc receptor for mouse IgG immunoglobulins (FcR) in mouse serum has been demonstrated. These molecules were defined, both by their ability to be recognized by a monoclonal antibody specific for mouse gamma Fc receptors (monoclonal antibody 2.4G2) and their functional capacity to be purified from Fc fragments of immunoglobulin. This discovery is extremely interesting, because this lymphokine is secreted with a rate which increases as a function of age (zero rate at birth, appearing around the fifth day in newborn mice and becoming systematically detectable around the seventh day), this rate is all the greater as there are infections or, more generally, as the immune system is stimulated (the mice being kept in an environment free of microbes having strictly zero rates throughout their life ), this rate being moreover relatively determined by genetic factors within a population of homozygous mice.

Furthermore, it has now been discovered, in particular in human serum, the existence of a lymphokine having the capacity to bind to the Fc fragments of immunoglobulin, namely the circulating serum soluble form of the low affinity gamma Fc receptors, also designated by the abbreviation Fc-gamma RLo, or also "CD16".

These receptors are present in biological fluids (in particular serum) in polymerized form, as has been observed by measurements of molecular weight which have, for example, provided a value greater than approximately 700,000 for the glycoprotein of which it has been shown to have a molecular weight, as a monomer, of 72,000 to 76,000.

The subject of the present invention is a soluble, low affinity Fc ɣ R type III receptor (at CD16), consisting of a glycoprotein of molecular mass 72000-76000 daltons, which is recognized in ELISA and in Western Blotting by the antibody. monoclonal anti-Leu 11b.

The subject of the invention is also a soluble, low affinity Fc ɣ R type III receptor (at CD16), characterized in that it consists of the product obtained by affinity chromoatography on a column coupled to 3G8 antibodies. or lectins (e.g. Lens Culinaris Agglutinin (LCA), wheat germ agglutinin, concanavalin) or to polyclonal anti-FcR receptor antibodies, of a biological fluid of human origin, then by gel filtration, the spectrum of said product, by electrophoresis on acrylamide gel in reducing condition, comprising a major band corresponding to a molecular mass of between 72,000 and 76,000 daltons, and a plurality of minor bands, the main ones of which correspond to. molecular masses respectively: - between 64,000 and 68,000 daltons

- between 51,000 and 55,000 daltons

- between 42,000 and 46,000 daltons

- between 33,000 and 37,000 daltons.

The invention also relates to an Fc ɣ R. type III receptor essentially comprising the molecular mass fraction 33000-37000 daltons, as it appears in electrophoresis on acrylamide gel, in the presence of a reducing agent and a detergent agent such as sodium dodecyl sulfate (SDS). The biological fluids discussed here include serum and plasma fluids, cerebrospinal fluids, urine and ascites, among others.

We can also characterize all these receptors by the fact that in Dot Blot, they recognize a polyclonal rabbit antibody anti-FcR receptor.

The invention finally relates to each of the fractions of the Fc ɣ R type III receptor, as defined above, as well as to all the possible combinations of these fractions. The present invention also relates to a method for identifying, detecting or assaying these lymphokines having the capacity to bind to the Fc fragments of immunoglobulin, in particular of the soluble human serum Fc receptor. The method according to the present invention for the identification or the determination of the soluble forms of Fc ɣ R type III low affinity receptor (or CD16), is characterized by the fact that it consists of:

(a) fix, on a solid phase, a first antibody, which is a monoclonal, or polyclonal antibody, or else a fraction of a monoclonal or polyclonal antibody

(for example, a Fab fragment), directed against a conformational epitope of the Fc ɣ receptor to be identified or to be assayed;

(b) washing said solid phase to remove said first uncoupled antibody;

(c) incubating the sample containing the Fc ɣ receptor to be assayed in the presence of the solid phase coated with said first antibody;

(d) washing to remove material not specifically attached to said first antibody;

(e) incubating, in the presence of the resulting solid phase, a second antibody, which is a monoclonal or polyclonal antibody, or else a fraction of a monoclonal or polyclonal antibody (for example a Fab fragment), and which is anti -Fc receptor recognizing the same category of Fc receptors as the first antibody, but by a completely different epitope;

(f) washing to remove said second non-specifically bound antibody; (g) incubating, in the presence of the resulting solid phase, a third antibody, which is an antibody capable of specifically recognizing said second antibody; (h) washing to remove the third antibody not specifically attached; and,

(i) assay the third fixed antibody and deduce the amount of Fc receptor initially present in the sample.

In step (a), use is notably made, as first antibody, of the monoclonal antibody 3G8. In step (e), a mouse IgM is used in particular as the second antibody consisting of the anti-Leu 11b monoclonal antibody. In step (g), a polyclonal antibody, namely a goat anti-mouse IgM antibody, is used in particular as the third antibody. In accordance with a particularly preferred embodiment of the method according to the invention, in step (g), a third antibody labeled with an enzyme is used and, in step (i), a calorimetric substrate of said enzyme, and, after having stopped the calorimetric reaction, for example by adding an aqueous solution of sulfuric acid, the colorimetric variation is read, from which the quantity of Fc receptor sought is deduced. In fact, the calorimetric variation is proportional to the quantity of the third antibody, and, therefore, proportional to the quantity of second antibody, and therefore of Fc receptor initially present in the sample.

Preferably, the enzyme is peroxidase, in particular horseradish peroxidase, and the colorimetric substrate of the peroxidase is orthophenylenediamine, in the presence of hydrogen peroxide, the colorimetric reading being carried out at 492 nm.

The incubation of step (a) for fixing the first antibody to the solid phase is carried out, for example, at a temperature of the order of 4 ° C., for a period of time ranging from 8 to 12 hours.

As for the incubations of steps (c), (e) and (g), they are carried out in particular at ambient temperature over a period of time ranging from 1 to 4 hours. Figure 7 of the accompanying drawing explains the dosage according to the invention. The lymphokine sought, which is specifically assayed by this method, comprises a first site for capture by the first antibody (3G8), and a second site for detection by the sequence oLeu 11b-goat anti-mouse IgM antibody and peroxidase. The present invention also relates to the kit of reagents necessary for the implementation of this process, this kit comprising: a solid support, in particular a microtiter plate, provided with a first antibody which is a monoclonal or polyclonal antibody, or another fraction of a monoclonal or polyclonal antibody, which is directed against a conformational epitope of the Fc ɣ receptor to be assayed; - a second antibody, which is a monoclonal or polyclonal antibody, or even a fraction of a monoclonal or polyclonal antibody, and which is an anti-Fc receptor, recognizing the same category of Fc receptors as the first antibody, but by a totally epitope different ; a third antibody, which is an antibody capable of specifically recognizing the second antibody; - a system for assaying said third antibody.

These three antibodies as well as the assay system for the third antibody have been defined above.

As background detector, that is to say, as negative control, an xenogenic sera are used (fetal calf, horse, goat).

As a standard for ensuring a positive qualitative control of the method, a known positive serum is used and, in order to be able to quantify the method, an extract of human polynuclear cells is used. These polymorphonuclear cells are purified according to conventional purification techniques, they are counted and they are then lysed by a mild detergent which does not destroy the conformational structures of the polymorphonuclear receptors which could no longer subsequently be recognized during the test by the antibody 3G8, and, knowing the number of Fc receptors per polynuclear, the number of polynuclear at the start and the different dilutions at which these polynuclear are tested, it is possible, by a simple relation, to determine the correspondence in soluble Fc receptors of the sera tested compared to a theoretical number of Fc receptors of polynuclear cells detected in the cell lysate. In particular, a human polynuclear lysate is used, purified according to conventional techniques, lysed only with an aqueous solution of octylthio beta d-glucoside 30 mM + 5 mM DFP at 4 ºC.

The applications of this method are essentially diagnostic and monitoring applications of the therapeutic use of these lymphokines. These are in fact substances whose variations are linked to the fine activation of the B lymphocyte system and / or of the macrophagic system, and whose variation of the rate makes it possible to determine the state of stimulation of this system. This is of major interest in infectious diseases, autoimmune diseases, transplant rejection, cancer and myeloma, and acquired immunodeficiency syndrome (AIDS). Far elsewhere, after injection of lympholcine (human Fc receptor), it is possible, thanks to this test, to measure the evolution of serum levels and, thereby, to manage with more efficiency and less toxicity the possibilities therapeutics of this lymphokine in human medicine. Indeed, such a lymphokine is capable, like its counterpart in vitro, of specifically inhibiting the secretion of a class of immunoglobulins and therefore of stopping an effector mechanism in certain pathologies, such as the abovementioned diseases, without, however, because of its specificity - indeed, an IgG receptor will only inhibit the secretion of IgG - cause an inhibition of the secretion of other classes of immunoglobulins, thus allowing the patient not to be for as immunosuppressed.

Similarly, by blocking the Fc fragments of immunoglobulins already engaged in a deleterious antigen-antibody reaction (as for anti-platelet autoantibodies in idiopathic thrombocytopenic purpura for example), this lymphokine can prevent the relationship between these immune complexes and the cells of the reticuloendothelial system which normally would have phagocytosed these antigens (platelets in the case of idiopathic thrombocytopenic purpura), resulting in the pathogenic effect (thrombocytopenia in the example chosen).

The invention also relates to a medicament containing an Fc ɣ R type III receptor or at least a fraction which composes it, as defined above.

A particular embodiment of the assay method of the invention will be described below.

(1) Preparation of microtiter plates. The microtiter plates used are 96-well poly (vinyl chloride), U-bottom plates (manufactured by the company "Dynatech").

The 3G8 monoclonal antibody is poured, at a rate of 3 μg per well, into 100 μl of carbonate buffer at pH 9.6. The incubation lasts 8 to 12 hours at a temperature of 4ºC.

(2) Washing

Wash 6 times with PBS (Phosphate Buffered Saline) containing 0.1% v / v of Tween 20. This washing solution, which we will have the opportunity to use again in the rest of this assay, will now be referred to more simply by the expression "PBS Tween".

(3) Deposit of the sample to be assayed 100 μl of the sample to be assayed, pure or diluted in PBS Tween, which is left for 4 hours at approximately 22 ° C (room temperature), is placed in each well. (4) Laying This washing is carried out in the same way as in step (2).

(5) Deposit of anti-Leu-11b antibodies 80 ng of anti-Leu-11b antibodies (from Beckton-Dickinson) diluted in 100 μl of PBS Tween are deposited in each well. The incubation is carried out for 2 hours at approximately 22ºC.

(6) Washing

This washing is carried out in the same way as in step (2).

(7) Deposit of goat anti-mouse IgM antibodies conjugated with horseradish peroxidase

The above-mentioned antibody is introduced (source: Jackson Immuno-Research Laboratories Inc.), diluted 1/5000 in PBS Tween making a total of 100 μl per well. They are incubated for 1 hour at 22ºC.

(8) Washing

This washing is carried out in the same way as in step (2).

(9) Deposition of the colorimetric substrate of the peroxidase: 150 μl are deposited per well of citrate phosphate buffer containing 4 mg / ml of orthophenylene diamine and 0.8 μl / ml of hydrogen peroxide. The colorimetric reaction is stopped by depositing 75 μl per well of a 10% aqueous sulfuric acid solution and the result is read at 492 nm in an ELISA reading device.

Thanks to the assay method according to the present invention, it has been demonstrated in human blood a molecule corresponding to a soluble serum form of the receptor of low affinity for the Fc fragment of IgC.

The characterization of the Fc ɣ R type III receptors of the invention will now be described.

A normal human serum (68 ml) is passed, in a first step, over a Sepharose column coupled to 3G8 monoclonal antibodies (2 mg of 3G8 antibody per ml of Sepharose; total volume of the column: 1 ml), to Room temperature. This column is then washed using 150 ml of a 0.1% solution of "Tween 20" in a salt phosphate buffer solution, and the adsorbed material is then eluted using a solution 1 / 2 M acetic acid, the fractions having a volume of 0.5 ml.

These fractions are immediately neutralized using a 3M sodium bicarbonate solution. The serum, before and after affinity chromatography, as well as the elution fractions, are tested for their content in soluble low affinity type III Fc receptor. The fractions which exhibit immunological reactivity are then pooled, and 0.2 ml of this pool of fractions is subjected to gel filtration, on a column of 25 ml by volume of "Superose 6" (Pharmacia); this column of "Superose 6" is balanced in a solution of ammonium bicarbonate

50 mM, pH 8. The fractions with a volume of 0.5 ml are collected and are tested using a direct ELISA assay method. This ELISA method consists in testing the elution fractions without going through the capture step using the 3G8 monoclonal antibody. These fractions are directly bonded to a solid support, such as a 96-well PVC plate, and are then, after washing, reacted with the anti-Leu 11b monoclonal antibody, followed, after 2 hours, by a second anti-leukocyte antibody. - Mouse IgM, labeled with peroxidase. Figure 1 of the accompanying drawing shows the results of this second purification step, therefore corresponding to gel filtration. Two curves appear in this figure. The abscissa shows the numbers of the elution fractions, as well as the elapsed time (expressed in minutes), and the ordinate shows the optical density, as it appears on the reading of the wells in direct ELISA. The curve in solid line corresponds to the chrσmatogram of elution of this gel filtration and gives only the quantity of proteins without giving their characteristics. The second curve (o - o) represents the immunoreactivity of each of the elution fractions, in terms of the purified soluble Fc receptor. It can therefore be seen that the peak between fractions 20 and 25 carries almost all of the purified soluble Fc type III receptor type immunoreactivity.

Fractions 20 and 22, which are the richest in soluble Fc receptor type immunoreactivity, since the optical density rises to around 2 units, were lyophilized, and they were subjected to acrylamide gel electrophoresis in presence of SDS and reducing agent. These fractions are shown in Figure 2, and show the existence of a main main band, characteristic, whose molecular weight is 72,000 to 76,000 daltons, but also slightly more minor bands, around 66,000, 53,000 , 43,000 and 35,000 daltons.

These bands are then transferred by a horizontal current on a nitrocellulose filter, according to the technique called "Western Blot"; this cellulose nitrate paper is then reacted with the anti-Leu 11b monoclonal antibody, and, after washing, this cellulose nitrate filter is reacted with the anti-mouse IgM polyclonal antibody, labeled with alkaline phosphatase. This is what is shown in FIG. 3. It can be seen that only one of these bands reacts in reality in Western Blot technique and therefore is always recognized by anti-Leu 11b antibody; this is the major band at 72,000-76,000 daltons.

Figures 4 and 5 illustrate the results of adsorption experiments on lectin columns. The experiment was as follows: 500 microliters of normal human serum were deposited twice successively, at room temperature, in an equivalent volume of agarose coupled with different lectins. In Figure 4, it is a lectin called Lens Culinaris Agglutinine (or LCA). After the column eluent has been collected, that is to say after all that which has not been retained by the column has been collected, the reactivity in Fc receptor was measured, using the technique d 'Indirect ELISA, as described above. The immunoreactivity of the sera was tested before passage through these lectins (column in solid line), and of the effluent, that is to say of what was not retained in the column, and there is almost 90% adsorption of the material according to the invention, representing the Fc receptor immunoreactivity on these columns. This proves that this material is glycoprotein in nature and contains glucose and / or mannose residues.

The material adsorbed on this column was then elected, that is to say detached; this is what is shown in Figure 5; this was achieved by applying to the column a solution which competes with the Fc receptor, namely a solution which contains the specific sugar of this lectin. In this case, the elution solution is a 0.5 M solution of o-methyl mannoside. It was thus possible to elute the Fc receptors from this lectin column; this is shown in FIG. 5. We see there the immunoreactivity as it is obtained using the classic ELISA technique in sandwich of the different elution fractions which are numbered on the abscissa, and on the ordinate, is indicated reactivity in ELISA in terms of optical density. We see that we have a classic elution curve, with a peak around the second and third fraction. This confirms once again that the Fc receptor according to the invention, as it circulates in the serum, is indeed a glycoprotein. Figure 6 illustrates the results of a so-called "Dot Blot" experiment. The Dot Blot technique is perfectly known and referenced in the literature. According to this technique, a cellulose nitrate filter is brought into contact with a solution containing the antigens to be assayed. These antigens are fixed on this cellulose nitrate paper by application of a vacuum on the other side of this porous filter. Thanks to the action of the vacuum, the antigenic molecules will be fixed on the cellulose nitrate paper. This paper is then reacted with different antibodies and makes it possible to observe chlorimetric reactions, thanks to the use of antibodies labeled with enzymes.

Here, there are three samples that have been tested in parallel:

• on the bottom line, it is an extract of cells, as also described above.

This cell extract is tested at four dilutions, from the left to the right of the figure, and shows a classic dilution curve with immunoreactivity corresponding to the calorimetric reaction observed on the nitrocellulose filter, which decreases in proportion to the dilution.

• On the intermediate line, instead of the cell extract, it is a serum considered positive according to the results of the ELISA sandwich according to the invention. Again, we see that the test shows a decrease in the optical density obtained, proportional to the dilution of serum which also goes from the left to the right of the figure, thus showing that we are dealing with a reaction as specific as that of the ELISA sandwich. . On the upper line, a serum was considered negative according to the results of the ELISA sandwich, that is to say that when it is reacted in a first step with a capture by 3G8 on PVC plate and secondarily with the anti-Leu 11b antibody pair and the polyclonal anti-mouse IgM antibody labeled with peroxidase, which normally gave a negative result, the presence was found, in this negative serum, of material antigenically recognized as an Fc receptor. The only difference here, compared to the sandwich ELISA, is firstly, that there is no capture step, therefore no need for this antigen to be recognized by the monoclonal antibody 3G8, and , secondly, that it is not the antibody Leu 11b and the anti-mouse IgM antibody which are used to reveal the Fc receptor, but directly a rabbit anti-Fc receptor polyclonal antibody, produced in the laboratory by immunization of a rabbit with Fc receptor purified according to the technique described in connection with FIG. 1, and a rabbit anti-immunoglobulin antibody labeled with peroxidase as the second antibody.

This shows that certain sera, considered to be negative by the use of the 3G8 / anti-Leu 11b pair, nevertheless carry soluble type III Fc receptors. This suggests that there is an antigenic polymorphism, some individuals having soluble plasma Fc receptors recognized by 3G8 / anti-Leu 11b, others having soluble soluble Fc receptors not recognized by the 3G8 / anti-Leu 11b pair.

Thanks to the assay and purification methods according to the invention, there is a molecule which can be used in humans in the treatment of various conditions: 1 - Monoclonal secretions of immunoglobulins, such

Multiple myeloma or Kahler's disease.

2 - Disease of immune complexes to diseases accompanied by the presence of tissue or serum immune complexes, such as lupus erythematosus and other autoimmune diseases.

3 - Lymphomas and leukemias, in particular lymphoma of

Burkitt.

4 - Rejection of a bone marrow or organ transplant (liver, kidney, heart for example) as a curative or preventive measure.

5 - Acquired immune deficits and, in particular, during infections by the HIV virus.

Claims

1 - Fc ɣ R type III receptor, soluble, of low affinity, (or CD 16), consisting of a glycoprotein of molecular mass 72000-76000 daltons, which is recognized in

ELISA and Western Blotting with the anti-Leu 11b monoclonal antibody.

2 - Fc ɣ R type III receptor, soluble, of low affinity, (or CD 16), characterized in that it consists of the product obtained by affinity chromatography on a column coupled to 3 G8 antibodies or to lectins or to polyclonal anti-FcR receptor antibodies, of a biological fluid of human origin, then by gel filtration, the spectrum of said product, by electrophoresis on acrylamide gel in reducing condition, comprising a major band corresponding to a mass molecular range between 72,000 and 76,000 daltons, and a plurality of minor bands, the main ones of which correspond to molecular masses respectively:

- between 64,000 and 66,000 daltons - between 51,000 and 55,000 daltons

- between 42,000 and 46,000 daltons

- between 33,000 and 37,000 daltons.

3 - Fc ɣ R type III receptor, according to claim 2, essentially comprising the molecular mass fraction 33000-37000 daltons, as it appears in electrophoresis on acrylamide gel, in the presence of a reducing agent and an agent detergent like sodium dodecyl sulfate (SDS).

4 - Fc ɣ R type III receiver, according to one of claims 1 to 3, characterized in that in Dot

Blot, it recognizes a polyclonal rabbit anti-FcR receptor antibody.

5 - Each of the fractions of the Fc ɣ R type III receptor, as defined in one of claims 2 to 4, and all the possible combinations of these fractions. 6 - Method for the identification or the determination of the soluble forms of the Fc ɣ R type III receptors, of low affinity, (or CD16), characterized in that it consists in: (a) fixing, on a solid phase, a first antibody, which is a monoclonal antibody to the polyclonal to the still a fraction of a monoclonal antibody to the polyclonal, and which is directed against a conformational epitope of the Fc ɣ receptor to be identified or to be assayed; (b) washing said solid phase to remove said first uncoupled antibody; (c) incubate the sample containing the Fc ɣ receptor to be assayed in. presence of the solid phase coated with said first antibody; (d) washing to remove material not specifically attached to said first antibody;

(e) incubating, in the presence of the resulting solid phase, a second antibody, which is a monoclonal or polyclonal antibody or a fraction of a monoclonal or polyclonal antibody, and which is an anti-Fc receptor antibody recognizing the same category of Fc receptors as the first antibody, but by a totally different epitope;

(f) washing to remove said second non-specifically bound antibody;

(g) incubating, in the presence of the resulting solid phase, a third antibody, which is an antibody capable of specifically recognizing said second antibody; (h) washing to remove the third antibody not specifically attached; and, (i) assay the third fixed antibody and deduce therefrom the amount of Fc receptor initially present in the sample. 7 - Process according to claim 6, characterized in that in step (a), the first monoclonal antibody 3G8 is used as the first antibody.

8 - Process according to claim 7, characterized in that in step (e), a second mouse IgM consisting of the anti-Leu 11b antibody is used as the second antibody.

9 - Method according to claim 8, characterized in that in step (g), as a third antibody, a polyclonal goat anti-IgM antibody, mouse.

10 - Method according to one of claims 6 to 9, characterized in that in step (g), using a third antibody labeled with an enzyme and in step (i) an adds a colorimetric substrate for said enzyme, and, after having stopped the calorimetric reaction, the calorimetric variation is read from which the quantity of Fc receptor sought is deduced.

11 - Process according to claim 10, characterized in that the enzyme is peroxidase, in particular horseradish peroxidase and the colorimetric substrate of peroxidase is orthophenylene diamine in the presence of hydrogen peroxide, the calorimetric reading being carried out at 492 nm. 12 - Method according to one of claims 6 to

11, characterized in that the incubation of step (a), with a view to fixing the first antibody on the solid phase, is carried out at a temperature of the order of 4 ° C, for a period of time ranging from 8 to 12 hours. 13 - Method according to one of claims 6 to

12, characterized in that the incubations of steps (c), (e) and (g) are carried out at ambient temperature, over a period of time ranging from 1 to 4 hours.

14 - Set of reagents necessary for the implementation of the method as defined in one of claims 6 to 13, characterized in that it comprises: a solid support, in particular a microtitration plate, provided with a first antibody, which is a monoclonal or polyclonal antibody, or also a fraction of a monoclonal antibody with the polyclonal, and which is directed against a confσrmational epitope of the receptor Fc ɣ to dose;

a second antibody, which is a monoclonal, polyclonal antibody, or even a fraction of a monoclonal or polyclonal antibody, and which is an anti-Fc receptor, recognizing the same category of Fc receptors as / the first antibody, but by a totally different epitope;

- a third antibody, which is an antibody capable of specifically recognizing the second antibody; - a system for assaying said third antibody.

15 - Box according to claim 14, characterized in that the first antibody is the monoclonal antibody 3G8, the second antibody is a monoclonal antibody consisting of a mouse IgM, namely the anti-Leu 11b antibody, and the third antibody is a goat anti-mouse IgM polyclonal antibody.

16 - Box according to one of claims 14 and 15, characterized in that the assay system of the third antibody comprises an enzyme conjugated to said third antibody, a colorimetric substrate associated with said enzyme and a substance intended to stop the calorimetric reaction.

17 - Box according to claim 16, characterized in that the enzyme is peroxidase, in particular horseradish peroxidase, the substrate associated with said enzyme is orthophenylene diamine in the presence of hydrogen peroxide and the substance intended to stop the colorimetric reaction is an aqueous solution of sulfuric acid.

18 - Application of the receptor Fc ɣ R type III, of the fractions which compose it and of the different possible combinations of these fractions, as defined in one of the claims 1 to 5, and of the method as defined in one of claims 6 to 13 for the diagnosis and monitoring of the therapy of diseases involving the Fc receptors, in particular infectious diseases, autoimmune diseases, transplant rejection, cancer and myeloma, and acquired immunodeficiency syndrome (AIDS).

19 - Application of the receptor Fc ɣ R type III, the fractions which compose it and the different possible combinations between. these fractions, as defined in one of claims 1 to 5, and of the method as defined in one of claims 7 to 13, for the study of human polymorphisms.

20 - Medicinal product containing an Fc ɣ R type III receptor or at least one fraction which composes it, as defined in one of claims 1 to 5.