Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
  • G01N33/6857—Antibody fragments

Definitions

• 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.
• infectious diseases infectious diseases, autoimmune diseases, graft rejection, diseases of human complexes, cancers, myelomas , and acquired immunodeficiency syndrome (AIDS)
• AIDS acquired immunodeficiency syndrome
• FcR Fc fragment of antibodies
• IBF immunoglobulin binding-factor
• 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).
• Fc receptor for mouse IgG immunoglobulins (FcR) in mouse serum has been demonstrated.
• 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.
• 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".
• 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 ⁇ 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 ⁇ 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
• the invention also relates to an Fc ⁇ 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).
• a reducing agent such as sodium dodecyl sulfate (SDS).
• SDS sodium dodecyl sulfate
• the biological fluids discussed here include serum and plasma fluids, cerebrospinal fluids, urine and ascites, among others.
• the invention finally relates to each of the fractions of the Fc ⁇ 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 ⁇ R type III low affinity receptor (or CD16), is characterized by the fact that it consists of:
• Fab fragment directed against a conformational epitope of the Fc ⁇ receptor to be identified or to be assayed;
• 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;
• step (a) use is notably made, as first antibody, of the monoclonal antibody 3G8.
• step (e) a mouse IgM is used in particular as the second antibody consisting of the anti-Leu 11b monoclonal antibody.
• step (g) a polyclonal antibody, namely a goat anti-mouse IgM antibody, is used in particular as the third antibody.
• 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.
• 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.
• 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.
• 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.
• 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 ⁇ 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.
• a solid support in particular a microtiter plate
• a first antibody which is a monoclonal or polyclonal antibody, or another fraction of
• an xenogenic sera are used (fetal calf, horse, goat).
• 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.
• 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.
• lymphokines 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).
• lympholcine human Fc receptor
• 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.
• 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 ⁇ R type III receptor or at least a fraction which composes it, as defined above.
• microtiter plates 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 ⁇ g per well, into 100 ⁇ l of carbonate buffer at pH 9.6.
• the incubation lasts 8 to 12 hours at a temperature of 4oC.
• step (3) Deposit of the sample to be assayed 100 ⁇ l 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).
• This washing is carried out in the same way as in step (2).
• the above-mentioned antibody is introduced (source: Jackson Immuno-Research Laboratories Inc.), diluted 1/5000 in PBS Tween making a total of 100 ⁇ l per well. They are incubated for 1 hour at 22oC.
• This washing is carried out in the same way as in step (2).
• 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.
• 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.
• 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
• 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 chr ⁇ matogram 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.
• FIGs 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.
• it is a lectin called Lens Culinaris Agglutinine (or LCA).
• LCA Lens Culinaris Agglutinine
• FIG. 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.
• 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.
• 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.
• lymphoma 3 - Lymphomas and leukemias, in particular lymphoma of

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• 1987
  • 1987-02-24 FR FR8702400A patent/FR2611913B1/fr not_active Expired - Fee Related
• 1988
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