FI95752B - Determination kit and method for immunological measurement of whole cells - Google Patents

Abstract

A kit for the assay of the surface antigens characteristic of a cell population or subpopulation comprising, as components: a) a solid support on which are fixed by covalent bonding or by physical adsorption one or more monoclonal antibodies directed against the surface antigens of the cellular population to be assayed; b) one or more solutions, each containing a monoclonal antibody specific for the cell population or subpopulation to be assayed, labelled with a radioisotope probe or an enzyme probe; c) in the case of antibodies marked with an enzyme probe, one or more solutions contributing the necessary reagents (substrate and chromogen) for detecting the enzyme activity. The invention also relates to a process for immunometric assay of the surface antigens of a cell population or subpopulation. The assay kit and the immunometric process according to the invention can be employed for the total assay of the population of T lymphocytes and/or of the subpopulations of T4 and T8 lymphocytes.

Description

- 95752 Assay kit and method for immunological assay of whole cells

The invention relates to a test kit and to an immunoassay method performed with this kit for the determination of surface antigens specific to cell populations or subgroups thereof. The immunoassay method and the corresponding kit can also be used to determine the cells themselves by their surface antigen assay. These . 10 assays are useful in diagnostics.

Knowledge of antigens or cell surface markers has progressed tremendously since the development of lymphocyte hybridization and the discovery of monoclonal antibodies by KÖHLER and MILSTEIN (Nature, 1975, 256, 495-497).

In particular, monoclonal antibodies can be used to detect and analyze surface markers or membrane antigens of various cells. These markers (or antigens) can be different: proteins, glycoproteins, or glycolipids. The characterizations studied are thus suitable mainly for the identification or typing of tissue or organ markers, markers of the state of differentiation or activation of normal cells, and normal or cancer cells. One particularly important area is the study of hematopoietic cell lines (erythrocytes, megakaryocytes, granulated cells, monocytes, lymphocytes).

Thus, for example, monoclonal antibodies can be used to determine the surface properties of T and B lymphocytes. Corresponding markers alone or in combination identify functional differentiation or specialization states of lymphocytes. By international agreement, surface markers of human leukocytes are classified into groups or differentiation classes (CDs) defined by the IUIS-OMS subcommittee in 1984 and are set forth in World Health Organization Publication 1984, 62 (5), 813-815.

35 The identification of these markers by monoclonal antibodies has made it possible to elucidate the structure and biological functions of the markers. For example, molecules of CD4 and CD8 markers are involved in leukocyte adhesion and have a helper and inducing function (CD4 marker) or a cytotoxic and suppressive function (CD8 marker) on the surface of T lymphocytes.

With these data and monoclonal antibodies recognizing markers, these markers can be used in the diagnosis and treatment of various pathological conditions, especially malignant hemopathies (leukemia, lymphoma, etc.) and immune system disorders such as autoimmune diseases, congenital or acquired immune disorders. AIDS, etc.). (BRETON-GORIUS and VAINCHENKER, Le Biologiste, 1987, XXI, no. 167 63-70, SHAW, Immunology Today, 1987, 8 (1), 1-3).

15 Monoclonal antibodies are now an indispensable tool in cellular assays in clinical biology.

There are cell counting methods that use labeling of cell surface antigens, but these methods are often lengthy, laborious, difficult to implement, and the results are sometimes random.

The well-known and used methods for determining the normal or altered expression of cell surface antigens can be divided into two groups. In the first group, antigens are determined by complex and specialized laboratory equipment based on flow cytometry (see PONCELET et al., J. Immunol.

Methods, 1985, 85, 65-74) or quantitative microscopic techniques (POULTER et al., J. Immunol. Methods, 1987, 98, 227-234). These methods for assaying cellular antigens are based on measuring a signal from anti-cellular antibodies directly or indirectly coupled to a reagent labeled with a fluorescent agent (or fluorochromes) such as fluorescein or rhodamine isothiocyanate, or an enzyme. Such as peroxidase or alkaline phosphatase. The use of these fluorescent or enzymatic reagents 3 - 95752 in combination with the necessary washing steps results only in fluorescence or color reaction caused by cell membranes and not by the environment. The widespread use of these methods in laboratories is limited by specialized and expensive equipment (fluorescent microscope that can be connected to an image analyzer, cryostat, flow cytometer). In addition, the analysis and interpretation of cell immunolabeling performed by these methods requires the expertise of a cytology specialist.

10 Another group of antigen assay methods is based on the quantitative evaluation of markers in the total cell population. With these methods, antigens can be determined by either direct or indirect labeling, which is thus often performed in two, three or four steps. In all cases, the reagent used in the final labeling step contains a label that is either an isotope, e.g., iodine 125, in immunoradiological measurements (BROWN et al., J. Immunol. Methods, 1979, 31, 201. STOCKER and HEUSSER, J. Immunol. Methods , 1979, 26, 87-95) 20 or enzyme in immunoenzymological measurements, most often peroxidase, alkaline phosphatase or beta-galactosidase, (VAN LEUVEN et al., J. Immunol. Methods, 1978, 23, 109-116, MORRIS Transplantation , 1983, 36 (6), 719, BAUMGARTEN, J. Immunol. Methods, 1986, 94, 91-98).

.. 25 The methods of this last group are quite cumbersome, laborious and uncertain, as they require several washes and centrifugations of the cell material; sometimes the colored solution resulting from the enzymatic reaction has to be transferred for the final spectrophotometric measurement; finally, the most commonly used chemical fixation of cells results in the irreversible degradation of some antigens that are sensitive, especially to conventional chemical fixatives such as glutaraldehyde or methanol (DROVER et al., J. Immunol. Methods, 1986, 90, 275-35281).

- 95752 4

It is known from the literature that an antigen having multiple antigenic determinants, i.e., multiple epitopes, can be determined by attaching this antigen from one of its epitopes to an antibody immobilized on a solid surface and binding to its other epitope another antibody to which an enzymatic or radioisotope marker for measurement.

One such method, often referred to as the sandwich technique, is explicitly disclosed in patents or. 10 patent applications FR 2 487 983, FR 2 500 166, EP 119 736. None of these documents discloses the use of this technique for whole cells, although the word "cell" appears in the list of antigens for which the method is suitable.

The various antigens of the examples set forth in the above patents are all exclusively protein molecules that are soluble in water and physiological fluids, such as hormones, enzymes or circulating tumor markers. On the contrary, it is clear that the cell is not a molecule and differs from it at least in its considerably larger size and because the cells are insoluble in physiological fluids. Therefore, to this day, sandwich technology has never been applied to whole cells.

,. In addition, immunocapture of cells on a solid support is disclosed in WO patent application 86/02091, which was intended to remove unwanted cells from bone marrow samples for transplantation. In this patent application, cells were attached to floating microcarriers, and therefore the antibody used had to be bound to a solid support with a complex macromolecular structure called a network relay to ensure proper orientation between the antibody and the corresponding cellular antigen.

There is no mention in this application of the use of a method for the quantitative determination of 35 antigens.

- WO 75752 5 WO patent application 84/03151 also discloses immunocapture of cells for analytical purposes. The purpose here was to identify tissue groups of cells to be examined (usually a method called HLA typing). The cells are fished with antibodies that have been bound and placed in a special shape on a very special medium (microscope slides). The results are obtained by a simple visual examination of the plate, and the result is "all or nothing". Thus, the previously described cell attachment methods cannot analytically quantify the antigens of some cell membranes. In addition, specificity can be a problem with all of these methods, as they rely on a single antibody for cell recognition.

The assay method of the present invention has significant advantages over previously known and used methods, as it allows all antigens in a single cell population to be quantified in a single assay. In this assay, cells are not treated chemically or physically and are in a physiologically intact state. In addition, the method of the invention has a very high specificity, which is characteristic of such an immunological dual recognition system with two different, specific antibodies against two different antigens of the same cell. This method is simple, fast and repeatable. It is very suitable for the analysis of large volumes of samples, which is why the method can be used for diagnostic purposes in clinical bio-30 biology laboratories.

Accordingly, the present invention relates to a kit for the immunological determination of at least one surface antigen typical of a cell population or subgroups thereof, comprising: a) a solid support to which one to 95752 6 or more monoclonal antibodies has been covalently attached or physically adsorbed; agents that are against a typical surface antigen of a cell population to be measured other than for the purpose of immobilizing on a carrier those cells of the subset having the antigen to be determined; (b) at least one solution containing a monoclonal antibody to a specific antigen of the cell population or subset thereof to be measured, which is radiolabeled or enzymatically labeled; C) when the antibody is labeled with an enzyme, the enzyme developer, i.e. one or more solutions containing the enzyme substrate, and, if desired, one or more reagents necessary for detecting the enzyme activity.

The term cell as used in this specification and the following claims means human and animal cells, protozoa and microorganisms (bacteria or fungi). Among blood cells, this invention relates to nuclear particles, such as leukocytes, and non-nuclear particles, such as red blood cells or platelets.

The assay method of this invention is applicable to whole cells, i.e., not lysed.

The cells are not treated in any way chemically or physically and are in a completely physiologically intact state in the assay. This will ensure that; The membrane markers to be assayed are best preserved intact.

As the solid support, all means suitable for handling cell suspensions can be used, and preferably tubes, certain types of magnetic media or hard or soft, well-hollow microtiter plates, which can be made of polyethylene, polystyrene, polyvinyl chloride or nitrocellulose. Monoclonal antibodies for cell immobilization can be attached to solid supports by either chemical covalent bonding or physical adsorption according to well-known classical methods, as described in STOCKER and EUSSER, J. IMMUNOL.

METHODS, 1979, Vol. 26, pp. 87-95. The carrier may preferably be impregnated with protein in advance.

According to the invention, monoclonal antibodies attached to or attached to a solid support can be used to bind a cell population containing it or those cell populations having the antigen to be measured. If this population consists of human cells, the most preferred monoclonal antibodies for binding are anti-10 HLA class I antibodies specific for a common portion of HLA-A, -B, and -C antigens present in leukocytes and several other cell lines in the body. Of the antibodies, a class I antibody marketed by BYOSIS is particularly preferred.

In some cases, when the test cells are human cells, and in all cases where the cells are not human cells, monoclonal antibodies suitable for the test cells may also be used in the immunocapture of the invention.

20 The term "radioisotope-labeled monoclonal antibody" means a monoclonal antibody to which is attached, either to a part of its natural structure, eg essential tyrosine residues, or to a suitable radical attached to it, .. a radioactive isotope with a radioactivity can be used to determine the amount of antibody bound.

By the term "enzymatically labeled monoclonal antibody" is meant a monoclonal antibody to which an enzyme has been coupled which, together with suitable reagents, allows the quantitative measurement of this monoclonal antibody.

The substrate and reagents are selected so that the end product of the reaction or chain of reaction between the enzyme and these compounds is: - either a colored or fluorescent compound which spreads in the fluid surrounding the cells and is the final spectrophotometric or fluorometric measurement, respectively, - or colored , an insoluble compound that settles on the cells and their anchoring walls, and which can be measured with a reflectance photometer or possibly evaluated visually by comparison with reference colors.

The assay kit may further comprise a wash buffer with which the solid support is washed after immobilization and after the cells have been labeled with the selected label-labeled antibody or antibodies.

The assay kit may also contain the necessary samples for assay calibration as well as quality control.

The present invention also relates to a method for the immunological determination of surface antigens of a cell population or a subset thereof, characterized in that: - the cell population is specifically immobilized or immunoprecipitated on a solid support in one step 20 using one or more monoclonal antibodies previously covalently attached or by physical adsorption to this medium, and which recognizes an antigen on the cell surface different from the antigen to be determined, and the co-immobilized surface antigen of the simultaneously immobilized cell population or any subset thereof is directly labeled with a monoclonal antibody specific for this antigen or labeled ; 30 - the test is incubated for a period of time during which immunocapture and labeling occur simultaneously; - the solid support is washed to remove unwanted unbound cells and excess radioisotope or enzyme-labeled antibody; 35 - measuring the amount of antigen to be determined in the cell population or subgroup thereof, either by counting the 9 95752 bound radioactivity or alternatively after treating the solution with an enzyme substrate and possibly one or more suitable additional reagents, measuring transmission or reflectance photometrically or measuring fluorescence .

The assay kit and method according to the invention are preferably used for the determination of human blood particles, such as in particular leukocytes, and in particular lymphocytes, T lymphocytes, T4 and T8 lymphocytes and B lymphocytes, as well as granulocytes, monocytes and platelets.

Another preferred use is in the determination of surface antigens of pathogenic microorganisms such as Candida albicans.

In addition, the assay kit and immunological method of the invention are well suited for the determination of surface antigens of tumor cells, especially urinary tract cancer cells and malignant hemopathic cells.

The assay kit and method of the invention can be used to measure signals (radioactivity or light absorption or emission) that depend on both the number of cells in the cell population being studied and the density of the antigen being measured on the cell surface. By measuring these signals, the total number of structural or functional antigen molecules contained in a cell population or subset thereof can be quantified.

For example, for hematologically particularly important Leu cytocyte markers, it is known that in the majority of healthy cases, the mean antigen density within the same cell population does not vary significantly from one sample to another. The cytological count of cells with the antigen in question correlates well with the signal measured according to the invention, which is proportional to the total number of antigen molecules in the test sample. In contrast, in some pathological conditions, the density of antigens on the cell surface may vary - 95752 10 within the same cell population without significantly varying the number or proportion of positive cells. These pathological conditions are detected more efficiently than the conventional cell counting method using the immunological method and kit of the invention.

By selecting a microtiter plate as a solid support, another application of the invention is obtained. The kit and immunological method of the invention can be advantageously used to determine different surface antigens specific to different subgroups of the cell population studied on a single plate. For this purpose, ready-to-use microtiter plates with one or more monoclonal antibodies capable of binding to all cells of the test population, on the one hand, and monoclonal antibodies with an appropriate marker, each of which can be used, can be used for this purpose. is specific for one of the antigens contained in the subgroups studied. Thus, the antigens required to characterize 20 quantitatively selected subgroups can be measured with a single treatment and the same carrier.

Such an application of the present invention is the characterization of antigenic structures of cells of clinical biological interest. One application is the determination of the tissue group of a subject to be examined, commonly referred to as HLA typing.

Another application is the typing of tumor cells, especially in malignant hemopathies such as leukemias and lymphoma. In this routine diagnosis, ka-30 is characterized by the type and origin of these patient tumor cells based on the presence or absence of an appropriately selected set of cell surface antigens.

Using an assay kit of the invention comprising a microtiter plate pre-attached with one or more monoclonal antibodies capable of binding to the cells of the cell population being studied, and 11,95,552 solutions containing various enzyme- or radiolabeled monoclonal antibodies. specific for a single tumor cell antigen, antigens specific to a patient's tumor cell population can be identified and quantified, and classified into one of a large group of cancers, and in particular a clinically characterized malignant hemopathy. Thus, the method according to the invention makes it possible to carry out a qualitative and quantitative analysis of the antigenic structures of tumor cells rapidly and on a single carrier.

Another application of the invention is human T lymphocytes, which specifically have two subgroups: lymphocytes characterized by a CD4 marker, referred to as T4-positive lymphocytes or simply T4 lymphocytes, and T4 lymphocytes. characteristic of a CD8 marker called T8-positive lymphocytes (or T8 lymphocytes).

20 The numerical measurement of the T4 / T8 ratio is very interesting for diagnostics and clinical biology.

In fact, it is known that the T4 / T8 ratio is altered by various disorders of the immune system, such as dysimmune diseases, chronic infections, viral infections, and especially infections caused by the HIV virus (AIDS virus).

The assay kit and immunological method of the invention can be used to determine antigens that are common to the T lymphocyte population and / or to the T4 and T8 lymphocyte subgroups. In this case, the T lymphocytes of the test sample are specifically immobilized on a solid support, and at the same time the surface antigens of the T4 lymphocytes are directly labeled with an anti-CD4 monoclonal antibody to which a radio-35 isotopic or enzymatic label is attached; similarly, T8 lymphocyte surface antigens are labeled with an anti-CD8 mono- 95752 12 clonal antibody with an appropriate label.

To measure the total number of T lymphocytes, anti-CD7 monoclonal antibody 5 (also called anti-T2) with a suitable label is preferably used.

For the specific immobilization of T lymphocytes in a sample, one or more monoclonal antibodies are preferably used as such or in admixture, to identify whole T cells in the sample, in the case of common anti-leukocyte antibodies (or anti-CD45), or when the antibodies together recognize Antibodies to a T population (called "general T"), such as anti-CD2 (or anti-T11), anti-CD5 (or anti-T1), anti-CD7 (or anti-T2) or other generalized T antibodies not yet classified into differentiation classes according to the criteria of 0MS.

The immunological method of the invention can be advantageously used to determine antigens specific for the T lymphocyte and T4 and T8 lymphocyte populations in several parts of the same solid support. A measurement based on radioactivity, light transmission or reflection, or fluorescence can be used to easily and directly determine the numerical value of the CD4 / CD8 ratio.

Similarly, T and B lymphocyte subgroups, which together form a lymphocyte line, can be determined with the same solid strain.

For example, a mixture of monoclonal antibodies against some or all of the surface antigens on T cells can be attached by adsorption to the walls of the wells. These monoclonal antibodies further immobilize all T cells in the test sample. The plates thus prepared can be lyophilized and stored preferably at 4 ° C. This step can be carried out on an industrial scale, and thus the finished plates can be used either in the total T lymphocyte assay li 13 95752 or in the assay kits used in the T lymphocyte subsets.

Samples containing the cells to be determined, derived from blood or other suitable, normal or pathogenic biological fluids, may be used as such or after preparation, such as density gradient centrifugation according to known methods, and in particular in a very dense solution, such as Pharmacia's commercial FICOLL-PAQUE. To determine blood lymphocytes, the blood sample to be examined can also be treated with lysis buffer, which degrades red blood cells.

Suitable samples of the cell suspension are contacted with a solid support, for example, in the wells of a pre-prepared microtiter plate, simultaneously with a solution of a monoclonal antibody specific for the intended cell population in the assay kit, wherein the monoclonal antibody is labeled with a suitable label, e.g. Thus, a radioisotope label can be prepared, e.g., by labeling a monoclonal antibody with iodine-125 or iodine-131, for example, in the presence of chloramine T according to a known method (FC GREENWOOD, WMHUNTER and Coll. Biochem. J., 1963, 89, 114); or an enzymatic label can be prepared by conjugating a monoclonal antibody to an enzyme such as alkaline phosphatase, peroxidase, beta-galactosidase or acetylcholinesterase according to known methods (see e.g.

M. O'SULLIVAN Methods in Enzymology 1981, 73, 147) or a modified method thereof. In some cases, enzymes are preferred over radioisotope labels in order to avoid the disadvantages associated with the handling of radioactive compounds and the disadvantages associated with the limited shelf life of these reagents.

The incubation time, i.e. the time required for cell immobilization and simultaneous labeling, is preferred. 95752 14 for a maximum of one hour. During this time, the solid support can optionally be centrifuged to promote cell immobilization. The solid support, e.g., a microtiter plate, is then washed to remove both non-adherent cells and excess enzyme- or radioisotope-labeled monoclonal antibody.

When a radioisotope label, such as iodine-125, is used, the radioactivity attached to the cells is measured with a gamma counter in a suitable manner and, for example, by first dissolving the cells in an alkaline solution (e.g.

NaOH) and collecting the radioactive solution with an absorbent buffer.

When the monoclonal antibody is enzymatically labeled, a colored or fluorescent compound 15 is formed by adding to the solid support to which a cell population containing the antigen to be measured is attached a solution containing the enzyme substrate and one or more reagents which ultimately yield a soluble or soluble reaction product. a soluble fluorescent compound as described above. From the samples thus treated, the light signal is then measured with the appropriate equipment for each case: a transmission or reflection photometer or a fluorometer, respectively. If the solid support is a microtite-25 plate, the light signals can be read sequentially from all wells of the same plate using automatic plate readers commonly used in biological laboratories, such as Titertek or Fluoroscan plate readers, for spectrophotometric or fluorometric measurement, respectively.

When alkaline phosphatase is used as the enzymatic label, this enzyme is coupled to a monoclonal antibody according to the method of Boehringer Mannheim Biochemica. Preferred sub-substrates for this enzyme are para-nitrophenyl phosphate for spectrophotometric measurement or 4-methylumbelliferyl phosphate for fluorometric measurement or 5-bromo-4-chloro-3-indolyl phosphate to give an insoluble colored compound. Beta-galactosidase, the preferred substrates of which are ortho-nitrophenyl-beta-D-galactopyranoside or 4-methylbelliferyl-beta-D-galactopyranoside, can also be used as an enzymatic label.

Monoclonal antibodies can be advantageously coupled to peroxidase. In this case, the coupling method is a variant of M.B. WILSON and P.K. In NAKANE 10 Immunofluorescence and Related Staining Techniques, W.

Knapp, K. Kolubar, G. Wicks, eds. Elsevier / North Holland. Amsterdam, 1978, pp. 215-224. The modifications used in the preparation of the enzyme conjugate compared to the original method are as follows: - the molar ratio of peroxidase / antibody is 3 when it was 2 in the method, - less efficient oxidation of carbohydrate moieties by peroxidase by reducing the proposed sodium periodate content by 33%.

Reagents used to determine peroxidase activity coupled to monoclonal antibodies include oxidized water, an enzyme substrate, and a suitable chromogen, for example, ortho-phenylenediamine or 2,2'-azino-bis- (3-ethylbenzoazoline-6-sulfonic) acid or ABTS: a to obtain a colored and environmentally soluble reaction product, or to obtain a reaction product insoluble in 3,3'-diaminobenzidine or 3-amino-9-ethylcarbazole or 4-chloro-alpha-naphthol, or to obtain a fluorescent and environmentally friendly solution of para-hydroxyphenylpropionic acid. to obtain the reaction product.

The kit of the invention for determining antigens specific for T, T4 and T8 lymphocytes preferably comprises: a) a microtiter plate having one or more anti-T lymphocyte monoclonal antibodies attached to the wells.

lfi 95752 lb b1) a solution containing at least one anti-T lymphocyte monoclonal antibody labeled with peroxidase.

b2) a solution containing at least one anti-5 CD4 antigen monoclonal antibody le peroxidase.

b3) a solution containing at least one anti-CD8 antigen monoclonal antibody labeled with peroxidase.

10 cl) a solution containing oxidized water and an enzyme substrate in a suitable buffer.

c2) a solution containing the chromogen used to detect the expression of enzyme activity.

Another preferred embodiment of the invention is the use of monoclonal antibodies coupled to acetylcholinesterase.

Acetylcholinesterase is preferably coupled to the antibody in FR patent application no. According to a variation of the method shown in 2,550,799 or by a method in which antibody fragments are prepared in a known manner, the enzyme is modified with a suitable heterobifunctional compound and finally the products thus obtained are coupled.

In this case, other known methods for preparing immunoenzymatic conjugates can also be used.

The enzyme activity specifically bound to the cell antigen observed with the acetylcholinesterase conjugate is preferably determined by a well-known method using acetylthiocholine as the enzyme substrate and Ellmann's reagent or 5,5'-dithio-2-nitrobenzoic acid as the chromogen, e.g. in Anal.

Chem. 57 (1985) 1170-1173.

Said chromogens can be used as such or as water-soluble salts.

17 95752

The results of the antigen assays of the invention may be reported in any manner appropriate to the assay performed. In more detail, results can be expressed either as the total number of particular antigen molecules (e.g., CD4 antigen) contained in a given volume of test sample, or as the ratio of the number of antigen molecules in a test sample to the number of other antigen molecules in the same sample (e.g., CD4 antigens and CD8 antigens or 10 CD4 / CD8 in a blood sample).

The molecular amount of a particular antigen in a test sample is preferably determined using a standard plot formed of cells or appropriate cell preparations containing the antigen to be determined and pre-calibrated by a known reference method. These standards are preferably either cells of similar origin to the cells of the sample to be determined, or cells of cell lines containing the antigen of interest, or preparations of these cells, e.g. membranes.

These standards are treated in the same way as the test samples. The signals given by them are used to draw up a standard graph against which the signals given by the samples to be examined are compared. Other calculations are 25 standard.

To determine the molecular ratio of the two antigens in the test sample, the standard method described above can be used and finally the desired ratio can be calculated. In many cases, it is simpler to directly calculate the specific signal provided by each antigen, correct it, if desired, with known factors, such as the ratio of the dimensions of the samples to be examined, and directly obtain the desired ratio.

The immunoassay method 35 of the invention is simple, rapid, and reproducible. Preferably, the total time included in the assay is at most one hour.

It can be easily used to analyze large volumes of samples. To understand its advantages over the other methods presented, it is suitable for analyzing several steps.

Immobilization of cells on a solid support is usually the most difficult or difficult step of the assay method. A commonly used means is chemical attachment of cells to glutaraldehyde or methanol-untreated or poly-L-lysine-treated wells (VAN LEUVEN F. et al., J. Immunol. Methods, 1978, 23, 109). However, these chemical fixations can reduce or even prevent the desired specific assay or, conversely, increase false positive results, which is a very serious drawback (DROVER and MARSHALL J. Immunol. Methods, 1986, 90, 275-281).

In addition, chemical attachment requires several steps: centrifugation of the cells, preparation of the attachment mixture, attachment, and washing of the attached cells.

Drying of the cells at 37 ° C as well as possible fixation of the wells with methanol has also been proposed (BAUM-GARTEN, J. Immunol. Methods, 1986, 94, 91-98). In fact, drying cells at 37 ° C can cause changes in some sensitive antigens and also make the peri-cellular plasma membrane permeable, which facilitates immunolabeling of intracytoplasmic antigens in addition to surface antigen labeling, thus leading to annoying background noise or false positives. 30 assays limited to surface antigens only.

In addition, the reproducibility of the method is uncertain; in fact, c decanting the cells into the wells of the plate and drying the cells may vary from experiment to experiment. Finally, this assay is lengthy, as the cells alone require at least two hours to dry.

19 95752

Lymphocyte populations have also been successfully immobilized by polyclonal antibodies adsorbed to the wells (STOCKER and HEUSSER J. Immunol. Methods, 1979, 26, 87-95). In addition to immobilizing the 5-lane cells in addition to the only population to be analyzed, polyclonal antibodies are cumbersome because they can react with the antigens to be measured with the labeled antibody, further reducing the final signal to be measured.

When adsorbing or attaching highly specific and affinity monoclonal antibodies to a solid support, and especially to the wells, only the desired cells can be captured, with other unbound cell populations removed during the washes performed after the antigen labeling step 15.

In addition, the properties of the antigens do not change during this step under the influence of any chemical or physical factor, as no chemical or physical methods are used to attach the cells.

Thus, in accordance with the present invention, it has been found that the immobilization of cells by monoclonal antibodies simplifies the step of immobilizing the antigen-containing cells to be determined and the reliability of the results.

. Immunological determination of these cell populations is usually performed by labeling the cells, usually by an indirect method in two or three consecutive steps, in which a label containing a specific signal is attached to the cells during the last labeling step. When using a two-step method ** to label cells, the main reagents used contain anti-immunoglobulin antibodies (anti-Ig) labeled with beta-galactosidase (COBBOLD et al. J. Immunol. Methods, 1971, 44, 125 -133) or alkaline phosphatase (HESSIAN J. Immunol. Methods, 1986, 20 95752 91, 29-34) or iodine-125 (SAVION J. Inununol. Methods, 1987, 97, 49-56). Labeling of peroxidase-anti-peroxidase reagent is a three-step method (VAN LEUVEN 1978).

Another three-step method also uses a monoclonal antibody specific for the antigen marker to be analyzed, followed by biotin-labeled anti-mouse Ig antibodies and finally a streptavidin-peroxidase conjugate (BAUMGARTEN, 1986) or a streptavidin-alkaline streptavidinase. conjugate (IGIETSEME et al. J. Immunol. Methods, 1987, 97, 123-131).

In the method of the invention, whole cells are immunocaptured without any physical or chemical treatment of the cells in one step at the same time, when some or all of the cells are labeled with one or more monoclonal antibodies to which a radioisotope or enzymatic label is directly attached. by itself for the first time to quantify the selected membrane antigen.

Direct labeling of immunologically immobilized cells according to the invention: - simplifies the assay method by reducing the following intermediate steps of the labeling steps in indirect labeling: cell centrifugations, labeling; 25 reagent removals, suspensions; - reduces reagent consumption; - improve reliability by reducing the number of manipulation steps; - saves time; 30 - allows the simultaneous processing of large volumes of samples with low use of materials and conventional equipment.

The incubation time required to immobilize the cell population and simultaneously label the antigens of the subgroups of cells to be determined is short. It is one hour for the determination of T lymphocytes and subgroups of T4 and T8 lymphocytes.

After washing the solid support, the above determination is performed by measuring an accurate and simple signal with conventional equipment: radioactivity, light absorption or emission.

The method set according to the invention thus has several advantages: it is fast, reliable, economical and simple.

The method of the invention can be used to determine surface antigens from cell populations whose cell concentrations can vary widely.

The sensitivity of the method per cell number depends on the antigen density of the cell population to be determined. If desired, the minimum concentration of antigen in moles that can be determined by the method of the invention can be determined for each antigen.

Thus, if the solid support is, for example, a microtiter plate, and the cells to be assayed are human lympho-20 cells, the assay is found to be significant when the number of cells to be analyzed is between a few hundred and about 200,000 cells in a volume of 200 microliters per well; however, their additional limitations are imposed by the density of the antigen to be measured on the surface of the test cells and the choice. However, the limitations of the detection method are known, so the limitations depend mainly on the dimensions and shape of the solid support. The same is true when the solid support is tubular.

It has been demonstrated that the recorded signals 30 (radioactive or photometric measurement) can be used to generate regular and acceptable standard plots as a function of the number of cells studied under normal processing conditions.

In addition, the sensitivity of the method can be improved, if necessary, by simultaneously attaching to the same surface antigen several different monoclonal antibodies specific for several different epitopes of the same antigen. This has been demonstrated by determining the CD4 antigens of the Ichikawa cell line (human T line) using 0 concomitant iodo-125-labeled 0KT4 and ST4 antibodies. The signal to be measured was amplified by about 50% compared to the signal obtained when each anti-CD4 antibody was used alone.

The following examples are used without mentioning the following terms or their abbreviations: BSA: bovine serum albumin PBS: phosphate buffered saline pH 7.4 POD: peroxidase

IgG: immunoglobulin G

IgM: immunoglobulin M

anti-T-IgG or anti-T: anti-T lymphocyte antibody anti-CD4 IgG or anti-CD4: anti-CD4 antigen antibody anti-CD8 IgG or anti-CD8: anti-CD8 antigen antibody substance 20 cpm: pulses per minute dpm: decomposition per minute

Example 1 Immunoenzymatic determination of molecular concentrations of CD4 and CD8 antigens in human blood perox-; 25 with a ligase-conjugated antibody.

A) Preparation of the assay kit a) Preparation of the plate Use a 96-well plastic microtiter plate from NUNC (Reference 64394). 200 μl of a solution containing purified anti-CD2 monoclonal antibody (designated ST 11) is pipetted into the wells, which is used to immobilize all T lymphocytes, i.e. to immunocapture them. This antibody, marketed by BI0SYS, Compiegne, France, reference ST 11, is used at a concentration of 35 pg / ml in PBS pH 7.4.

23 95752

The monoclonal antibody is adsorbed at 4 ° C for 12 hours. Excess antibody is removed by inverting the plate.

Prepare a solution containing 0.1% gelatin and 0.5% BSA in PBS. 250 μΐ of this buffer is pipetted into the wells and kept there for 1 hour at 37 ° C, which saturates the surface of the wells with protein; the plate is washed 3 times with PBS. The plates thus prepared are lyophilized and stored at 4 ° C in sealed plastic bags.

b) Preparation of peroxidase-conjugated antibody solution Peroxidase (POD) prepared by Boehringer Mannheim Biochemica (ref. 814 393) is used.

The method for coupling between antibody and peroxidase is described in M.B.

WILSON and P.K. NAKANE, Immunofluorescence and Related Techniques (W. KNAPP, K. KOLUBAR, G. WICK ed., 1978,

Elsevier / North Holland, Amsterdam - pp. 215-224), with the exception of the use of 1.5 mg POD in 0.36 ml of distilled water for peroxidase oxidation and the addition of 50 microliters of 0.2 M sodium periodate solution. The product thus obtained is coupled with 2 mg of anti-CD4 IgG in 500 microliters of carbonate buffer. After treatment with sodium borohydride and dialysis against PBS, the IgG-POD conjugate is sterile filtered through a 0.22 membrane into a glass tube under sterile conditions at an IgG concentration of 0.5 mg / ml at 4 ° C. The reagent remains stable for at least one year.

The anti-CD8-POD conjugate is prepared in the same manner. IgM-POD conjugates can also be prepared in a similar manner.

(c) Preparation of detection reagent

The detection reagent is obtained as follows: Prepare 0.1 M citrate buffer by dissolving citric acid monohydrate in water to a concentration of 2.1% 24 95752 and adjusting the pH to 5 by adding 7N NaOH. 30 mg of orthophenyleneamine dichlorohydrate are then added to 20 ml of the prepared citrate buffer, followed by the last minute addition of 40 microliters of oxidized water (enzyme substrate) at a rate of 30% per 20 ml of citrate buffer containing orthophenyleneamine.

B) Immunological method a) Separation of cells

Take 2 ml of the blood sample to be examined. Mix 10 with 2 ml of PBS buffer, and precipitate it with 3 ml of FI-COLL-PAQUE (marketed by Pharmacia). The sample is centrifuged at 400 x g for 30 minutes at ambient temperature, the suspension ring formed containing mononuclear cells. This suspension, 15 ml in volume, is collected and the solution from the suspension is dispensed 6 x 100 μΐ into six wells of a previously prepared plate.

b) Incubation of cells

The POD-antibody conjugate prepared above is diluted 1: 100 in PBS containing 1% protein-20 such as BSA or milk powder and 100 microliters of this solution is pipetted into the wells, i.e.: 2 wells are filled with 100 μΐ / well of POD-anti-CD4 conjugate.

- 2 wells are filled with 100 μΐ / well POD-anti-CD8 conjugate.

- Fill 2 wells with 100 μΐ / well of 1% protein solution (reaction blank)

To promote attachment of the cells to the support, the plate is centrifuged at 150 x g for 3 minutes after standing at ambient temperature for one hour.

(c) Detection and measurement of attached enzyme

The wells are emptied by turning the plate. The wells are washed 4 times with 200 μl PBS. Pipette 200 μΐ of freshly prepared detection reagent into each well. The plate is incubated for 20 min at ambient temperature

It 25 95752 divided. The optical density is measured with a spectrophotometer at a wavelength of 492 nm (Titertek Multiskan, type 310 C - Flow Laboratories).

(d) Standardization and expression of results.

To standardize these experiments, the CD4 and CD8 antigens of PONCELET et al. (J. Immunol. Methods 1985, 85, 65-74) cytofluorometrically calibrated human total lymphocytes. Samples from this reference preparation were used to determine 2 standard curves for CD4 and CD8 antigens, which were used to calculate the corresponding antigen concentration in each sample.

The following Table 1 shows the optical densities, molecular amounts of CD4 and CD8 antigens determined from blood samples from 20 blood donors, and the ratio of the amounts of these anti-15 genes (CD4 / CD8).

\ •> «· 26 95752 TABLE 1 CD4 antigen CD6 antigen - CD4 / CD8 donor Antigen-Antigen molar number number of molecules number of rinen

Optical (in millions Optical (in millions density in p1 blood) density in jxL blood) ratio: 1: 0.64: 23: 1.29: 92: 0.25:: 2: 0.71: 27: 0.75 : 37: 0,73:: 3: 0,82: 32: 1,10: 81: 0,39:: 4: 0,49: 17: 0,55: 23: 0,74:: 5: 0, 74: .29 0.89: 52: 0.56:: 6: 0.88: 38: 0.92: 55: 0.69:: 7: 0.74: 29: 0.72: 36: 0, 80:: 8: 0.75: 29: 0.69: 33: 0.88:: 9: 0.48: 16: 0.80: 41: 0.39:: 10: 1.00: 50: 1 , 08: 80 s 0.62: 11: 0.61: 22: 0.69: 33: 0.67: 12: 0.73. : 28: 0.85: 46: 0.61:: 13: 0.51: 18: 0.66: 30: 0.60:: 14: 0.65: 24: 1.06: 76: 0.31 :: 15: 0.92: 42: 0.87: 48: 0.87:: 16: 0.77: 31: 0.98: 63: 0.49:: 17: 0.68: 25: 1, 01: 67: 0.37: ”: 18: 0.85: 36: 0.96: 61: 0.59:: 19: 0.91: 41: 0.90: 52: 0.79:: 20: 1.00: 50: 1.02: 69: 0.72: 27 95752

Example 2 Immunoenzymatic determination of molecular concentrations of CD4 and CD8 antigens in human blood using an antibody conjugated to alkaline phosphatase.

5 The assay is performed according to Example 1. The conjugate of monoclonal antibody and alkaline phosphatase is prepared according to the instructions of Boehringer Mannheim - Biochemica (Ref. 567 744). The cells to be determined and the conjugate are incubated for 1 hour, after which the activity of the immobilized enzyme is determined by adding a solution of 1 mg / ml paranitrophenyl phosphate in a buffer containing 1,2% diethanolamine in distilled water, the pH of which is adjusted to 9,8 with dilute hydrochloric acid. After two hours at 37 ° C, the optical density is measured with a spectrophotometer at 405 nm.

The results are calculated and reported in the same way as in Example 1.

Example 3 Immunoenzymatic Assay of Molecular Concentrations of CD4 and CD8 Antigens from Human Blood Using an Antibody Conjugated to Iodine-125.

Iodine-125-labeled antibody is prepared as described in F.C. Greenwood, V.M. Hunter et al., Biochem. J., 1963, 89, 114.

• 25 50 anti-CD4 or anti-CD8 monoclonal antibody in 50 phosphate buffered saline pH 7.2 is mixed with 37 MBq of iodine-125 in the form of the sodium salt, and a further 30 μΐ T -chloramine solution containing 0.33 mg of T-chloramine in 30 ml of PBS buffer. After stirring for 1 minute, the labeling reaction of the monoclonal antibody is stopped by adding 100 μ 100 sodium metabisulfite solution at a dose of 2.5 mg / ml. The solution thus prepared is passed through a PD-10 column (Pharmacia - Sephadex G25M), and eluting with a fraction containing the radiolabeled antibody. In the assay, 100 μl of a cell suspension prepared according to Example 1 of 95 95752 containing human blood mononuclear cells is pipetted into four wells. Then pipette 100 μΐ of the radioactive antibody dilution in PBS buffer containing 5% BSA so that 150 000 cpm enters each well; two wells are each loaded with 100 μ CD of anti-CD4-I-125 conjugate, and two wells with 100 μΐ of anti-CD8-I-125 conjugate each. Cell attachment is promoted by centrifuging the plate for 3 min at 150 x g, after incubating the plate for 1 hour at ambient temperature.

The wells are emptied by turning the plate. The wells are washed 4 times with 200 PBS / well. 75 μΐ IM of NaOH is then pipetted into each well. After 10 min, the contents of each well are collected in absorbent buffer, and the radioactivity is measured with a gamma counter (LKB multiwell counter).

The results are calculated and reported according to Example 1, but the optical densities are replaced by the counter results in dpm.

20 Example 4

Certificate of validity of the method.

CD4 and CD8 antigens of human T cells were determined from 20 human blood samples by the peroxidase immunological method described in Example 1. From the same blood samples, the ratio of the numbers of T4-positive and T8-positive cells was further determined by cytological calculation using a conventional technique (W.W. ERBER et al. Lancet, 1984, (8385), 1042-1045).

The correlation coefficient for the immunoenzymatically determined CD4 / CD8 ratio and the cytologically calculated T4 / T8 ratio is r = 0.72.

«·

In addition, from seven other human blood samples, the CD4 / CD8 ratio obtained by the immunoradiometric method of this invention and the iodine-125 label was compared with the T4 / T8 cell ratio obtained by cytological calculation. The correlation coefficient is r = 0.87.

95752 29

The correlation coefficients obtained in the two cases show that, despite the satisfactory consistency between the results obtained by the two methods, the information given for the two is not equivalent, which is obvious since the antigen assay method of the present invention takes into account not only the number of positive cells. but still further on the surface of the positive cells contained in each sample of that antigen density. Thus, the information obtained by the method of the present invention is considerably more complete than the information provided by the conventional reference method (W.W. ERBER et al. Lancet, 1984, (8385), 1042-1045).

Example 5 Immunoenzymatic determination of molecular concentrations of CD4 and CD8 antigens in T lymphocytes from human blood using a peroxidase-conjugated antibody.

The purpose of this example is to show that the time required for the assay can be reduced compared to the conditions shown in Example 1 by modifying the cell separation process on the one hand and the incubation times necessary in the immunocapture and cell labeling steps on the other hand.

A) Effect of total blood cell separation process.

. 25 a) Method based on short centrifugation: 0.5 ml of the blood sample to be determined is mixed with 1.5 ml of PBS buffer. 1.5 ml of Pharmacian Ficoll-Paque is added to a 5 ml hemolysis tube, and a blood sample diluted in PBS is added to the surface of the Ficoll layer. After centrifugation for 5 minutes 30, a 0.5 ml suspension ring containing mononuclear cells formed at 900 x g at ambient temperature is collected. To this sample is added 1.5 ml of PBS buffer.

(b) Erythrocyte lysis method: 35 Another rapid method for separating blood cells is to use a buffer that lyses red blood cells. The composition of the lysis buffer used is as follows: 95752 ammonium chloride: 8.29 g of potassium hydrogen carbonate: 1 g of the two sodium salts of ethylenediaminetetraacetic acid: 0.0307 g per liter of distilled water, pH adjusted to 7.3.

Mix 5 ml of lysis buffer and 0.250 ml of blood.

After stirring for 10 min, the mixture is centrifuged at 600 x g for 10 min. The resulting cell pellet is taken up in 1 ml of PBS buffer.

By determining the CD4 and CD8 antigens by the method of Example 1, and by comparing the reference method in Example 1 to the isolation of blood mononuclear cells, it has been demonstrated that both methods for isolating whole blood mononuclear cells can collect a total number of blood samples examined.

B) Effect of incubation time on cell immunosuppression and labeling.

To determine if the 1 hour delay used in Special Mark 1 20 can be reduced to speed up the assay, the results obtained from identical samples containing 20,000 mononuclear cells / well were compared when the delay of the cell capture and labeling steps was varied between 10 min and 1 hour.

. 25 Otherwise the conditions are the same as in Example 1, the results obtained are shown in Table 2.

li 3i 95752 TABLE 2 Optical density

Incubation -------------------------------------- 5 time CD4 antigen: CD8 antigen (in minutes) ----------------------------------------

Experiment: Blank *: Experiment: Blank * 10 0.322: 0.037: 0.329: 0.023 10 20 0.438: 0.049: 0.401: 0.025 30 0.620: 0.055: 0.498: 0.027 * These optical density values correspond to the reagent blank without cells. Reference values obtained with cells but without either conjugate or substrate are not greater than those shown.

These results show that: The non-specific signal (reagent blank) is weak, the lower the shorter the incubation time of the conjugate.

- Within 10 minutes, the specific signal reaches an analytically usable, accurate and reproducible value. This shows that it is possible to significantly shorten the assay time compared to the conditions of Example 1, without greatly reducing the accuracy of the results. In practice, considering the total time savings that can be achieved in blood sample preparation and immunocapture, the test kit and method of the invention allow the determination of CD4 and CD8 antigens in a whole blood-30 sample, 10 or 20 samples per 96-well plate, in less than 1 hour ). This assay time is shorter than can be achieved by any of the methods known today.

32 95752

Example 5

Immunoenzymatic determination of molar concentrations of CD5 antigens in human T lymphocytes from human blood using an acetylcholinesterase-conjugated antibody.

A) Preparation of assay kit a) Solid support.

A microtiter plate prepared according to Example 1 is used.

B) Acetylcholinesterase conjugate of a monoclonal antibody.

Electrophorus electricus acetylcholinesterase, prepared according to FR patent application no.

2,550,799.

15 This enzyme is coupled to an anti-CD5 antibody called ST 1, marketed by BIOSYS. The coupling is performed in YOSHITAKE et al., Eur.

J. Biochem. 101 (1979), 395-399.

20 c) Detection reagent.

This reagent simultaneously contains both the enzyme substrate (acetylthiocholine) and the Ellmann reagent and has the following composition:

- Acetylthiocholine 7.5 x 10'4 M

25 - 5,5'-Dithio-2-nitro benzoic acid (DTNB) 5 x 10'4M

% ·, IM in sodium phosphate buffer, pH 7.4 This solution is diluted 1/100 with distilled water before use.

B) Immunological method.

A) Mononuclear cells are separated using * the method described in Example 1.

b) Incubation of cells for immunocapture and labeling takes 20 minutes as in Example 5.

c) Detection and measurement of the coupled enzyme.

35 Add 100 μΐ of detection reagent to each well. The absorbance is measured after 45 min of incubation at 412 nm.

li 33 95752

Table 3 shows the measured optical densities for samples containing a known number of T cells. Under these experimental conditions, the blank of the reagents is particularly weak, corresponding to an optical density of 0.002 to 0.003.

5 TABLE 3 Number of T lymphocytes per well, and number of CD5 antigen molecules per 10 million wells 540 1,080 2,160 4,320 8,640 17,280 (32) (65) (130) (260) (520) (1040) 15 Optical density 0.030 0.060 0.138 0.260 0.470 0.850

The results show the special sensitivity of the method thus performed. In fact, an optical density of 0.030 obtained from a well containing 540 T lymphocytes (i.e., 32 million CD5 antigen-20 molecules), which is accurately measurable and 10 times higher than the background noise, corresponds to about 5 · 10'17 moles. CD5 antigen per assay well. This sensitivity is in the same range as the best known immunological assays using the most sensitive radiolabels.

Example 7

Determination of various surface antigens of activated human T lymphocytes.

T lymphocyte activation is a physiological process that starts early whenever the immune system. as in infectious pathological conditions, transplants and some dysimmune diseases. This natural process is also often initiated in vitro under laboratory conditions in a number of tests, such as mixed lymphocyte reactions or lymphoblastic

• · I

34 95752 in food transformations. In the latter case, polyclonal activation is induced using e.g. phytohemagglutinin (PHA), concanavalin-A or other lectins. Activation of T lymphocytes results in a marked increase in the expression of several membrane markers, especially for CD25 antigen (interleukin-2 receptor) or CD2 antigen. These antigens are excellent markers for the expression of the state of activation and their determination is of great interest both for clinical biology and for the laboratory assays described above, since in each case the use of radioactive reagents can be avoided.

Antigens are determined using the method set forth in Example 1 according to the specifications set forth in Table 4.

TABLE 4

Determination of surface antigens of activated T lymphocytes 20 To be determined Antibody used in immunocapture with peroxidase antigen, labeled antibody, marketer marketer

CD 2 ST 1, BIOSYS ST 11, BIOSYS

25 CD 25 ST 11, BIOSYS IOT 14, IMMUNOTECH

The following Table 5 shows the optical densities measured for 251103 mononuclear cells at 450 nm and has been compared without PHA stimulation and after 3 days of stimulation with the same cells. values obtained.

• · • · 35 95752 TABLE 5

Optical densities 5 ---------------------------------------------- ---------

Non-activated Activated Reagent-Antigen cells cells, 3 days blank CD 25 0.064 0.529 0.044 10 CD 2 0.129 0.768 0.027

The results show that the specific signal given by the 2 antigens studied is significantly increased by activation.

Example 8 Determination of CD22 and HLA-Dr (or HLA class II) antigens on the surface of B lymphocytes.

This example shows the determination of cell surface antigens for cells of the RAJI cell line which is J. Clln of PULVER-20 TAFT. Patol., 1965, 18, 261-274, a human B-lymphoid cell line.

For immunocapture of cells, either class II S antibody (BIOSYS) is determined in the assay wells according to Example 1 to determine CD22 antigens or. 25 SB4 antibody (BIOSYS) for the determination of class II HLA (or HLA-Dr) antigens.

a) CD22 antigen assay: SB22 (BIOSYS) antibody is labeled with iodine-125 according to the method described in Example 3. The CD22 anti-30 genes are determined as described in Example 3 for CD4 and CD8 antigens, by adding to the wells in the order:

RAJI line 5 · 104 cells followed by 105 cpm labeled SB

22 antibody. After one hour at 4 ° C, the wells are emptied by inverting the plate, after which each well is washed 4 times with 200 PBS buffer. Radioactivity bound to the cells is collected and measured according to Example 3. This gives a reading of 760 dpm for a well containing 5 x 104 RAJI cells and a reading of 50 dpm for the reagent blank (without cells).

5 b) HLA-Dr antigen assay:

105 cells and a peroxidase-labeled class II S antibody conjugate according to Example 1 are added sequentially to the wells. The assay is performed as described in Example 1. The value of the optical density is 10,840 and for the reagent blank without cells the value is 0.105.

Example 9

Use of the method according to the invention for the determination of antigens of T and B lymphocytes in human blood.

A) Preparation of plates: Microtiter plates prepared according to Example 1 are used, with the exception that at least most T-lymphocytes (BlOSYS class IS) and B-lymphocytes (BIOSYS class II) are adsorbed simultaneously to all wells of the plate. S) monoclonal antibodies capable of attachment, each used at a concentration of 5 pg / ml.

B) Immunological assay:

Mononuclear cells are separated from whole blood by Fi-coll under the conditions of Example 1 or 5, after which 80,000 mononuclear cells in 75 PBS buffer are added to each well. T lymphocytes are assayed in a portion of the wells with BiOSYS ST 11 antibody (anti-CD 2); B lymphocytes are determined from the remaining wells with BI0SYS SB 3 antibody (anti-CD 37), which recognizes all peripheral B lymphocytes. These antibodies are: pre-coupled to peroxidase according to the method set forth in Example 1. The actual assay is performed according to Example 1.

C) Results 35 The absolute numbers of T and B lymphocytes in the test sample are determined from standard

II

37 95752 formed by identical treatment of mononuclear cells from which T and B lymphocyte counts have been measured by a reference method.

Alternatively, the results of the test sample can also be expressed as molar concentrations of CD2 and CD37 antigens by appropriate standards.

Example 10

Determination of human platelet GpIIb-IIIa and CD9 antigens.

The assay is performed according to Example 3 using an iodine-125-labeled monoclonal antibody.

Platelets are separated from human blood by centrifugation in the presence of PLASMION perfusion solution (laboratory R. Bellon). Mix 5 ml of blood and 5 ml of PBS buffer and 10 ml of PLASMION in a test tube. The test tube is then centrifuged for 10 minutes at 1500 rpm. Platelets collected in the supernatant are collected by aspiration with a pipette. They are washed once with PBS 20 by mixing 1 volume of the flake suspension with 10 times the volume of PBS, after which the solution is centrifuged for 10 min at 1000 xg. Finally, the flake precipitate is collected and resuspended in PBS (2 ml). The flakes are counted and their concentration is adjusted to 2.5 million / ml PBS buffer.

In the GpIIb-IIIa antigen assay, platelets are immunocaptured with a 10B monoclonal antibody (Immunotech), and the GpIIb-IIIa antigen is labeled with a P2 monoclonal antibody (Immunotech).

In the CD9 antigen assay, a P2 monoclonal antibody is used for immunocapture, and a 10B2 monoclonal antibody is used for labeling. The dpm of a sample containing 200,000 platelets is measured.

The results are shown in Table 6.

38 95752 TABLE 6

Measured Comparison, Sample to be determined without cells 5 antigen (dpm) (dpm) CD 9 1 927 113

GpIIb-IIIa 2,891,238 10 Example 11

Determination of CD15 antigens in human granulocytes.

The CD15 antigen is a good specific marker for human-derived granulocytes (let alone polynuclear-15 set). This antigen allows these cells to be determined from the blood, as can all other subtypes of leukocytes. This antigen is also useful in the determination of urinary granulocytes, e.g. in urinary tract infections such as cystitis or pyelonephritis.

In this example, the assay is performed on whole blood granulocytes isolated as described below.

Granulocytes, as well as some mononuclear cells, are separated from erythrocytes as described in Example 10 for platelets, with the exception that the cell-containing test tube is allowed to stand at ambient temperature for 45 minutes and the leukocyte suspension is collected in 500 μl aliquots from the liquid surface.

Granulocytes are immobilized in the wells with a monoclonal antibody specific for CD45 antigen on the cell membrane of all leukocytes: »« anti-LCA antibody (BIOSYS) adsorbed on the wells of the titration plate according to Example 1 is used.

The assay uses an anti-CD15-SMY-15a monoclonal antibody (BIOSYS) labeled with peroxidase according to Example 1.

The total number of 12,500 cells thus added to the wells gives a granulocyte-specific signal for CD15 antigen of 0.616, after correcting for a coarse optical density ar-5 of 0.100 obtained with the cells and without entzyme jug. due to endogenous peroxidases of granulocytes.

Example 12

Evaluation of leukemia phenotype by immunoenzyme-10 method.

Phenotyping of tumor cells in a leukemia patient is a systematically performed diagnostic study to characterize the type and origin (T, B, granulocytes, myeloblasts, etc.) of the patient's leukemia cells. This study is classically performed by the immunofluorescence method using a selection of monoclonal antibodies, as well as by microscopy and counting of positive cells. Flow cytometry is also used to study cell labeling.

The use of monoclonal antibodies in the method of the invention allows the identification of large groups of clinically significant acute or chronic leukemias by providing quantitative information on the relative densities of the various antigens of interest.

• · - In this example, six peroxidase-conjugated monoclonal antibodies specific for antigens on the surface of leukemia cells were prepared: 30 - ST 1 and ST 11 (BIOSYS): anti-CD5 and anti-CD2 - SB 3 (BIOSYS): anti -CD37 - S-CALLA (SANOFI): anti-CALLA (or anti-CD10) - Class II S (BIOSYS): anti-HLA-Dr (or anti-HLA, class II) 35 - SMY 15 (BIOSYS): anti -CD15 • · 40 95752

A microtiter plate was prepared by pre-adsorbing on the plate a mixture of anti-CD45 (S-LCA, BIOSYS) and anti-HLA class I (S-class I, BIOSYS) monoclonal antibodies.

5 Using the six monoclonal antibodies selected above, the phenotype of chronic B-lymphoid leukemia (LLC-B) was evaluated and further characterized by a conventional method.

Table 7 below shows the optical densities measured at 492 nm for a total of 80,000 mononuclear cells in one well, or for control wells without cells. For control wells containing cells but no peroxidase-labeled antibody, an optical density of 0.110 was obtained.

15 TABLE 7

Optical density

Monoclonal Anti- --------------------------------- 20 Substance and Equivalent Sample of Cells Comparison Without Antigen with Cells of ST 11 ( CD2) 0.270 0.030 ST 1 (CD5) 1.350 0.014 25 SB 3 (CD37) 0.850 0.005 • ·

Thus, class II S (HLA-Dr) 1,640 0,010 SMY 15 (CD15) 0,235 0,007 CALLA (CD10) 0,160 0,002 30 The leukemia cells examined are rich in CD5,: CD37 and H LA class II antigens but do not contain or contain very low levels of CD15, CALLA and CD2 antigens, which is typical of LLC-B.

M

41 95752

Example 13

Determination of antigens associated with urinary tract cancers.

The assay kit and methods of this invention have been used in particular for the detection of tumor cells, especially in urinary tract tumors, and are well suited for mass studies of at-risk groups, e.g., chemical workers or other exposed groups.

This example demonstrates the application of the invention to the antigen assay of RT4 cell cells associated with bladder cancer due to human urinary papilloma (C.C. Rigby and L.M. Franks, Brit. J. Cancer, 1970, 24, 746-754).

Plates for immunosuppression of cells are prepared according to Example 1 by adsorbing into the wells a class I (BIOSYS) monoclonal antibody which recognizes a class I HLA antigen and which is capable of binding all epithelial cells.

Labeling conjugates are prepared by the method of Example 1 (enzymatic labeling) or Example 3 (radioisotopic labeling) from a 12F 6 monoclonal antibody (Sanofi) that recognizes a bladder cancer-associated antigen.

In the assay, 75 μΐ of cell suspension (i.e., 5011O3 cells / well) is dispensed into four wells. Peroxidase-coupled 12 F 6 monoclonal antibody conjugate (SANOFI) is added to two wells, and 12 F 6 antibody coupled to iodine-125 (100,000 30 cpm / well) is added to the other two wells. Table 8 shows the results obtained by immunoenzymate or radioimmunological method.

· «42 95752 TABLE 8

Labeled Optical density (492 nm) or cpm Positive monoclonal ------------------------------ and non-specific Sample 5 Comparison fisen sig antibody nal ratio 12 F 6 0.782 0.078 10.0 peroxidase 10 12 F 6 1 612 350 4.6 iodine 125

Example 14 Enzyme-linked immunosorbent assay for Candida albicans yeast membrane antigen.

Candida albicans (serotype 17) cells are from the collections of the Pasteur Institute in Paris. Yeast cells were grown for 18 hours on conventional solid synthetic medium. Cells were counted and a cell suspension was prepared containing 105 cells / ml PBS buffer.

The anti-Candida albicans monoclonal antibodies used were prepared by lymphocyte hybridization according to the study of T. Chardes, Faculty de Pharmacie,. 25 University de Montpellier I, 1988.

* ·

CA4 antibody was used for immunocapture, while peroxidase-labeled CA12 antibody was used for the assay under the conditions of Example 1.

Optical density measured at 492 nm for 30 wells containing cells and a control; the values were 1.025 and 0.080, respectively.

Example 15

Immunoenzymatic assay with magnetic beads: determination of CD5-an-35 genes of human T lymphocytes with acetylcholinesterase-labeled 1

II

i * 43 95752 antibody conjugate on a special medium consisting of magnetic beads.

The medium used to capture the cells consisted of magnetic beads, the mark DYNABEADS. BIOSYS market-5 spheres, ref. DYN-11101, a surface anti-CD2 antibody, was able to attach to all T lymphocytes in the test sample. Prior to use, the beads were further treated with anti-CD2 antibody solution to improve test performance.

To this end, 1 ml of antibody solution at 25 pg / ml in PBS buffer was mixed with 10 μl of the bead suspension, and the solution was gently stirred for 1 hour.

The beads were then washed 4 times with PBS buffer and stored in 4 ml of PBS.

In the assay, the following was added to a 5 ml tube in the following order: 200 μΐ of bead suspension, 80 μΐ of Ficoll-Paque (PHARMACIA) whole blood mononuclear cells, and another 280 μΐ of ST1 antibody conjugate labeled with acetyl-20 cholinesterase as used in Example 6. .

The mixture was gently stirred for 1 hour, after which the beads were separated with a magnet and the cells attached to the beads were washed with PBS buffer (5 washes).

The acetylcholinesterase bound to the cells was tasted in the same manner as in Example 6. The signal value obtained was 0.168 in the presence of labeled cells; the blank obtained without cells was 0.062.

i * 4

> · I

Claims (37)

95752 An assay kit for the immunological determination of a surface antigen specific to a cell population or a subset thereof, characterized in that its components are: a) a solid support to which one or more monoclonal antibodies have been attached by covalent bonding or physical adsorption; a cell population to be measured against something other than said typical surface antigen; b) one or more solutions, each containing a monoclonal antibody labeled with a radiolabel or enzymatic label specific for an antigen specific to the cell population or subset thereof to be measured; (c) if the antibody is labeled with an enzyme, one or more solutions containing the necessary reagents for detecting the activity of the enzyme; (d) possibly an additional component consisting of wash buffer and / or samples to compare and control the quality of the measurement. Kit according to Claim 1, characterized in that component (a) is a solid support which is a microtiter plate in which antibodies or substances are intended to immobilize the cells containing the antigen to be determined from among the populations or subgroups. Putty according to Claim 1, characterized in that component (a) is a special magnetic carrier. Kit according to one of Claims 1 to 3, characterized in that component (a) is a solid support to which one or more anti-HLA class I monoclonal antibodies have been attached. Kit according to one of Claims 1 to 3, characterized in that the monoclonal antibodies of component (b) 95752 are labeled with a radioisotope label, which is Iodine-125 or Iodine-131. Kit according to one of Claims 1 to 3, characterized in that the monoclonal antibodies of component (b) are labeled with an enzyme label. Kit according to Claim 6, characterized in that the monoclonal antibodies of component (b) are labeled with peroxidase and component (c) definitely contains oxidized water and a chromogen, which is ortho-phenylenediamine, 2,2'-azino- bis- (3-ethylbenzothiazoline-6-sulfonic) acid, 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole or a water-soluble salt thereof. Kit according to Claim 6, characterized in that the monoclonal antibody of component (b) is labeled with alkaline phosphatase and component (c) contains para-nitrophenyl phosphate, 4-methylumbelliferyl or 5-bromo-4-chloro -3-thia-indolyylifosfaat. Kit according to Claim 6, characterized in that the monoclonal antibody of component (b) is labeled with beta-galactosidase and component (c) contains ortho-nitrophenyl-beta-D-galactopyranoside or 4-methylumbelliferyl-beta-D -galactopyrano-25. Kit according to Claim 6, characterized in that the monoclonal antibody of component (b) is labeled with acetylcholinesterase and component (c) must contain acetylthiocholine and 5,5'-dithio-2-nitrobenzoic acid or one of their water-soluble salts. . Kit according to Claim 1, characterized in that it is suitable for the determination of any one or more surface antigens from human blood particles, such as leukocytes, lymphocytes, T lymphocytes, B lymphocytes, granulocytes and platelets. Kit according to Claim 11, characterized in that it is suitable for the determination of CD4 or CD8 antigens from human T lymphocytes containing these antigens, called T4 or T8 lymphocytes. Kit according to Claim 1, characterized in that it is suitable for determining any one or more surface antigens from a pathogenic microorganism. Kit according to Claim 13, characterized in that the pathogenic microorganism is Candida albicans. The kit of claim 1, characterized in that it is suitable for the determination of any one or more membrane antigens from tumor cells. Kit according to Claim 15, characterized in that the tumor cells are urinary tract cancer cells or malignant haemopathic cells. Kit according to Claim 1 or 2, characterized in that component (a) is a microtiter plate in which one or more monoclonal antibodies specific for surface antigens of a cell population are attached to the wells and component (b) consists of a plurality of solutions, each of which contains a monoclonal antibody specific for a cell antigen surface antigen, which is the major surface antigen of the immobilized cell population. A kit according to claim 17, characterized in that it is suitable for the determination of specific antigens of subgroups of T, T4, T8 and B lymphocytes. Kit according to Claim 17, characterized in that it is suitable for characterizing and determining the various antigens which form the surface antigenic epitope of malignant haematopathic tumor cells. 20. A method for the immunological measurement of a surface antigen specific to a cell population or subgroup 5, characterized in that the method comprises: a) immobilizing a cell population or subgroup thereof containing an antigen to be determined on a solid support using one or more monoclonal antibodies; , which is fixed in advance to the covalent bond or physical adsorption to the carrier, and which identifies an antigen in the cell population that is not the same as the antigen to be determined, and at the same time the cell population or subset to be determined is labeled with at least one monoclonal antibody specific for the antigen to be determined, radiolabeled, ) the incubation time is monitored; c) washing the support to remove unbound cells and excess antibody; (d) when the antibody is labeled with the enzyme, add the necessary or necessary reagents (substrate or chromogen) to detect enzyme activity; (e) read the results either by determining radioactivity or by measuring with a light signal (color formation or fluorescence) and possibly by comparison with a standard image. Assay method according to Claim 20, characterized in that the solid support is as defined in Claims 1 or 2. The assay method according to claim 20 or 21, characterized in that the monoclonal antibody used for the assay contains an enzyme label. The assay method according to claim 22, characterized in that the enzymatic label 95752 is peroxidase and the enzyme activity is measured with oxidized water and a chromogen which is ortho-phenylenediamine, 2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulphate). -phonic) acid, 3,3'-diaminobenzidine, 3-amino-9-ethyl-5-carbazole or a water-soluble salt thereof. The assay method according to claim 22, characterized in that the enzymatic label is alkaline phosphatase and the enzyme activity is measured with para-nitrophenyl phosphate, 4-methylumbelliferyl 10 or 5-bromo-4-chloro-3-indolyl phosphate. The assay method according to claim 22, characterized in that the enzymatic label is beta-galactosidase and the enzyme activity is measured with ortho-nitrophenyl-beta-D-galactopyranoside or 4-methylbelliferyl-beta-D-galactopyranoside. Assay according to Claim 22, characterized in that the enzymatic label is acetylcholinesterase, the enzyme activity is measured with acetylthiocholine and 5,5'-dithio-2-nitrobenzoic acid or one of its water-soluble salts is used as chromogen. Assay method according to Claim 20 or 21, characterized in that the monoclonal antibody used for the assay is labeled with a radioisotope such as iodine-125 or iodine-131. The method of claim 20, wherein the monoclonal antibodies bound or bound to the solid support are HLA class I antibodies. A method according to claim 20, characterized in that it is suitable for the determination of one or more surface antigens from human blood particles. 30 for the determination of specific antigens of B lymphocyte subsets. A method according to claim 20, characterized in that the human blood particles are leukocytes, lymphocytes, T lymphocytes, T lymphocytes containing a CD4 marker, called T4 lymphocytes, T lymphocytes containing a CD8 marker. , called T8 lymphocytes, B lymphocytes, granulocytes, or platelets. The method according to claim 20, characterized in that it is suitable for the determination of any one or more surface antigens from a pathogenic microorganism. The method according to claim 31, characterized in that the pathogenic microorganism is Candida albicans. The method of claim 20, characterized in that it is suitable for the determination of any one or more membrane antigens from tumor cells. The method of claim 33, characterized in that the tumor cells are urinary tract cancer cells or malignant hemopathic cells. A method according to claim 20 or 21, characterized in that the cell population is immobilized on a microtiter plate with one or more monoclonal antibodies specific for the surface antigen of this population and at the same time a subset of the attached population is directly labeled with several solutions, each containing one assay. a monoclonal antibody specific for one surface antigen of the subgroup. A method according to claim 35, characterized in that it is suitable for T, T4, T8 and 37. A method according to claim 35, characterized in that it can be used to characterize and determine various antigens which form a surface antigenic epitope of malignant haemopathic tumor cells. 95752