DK174032B1 - Kit as well as immunometric dosing method that can be applied to 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

in DK 174032 B1

The present invention relates to an equipment and method for immunometric determination using this equipment for the determination of surface antigens characteristic of cell populations or cell subpopulations.

The method of immunometric determination and the corresponding equipment is also intended for the determination of the cells even by intermediate determination of their surface antigens. These provisions are useful in diagnostics.

10 The knowledge of antigens or markers on the cell surface has made great progress in the practical application of lymphocyte hybridization and the discovery of monoclonal antibodies by Koehler and Milstein (Nature, 1975, 256, 495-497). In particular, the monoclonal antibodies have enabled visualization and analysis of surface markers or membrane antigens on cells of the most diverse origins. These markers (or antigens) can be of different character: proteins, glycoproteins or giyocipids. The desired characterizations mainly apply to tissue or organ markers, 20 markers of differentiation or activation states of normal cells, and identification or typing of normal cells or cancer cells. A particularly important area of application is the study of blood cell lines (erythrocyte, megakaryocyte, granulocyte, monocyte, lymphocyte).

25

Thus, the monoclonal antibodies have e.g. enabled to determine the respective surface properties of lymphocytes T and B. The corresponding markers alone or in combination identify differentiation stages and functional specializations of lymphocytes. According to international convention, the surface markers of leukocytes have been classified into groups or classes of differentiation (CD) defined by subcommittee IUIS-OMS, 1984 and described in the World Health Organization Bulletin 1984, 62 (5), 813-815.

35

The identification of these markers, made possible by monoclonal antibodies, has made it possible to arrive at their structure and their biological functions. Molecules of the markers CD4 and CD8 take e.g. part in 1 eukocyte adhesion functions and located on the surface of T lymphocytes and have auxiliary and induction function (marker CD4) or a cytotoxic and suppressive function 5 (marker CD8, respectively).

The establishment of this knowledge has made it possible to utilize these markers thanks to the antibodies they recognize to diagnose and follow various pathologies, especially possible hemopathies (leukemias, lymphomas, etc.) and conditions of immune system dysfunction (auto immune disorders , innate or acquired immunological deficiencies, such as SIDA, etc.) (BRET0N-G0RIUS et VAINCHENKER, Le Biologiste, 1987, XXI, nos. 167, 63-70, SHAW, Immunology today, 1987, 8 (1), 1-3) .

15

Monoclonal antibodies are today irreplaceable tools for the clinical biology used for cell analysis.

Methods exist for counting cells that utilize the labeling of their surface antigens, but these methods are often lengthy, complicated and difficult to perform, and their results are sometimes uncertain.

The known methods used to measure the normal or modified expression of the antigens on the surface of the cell can be divided into two groups. In the first group, the antigens are measured by complicated and specialized laboratory equipment based on flow cytometry (see, in particular, PONCELET et al., J. Immunol. Methods, 1985, 85, 65-74) or the quantitative microscopy techniques. (POULTER et al., J. Immunol. Methods, 1987, 98, 227-234). The performance of these assays for cell antigens is based on the measurement of signals carried by anti-cell antibodies coupled directly or indirectly to a reactive marker with fluorescent substances (or fluorochromes) such as fluorescein sothiocyanate or rhodamine, or enzymes such as peroxydase or alkaline phosphatase. Use of these fluorescent reagents or enzymatic reagents together with appropriate washing steps then results in the emergence of fluorescents or stains that are strictly confined to the cell membranes and do not disperse in the surrounding environment. The current use of these laboratory methods is limited by the necessity of a costly and specialized apparatus (fluorescence microscope, optionally together with a bi 1 joint analyzer, cryostat and flow cytometer). Furthermore, the analysis and interpretation of the immuno-markers of the cells by these methods necessitates the competence of a cytological specialist.

10

Another group of methods for measuring antigens is based on the quantitative assessment of markers of the total cell population. These methods allow the measurement of antigens with a label either directly or indirectly, which is then most frequently performed in two, three or four steps. In all cases, the reagent used from the last marking step carries a probe which is either isotopic in nature e.g. iodine 125 for a radioimmunoassay (BROW et al., J. Immunol. Methods, 1979, 31, 201 - STOCKER et HEUSSER, J. Immunol. Methods, 1979, 20 26, 87-95) or an enzyme for the determination of enzyme geometry. type, most commonly peroxidase, alkaline phosphatase, or β-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 latter group are rather impractical, cumbersome and dangerous to use due to the necessity of numerous washes and centrifugations of cell material. It is sometimes necessary to extract the colored medium resulting from the enzymatic reaction for the final spectrophotometric measurement. Finally, the chemical fixation of the cells most commonly used causes irreversible destruction of certain antigens that are particularly sensitive to conventional chemical fixatives such as glutaraldehyde or methanol (DROVER et al., J. Immunol. Methods, 1986, 90 , 275-281).

It is known from the literature that an antigen carrying multiple antigenic determinants can be determined, viz. several epitopes by fixating this antigen with one of its epitopes by virtue of an antibody immobil1 in the sera of a solid support, and by binding to another epitope of the antigen another antibody carrying an enzymatic marker or radioisotope marker enabling measurement.

Such a technique, often called sandwich technique, is particularly described in French Patent Application Nos. 2,487,983, 2,500.

165 and EP 119,736. None of these documents discloses the application of this technique to whole cells, although the word "cell" is sometimes included in the list of antigens to which the process may be used.

In the above patents, the various antigens subject to the examples are all exclusively protein molecules that are soluble in water and physiological fluids such as hormones, enzymes or circulating tumor markers. In contrast, it is clear that a cell is not a molecule and differs at least by a significantly larger size 20 and in that it is not soluble in physiological media. To this day, the sandwich technique has therefore never been applied to whole cells.

In addition, immunoprecipitation of cells on a solid support is described in patent application WO 86/02091, the purpose of which is to remove unwanted cells in bone marrow specimens intended for transplants. According to this patent application, the capture of the cells is carried out on liquid microspheres and necessitates that the antibody used be associated with the solid carrier with a complex macromolecular structure called net relay, which is capable of ensuring a favorable orientation of the antibody relative to it. corresponding cell antigen. In this application no application of the technique for quantitative determination of an antigen has been demonstrated.

35

Immune capture of cells is also described in application WO 84/03151 for an analytical application. In this application, the purpose is to identify tissue types to which the examined cells belong (an operation commonly referred to as typing HLA). The capture of the cells is by virtue of antibodies located according to a particular geometry of highly specialized carriers (in microscope 1ame11s). The results are obtained by simple visual observation of the carrier and leading to the answers all or nothing. Thus, the previously described systems for immunoprecipitation of cells do not lead to analytical applications that allow quantitative determination of an antigen expressed on the membrane of particular cells. Furthermore, all of these systems may lack specificity because they are based on recognition of cells by a single antibody.

The assay method which is the subject of the present invention has significant advantages over all the prior art and prior art techniques, because it allows quantitatively to measure all antigen in a cell population in a single assay step. This assay is performed on cells which are not subjected to any chemical or physical intervention and which are in their complete physiological state. Furthermore, the assay method of the invention has the properties of very high specificity, which is peculiar to dual immunological recognition systems utilizing 2 different antibodies specific for 2 different antigens carried by the same cell. This process is simple, fast and reproducible. It is well suited for the analysis of a large number of samples, enabling its use for diagnostic purposes in clinical, biological 1 aborator i s in high yield.

Thus, the present invention relates to an equipment for immunometric determination of at least one surface antigen which is characteristic of a population or subpopulation of cells and comprising as constituents *.

a) a solid carrier upon which, by covalent binding or by physical adsorption, one or more monoclonal antibodies directed against surface antigens of the cell population examined, in addition to said characteristic antigen, are intended to immobilize the carrier, including the cells contained in 6 DK 174032 B1 cells of the subpopulation carrying the antigen to be determined; b) at least one solution comprising a specific monoclonal antibody for the characteristic antigen in the cell population or cell subpopulation carrying the antigen to be determined and labeled with a radioisotope probe or enzymatic probe; (C) in the case of monoclonal antibodies labeled with an enzymatic probe, an enzyme assay, namely one or more solutions containing the enzyme substrate and, where appropriate, one or more reagents necessary to detect the activity of the enzyme.

15

The term cell used in the present specification and claims includes human cells, animal cells, protozoa cells and microorganism cells (bacteria or fungi). As far as the blood cells are concerned, the invention includes nucleated shaped elements such as leukocytes and nucleated shaped elements such as red blood cells or platelets.

The assay method of the invention is applicable to whole cells, i.e. non-lighted cells.

25

The cells have not undergone any physical or chemical intervention and are used in a state of complete physiological wholeness. This situation provides the best guarantee for the integrity of the membrane markers chosen as the destination.

30

As a solid support, any system suitable for manipulating cell suspensions and preferably test tubes, particulate magnetic carriers or rigid or soft microtitration plates of polyethylene, polystyrene, polyvinyl chloride or nitrocellulose having micro depressions can be used.

The monoclonal antibodies intended for immobilization of the cells may be fixed on the solid supports either by covalent chemical bonding or by physical adsorption according to the conventional well known technique such as that described by STOCKER and HEUSSER, J. Immunol. Methods, 1979, Vol. 26, pages 87-95. Advantageously, the carrier may be pre-saturated by a protein.

According to the invention, the monoclonal antibody (s) fixed on the solid support should enable capture of the cell population in which is located the cell population (s) carrying the antigens to be determined. When this population is made up of human cells, the preferred 10 monoclonal antibodies for capture are the class I anti-HLA antibodies, which are specific for the common portion of the antigens HLA A, B and C found on the 1 eucocytes, and for numerous others. of the cell lines of the organism. Among these antibodies, especially those called S-class I, which are marketed by BIOSYS, are preferred.

In other cases where the cells examined are human cells and in all cases when the cells are not human, monoclonal antibodies suitable for the cell type examined can also be used for immuno-capture of the invention.

The term "a monoclonal antibody labeled with a radioisotope probe" means that the monoclonal antibody carries either an appropriate element of the structure, e.g. the incoming tyrosine residues or on a suitable radical which has been fixed thereon a radioactive isotope which allows it to be determined by counting the associated radioactivity.

The term "a monoclonal antibody labeled with an enzymatic probe" means that the monoclonal antibody is coupled with an enzyme which, together with the use of appropriate reagents, allows a quantitative measurement of this monoclonal antibody.

The substrate and reagents are selected such that the final reaction product or final product from the sequence of reactions elicited by the enzyme using these substances is either a colored or fluorescent substance which diffuses into the liquid medium. which surround the cells and which are subject to the final spectrophotometry or fluorescence metric measurement, or a colored substance which is insoluble and which settles on the cells and walls on which they are fixed and which can be subjected to for either a photometric: reflection measurement or a visual assessment, possibly in relation to a calibrated color scale.

The assay equipment may contain as a further component a buffer solution designed for washing the solid support after immobilization and labeling the cells with the antibody (s) carrying the selected probe.

The assay equipment may also contain as additional constituents the samples necessary for calibrating the assay and for checking the quality of the assay.

20

The invention also relates to a method for immunometric determination of surface antigens on a cell population or cell subpopulation, characterized in that it comprises: a single step for specific immobilization or immunoprecipitation of a cell population on the solid support using of one or more monoclonal antibodies pre-fixed by covalent binding or by physical adsorption on the carrier and capable of recognizing an antigen present on the surface of the cells, in addition to the antigen to be determined and simultaneously direct labeling of the surface antigen, to be determined and belonging to the immobia 1 in the serous cell population or to one of its subpopulations, with a monoclonal antibody specific for this antigen to be determined, which monoclonal antibody carries a radioisotope probe or alternative enzymatic probe, 9 DK 174032 B1 an incubation period to allow immuno-capture and as well Identical labeling, washing the solid support to remove unwanted cells or non-immobia 1 in serous cells, and excess monoclonal antibody bearing the radioisotope or enzymatic probe, actual measurement of the antigen to be determined in the labeled cell population or cell subpopulation by counting the fixed radioactivity or, alternatively, after treating the medium with the substrate for the enzyme and optionally one or more appropriate auxiliary reagents, by transmission photometric or reflection photometric measurement or by measuring fluorescence emission.

15

The assay equipment and immunometric method of the invention are preferably used to determine surface antigens of shaped elements of human blood, especially leukocytes, and especially lymphocytes, T lymphocytes, T4 lymphocytes, T8-1 lymphocytes, B lymphocytes and granulocytes. , monocytes and platelets.

Another preferred application is the determination of the surface antigens on pathogenic microorganisms, e.g. Candida albi-cans.

In addition, the assay equipment and immunometric method of the invention are especially useful for the determination of surface antigens on tumor cells, especially cancer cells from the urinary tract and malignant hemopathic cells.

The assay equipment and the immunometric method of the invention make it possible to measure signals (radioactivity or absorbed or emitted light) which depend both on the number of cells found in the cell population examined and on the measured antigen density on the surface of the cells. Measurement of these signals allows a quantitative assessment of the total number of molecules of this antigen carried by the cell population or cell subpopulation examined in which this antigen has a structural or functional role.

For example, in the case of leukocyte markers that are particularly important in hematology, for example, in healthy individuals, in most situations, the mean value of antigen density does not vary greatly from one sample to another for the same cell population. . There is then a good correlation between the cytological count of the cells carrying the antigen in question and the signal measured according to the invention which is proportional to the number of antigen molecules present in the assay examined. On the contrary, in certain pathological conditions, the density of surface antigens may vary for the same cell population, without the number or amount of positive cells varying considerably. Such pathological conditions are more effectively detected using the immunometric method of the invention or using the equipment of the invention than by a conventional method of cytologic counting.

20

Another application of the invention is obtained by selecting as a solid carrier a microtiter titre plate. The assay equipment and immunometric method of the invention can then be advantageously utilized for the determination on a single plate of a series of 25 surface antigens characteristic of various subpopulations constituting the cell population examined. For this use, one can use microtitration plates prepared for use on which are pre-fixed one or more monoclonal antibodies capable of retaining all the cells of the studied population and, on the other, a series of monoclonal antibodies, coupled to a suitable marker, each specific to an antigen characteristic of one of the subpopulations to be evaluated. Thus, by a single manipulation and 35 on the same carrier, quantitative determination of all the antigens needed to characterize the selected subpopulation in ounces can be obtained.

11 DK 174032 B1

This application of the present invention is illustrated by the characterization of the antigenic equipment of cells of interest in clinical biology. A first case is represented by the determination of tissue groups which characterize a given individual and are known under the usual term typing HLA.

Another case is represented by the typing of tumor cells, especially for sufferers who reach malignant hemopathies such as leu-10 germs and lymphomas. This diagnostic study, which is systematically practiced, consists in characterizing the type and origin of the patient's tumor cells in the presence or absence on the cells of a variety of surface antigens that are appropriately selected.

15

By using the equipment of the invention, which comprises a microtitration plate on which is pre-fixed one or more monoclonal antibodies capable of fixing all the cells of the studied population, and solutions of various monoclonal antibodies labeled with a enzymatic or radioisotope probes, each specific to an antigen found on the tumor cells, can be identified and quantitatively assessed for the antigens characteristic of the patient's population of tumor cells, thereby attributing them to one of the major cancer groups and especially malignant hemopathies which is characterized clinically. Thus, application of the method of the invention enables a single carrier to perform a qualitative and quantitative study of the antigenic equipment of the tumor cells quickly.

30

To illustrate another application of the invention may be mentioned the case of human T lymphocytes, of which there are in particular 2 subpopulations of cells: lymphocytes characteristic of the presence of marker CD4, which is called positive T4 lymphocytes or merely T4 lymphocytes and lymphocytes that are characteristic of the presence of the marker CD8 and which are called positive T8 lymphocytes (or T8 lymphocytes).

12 DK 174032 B1 Measurement of the numerical relationship T4 / T8 has great diagnostic interest in clinical biology. Indeed, changes in the T4 / T8 ratio are known to occur through various influences of the immune system, such as immune-disruptive diseases, 5 chronic infectious diseases, viral infections, and especially HI V diseases (SIDA virus).

The assay equipment and immunometric method of the invention can be used to determine antigens which are collectively characteristic of the population of T lymphocytes and / or antigens characteristic of subpopulations of T4 and T8 lymphocytes. In this case, specific immobilising of T lymphocytes from the tested sample is performed on a solid support, and at the same time direct labeling of surface antigens on the T4 lymphocytes by a monoclonal antibody anti-CD4 carrying a radio in soot or enzymatic probe. Similarly, direct labeling of surfactant in genes on T8 lymphocytes is performed with a monoclonal antibody anti-CD8 carrying a suitable probe.

20

To determine total T lymphocytes, a monoclonal antibody anti-CD7 (also referred to as anti-T2) is preferably used which carries a suitable probe.

To obtain specific immobilization of the T lymphocytes of the sample, one or more monoclonal antibodies capable of recognizing all or the T cells of the sample alone are preferably used, as is the case with the common anti-leukocyte antibodies (or anti-leukocyte antibodies). CD45) or antibodies that recognize the entire T population (called "pan T"), such as the anti-CD2 (or anti-T11), anti-CD5 (or anti-T1), anti-CD7 (or anti-T1) antibodies. T2) or other pan-T antibodies that have not yet been assigned to a differentiation class according to the OMS criteria.

Advantageously, the immunometric method of the invention can be used to determine antigens characteristic of the population of T lymphocytes and T4 and T8 lymphocytes on several portions of the same solid support. Measurement of the signals by counting radioactivity, photometric measurement in transmission or reflection or fluorescence measurement allows the numerical ratio 5 CD4 / CD8 to be calculated easily and directly.

Similarly, on the same solid support, the subpopulations called T-1 lymphocytes and B-1 lymphocytes, which make up the entire lymphocyte range, can be determined.

10

One can, for example. upon adsorption, a monoclonal antibody or a mixture of monoconal antibodies specific for all the surface antigens on T cells fix onto the walls of myocardial depressions. These monoclonal antibodies make it possible later to immobilize in the cropland the entire population of T cells in the sample under study. Plates made in this way can be lyophilized and stored preferably at 4 ° C. This step can be carried out industrially, and thus one can have plates ready for use in 20 assay equipment applicable either to total T lymphocytes or to any subpopulation of T lymphocytes.

Samples containing cells to be determined and derived from blood or other appropriate biological fluid, whether normal or pathological, may be used as they are or after preparation, especially by density gradient centrifugation in known ways and especially in medium of heavy density, such as FICOLL-PAQUE sold by Pharmacia. For the determination of blood lymphocytes, one may also treat the blood sample to be determined with a solution called a light cushion which illuminates the red blood cells.

Equal portions of the cell suspension are placed in contact with the solid support, e.g. in micron wells in a pre-prepared microtiter plate at the same time as the solution forming part of the assay equipment containing the monoclonal antibody specific to the intended cell population and carrying a suitable probe, i. a radioisotope or enzymatic tracer. Thus, a radioisotope probe can be realized e.g. by labeling the monoclonal antibody with iodine 125 or iodine 131, e.g. in the presence of chloroamine T in a known manner (FC GREENWOOD, WM HUNTER et al., 5 Biochem. J., 1963, 89, 114), or an enzymatic probe may be realized by linking the monoclonal antibody to an enzyme such as alkaline phosphatase, peroxidase, β-ga1, act in dase or acetylcholinesterase in ways known per se (see, for example, M. O'SULLIVAN, Methods in Enzymology, 1981, 73, 147) 10 or by a method derived thereof. In order to avoid certain inconveniences associated with the manipulation of radioactive substances and the limited duration of the validity of the reagents, the enzymes are used in some cases over radioisotope probes.

15

The incubation period, i.e. the time required for the immobilization and simultaneous labeling of the cells is preferably less than or equal to 1 hour. During this time, the solid support may optionally be centrifuged to improve the immobilisation of the cells. The solid support, e.g. in the scrap plate, is then washed to simultaneously remove unfixed cells and excess monoclonal antibody carrying an enzymatic or radioisotope probe.

When using a radioisotope probe e.g. iodine 125, the radioactivity associated with the cells is counted in a gamma counter in any appropriate manner, and e.g. after solubilization of the cells with an alkaline solution (e.g., a sodium hydroxide solution) and recovery of the solution in resorbing radioactivity by means of an absorbent buffer.

When using an enzymatic probe for the monoclonal antibody, a colored or fluorescent product is obtained by attaching to the solid carrier, the cell population carrying the antigen to be determined, a solution containing the enzyme substrate and one or more auxiliary reagents which ultimately enable, as a reaction product, to obtain either a colored product which is soluble in the medium or a colored product which is insoluble or a soluble fluorescent product, as is explained above. The light signal derived from the samples thus treated is then measured by apparatus suitable for each case: transmission photometer or reflection photometer or fluorimeter, respectively. When the solid carrier is less than one meter in length, the reading of the illuminating signals can be carried out sequentially in all the grooves in the same plate using automated conventional type readers in biology laboratories, e.g. the Titer-tek plate reader or the Fluoroscan plate reader for spectrophotometric or fluorometric readings, respectively.

15 When used as an enzymatic probe of alkaline phosphatase, the coupling of this enzyme is effected by the monoclonal antibody following the method proposed by Boehringer Mannheim - Biochemistry. The preferred substrates for this enzyme are paranotrophenyl phosphate for a final spectrophotometric reading 20 or 4-methyl umbel 1 in ferry 1 phosphate for a liquid metric reading or 5-bromo-4-chloro-3-indolyl phosphate to obtain an insoluble dye. reaction product. It is also possible to use as an enzymatic probe β-ga1 actos idase, the preferred substrates of which are orthonitrophenyl-β-D-galactopyranoside or 4-methylbum 1 in feryl / 3-D-galacto py ranoside.

Preferably, the monoclonal antibodies can be coupled to the peroxidase. In this case, the coupling process is derived from that described by M.B. WILSON and P.K. NAKANE in Immunofluoro-rescence and Related Staining Techniques. W. Knapp, K. Kolu-bar, 6. Wick Publishers Elsevier / North Holland, Amsterdam, 1978, pages 215-224. The modifications introduced in relation to the first advancement in enzyme conjugation protocol relate to the following points: the molar ratio peroxydase / antibody is equal to 3, as opposed to 2 in the protocol, a less advanced one. oxidation of the peroxydase glue moieties by a 33¾ decrease of the proposed sodium periodate concentration.

The reagents used to detect peroxydase conjugated with monoclonal antibody include oxygenated water, enzyme substrate and a suitable chromogen, e.g. ortho-phenylenediamine or 2,2'-azino-bis- (3-ethyl-6-benzothiazoline sulfonic acid) or ABTS to obtain a colored s 1 reaction product which is soluble in the medium or 3,3 '-diaminobenzidine or 1' -amino-3-ethyl-9-carbazole or 4-chloro-α-naphthol to obtain a final insoluble reaction product or parahydroxyphenyl1-propionic acid to obtain a fluorescent reaction product soluble in the medium.

In a preferred embodiment, the equipment of the invention for the determination of antigens characteristic of T lymphocytes, T4-1 lymphocytes and T8 lymphocytes comprises: a) a microtitration plate in recesses fixed to one or more monoclonal antibodies, anti-lymphocytes T.

bl) A solution containing at least one anti-lymphocyte monoclonal antibody T labeled with peroxidase.

B2) A solution containing at least one monoclonal antibody anti-antigen CD4 labeled with peroxidase, b3) A solution containing at least one monoclonal antibody 30 anti-antigen CD8 labeled with peroxidase.

cl) A solution containing oxygenated water, enzyme substrate in a suitable buffer.

C2) A solution containing the chromogen used to reveal the expression of the enzyme's activity.

Another preferred embodiment of the invention is the use of monoclonal antibodies coupled with acetylcholine in esterase.

Acetylcholinesterase is coupled to the antibody, preferably using a method derived from that described in 5 French Patent No. 2,550,799, which proceeds schematically as in preparing fragments of the antibody by a prior art, modifying the enzyme by reaction with a suitable heterobifunctional agent and finally coupling of the products thus produced. Other known methods for constructing immunoenzymatic conjugates may also be used in this case.

The detection of the enzymatic activity specifically bound to the cell antigen, recognized by the conjugate with acetylcholinesterase, is preferably carried out by the well-known technique using acetylthiocholine as enzyme substrate and Ellman's reagent or 5,5'-dithio-2-nitrobenzoic acid as chromogen. according to any variant suitable for the particular case, e.g. the one described Pradelles and others, Anal. Chem., 57, (1985) 1170-1173.

Said chromogens are used as they are or in the form of salts which are soluble in water.

Determining the results of antigens according to the invention can be expressed in any manner appropriate to the assay performed. Specifically, the results can be expressed either in the total number of molecules of a given antigen (e.g., antigen CD4) found in a given volume of assay examined (e.g., in microliters of blood), or by the ratio of the number of molecules of a antigen and the number of molecules of another antigen in the assay examined (for example, the ratio of the CD4 antigens to the CD8 antigens or the CD4 / CD8 ratio in the blood sample examined.

To determine the number of molecules of a given antigen in the assay examined, a calibrated scale consisting of cells or cell preparations carrying the antigen to be determined and calibrated prior to a known calibration is preferably used. reference method. These standards are preferably constituted either by cells which are identical in origin to the cells which are subject to determination or by cells 5 from established cell lines carrying the desired antigen or by preparations e.g. of membranes originating from the cells.

These standards are treated as accurately as the samples to be examined. The resulting signals serve to construct a calibration scale that compares the signals measured with the samples to be examined. The final calculations are classic.

In order to determine the ratio of the number of molecules of two antigens in the sample to be examined, one can use the calibration system described above and finally calculate the ratio sought. More simply, in numerous cases, the ratio of specific signals obtained with each of the antigens sought can be calculated directly, where appropriate, corrected with known factors such as the ratio of the dimensions of the samples taken into use and obtained directly it sought relationship.

The immunometric assay method of the invention is simple, fast and reproducible. Its use is perfectly suitable for analyzing a large number of samples. In order to understand the advantages over other described methods, it is appropriate to analyze the various steps.

30

The immobilization of the cells on the solid support is the determination phase that traditionally presents most difficulties or is most critical to perform. The agent commonly used is chemical fixation of the cells with gutaraldehyde 35 or methanol in dishes treated or not treated with poly-L-lysine (VAN LEUVEN et al., J. Immunol. Methods, 1978, 23, 109 ). Nevertheless, the chemical fixations thus performed may diminish or even suppress the specific detection sought or, on the contrary, induce false positive markings of cells, which is a very serious drawback (DROVER and MARSHALL, J. Immunol. Methods, 1986, 90, 275 -281).

Further, the process of chemical fixation requires several steps: centrifuging the cells, preparing the mixture of fixative, fixing and then washing the fixed cells.

Drying of the cells at 37 ° C, optionally followed by a fixation with methanol in micro-wells, has also been suggested (BAUMGARTEN, 0. Immunol. Methods, 1986, 94, 91-98).

Dehydration sees in some of the cells at 37 ° C in addition to being able to alter certain fragile antigens, permeabilizes the piasm membrane around the cells, facilitating immunomarking of intracytoplasmic antigens more than labeling surface antigens, and thus causing annoying noise which is annoying. or false positive results when you want a measurement that is limited to surface antigens.

20

Furthermore, the reproducibility of this method is uncertain. The decantation of the cells in the mole depths to determine and dry the cells may vary from one experiment to another. In addition, this determination is long-lasting to perform for the drying step of the cells itself requires a time of more than 2 hours.

The immobilisation of lymphocyte populations has also been achieved by the use of polyclonal antibodies adsorbed in mycorrhizal tubules (STOCKER and HEUSSER, J. Immunol. Methods, 1979, 26, 87-95). In addition to allowing immobilization of cells that are foreign to the only population to be assayed, the polyclonal antibodies also present the disadvantage of reacting with the antigens destined to be measured with labeled antibody, which reduces as much the final measured signal.

Use of highly specific and related monoclonal antibodies adsorbed or fixed on the solid support, and in particular in assays, allows exclusive capture of the targeted cells, removing the other non-retained cell populations during washes performed to label the antigens to be determined. . Moreover, no chemical or physical agent modifies the characteristic properties of the antigens at this stage, for the various operations intended to chemically or physically fix the cells to the carrier have been abolished.

Thus, according to the present invention, it has been found that the immobilization of the cells with monoclonal antibodies is a method which allows the immobilization step of the cells carrying the antigen to be determined while simplifying the results more reliably.

15

The immunometric assays applied to cell populations are generally performed by labeling the cells by an indirect method in 2 or 3 consecutive steps, the probe carrying the specific signal being fixed to the cells during the last 20 steps of the labeling process. In the labeling of the cells in two steps, the main reagents used employ antibody anti-immunoglobulins (anti-Ig) coupled to β-ga1 actose in dase (COBB0LD et al., J. Immunol. Methods, 1971, 44, 125-133) or alkaline phosphatase (HESSIAN, J. Immunol. Methods, 1986, 91, 29-34) or labeled with iodine 125 (SAVION J. Immunol. Methods, 1987, 97, 49-56). The labeling technique with the peroxidase anti-peroxidase reagent is performed in 3 steps (VAN LEUVEN 1978).

Another method, also in 3 steps, uses a monoclonal antibody specific for the antigenic marker to be assayed and then antibody anti-Ig from mice carrying biotin and finally a conjugate streptavidin peroxidase (BAUMGARTEN 1986) or streptavidi n. alkaline phosphatase (IGIETSEME et al., J, Immunol. Methods, 1987, 97, 123-131).

The method of the invention simultaneously involves the use in a single step of a multicell capture process of whole cells without physical or chemical intervention in the cells, and labeling all or part of the cells with one or more monoclonal antibodies which directly carries a radioisotope probe or enzymatic probe, which for the first time allows quantitative determination on the cells themselves of selected membrane antigens.

5

According to the invention, direct labeling of immunological immobility in serous cells allows: simplification of the method of determination by eliminating intermediate repetitive manipulations between the successive steps of labeling, where indirect labeling is concerned: centrifugation of the cells, removal of labeling reagents, resumption in suspension, an economy of reagents, an improved reliability by reducing the number of steps and manipulations, 20 a time gain, the ability to simultaneously process a large number of samples using only ordinary materials and apparatus.

25

The incubation period to immobilize the cell population and at the same time direct labeling of the antigens in the cell subpopulation to be determined is short. It is less than or equal to 1 hour in the determination of T lymphocytes and subpopulations of T4 and T8 lymphocytes.

After washing the solid support, the actual determination is made by observing a signal that is accurate and easy to measure with conventional apparatus: radioactivity, absorption, or emission of light.

Thus, the entire process of the invention offers numerous advantages: it is fast, reliable, economical and simple.

22 DK 174032 B1

The method of the invention makes it possible to determine surface antigens in a cell population within a wide range of cell concentrations.

The sensitivity of the method to the number of cells depends on the antigen density of the particular cell population. For each antigen, if desired, the minimum molar concentration of antigen which can be measured by the method of the invention can be defined.

10 For example. the selected solid carrier is a mi scrap iteration plate, and when the cells examined are human lymphocytes, it has been observed that significant measurements are obtained when the number of cells analyzed is between a few hundred and ca.

15 200,000 per micro-indentation of 200 μΐ, the lower limit being determined by the measured density of antigen on the cells examined and the sensitivity of the detection technique selected, while the upper limit essentially depends on the dimension and geometry of the solid support. It is the same when the solid 20 support is made of test tubes.

It has been confirmed that the recorded signals (counting radioactivity or photometric measurements) allow calibration curves to be regular and satisfactory as a function of the number of cells used under the usual manipulation conditions.

Furthermore, the sensitivity of the method can be improved if necessary by simultaneously fixating on the same surface antigen several different monoclonal antibodies specific for several different epitopes of the same antigen.

This has been confirmed by the determination of CO 4 anti-genes on cells of the Ichikawa line (human T-line), to which the antibodies OKT4 and ST4 labeled with iodine 125 have been used simultaneously.

35 The measured signal is increased approx. 50¾ relative to the signals obtained with each of the antibodies anti-CD4 used alone.

The following examples are used without distinction the following terms or their abbreviations: · 23 DK 174032 B1 BSA: bovine serum1 bum in PBS: saline phosphate buffer with pH 7.4 POD: peroxidase

IgG: immunoglobulin G

IgM: immunoglobulin M

IgG anti-T or anti-T: antibody anti-lymphocyte T

IgG anti-CD4 or anti-CD4: antibody anti-antigen CD4

IgG anti-CD8 or anti-CD8: antibody anti-antigen CD8 10 cpm: stroke per minute ppm: disintegrations per minute minute.

Example 1 Immunoenzymometric determination of the molecular concentration of the antigens CD4 and CD8 from human blood samples comprising a labeling of antibodies with peroxidase.

A) Manufacture of the determination equipment.

20 a) Preparation of the plate.

A plastic microtiter titration plate sold by NUNC (reference 64393) with 96 micro-recesses is used. 25 µl of 200 µl of a solution containing the purified anti-CD2 monoclonal antibody (called ST11) used to immobilize total T lymphocytes, i. to induce their immune capture. This antibody sold by BIOSYS, Compiégne, France under the reference ST11 is used at a concentration of 10 pg / ml in a saline phosphate buffer of pH 7.4 (PBS). The adsorption of the monoclonal antibody occurs at 4 ° C for 12 hours. Excess antibody is removed by inverting the plate.

A solution containing 0.1% gelatin and 0.5% BSA in a saline phosphate buffer is prepared. 250 μΐ of this solution is introduced per day. micro-indentation to saturate the surfaces of the indentations with protein, which is obtained after 1 hour at 37 ° C. Wash the plates 3 times with saline phosphate buffer. The plates thus produced are lyophilized and stored at 4 ° C in a sealed plastic bag.

B) Preparation of the solution of monoclonal antibody and peroxidase conjugate.

Peroxidase (POD) sold by Boehringer Mannheim Biochemica is used (reference 814 393).

10

The coupling process between the antibody and the peroxidase is that described by M.B. WILSON and P.K. NAKANE in Immunofluorescence and Related Techniques (W. KNAPP, K. KOLUBAR, G. WICK Publishers 1978, Elsevier / North Holland, Amsterdam, pages 215-15 224) except that 1.5 mg POD is used for the oxidation of the peroxidase. in 0.36 ml of distilled water and adding 50 μΐ of a 0.2 M solution of sodium periodate. The product thus obtained is coupled with 2 mg IgG anti-CD4 contained in 50 μΐ carbonate buffer. After treatment with sodium borohydride and dialysis against PBS, the IgG-POO conjugate is sterilized by filtration on membrane 0.22 µm and stored under sterile conditions at a concentration of 0.5 mg IgG per ml. ml at 4 ° C in test tubes. The reagent is stable for at least 1 year.

In the same way, the conjugate IgG-anti CD8-POD is prepared. Similarly, the IgM-POD conjugate can be prepared.

c) Preparation of the detection reagent.

The precipitate in the reagent is obtained as follows: a 0.1 M citrate buffer is prepared by dissolving the citric acid monohydrate in an amount of 2.1% in water and adjusting to a pH of 5 by the addition of 7N sodium hydroxide. Then, 30 mg of orthophenyl1 than iamine dichlorohydrate is added to 20 ml of citrate buffer and then 40 μΐ of 30% oxygenated water (enzyme substrate) is added to 20 ml of citrate buffer containing ortho-phenylendi amine at the last minute.

B) Immunometric method, a) Separation of cells.

5 Take 2 ml of blood sample to be determined. Mix with 2 ml of saline phosphate buffer and place this mixture on top of 3 ml of FICOLL-PAQUE (marketed by Pharmacia).

By centrifugation at 400 x g for 30 minutes at ambient temperature, a ring of suspension containing the 10 enriched cells is formed. This whole suspension is recovered in a volume of 1 ml and 6 x 100 μΐ of the resulting suspension is distributed in 6 ml of indentations on the previously prepared plate.

B) Incubation of the cells.

One-hundredth of the previously prepared conjugate POD antibody is diluted with PBS containing 1% protein, such as BSA or skimmed milk powder, and 20 100 µl of this solution is applied per day. micro-recess namely: 2 m in the recess depth is filled with every 100 μΐ of the solution of conjugate POD-anti-CD4.

25 2 ml of corpuscles are filled with every 100 μΐ of the solution of conjugate POD-anti-CD8.

2 ml of crustal depths for control are filled with 100 ml of solution with 1% protein (blind reaction) every 100 ml.

30

To improve the fixation of the cells on the support, the plate is centrifuged for 3 minutes at 150 x g after waiting 1 hour at ambient temperature.

35 c) Detection of fixed enzyme and measurement.

Empty the innards by reversing the plate. Microprocessing was washed 4 times with 200 μΐ PBS. 26 DK 174032 B1 is added to each well 200 μΐ of 1 earring reagent prepared for immediate use. Allow to incubate for 20 minutes at ambient temperature in subdued light. The optical density is measured with a spectrophotometer at 492 nm (the Titertek Multi-5 scan type 310 C - Flow Laboratories apparatus).

d) Calibration and expression of results.

To perform the calibration of these measurements, a preparation of total human lymphocytes pre-calibrated for CD4 and CD8 antigens is used in the cytofluorometric technique of PONCELET et al. (J. Immunol. Methods, 1985, 85, 65 -74). Taking equal known portions of this comparative preparation serves to constitute the two calibration scales for antigen CD4 and antigen CD8, respectively, relative to which the number of antigens corresponding to each sample is calculated.

By way of example, the following Table 1 for 20 human blood samples 20 derived from healthy donors shows the values of the optical densities obtained, the number of molecules of antigen CD4 and CD8, and the resulting ratio of number of antigens (CD4 / CD8 ratio).

25 30 35 27 DK 174032 B1

Table 1

Antigen CD4 ~~ Antigen CD8

Number of antigen- Number of antigen- for-

Donor Optical Molecules (in Optical Molecules (in team 5 no density millions) density millions) CD4 /

___pr. μΐ blood _ per ul blood | C08 I

1 0.64 23 1.29 92I 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 1 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 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 15 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 20

Example 2

Immunoenzymetry determination of the molecular concentration of the antigens CD4 and CD8 in human blood samples comprising a labeling of antibody with alkaline phosphatase.

This assay is carried out as in Example 1. The preparation of the monoclonal antibody and alkaline phosphatase conjugate is carried out by the method of Boehringer Mannheim - Biochemica (reference 567 744). After 1 hour of incubation of the cells to be determined with this conjugate, fixed enzyme is revealed by adding a solution of pairs of itrophenyl phosphate 1 mg / ml in a 1.2% diethanolamine buffer in distilled water adjusted to pH 9.8 with a dilute solution of hydrochloric acid. After 2 hours at 37 ° C, the optical density is measured with the spectrophotometer at 405 nm.

The results are calculated and expressed as in Example 1.

28 DK 174032 B1

Example 3

Immunoradiometric determination of the molecular concentration of the antigens CD4 and CD8 in human blood samples comprising a label of iodine 125 antibody.

The preparation of antibody labeled with iodine 125 is carried out by the technique described by F.C. GREENWOOD, V.M. HUNTER and others, Biochem. J., 1963, 89, 114.

10

50 µg monoclonal antibody anti-CD4 or anti-CD8 contained in 50 μΐ saline phosphate buffer pH 7.2 with 37 MBq iodine 125 in the form of sodium iodide and 30 μΐ of a solution of chloramine T in a saline phosphate buffer containing 15 µg 0.33 mg of chloramine T per ml. ml. After 1 hour with stirring, the labeling reaction of the monoclonal antibody is stopped by adding 100 μΐ of a solution of sodium metha bisulfite with 2.5 mg / ml. The solution thus prepared is placed on a PD 10 column (Pharmacia - Sephadex G25M) and the fraction containing the radiolabelled antibody is recovered in the drain. The determination is carried out by introducing in a volume of 4 ml of cropland a volume of 100 μΐ of cell suspension containing the single-blooded cells of human blood prepared as in Example 1.

100 μΐ of dilute solution of radioactive antibody is then placed in a PBS buffer containing 5% BSA, so that 150,000 cpm is introduced. recess. 2 micro-depressions are filled with each 100 μ hver of the anti-CD4-125-I conjugate solution, 2 micro-depressions are filled with every 100 μΐ of the anti-CD8-125-I conjugate solution. The fixation of the cells is improved by centrifuging the plate for 3 minutes at 150 x g after 1 hour incubation at ambient temperature. The micropores are emptied by inverting the plate. The micro-wells were washed 4 times with 200 μΐ PBS per ml. recess. 75 μΐ of 1 M sodium hydroxide solution is then introduced into each for-depth of 5 to 5 ng. After 10 minutes, the contents of each well are recovered with an absorbent buffer before counting the radioactivity with a γ counter (multi-counter LKB).

29 DK 174032 B1

The results are calculated and expressed as in Example 1, replacing the optical density values with the results of counting in ppm.

Example 4

Confirmation of the validity of the method.

On 20 human blood samples, the antigens 10 CD4 and CD8 are measured on human T cells following the peroxydase immunometric method described in Example 1. In addition, the same blood tests are determined using the usual technique {W.W. ERBER et al., Lancet, 1984, (8385), 1042-1045) to cytological count the ratio of the number of positive T4 cells to the number of positive T8 cells.

The correlation coefficient between the ratio of CD4 / CD8 obtained by immunoenzymometric determination and the ratio T4 / T8 obtained by cytological count is r = 0.72.

20 On 7 other human blood samples, the ratio of CD4 / CD8 determined by the immunoradiometric method is also compared using iodine 125 labeling in the method of the present invention and the ratio of cells T4 / T8 determined by counting the cells. The correlation coefficient is r = 0.87.

In the 2 cases, the correlation coefficients obtained show that despite an overall satisfactory agreement between the results of the 2 techniques, the information provided by the 2 ratios is not equivalent, which is clear since the determination of the antigens of the invention does not take into account not only the number of positive cells as in the cytological method, but also the density of the particular antigen on the positive cells of each sample. The information given by the technique described in the present invention is therefore more complete than that provided by the usual comparative technique (W.W. ERBER et al., Lancet, 1984, (8385), 1042-1045).

30 DK 174032 B1

Example 5

Immunoenzymometric determination of the molecular concentration of the antigens CD4 and CD8 on T lymphocytes in samples of 5 human blood comprising a labeling of antibody with peroxidase.

This example is intended to show that the required duration of the assay can be reduced relative to the conditions described in Example 1 by modifying, on the one hand, the cell separation procedure and, on the other, the incubation time required in the immuno-capture step and labeling of the cells, 15 A) Influence of method for separating cells from whole blood.

a) Short-spin centrifugation technique: 0.5 ml of blood sample is to be determined, which is mixed with 1.5 ml of saline phosphate buffer. Into a 5 ml high-vial glass, 1.5 ml of Fico11-Paque, marketed by Pharmacia, is introduced and the blood sample diluted in PBS is placed on the surface of Ficoll. After 5 minutes centrifugation at 900 x g at ambient temperature, the ring of suspension containing the single-core cells is taken out in a volume of 0.5 ml. This sample is added to 1.5 ml of PBS.

b) Erythrocyte lysis technique: 30

Another rapid method of separating blood cells is the use of a buffer that brightens the red blood cells. The light cushion used as a non-limiting example has the following composition: 35 ammonium chloride: 8.29 g of acidic potassium carbonate: 1 g of disodium salt of ethylene diamine tetraacetic acid: 0.0307 g to 1 liter of distilled water whose pH is adjusted to 7.3.

31 DK 174032 B1

Mix 5 ml of light buffer and 0.250 ml of blood. After stirring for 10 minutes, centrifuge at 600 x g for 10 minutes. The resulting precipitate of cells is taken up in 1 ml buffer PBS.

5

Then, by performing the assay of the antigens CD 4 and CD8 under the conditions of Example 1, and by comparison with the reference protocol described in Example 1 for isolating single-core blood cells, it has been confirmed that one and the other 10 of these 2 variants of the method for separation of single-core cells from total blood has allowed the collection of all the lymphocytes in the blood samples examined.

B) Influence of incubation time on immunoprecipitation and labeling of the cells.

To confirm whether the 1 hour time used in Example 1 cannot be reduced to speed up the determination, the results obtained for identical 20 samples containing 20,000 single-core cells per 20,000 cells were compared. recess when allowing the time in the capture and labeling step to vary from 10 minutes to 1 hour. Other operating conditions are as in Example 1, and the results obtained are shown in Table 2.

25

Table 2 I I _ Optical density_ | 30 In Incubation In Antigen CD4_ | _Antigen CD8_ |

1 time (min) ._ 1 Sample \ Blind Sample * | Sample I Blind Sample * I

I 10 I 0.322 1 0.037 | 0.329 | 0.023 J

I 20 I 0.438 1 0.049 | 0.401 | 0.025 | I 30_1 0.620 1 0.055_1 0.498 1 0.027_ 35 These optical density values correspond to blind tests performed in the absence of cells. The control values obtained in the presence of cells and omitting either the conjugate or substrate are no better than the values shown.

32 DK 174032 B1

These results show that: the nonspecific signal (blank) remains at values which are always low and the lower the shorter the time of presence of conjugate.

The specific signal, after 10 minutes of contact, achieves a value that can be used analytically with accuracy and reproducibility. This shows that, relative to the conditions of Example 1, it is possible to significantly reduce the duration of execution of the assay without significant loss of accuracy in the results.

In practice, and if you gather the time gain that can be obtained in the preparation of the blood sample and in the immuno-capture, it is possible with the equipment and method of the invention to determine the antigens CD4 and CD8 (as non-limiting examples) in samples of total blood in a blood sample. amount of 10 or 20 samples per 96-well plate at a total time (after receiving 20 samples in the laboratory) that does not exceed 1 hour. This level of productivity is better than any alternative technique known to date.

Example 6 25

Immunometric determination of the molecular concentration of the antigens CO5 on human T lymphocytes in samples of human blood including labeling of antibody with acetylcholinesterase.

30 A) Manufacture of the determination equipment.

a) Solid carrier.

A microtitration plate prepared in the manner described in Example 1 is used.

b) Monclonal antibody conjugate and acetylcholinesterase.

33 DK 174032 B1

Acetylcholinesterase from E1ectrophorus electricus made by the technique described in French Patent No. 2,550 is used.

799th

5 This enzyme is coupled with the antibody anti-CD5 called STI and marketed by BIOSYS. The coupling is performed by the method described by YOSHITAKE and others, Eru. J. Biochem., 101 (1979), 395-399.

C) Detection reagent.

This reagent comprises both the enzyme substrate (acetylthiocholine) and Ellman's reagent and has the following composition:

Acetylthiocholine 7.5 x 10 "* M

5,5'-dithi o-2-nitrobenzoic acid (DTNB) 5 x 10 104M in a sodium phosphate buffer 1M pH 7.4.

This solution is diluted to 1 / 100e in distilled water before use.

B) Iromunometry method.

a) The enriched cells are separated using the method described in Example 1.

b) The incubation of the cells in the immuno-capture and labeling step takes 20 minutes as in Example 5.

30 c) Detection of fixed enzyme and measurement.

100 μΐ detection reagent is added to each ml of immersion well. The absorbance is measured at 412 nm after 45 minutes of incubation.

The optical densities measured for samples containing known numbers of T cells are shown in Table 3. Under these test conditions, the blank is particularly weak and corresponds to an optical density of 0.002 to 0.003.

34 DK 174032 B1

Table 3 | | Number of T lymphocytes introduced per recess and | 5 I In number of molecules of antigen CD5 present per for · * 1 I 1 dip in nq j mill ions_1 I I 540 j 1,080 I 2,160 j 4,320 | 8.640 | 17,280 j I_1 (323-1 (61L ___ Ll.l3 ". (260J_L (5.20) 1 (1,040)] I Optical I I I I I I j 10 I density I 0.030 I 0.060 | 0.138 | 0.250 | 0.470 1 0.850 | I_1_I_____J___L______J___I____1

These results illustrate the great sensitivity of the technique thus prepared. The optical density of 0.030 obtained in 15 wells containing 540 T lymphocytes (ie 32 million molecules of antigen CD5) which can be accurately measured and is equal to 10 times the background noise corresponds to approx. 5 x 10 ~ 17 moles of antigen CD 5 pr. recess. Such foals are of the same order of magnitude as the best known immunoassays, which utilize the most sensitive radioactive tracers.

Example 7

Determination of various antigens expressed on activated 25 human T lymphocytes.

Activation of T-lymphocytes is a physiological process that intervenes prematurely every time the immune system is stimulated, e.g. in infection pathologies, in organ transplantation, and in certain diseases with a failing immune system. This natural process is also commonly effected in vitro in the laboratory in samples, such as, in particular, mixed lymphocyte reactions in onions or samples of 1-lymphoblast in conversion. In the latter case, the polyclonal activation is achieved by the use of agents such as phytohaemaglutinin (PHA), concanavalin A or other lectins. The activation of T lymphocytes is accompanied by significant growth in the expression of several membrane markers and in particular the antigen CD25 (receptor for interleukin 2) or the antigen CD2. Therefore, these antigens are excellent markers for the state of activation and their determination is of great interest both in clinical biology and to the aforementioned laboratory work, avoiding in all cases the use of radioactive reagents.

5

These antigens are measured using the method described in Example 1 with the specifications specified in Table 4.

1 ° Table 4 Measurement of surface antigen on activated T lymphocytes__] I Measured I Antibody used for | Antibody labeled with | In antigen 1 immuno capture [peroxidase | 1_I negotiated by_1 negotiated by_ | I CD2 j ST 1 BIOSYS | ST 11 BIOSYS |

1 CD2 5 I ST 11 BIOSYS 1 IOT 14 IMMUNTECH_J

20 The optical density values measured at 450 nm for the 25 κ 103 single-core cells used are shown in Table 5 below, and compare the same cells without stimulation with PHA and after 3 days of stimulation.

25 Table 5 _1_Optical Densities__] I I Non-activated | Cells activated | Blind | 30 1 Antigen I cells_I for 3 days ___ [Prove__ | I CD25 I 0.064 I 0.529 | 0.044 |

I C02_1_0.129 ____ J ____ 0.768_j 0.027 I

Oisse results show a significant growth in the specific signals of the 2 antigens studied when active ringing occurs.

Example 8 36 DK 174032 B1

Determination of the antigens CD22 and HLA-Dr (or class II HLA) found on B-1 lymphocytes.

5

This example describes the determination of these antigens on cells of the RAJ I line, which is a human B lymphoid line, described by PULVERTAFT in J. Clin. Pathol., 1965, 18, 261-274.

For assay immunoprecipitation of cells, assays, as shown in Example 1, either antibody S class II (BIOSYS) to determine the antigens CD22 or antibody SB4 (BIOSYS) to determine the class II HLA (or HLA-Dr) antigens, have been adsorbed.

A) Determination of antigen CD22:

Antibody SB22 (BIOSYS) has been labeled with iodine 125 by the method described in Example 3. The determination of antigen CD22 is carried out in the manner shown in Example 3 for antigens CD4 and CD8 by introducing into the groove indentations in sequence: 5 x 10 4 cells of the RAJI line and then 105 cpm of labeled antibody SB22. After 1 hour at 4 ° C, the micro-depressions are emptied by inversion of the plate, and then washed 4 times with 200 μΐ PBS 25 per ml. recess at each wash. The radioactivity fixed on the cells is recovered and counted as in Example 3. This gives a count of 760 ppm. well containing 5 x 10 4 cells RAJI with a blank (without cells) of 50 ppm.

30 b) Determination of antigen HLA-Dr s

105 cells and the conjugate antibody S class II labeled with peroxidase are sequentially introduced in the manner described in Example 1. The assay is performed following the same protocol as in Example 1. This gives an optical density of 1.840 and a blank sample without cells of 0.105.

Example 9 37 DK 174032 B1

Use of the method of the invention for measuring antigens carried by T-1 lymphocytes and B lymphocytes from human blood.

A) Manufacture of sheets:

Microtitration plates prepared as in Example 10 are used except that in each well of the microplate, both monoclonal antibodies capable of fixating at least all T lymphocytes (S class I from BIOSYS) and B lymphocytes ( S class II from BIOSYS), each used at a concentration of 5 pg / ml.

B) Immunometric determination:

The single-celled cells are separated from total blood by Fi-coll under the conditions of Example 1 or Example 5, and then 80,000 single-celled cells are introduced per cell. immerse in ng in 75 μΐ buffer PBS. The T-1 lymphocytes are measured in certain wells with antibody ST11 (anti-CD2) from BIOSYS. The B lymphocytes are measured in other wells with antibody SB3 (anti-CD37) from BIOSYS, which recognizes all the peripheral B lymphocytes. These antibodies are pre-coupled with peroxidase according to the protocol described in Example 1. The actual assay is performed as in Example 1.

C) results.

30

The absolute number of T-lymphocytes and B-lymphocytes contained in the sample tested is determined by following calibration curves established by processing identical single-celled cells in which the T and B lymphocytes have been counted by a comparison technique.

Alternatively, using appropriate standards, one can also express the results in molar concentrations of antigens CD2 and CD37 in the sample under study.

Example 10.

38 DK 174032 B1

Determination of the antigens GpIIb-IIIa and CD9 on human blood p 1 veins.

5

The assay is performed according to Example 3 using monoclonal antibodies radiolabelled with iodine 125.

The blood vessels are isolated from human blood by centrifugation in the presence of perfusion fluid PLASMION (Laboratory R. Bel.

Ion). He mixes in a test tube 5 ml of blood + 5 ml of saline phosphate buffer (PBS) and 10 ml of PLASMION. The glass is then centrifuged for 10 minutes at 1500 rpm. Platelets collected in the overlying fluid are removed by suction with 15 pipettes. They are washed once in PBS by mixing 1 volume bl odp 1 adesuspens ion with 10 ml PBS and then centrifuging for 10 minutes at 1000 x g. Finally, the platelet precipitate is collected, which is brought into suspension again in PBS (2 ml). . Platelets are counted and their concentration adjusted to 2.5 mil-20 l ions per ml. ml of PBS.

To determine the antigen GpIIb-IIIa, immunoprecipitation of the platelets with the monoclonal antibody IOB2 (Immunotech) and labeling of the antigen GpII-IIIa with the monoclonal antibody P2 (Immunotech) were performed.

To determine the antigen CD9, immunoprecipitation is effected with the monoclonal antibody P2 and labeling with the monoclonal antibody IOB2. The number of ppm per meter is measured. sample approximately 30,000 platelets.

The results are shown in Table 6.

35 39 DK 174032 B1

Table 6] Measured antigen | Sample measured | Control without cells \

5 1 _] _____ Ldpm] ___ I_ (ppm) ____ J

CD9_1 1.927_I_ 113_I

I GpIIb-lIIa__] 2.S91_J_238_ |

Example 11 10

Determination of antigens CD15 carried by human granulocytes.

*

Antigen C015 is a good specific marker for human granulocytes (also called multicellular bodies). The assessment of these 15 cells by virtue of this antigen can be practiced in blood, as for any other leukocyte subpopulation. It is also used to assess the presence of granulocytes in urine e.g. in cases of urinary tract infections, such as cyst for ten s or pyelonephrites. In the present example, the assay 20 is carried out on granu l ocytes derived from total blood and separated as shown below.

The granulocytes are simultaneously separated from red blood cells by certain enriched cells by a method analogous to that described in Example 10 for platelets, except that the glass containing the cells is left for 45 minutes at ambient temperature and suspension of The 1 eukocytes in the 500 μΐ outlet at the surface of the fluid.

30

The granulocytes are immobilized in wells with a monoclonal antibody specific for antigen CD45 that is found on the cell membrane of all 1 eukocytes: the anti-drug anti-LCA (BIOSYS) adsorbed in the mucosal depths of the titration plate is used as shown. in Example 1.

For the determination, the monoclonal antibody anti-CD15 SMY-15a (BIOSYS) labeled with peroxidase is used in the technique described in Example 1.

40 DK 174032 B1

With 12,500 total cells introduced per cell. Thus, the specific signal of antigen CD15 from the granulocytes is 0.616 after correction of the unprocessed optical density for a blank of 0,100 obtained in the presence of cells and in the absence of enzymatic conjugate and for the endogenous peroxidase water of the granulocytes.

Example 12 Evaluation of a Leukemia Phenotype by the Immunoenzymometric Procedure.

-J

The phenotype determination of tumor cells in a 1 eukemia patient is a systematic diagnostic study performed to characterize the type and origin of the patient's 1 eukemia cell1 (T, B, granulocytes, myeloblasts, etc.). This study is traditionally performed in the mmofluorescence technique using a battery of monoclonal antibodies and observing and counting the positive cells in the microscope. 20 flow cytometry is also used to analyze the labeling of the cells.

The use of monoclonal antibodies in the method of the invention makes it possible to identify the large clinically significant groups of acute or chronic leukemia 25 by additionally using a quantitative information on the relative densities of the various antigens investigated.

In this example, 6 monoclonal antibodies prepared with peroxidase specific for antigens found on leukemia cells are prepared: STI and ST11 (BIOSYS): anti-CD5 and anti-CD2 SB3 (BIOSYS): anti-CD37 S -CALLA (SAN0FI): anti-CALLA (or anti-CD10) S-class II (BIOSYS): anti-HLA-Dr (or anti-HLA of class II) SMY15 (BIOSYS): anti-CD15.

41 DK 174032 B1

The microtitration plate is prepared by prior adsorption of a mixture of the anti-CD45 monoclonal antibodies (S-LCA, BIOSYS) and class I anti-HLA (S-class I, BIOSYS).

5 Using the above 6 selected monoclonal antibodies, the phenotype of a chronic lymphoid leukemia B (LLC-B), which was otherwise characterized by the usual method, was evaluated.

For a total of 80,000 single-celled cells introduced per The optical densities measured at 492 nm are given in Table 7. For control wells with cells, but without antibody labeled with peroxy-dase, the optical density is 0.110.

15

Table 7 I I Optical density |

20 In Monoclonal Antibody | ____________ J

I and equivalent | Try with | Control without | 1 antigen_1 cells__1 ..... cells ____________ | I ST11 (CD2) I 0.270 | 0.030 | I STI {CD5) I 1.350 I 0.014 | 25 In SB3 (CD3 7) | 0.850 | 0.005 | In S Class II (HLA-Dr) | 1,640 | 0.010 | I SMY15 (CD15) I 0.235 | 0.007 |

I CALLA (C010) _1 0.160_I 0.002_ I

30

Thus, the investigated eukemia mice11 carry plenty of the class II antigens CD5, CD3 7 and HLA, but they do not or only very weakly carry the antigens CD15, CALLA and CD2, which is characteristic of an LLC-B.

35

Example 13 42 DK 174032 B1

Determination of antigens associated with urinary tract cancer.

5

The equipment and methods of the invention are particularly adapted for the detection of tumor cells, especially tumors of the urinary tract, and are well suited for mass detection in high-risk groups, e.g. workers in the chemical industry or other very disadvantaged groups.

The present example illustrates the use of the invention for the determination of an antigen associated with bladder cancer on the cells of the RT4 lineage derived from a human napkin 11 human clock (CC Rigby and LM Franks, Brit. 0. Cancer, 1970, 24, 746-754).

The plates intended for immunoprecipitation of the cells are prepared as in Example 1 by adsorbing in the wells the S-Class I monoclonal antibody S (BIOSYS), which recognizes a class I antigen HLA, and is capable of retaining all the epithelial cells.

Markers in the conjugates are obtained by the methods of Example 1 25 (for enzymatic labeling) or Example 3 (for radioisotope labeling) with the monoclonal antibody 12F6 (SAN0FI) which recognizes an antigen associated with bladder cancer.

For the determination, in 4 ml of cropland depths, 75 μΐ of the cell-30 lesion suspension is distributed (i.e., 50 x 10 6 cells per well). In 2 wells, the conjugate of monoclonal antibody 12F6 (SANOFI) coupled with peroxydase is introduced, in 2 other wells the antibody 12F6 labeled with iodine 125 (100,000 cpm / well). The results obtained using the immunoenzymometric method or the radioimmunometric method are shown in Table 8.

Table 8 43 DK 174032 B1 | The brand mono- | In Relationship | 5 | clonal anti- | Optical density (at 492 nm) | patient sig- | In substance 1 or com _1 nal / unspecific | I I Sample which must 1 Check | fictitious signal |

1_I is determined ___ J ___ L —-- J

I 12F6 peroxidase J 0.782 | 0.078 | 10.0 | 10 1 12F6 iodine 125 I 1.612_1 350 1 *> 6_1

Example 14

Immunoenzymometry determination of a membrane antigen on the yeast 15 Candida albicans.

The cells of Candida albicans (serotype 17) originate from the collection at Institut Pasteur, Paris. The yeast is grown for 18 hours in the usual manner on a solid synthetic medium. The cells are counted and one cell suspension containing ID5 cells per cell is prepared. ml solution in buffer PBS.

The anti-Candida albicans monoclonal antibodies used are prepared by lymphocyte hybridization, as described in the thesis of T. Chardes, Faculty of Pharmacie, University of Montpellier I, 1988.

The antibody CA4 is used for the immunoprecipitation, while the antibody CA12 labeled with peroxidase is used for determination 30 under the conditions of Example 1.

Oe at 492 nm measured optical densities for the wells containing the inserted cells and for the control sample without cells are 1.025 and 0.080, respectively.

35

Example 15 44 DK 174032 B1

Immunoenzymometric assay performed on magnetic beads: determination of antigen CD5 on human T lymphocytes with a conjugate of antibody labeled with acetylcholinesterase and a particulate carrier formed by magnetic beads.

The carrier used for trapping the cells is made up of DYNABEADS magnetic spheres. The beads designated 10 DYN-11101, which are marketed by BIOSYS, carry on their surface an antibody anti-CD2 capable of fixing all the T-1 lymphocytes in a tested sample. These beads are further treated with a solution of antibody anti-CD2 before use to improve the performance of the assay.

15

To this end, mix 10 μΐ of the bead suspension with 1 ml of antibody solution with 25 pg / ml in buffer PBS for 1 hour with gentle stirring. The balls are then washed 4 times with buffer PBS and then stored in 4 ml buffer PBS.

20

For determination, sequentially introduce into a 5 ml glass: 200 μΐ ball suspension, 80 μΐ suspension of total blood secreted by total blood using Ficoll-Paque (Pharmacia), and then 280 μΐ conjugate of antibody STI labeled 25 with acetylcholinesterase is added. identical to that used in Example 6.

After 1 hour with gentle stirring, the beads are separated by a magnet and the cells fixed on the beads are washed with a PBS buffer (5 washes).

The acetylcholinesterase fixed on the cells is revealed in the same manner as in Example 6. The resulting optical density signal is 0.168 in the presence of the labeled cells and the blank 35 without cells shows 0.062.

Claims (40)

Equipment for immunometric determination of a surface antigen which is characteristic of a population or subpopulation of cells, characterized in that it comprises: a) a solid support which is fixed by covalent bonding or by physical adsorption one or more monoclonal antibodies directed against the surface antigens of the cell population to be determined in addition to said characteristic antigen; b) one or more solutions, each containing a monoclonal antibody specific for said antigen characteristic of the population or subpopulation of cells to be determined and labeled with a radioactive probe or enzymatic probe; (c) in the case of antibody labeled with an enzymatic probe, one or more solutions containing the reagents which are (d) optionally, an additional component constituted by a buffer wash solution and / or samples that enable calibration and control of the quality of the determination. Equipment according to claim 1, characterized in that the component (a) is a solid support constituted by a microtitre plate in which wells are fixed the antibody (s) intended to immobilize the cells, among which there are cells of the population or subpopulation that carry the antigen to be determined. Equipment according to claim 1, characterized in that the component (a) is a particulate magnetic carrier. Equipment according to claims 1 to 3, characterized in that the component (a) is constituted by a solid support on which one or more class I monoclonal antibodies HLA are obtained. Equipment according to claim 4, characterized in that the fixed monoclonal antibody is antibody called S-class. 5 I. Equipment according to claims 1 to 3, characterized in that the monoclonal antibodies in component (b) are labeled with a radioisotope probe of iodine 125 or iodine 131. Equipment according to claims 1 to 3, characterized in that the monoclonal antibodies in component (b) are labeled with an enzymatic probe. Equipment according to claim 7, characterized in that the monoclonal antibodies in component (b) are labeled with peroxidase and that component (c) comprises substantially oxygenated water and a chromogen selected from orthophenylenediamine, 2. 2'-azino-bis (3-ethyl-6-benzothiazolinisulphonic acid), 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole or one of their water-soluble salts. Equipment according to claim 7, characterized in that the monoclonal antibodies in component (b) are labeled with alkaline phosphatase and that component (c) is constituted by paran itrophenyl-phosphate, 4-methyl umbel 1 in ferry 1 phosphate or 5-bromo -4-chloro-3-indolyl phosphate. Equipment according to claim 7, characterized in that the monoclonal antibodies in component (b) are labeled with β-galactosidase and that component (c) is constituted by orthonitrophenyl-β-D-galactopyranoside or 4-methylumbeliferyl -β-D-ga-1actopyranosi d. 35 li. Equipment according to claim 7, characterized in that the monoclonal antibodies in component (b) are labeled with acetylcholinesterase and that component (c) comprises substantially acetylthiocholine and 5,5'-dithio-2-nitrobenzoic acid or one of their water-soluble salts. . DK 174032 B1 Equipment according to claim 1 for determining one or more surface antigens on shaped elements in human blood, such as leukocytes, lymphocytes, T-1 lymphocytes, B lymphocytes, granulocytes and platelets. 5 Equipment according to claim 12 for the determination of the antigen CD4 or CD8 on human T lymphocytes carrying this antigen called T4 lymphocytes or T8 lymphocytes. Equipment according to claim 1 for determining one or more surface antigens on a pathogenic microorganism. Equipment according to claim 14, characterized in that the pathogenic microorganism is Candida albicans. 15 The apparatus of claim 1 for determining one or more membrane antigens on tumor cells. Equipment according to claim 16, characterized in that the tumor cells are urinary cancer cells or one of the cells in malignant hemopathies. Apparatus according to claim 1 or 2, characterized in that the component {a) is a microtitration plate in which recesses are fixed one or more monoclonal antibodies specific for surface antigens on a cell population and the component (b} is made up of several solutions, each containing a monoclonal antibody specific for a surface antigen on a cell subpopulation in on, which constitutes a substantial portion of the fixed cell population. The equipment of claim 18 for the determination of antigens specific to the lymphocyte subpopulations T, T4, T8 and B. The apparatus of claim 18 for characterizing and determining the various antigens constituting the antigenic equipment on the surface of tumor cells of malignant hemopathies. DK 174032 B1 A method of immunometric determination of surface antigens on a population or subpopulation of cells, characterized in that it consists of: a) immobilizing the cell population carrying the antigen to be determined or a cell population comprising the subpopulation carrying the antigen; to be determined on a solid support using one or more monoclonal antibodies pre-fixed by covalent binding or by physical adsorption on the support and capable of recognizing an antigen found on the surface of the cells in addition to the antigen as must be determined, and in the same step directly labeling the population or subpopulation of cells to be determined by at least one monoclonal antibody specific for the antigen to be determined, which antibody carries a radioisotope probe or enzymatic probe, 20 b) adhere to an incubation period, c) wash the carrier to remove the non-immobic 1 in serous cells, and (d) in the case of antibody carrying an enzymatic probe, to add the necessary reagent (s) (substrate and chromogen) to detect enzyme activity; (e) to read the results either by counting the radioactivity 30 or by measuring light signals (staining or fluorescence), possibly comparing with a calibration scale. The method according to claim 21, characterized in that the solid support is as defined in one of claims 1 or 2. The method of claim 21 or 22, characterized in that the monoclonal antibodies determined for labeling carry an enzymatic probe. DK 174032 B1 Process according to claim 23, characterized in that the enzymatic probe is peroxydase and that the activity of the enzyme is detected with oxygenated water and a chromogen selected from orthophenylenediamine, 2,2'-azino-bis (3-ethyl-6-benzothiazoline-sulfonic acid). ), 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole or one of their water-soluble salts. Process according to claim 23, characterized in that the enzymatic probe is alkaline phosphatase and that the enzyme activity is detected with paranitrophenyl phosphate, 4-methyl umbel 1 in feryl phosphate or 5-bromo-4-chloro-3- . Process according to claim 23, characterized in that the enzymatic probe is / - 3 g 1 ac tos in phase and that the enzyme activity is detected with orthonitrophenyl-β-D-galactopyranoside or 4-methyl umbel liferyΙ-β-D galactopyranos id. Process according to claim 23, characterized in that the enzymatic probe is acetylcholinesterase and that the enzyme activity is revealed with acetylthiocholine and as chromogenic 5,5 "-dithio-2-nitrobenzoic acid or one of their water-soluble salts. A method according to claim 21 or 22, characterized in that the monoclonal antibody for marking carries a row of 0 in the soot top probe of Iodine 125 or Iodine 131. Method according to claim 21 or 22, characterized in that the total duration of the execution of the determination is equal to or less than 1 hour. Method according to claim 21, characterized in that the monoclonal antibodies fixed to the solid support are class I antibodies HLA. Method according to claim 30, characterized in that the monoclonal antibody fixed to the solid support is the antibody called S class I. DK 174032 B1 The method of claim 21 for determining one or more surface antigens on shaped elements in human blood. The method of claim 21, characterized in that the shaped elements of human blood are leukocytes, lymphocytes, T lymphocytes, T lymphocytes carrying the marker CD4 called T4 lymphocytes, T lymphocytes carrying the marker C08 called T8. lymphocytes, B-1 lymphocytes, granulocytes, platelets. 10 The method of claim 21 for determining one or more surface antigens on a pathogenic microorganism. The method according to claim 34, characterized in that the pathogenic microorganism is Candida albicans. The method of claim 21 for determining one or more membrane antigens on tumor cells. The method according to claim 26, characterized in that the tumor cells are cancer cells in the urinary tract, or cells of malignant hemopathies. A method according to claims 21 and 22 comprising immobilizing a cell population on a microtiter graft plate with one or more monoclonal antibodies specific for surface antigens on this population, and at the same time directly labeling cell subpopulations in onions which are a substantial portion of the fixed population, by means of multiple solutions, each containing a monoclonal antibody, each specific for a surface antigen of a cell subpopulation to be determined. The method of claim 38 for the determination of antigens 35 specific for lymphoid subpopulate in the onions T, T4, T8 and B. The method of claim 38 for characterizing and determining various antigens constituting the antigenic armor on the surface of tumor cells of malignant hemopathies. 5 10 15 20 25 30 35