NO177444B - Method for immunometric determination of an antigen, as well as equipment package for such determination - 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

The present invention relates to a method for the immunometric determination of surface antigens that are characteristic of populations or subpopulations of cells, and an equipment package for the determination of such genes. The procedure for immunometric determination and the corresponding equipment package are similarly intended for the measurement of the cells themselves in the determination of their surface antigens as intermediates. These measurements/determinations are applicable in diagnostics.

The knowledge of antigens or markers of the cellular surface has made enormous progress with the clarification of the lymphocytic hybridization and the discovery of monoclonal antibodies by KOEHLER AND MILSTEIN, (Nature, 1975, 256, 495-497).

The monoclonal antibodies have particularly enabled the appearance and analysis of markers of surface or membrane antigens for cells from the most varied origins. These markers (or antigens) can be of different nature: proteins, glycoproteins or glycolipids. The studied characteristics therefore mainly relate to markers for tissues or organs, markers for differentiation states or for activation of normal cells and for identification or for type determination of normal or cancerous cells. A particularly important area of application is the study of hematopoietic cell lines (erythrocytic, megacytic, granulocytic, monocytic, lymphocytic).

Thus, for example, the monoclonal antibodies have enabled accurate indication of surface characteristics for T and B lymphocytes respectively. The corresponding markers, alone or in combination, identify stages of differentiation and functional specialization of lymphocytes. By international agreement, the surface markers for human leukocytes have been classified into groups or differentiation classes (CD) defined according to subcommittee IUIS-OMS, 1984 and described in the journal of the World Health Organization 1984, 62 (5), 813-815.

Identification of these markers, made possible thanks to monoclonal antibodies, has made it possible to arrive at their structure and their biological functions. For example, the molecules of markers CD4 and CD8 participate in leukocyte adhesion functions and are located on the surface of T-lymphocytes with a helping and inducing function (marker CD4) or with a cytotoxic and suppressive function (marker CD8).

The acquisition of this knowledge has enabled the use of these markers, thanks to antibodies that recognize them, for the diagnosis and investigation of various pathological conditions, of which in particular malignant hemopathies (leukemias, lymphomas, etc.) and conditions with dysfunction of the immune system (autoimmune diseases, congenital or acquired immunological defects such as AIDS etc.) (BRETON-GORIUS and VAINCHENKER, Le Biologiste, 1987, XXI, no. 167 63-70, SHAW, Immunology today, 1987, 8 (1), 1-3)

Monoclonal antibodies are today irreplaceable tools in applied clinical biology for cellular analyses.

There are methods for counting cells that use labeling of the surface antigens, but these methods are often long, labor-intensive, difficult to implement, and the results are often uncertain.

The methods that are known and used to measure normal or modified expression of surface antigens in the cell can be divided into two groups. In the first group, the antigens are measured using complicated and specialized laboratory equipment, on the basis of flow cytometry (see especially PONCELET and Coll., J. Immunol. Methods, 1985, 85, 65-74) or quantitative microscopy techniques (POULTER and Coll. J . Immunol. Methods, 1987, 98, 227-234. The implementation of these methods for the evaluation of antigens in the cell is based on the measurement of signals brought by anti-cellular antibodies bound in a direct or indirect way to a reagent labeled with fluorescent substances ( or fluorochroming) such as the isothiocyanate of fluorescein or rhodamine, or enzymes such as peroxidase or alkaline phosphatase. The use of these fluorescent or enzymatic reagents, coupled with appropriate washing steps, thus results in the appearance of fluorescence or staining that is strictly confined to the cellular membranes and does not diffuse into the environment The ongoing application of these procedures in the laboratory remains b limited by the need for specialized and expensive equipment (fluorescence microscope possibly connected to an image analyser, cryostat, flux cytometer). Moreover, the analysis and interpretation of the immunological markings of the cells by means of these methods requires the expertise of a specialist in cytology.

Another group of methods for measuring the antigens is based on the quantitative evaluation of markers for the overall cell population. These methods enable the measurement of anti-genes by means of labeling either directly or indirectly, which is therefore most often carried out in 2, 3 or 4 steps. In all cases, the reagent used during the first labeling step carries a marker which is either isotopic in nature, for example iodine 125, for the determination of radioimmunometric type BROW and Coll. J. Immunol. Methods, 1979, 31 201 - STOCKER et HEUSSER J. Immunol. Methods, 1979, 26, 87-95) or an enzyme for determining immunoenzymometric type, most often peroxidase, alkaline phosphatase or β-galactosidase, (VAN LEUVEN et Coll., J. Immunol. Methods, 1978, 23, 109-116 - MORRIS Transplantation, 1983, 36(6), 719 - BAUMGARTEN J. Immunol. Methods, 1986, 94, 91-98).

The procedures in this last group are rather cumbersome, laboriously demanding and risky to use due to the necessity of numerous rinsings and centrifugations of the cell material; it is sometimes necessary in advance to remove the colored environment resulting from the enzymatic reaction in view of the final spectrophotometric measurement; finally, the chemical fixation of the cells, which is the most commonly used, is the basis for irreversible destruction of these antigens which are particularly sensitive to common chemical fixation agents, such as glutaraldehyde or methanol (DROVER et Coll. J. Immunol. Methods, 1986, 90 , 275-281).

It is known in the literature that one can measure an antigen that carries several antigenic determinants, i.e. several epitopes, fixing this antigen with the help of one of its epitopes with the help of an antibody immobilized on a solid support and while going to another epitope to the antigen binds another antibody carrying an enzymatic radioisotopic marker which enables determination.

Such a technique, often called the sandwich technique, is particularly described in the patent or patent applications FR 2487 983,

FR 2 500 166, EP 119 736. None of these documents describe the application of this technique to whole cells, although the word "cell" is occasionally included in the list of antigens used by this method.

In the above patents, the various antigens, which are targets of the described examples, are all and exclusively protein molecules, soluble in water and physiological fluids, such as hormones, enzymes or circulating tumor markers. Conversely, it is clear that a cell is not a molecule and that it differs from this by at least a considerably larger size and by the fact that it is not soluble in the physiological environment. Thus, to this day, the sandwich technique has never been applied to whole cells.

Furthermore, immunological collection of cells on a solid support is described in patent application WO 86/02091 in which the aim is to eliminate unwanted cells in bone marrow samples intended for transplantation. In the aforementioned patent application, the capture of cells was carried out on a liquid microsphere, and makes it necessary that the antibodies used are bound to a solid carrier by means of a complex macromolecular structure, called a network relay, able to ensure a favored orientation of the antibody as the cellular antigen corresponds to. No application of this technique for quantitative measurement of an antigen is indicated in this application.

The immunological capture of cells is similarly described in patent application WO 84/03151 for an analytical application.

In this patent, the aim is to identify tissue groups to which the examined cells belong (operation which is mainly called HLA-type indication). Capture of cells is carried out using antibodies placed in a special geometry on highly specialized supports (microscope slides). The results are obtained by simple visual observation of the wearer and lead to "all or nothing" responses. Thus, the systems for immunological capture of cells described to date do not lead to analytical applications that enable quantitative determination of an antigen expressed on the membrane of certain cells. Moreover, all these systems may lack specificity, as they rely on the recognition of cells by means of a unique antibody.

The method which is the object of the present invention has considerable advantages compared to all the previously known and used techniques, as it enables quantitative measurement of all antigens in a cell population in a single analysis time. This determination is performed on cells that have not undergone any chemical or physical intervention and are in their state of physiological integrity. Moreover, the method according to the invention has characteristics of very high specificity, particularly for systems with double immunological recognition, using two different antibodies specific for two different antigens carried on the same cell. This procedure is simple, fast and reproducible. It is fully adapted to the analysis of a large number of samples, enabling its use for diagnostic purposes in large-capacity clinical geology laboratories.

Thus, the present invention describes a method for the immunometric determination of surface antigens from a cellular population or subpopulation selected from among tumor cells and blood cells, the method being characterized in that it consists of: a) immobilization of the cell population carrying the antigen to be determined or a cell population comprising the subpopulation which carries the antigen to be determined, on a solid support, by using one or more monoclonal antibodies previously fixed by covalent binding or by physical absorption on said support and capable of recognizing an antigen present on the surface of cells different from the antigen to be determined, and,

in the same step, direct labeling of the cellular population or subpopulation to be determined, using at least one monoclonal antibody specific for the antigen to be determined, said antibody carrying a radioisotopic

or enzymatic marker/

b) observation of an incubation period; c) washing the support to eliminate unimmobilized cells and excess antibody; d) in the case where the antibody carries an enzymatic marker, addition of the necessary reagent(s) (substrate and

chromogen) to reveal the activity of the enzyme,

e) reading the results, either by counting radioactivity or by measuring light signals (colouring or fluorescence)

referring to a standard form if necessary.

Furthermore, the present invention describes an equipment package for use in the method according to the invention, which is characterized in that it comprises the following components: a) a solid support on which, by covalent binding or by physical adsorption, one or more monoclonal

antibodies directed against surface antigens of the cell population to be determined different from it

characteristic antigen to be determined;

b) one or more solutions each containing a monoclonal antibody specific for said antigen which is characteristic of the cellular population or subpopulation to be determined, labeled with a radioactive marker or a

enzymatic marker;

c) in the case of antibody labeled with an enzymatic marker,

one or more solutions containing the reagents that are

necessary to reveal the activity of the enzyme;

d) possibly an additional component consisting of a buffer solution for washing and/or samples that enables standardization and

quality control of the provision.

The term cell, as used in the present specification and in the patent claims that follow, includes human cells, animal cells, cells of protozoa and cells of microorganisms (bacteria or fungi). Regarding blood cells, the present invention includes the nuclear-appearing elements, such as leukocytes, and the anuclear-appearing elements, such as the red blood cells or platelets.

The method according to the invention uses whole cells, i.e. did not light up.

These cells have not undergone any physical or chemical intervention, and they are used in a state of complete physiological integrity. This situation constitutes the main guarantee before the integrity of membrane markers chosen as targets for determination.

As a solid carrier, one can use all devices adapted to handling cellular suspensions and preferably tubes, special magnetic carriers or solid or soft microtiter plates of polyethylene, polystyrene, polyvinyl chloride or nitrocellulose which have microwells. The monoclonal antibodies intended for immobilization of the cells can be fixed on a solid support, either by chemical covalent binding or physical absorption according to classical techniques well known to those skilled in the art, such as those described by STOCKER et HEUSSER J. immunol. Methods, 1979, vol. 26 pp. 87-95. Advantageously, the carrier can be saturated in advance with the help of a protein.

According to the invention, the monoclonal antibody(s) fixed on the solid carrier must enable immunological capture of the cell population in which the cell population(s) carrying the antigens to be determined are located. When this population consists of human cells, the preferred monoclonal antibodies for immunological capture are the class I anti-HLA antibodies specific for the common group of antigens HLA A, B and C present on leukocytes and numerous other cellular lineages in the organism. Among these antibodies, what is called S-class I, marketed by BIOSYS, is particularly preferred.

In other cases where the investigated cells are human cells and in any case when the cells are not human, the monoclonal antibodies which suit the investigated cell types can be used in the same way in the method according to the invention.

The term "a monoclonal antibody labeled with a radioisotope marker" denotes that the monoclonal antibody carries either on a pure element of its structure, for example the residues of tyrosine constituents, or on a suitable radical fixed thereon, a radioactive isotope which enables determination by counting of the radioactivity associated with it.

The term "a monoclonal antibody labeled with an enzymatic marker" denotes that the monoclonal antibody is bound to an enzyme which, associated with the use of appropriate reagents, allows quantitative measurement of this monoclonal antibody.

The substrate and reagents are chosen in such a way that the final product of the reaction or sequence of reactions caused by the enzyme and using these substances is: either a colored or fluorescent substance that diffuses in the liquid environment of the cells and which is the target for the respective spectrophotometric or fluorimetric final measurement,

or a colored, insoluble substance that settles on the cells separates them from those that are fixed to and which can be the target either for a photometric measurement by reflection or for a visual evaluation, possibly in relation to a standardized color scale.

As an additional component, the equipment package may contain a buffer solution intended for rinsing the solid after immobilization and labeling of cells with one or more antibodies that carry the selected marker.

The equipment package can also contain, as additional components, the samples necessary for standardization of the determination as well as quality control of the determination.

The equipment package and the immunometric method according to the invention are used in a preferred way for the determination of surface antigens of elements that appear in human blood, in particular leukocytes, and more particularly lymphocytes, T lymphocytes, T4 lymphocytes, T8 lymphocytes, B lymphocytes , such as granulocytes, monocytes and platelets.

Another, preferred application is the determination of surface antigens for pathogenic microorganisms, for example Candida albicans.

Moreover, the equipment package and the immunometric method according to the invention are particularly applicable for determining surface antigens for tumor cells, in particular cancer cells from the urinary tract and cells in malignant hemopathies.

The equipment package and the immunometric method according to the invention allow the measurement of signals (radioactivity or absorbed or emitted light) which at the same time depend on the number of cells present in the investigated cellular population and the density of the measured antigen on the surface of these cells. The measurement of these signals enables a quantitative evaluation of the total number of molecules of this antigen carried by the examined cellular population or subpopulation, this antigen playing a structural or functional role.

For example, in the case of leukocyte markers, which are particularly important in hematology, it is known that in healthy individuals,

in the majority of situations, the mean value of the antigenic density does not vary significantly from one sample to another for the same cellular population. There is thus a good correlation between the cytological count of cells carrying the antigen in question and the signal measured according to the invention, which is proportional to the total number of antigen molecules present in the examined sample. In contrast, in certain pathological conditions, the density of surface antigens may vary for the same cellular population without the number or proportion of positive cells varying appreciably. Such pathological conditions are clarified more effectively by using the immunometric method according to the invention or by using the equipment package according to the invention, than by a conventional method with cytological counting.

Another application of the invention emerged when a microtiter plate was chosen as a solid support. The equipment package and the immunometric method according to the invention can therefore be advantageously used for the determination on a single plate of a number of surface antigens which are characteristic of different subpopulations which make up the investigated cell population. For this purpose, one can, on the one hand, use ready-to-use microtitration plates, on which one or more monoclonal antibodies capable of retaining all cells in the examined population have been previously fixed, and, on the other hand, place a number of monoclonal antibodies bound to a appropriate marker and each specific for an antigen characteristic of one of the subpopulations to be evaluated. Thus, in a single operation and on the same carrier, the quantitative determination of all the antigens necessary for the characterization of the selected subpopulations can be achieved.

This application of the present invention is illustrated by characterization of the antigenic equipment for cells of interest in clinical biology. A first case is represented by the determination of tissue groups that characterize a given individual, known under the common type designation HLA.

Another case is represented by the type indication of tumor cells, especially for patients affected by malignant hemopathies, such as leukemias or lymphomas. This diagnostic examination, systematically practiced, consists in characterizing the type and origin of the patient's tumor cells by the presence or absence on the cells of a number of selected, conventional surface antigens.

By using the equipment package according to the invention, which comprises a microtitration plate on which one or more monoclonal antibodies capable of fixing all the cells in the investigated population and solutions of different monoclonal antibodies labeled with an enzymatic or radioisotopic marker are fixed in advance, each specific for an antigen that is present on the tumor cells, one can identify and evaluate quantitatively the antigens that are characteristic of the patient's tumor cell population, and thus link them to one of the large groups of cancers and particularly malignant hemopathies, characterized clinically. The application of the method according to the invention also enables rapid execution, and a single carrier, qualitative and quantitative examination of the antigenic equipment of the tumor cells.

To illustrate another application of the invention, cases with human T-lymphocytes can be mentioned, for which there exist in particular 2 subpopulations of cells: lymphocytes which are characterized by the presence of the marker CD4, and which are called T4-positive lymphocytes or simply T4- lymphocytes and lymphocytes characterized by the presence of the marker CD8, which are called T8-positive lymphocytes (or T8 lymphocytes).

The measurement of the number ratio T4/T8 is of great diagnostic interest in clinical biology. It is actually known that changes in the ratio T4/T8 occur in several disorders of the immune system, such as the dysimmune diseases, chronic infectious diseases, viral infections and especially disorders due to HIV (virus from

AIDS).

The equipment package and the immunometric method according to the invention can be used to determine antigens in their entirety, which are characteristic of the population of T lymphocytes and/or antigens characteristic of subpopulations of T4 and T8 lymphocytes. In this case, specific immobilization of T-lymphocytes in the examined sample is carried out, on a solid support, and at the same time direct labeling of surface antigens on T4-lymphocytes is carried out by a monoclonal antibody anti-CD4 carrying a radioisotopic or enzymatic marker; similarly, the direct labeling of surface antigens of T8 lymphocytes is performed by a monoclonal antibody anti-CD8 carrying an appropriate marker.

For the determination of total T-lymphocytes, the monoclonal antibody anti-CD7 (also called anti-T2) which carries a suitable marker is preferably used.

In order to achieve specific immobilization of T-lymphocytes in the sample, one or more monoclonal antibodies are preferably used which alone or in combination are able to recognize the entirety of T-cells in the sample, which is the case with the known anti-leukocyte antibodies (or anti-CD5) or antibodies that recognize the pool of population T (called "pan T") such as antibody anti-CD2 (or anti-Tll), anti-CD5 (or anti-Tl), anti-CD7 (or anti -T2) or other antibodies pan-T which are not yet linked to a differentiation class according to the OMS criteria.

One can advantageously use the immunometric method according to the invention to determine antigens characteristic of the population of T lymphocytes and T4 and T8 lymphocytes on several areas of the same solid carrier. The measurement of the signals by counting radioactivity, photometric measurement in transmission or reflection, or measurement of fluorescence allows easy and direct calculation of the number ratio CD4/CD8.

In the same way, the subpopulations called T-lymphocytes and B-lymphocytes, which make up the collection of the lymphocytic line, can be determined on the same solid carrier.

One can, for example, by absorption fix a monoclonal antibody or a mixture of monoclonal antibodies specific for the total quantity of surface antigens of T cells on the side walls of microwells. The monoclonal antibodies enable subsequent immobilization in the microwells of the entire population of T cells in the examined sample. The flakes produced in this way can be lyophilized and made into stick solutions, preferably at 4°C. This step can be carried out industrially, and plates can thus be placed ready for use in the equipment packages which are applicable either to total T-lymphocytes or to the entire subpopulation of T-lymphocytes.

The samples containing cells to be determined and which come from blood or other suitable biological fluid, normal or pathological, can be used as such or after treatment, in particular by density gradient centrifugation according to already known methods and in particular in an environment with strong density such as FICOLL-PAQUE, sold by Pharmacia. For the measurement of lymphocytes in blood, the blood sample to be determined can be treated in the same way with a solution called lysis buffer which lyses the red blood cells.

Aliquots of the appropriate cellular suspension are placed in contact with the solid support, for example in micro-wells of a microtiter plate prepared in advance, at the same time as the solution which forms part of the kit and which contains the specific monoclonal antibody for the intended cellular population and which carries an appropriate marker, i.e. a radioisotopic or enzymatic marker. Thus, a radioisotopic marker can be prepared, for example, by labeling the monoclonal antibody with iodine 125 or iodine 131, for example in the presence of chloramine T according to a known method (F.C. GREENWOOD, W.M. HUNTER et Coll, Biochem. J., 1963, 89, 114); or alternatively, an enzymatic carrier can be prepared by conjugating the monoclonal antibody with an enzyme such as e.g. alkaline phosphatase, peroxidase, β-galactosidase or acetylcholinesterase, according to known methods (see for example M. O'SULLIVAN Methods in Enzymology 1981, 73., 147 or according to a method derived from it. In these cases , to avoid certain disadvantages linked to the handling of radioactive substances and to the duration which is limited to the effectiveness of the reagents, the enzymes will preferably be used with radioisotopic markers.

The incubation period, i.e. the duration required for simultaneous immobilization and labeling of cells is preferably less than or equal to one hour. During this time period, the solid support can optionally be centrifuged to improve the immobilization of the cells. The solid support, for example the microtiter plate, is then rinsed to simultaneously eliminate the unfixed cells and the monoclonal antibody carrying an enzymatic or radioisotopic marker in excess.

When using a radioisotopic marker, such as iodine 125, the radioactivity associated with the cells is counted in a gamma counter according to any suitable method and, for example, after solubilizing the cells with an alkaline solution (for example, a sodium hydroxide solution) and collecting of the solution containing radioactivity using an absorbent tampon.

When applying an enzymatic carrier to the monoclonal antibody, the appearance of a colored or fluorescent product will be achieved by adding to the solid carrier on which the cellular population bearing the antigen to be measured is fixed a solution containing the enzyme substrate and one or more auxiliary reagents which make it possible to finally obtain as a reaction product either a colored product soluble in the environment or a colored, insoluble product, or a soluble fluorescent product as has been explained before. The light signal coming from the samples that have been treated in this way is further measured using the equipment adapted to each case: photometer by transmission or reflection or fluorimeter respectively. When the solid carrier is a microtiter plate, the reading of light signals can be carried out sequentially in all the wells of the same plate by using automated readers for ongoing use in biology laboratories, such as, for example, the Titertek plate reader or the Fluoroscan plate reader, for spectrophotometric or fluorometric readings, respectively.

When alkaline phosphatase is used as an enzymatic carrier, the binding of this enzyme to the monoclonal antibody is carried out according to the method proposed by Boehringer Mannheim - Biochemica. The preferred substrates of this enzyme are paranitrophenyl phosphate for a spectrophotometric final reading or 4-methyl-umbelliferyl phosphate for a fluorimetric reading or 5-bromo-4-chloro-3-indolyl phosphate to obtain a product with an insoluble color reaction. In the same way as an enzymatic probe, /3-galactosidase can be used, for which the preferred substrates are then orthonitrophenyl beta D-falactopyranoside or 4-methyl-umbelliferyl-£-D-galactopyranoside.

Preferably, the monoclonal antibodies can be bound to peroxidase. In this case, the binding procedure is derived from that described by M.B. WILSON and P.K. NAKANE in Immunofluorescence and Related Staining Techniques, W. Knapp, K. Kolubar,

G. Wicks eds, Elsevier/North Holland. Amsterdam, 1978, pp. 215-224. The modifications introduced with regard to the initial protocol for the preparation of the enzymatic conjugate have the following points: the molar peroxidase/antibody ratio is equal to 3, against 2 in

the protocol,

oxidation reduces the pressure of glucose units in the peroxidase by reducing 33% of the proposed concentration of sodium periodate.

The reagents used to reveal the peroxidase conjugated to monoclonal antibodies contain oxygenated water, enzyme substrate and an appropriate chromogen, for example orthophenylenediamine or 2,2'-azino to (3-ethyl-6-benzothiazoline sulfonic acid) or ABTS to obtain a final reaction product that was colored and soluble in the environment or also 3,3' diamino benzidine or 3-amino-9-ethyl-carbazole or 4-chloro-alpha-naphthol to obtain an insoluble final reaction product, or also parahydroxyphenylpropionic acid to obtain a fluorescent reaction product which is soluble in the environment.

According to a preferred embodiment, the equipment package according to the invention, for the determination of antigens characteristic of T-, T4- and T8-lymphocytes, comprises: a) a microtiter plate in whose wells one or more monoclonal antibodies, anti-lymphocytes T.

bl) a solution containing at least one monoclonal antibody

antilymphocytes T labeled with peroxidase.

b2) a solution containing at least one monoclonal antibody

antigen-CD4 labeled with peroxidase.

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

cl) a solution containing oxygenated water, enzyme substrate, i

a suitable buffer.

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

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

Acetylcholinesterase is bound to antibodies by preferably using a method derived from that described in French Patent No. 2,550,799 or a method which schematically comprises the preparation of fragments of antibodies by means of a known technique, modification of the enzyme by reaction with a suitable heterobifunctional agent and finally binding of the products thus obtained. Other known methods for the construction of immunoenzymatic conjugates can also be used in this case.

The revelation of the enzymatic activity specifically linked to the cellular antigen recognized by the conjugate with acetylcholinesterase is preferably carried out according to the well-known technique using acetylthiocholine as substrate for the enzyme and Ellman's reagent, or 5,5-dithio-2-nitrobenzoic acid as chromogen , according to any variant adapted to the investigated case, for example the one described by Pradelles et al. Anal. Chem. 57 (1985) 1170-1173.

The mentioned chromogens are used as such or in the form of salts soluble in water.

The results of the determination of antigens according to the invention can be expressed in any way that is appropriate for the investigation carried out. More particularly, one can express these results either in total number of molecules of a particular antigen (for example, antigen CD4) presented in a volume of the examined sample (for example, per microliter of blood), or in relation to the number of molecules of an antigen or the number molecules of another antigen in the examined sample (for example the ratio between antigen CD4 and the antigen CD8 - or the ratio CD4/CD8 in the examined blood sample.

To determine the number of molecules in a particular antigen in the examined sample, one will preferably use a calibration scale consisting of cells or suitable cell preparations, which carry the antigen to be determined and which has been calibrated in advance using a known reference method. These standards will preferably consist of either cells that are identical in origin to cells that are the target of the determination, or of cells from established cell lines that carry the sought-after antigen, or of preparations, for example of membranes, that originate from these cells.

These standards are therefore treated exactly like the samples to be examined. The signals coming from these serve to construct a standardization core to which the signals measured with the samples to be examined are returned. The further calculations are conventional.

To determine the ratio between the number of molecules in the two antigens in the sample to be examined, one can use the standardization system described above and finally calculate the sought ratio. In a simpler way, one can in numerous cases directly calculate the ratio between specific signals obtained with each of the sought antigens, correct the resulting cases with known factors such as the ratio between dimensions of used experimental samples, and one directly obtains their sought ratio.

The immunometric measurement method according to the invention is simple, fast and reproducible. Its application is fully adapted to the analysis of a large number of samples. To understand the advantages of comparison with other methods described, it is appropriate to analyze the different steps.

Immobilization of the cells on the solid support is the measurement phase that will usually present the most difficulties or is the most critical to perform. A frequently used method is chemical fixation of the cells by means of glutaraldehyde or methanol in dishes which may or may not be treated with poly-L-lysine (VAN LEUVEN F. et Coll., J. Immunol, Methods, 1978, 23, 109 ). Nevertheless, the chemical fixations thus used may diminish or even suppress the sought specific detection or conversely induce false positive labeling of cells, which is a very serious disadvantage (DROVER et MARSHALL J. Immunol. Methods, 1986, 90, 275- 281).

Moreover, carrying out the method by chemical fixation necessitates several steps: centrifugation of the cells, preparation of the fixative mixture, fixation and then rinsing of the fixed cells.

Drying of the cells at 37°C, followed or not by a fixation in methanol in the microwells has also been suggested (BAUMGARTEN, J. Immunol. Methods, 1986, 94, 91-98). In fact, dehydration of the cells at 37°C, besides altering certain fragile antigens, will penetrate the pericellular plasma membrane, facilitating immunological labeling of intracytoplasmic antigens in addition to labeling of surface antigens, thus leading to annoying "background noise" " or to false positive results when one wants a target limited to the surface antigen.

Moreover, the reproducibility of this method is uncertain; in fact, the decantation of the cells in the microwells for the determination and the drying of the cells may vary from one experiment to another. Finally, this determination takes a long time to perform as the actual drying step of the cells alone necessitates a time period of more than two hours.

The immobilization of lymphocytic populations has also been achieved thanks to the use of polyclonal antibodies absorbed in microwells (STOCKER et HEUSSER J. Immunol. Methods, 1979, 26, 87-95). Besides allowing the immobilization of cells that are foreign to the one population that one wishes to analyse, the polyclonal antibodies also present the disadvantage that they react with the antigens to be measured by means of the labeled antibody, which equally reduces the signal that was finally measured.

The use of highly specific and delimited monoclonal antibodies absorbed or fixed to the solid support and especially in measurement wells, allows the exclusive capture of sought after cells, as the other non-retained cell populations are eliminated during the rinses that are performed at the end of the labeling of the antigens to be determined. Moreover, there is no chemical or physical agent that changes the characteristics of the antigens in this step, since the various operations destined to fix the cells chemically or physically to the carrier are suppressed.

Thus, according to the present invention, it has been found that the immobilization of cells by means of monoclonal antibodies is a method that enables the simplification of the immobilization step of cells carrying the antigen to be determined, and all this makes the results more reliable.

The immunometric measurements that are applied to the cell populations are mainly carried out by labeling cells according to the indirect method in 2 or 3 successive steps, the marker that brings the specific signal being fixed on the cells during the last step of the labeling procedure. During the labeling of the cells in two steps, the main reagents used are antibodies, anti-immunoglobulins (anti-Ig) bound to β-galactosidase (COBBOLD and Coll. J. Immunol. Methods, 1971, 44, 125-133) or to alkaline phosphatase (HESSIAN J. Immuno. Methods, 1986, 91, 29-34) or labeled with iodine 125 (SAVION J. Immunol. Methods, 1987, 97, 49-56). The technique of labeling with reactive peroxidase anti-peroxidase is performed in 3 stages (VAN LEUVEN 1978).

Another method, this one also in 3 steps, introduces a monoclonal antibody that is specific for the antigenic marker to be analyzed, then antibody anti-Ig from mice carrying biotin and finally a conjugated streptavidin-peroxidase (BAUMGARTEN, 1986) or streptavidin -alkaline phosphatase (IGIETSEME and Coll. J. Immunol. Methods, 1987, 97, 123-131).

The method according to the invention which comprises simultaneous application in a single step of an immunocapture process of whole cells, without physical or chemical intervention on the cells and labeling of all or part of these cells with one or more monoclonal antibodies that directly carry a radioisotopic or enzymatic marker, enables for the first time quantitative measurement on the cells themselves of selected membrane antigens.

According to the invention, it enables the direct labeling of the immobilized cells immunologically: simplification of the method of determination by the omission of repeated intermediate handling between the subsequent labeling steps in the case of indirect labeling: centrifugation of the cells,: elimination of reagents for labeling, resuspension;

savings of reagents; - an improved reliability by reducing the number of steps and handling;

gain time;

possibility of simultaneously processing large numbers of samples using only common materials and equipment.

The incubation time for immobilization of the cell population and simultaneous direct labeling of antigens in the cellular subpopulation to be dosed is short. It is less than or equal to one hour in the case of determination of T-lymphocytes and sub-populations of T4 and T8 lymphocytes.

After rinsing the solid carrier, the dosage is, in the true sense, carried out by observing a signal that is accurate and easy to measure, with ordinary equipment: radioactivity, absorption or light emission.

Thus, the entirety of the method according to the invention presents numerous advantages: it is fast, reliable, economical and simple.

The method according to the invention enables the determination of surface antigens in a cell population in a large scale of cell concentrations.

The sensitivity of the method vis-à-vis the cell count depends on the antigenic density and the measured cell population. For each antigen, one can, if desired, define the minimum molar concentration in antigen that can be measured by the method according to the invention.

Thus, for example, when the solid carrier chosen is a microtiter plate and when the cells examined are human lymphocytes, it has been observed that significant measurements are obtained when the number of analyzed cells is included between a few hundred and approximately 200,000 per /il as the lower limit is determined by the density of the antigen measured on the examined cells and the sensitivity of the chosen detection technique, while the upper limit depends mainly on the dimension and geometry of the solid support. The same applies when the fixed carrier consists of pipes.

It has been verified that the recorded signals (counting of radioactivity or photometric measurements) enable the achievement of regular and satisfactory standardization curves as a function of the number of cells used, under normal handling conditions.

Moreover, one can improve the sensitivity of the method if necessary by simultaneously fixing on the same surface antigen several different monoclonal antibodies, specific for several different isotopes in the same antigen. This has been verified by determining CD4 antigens on cells from the Ischikawa line (human T line) for which the antibodies 0KT4 and ST4 labeled with iodine 125 were used at the same time. The measured signal was amplified by approximately 50% in relation to signals obtained with each of the anti-CD4 antibodies used alone.

In the examples that follow, the following terms or their abbreviations are used indifferently: BSA: albumin from bovine serum or bovine serum albumin

PBS: phosphate buffered saline 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

cpm: counts per minute dpm: splits per minute

EXAMPLE 1

Immunoenzymometric determination of the molecular concentration of antigen CD4 and CD8 from samples of human blood which includes labeling of antibodies with peroxidase.

A) Preparation of the equipment package for determination

a) Production of the plate

A commercially available plastic microtitration plate is used

of NUNC (reference 64394) which holds 96 micro-wells. One places in each microwell 200 ( Ml) of a solution containing the purified monoclonal antibody anti-CD2 (called ST 11) used to immobilize the total number of T-lymphocytes, i.e. to perform their immunological capture. This antibody sold by BIOSYS/ Compiegne, France under reference ST 11, is used in a concentration of 10 nq/ial in a phosphate buffer salt solution at pH 7.474 (PBS).

The adsorption of the monoclonal antibody is carried out by

4°C for 12 hours. Excess antibody is eliminated by rotating the plate.

A solution containing 0.1% gelatin and 0.5% BSA in a phosphate-buffered saline solution is prepared. 250 il of this solution is added per jug, to saturate the surface of the wells with protein, which is achieved after one hour at 37°C: the plates are washed 3 times with phosphate-buffered saline solution. The plates produced in this way are lyophilized and

put in stock solution at 4°C in a small, closed plastic bag.

b) Preparation of the solution of conjugated monoclonal antibody peroxidase

Peroxidase (POD) sold by Boehringer is used

Mannheim Biochemica (reference 814 393).

The procedure for binding between antibody and peroxidase is described by M.B* WILSON and P.K. NAKANE in Immunofluorescence and Related Techniques (W. KNAPP,

K. KOLUBAR, G. WICK ed., 1978, Elsevier/Nord Holland, Amsterdam - pp. 215-224), with the exception that 1.5 mg of POD in 0.36 ml of distilled water is used for the oxidation of peroxidase and that one adds 50 jixl of. a 0.2 M sodium periodate solution. The product thus obtained is bound to 2 mg of IgG-anti-CD4 contained in 500 µl of carbonate buffer. After treatment with sodium borohydride and dialysis against PBS, the conjugate IgG-POD is sterilized by filtration on a membrane 0.22 µm and preserved under sterile conditions at a concentration of 0.5 mg IgG per ml at 4°C in glass tubes. The reagent is stable for at least one year.

Conjugate IgG-anti-CD8-POD is prepared in the same way.

Conjugates IgM-POD can be prepared in the same way.

c) Preparation of the disclosure reagent.

The disclosure reagent is obtained in the following way: Mon

prepares a 0.1 M citrate buffer by dissolving citric acid monohydrate at 2.1% in water and adjusting to pH 5 by adding 7N sodium hydroxide. 30 mg of orthophenylenediamine dichlorohydrate is then added to 20 ml of the obtained citrate buffer, then 40 jul is added at the last moment

oxygenated water (substrate for enzymes) to 30% per 20 ml of citrate buffer containing orthophenylenediamine.

B) Immunometric method

a) Separation of the cells

2 ml of the blood sample to be dosed is taken. Mon

it is mixed with 2 ml of the phosphate-buffered saline solution and this mixture is deposited on 3 ml of FICOLL-PAQUE (sold by Pharmacia). Upon centrifugation at 400 x g for 30 minutes at room temperature, a ring of suspension containing the mononuclear cells is formed. This entire suspension is collected in a volume of 1 ml and 6 x 100 µl of the thus obtained suspension is distributed into 6 microwells in the

pre-manufactured plate,

t») Incubation of the cells.

The conjugated POD antibody obtained above is diluted to one hundredth with PBS containing a 1% protein-containing substance such as BSA or skimmed dry milk and 100 µl of this solution is added per microwell, i.e.: 2 microwells are each filled with 100 µl of the solution of conjugate POD-anti-CD4. 2 microwells are each filled with 100 µl of the solution of conjugate P0D-anti-CD8. 2 control microwells are each filled with 100 μl of the solution with 1% proteinaceous substance (blind to reaction)

To improve the fixation of cells on the support, the plate is centrifuged for 3 minutes at 150 x g after resting in

one hour at room temperature.

c) Disclosure of the fixed and measured enzyme.

The microwells are emptied by rotating the plates. Mon

rinse the microwells 4 times with 200 µl PBS. 200 ul of the revelation reagent prepared for the purpose just before is added to each well. Incubate for 20 min. at room temperature in dim lighting. The optical density is measured in a spectrophotometer at 492 nm (Titertek Multiskan type 310 C - Flow

Laboratories).

d) Standardization and expression of results.

In order to carry out the standardization of these experiments, a

preparation of total human lymphocytes previously calibrated in anti-genes CD4 and CD8 by the cytofluorometric technique of PONCELET et al. (J. Immunol. Methods 1985, 85, 65-74 applied. The pre-taken, known aliquot amounts from this reference preparation served to constitute the two standardization schemes in relation to antigen-CD4 and antigen-CD8 respectively, in relation to the number of antigens that responded to each sample was calculated.

In an indicative way, Table 1 which follows hereafter shows 20 human blood samples coming from healthy donors, the optical density values obtained, the number of molecules of the corresponding antigens CD4 and CD8 and the ratio between the antigen numbers derived from these (ratio CD4/CD8 ).

EXAMPLE 2

Immunoenzymometric dosing of the molecular concentration of the antigens CD4 and CD8 from human blood samples that includes labeling of antibodies with alkaline phosphatase.

This dosage is carried out as in example 1. Preparation of the monoclonal antibody-alkaline phosphatase conjugate is carried out according to the method indicated by Boehringer Mannheim - Biochemica (Ref. 567 744). After an hour's incubation of the cells to be dosed with this conjugate, the fixed enzyme is revealed by adding a solution of 1 mg/ml paranitrophenyl phosphate in a 1.2% diethanolamine buffer in distilled water adjusted to pH 9.8 with a dilute hydrochloric acid solution . After 2 hours at 37°C, the optical density is measured in a spectrophotometer at 405 nm.

The results are calculated and expressed as in example 1.

EXAMPLE 3

Immunoradiometric measurement of the molecular concentration of antigen CD4 and CD8 from human blood samples that includes labeling of antibodies with iodine 125.

Preparation of labeled antibodies with iodine 125 is carried out according to the technique described by F.C. Greenwood, V.M. Hunter and Coll., Biochem. J., 1963, 89, 114.

50 µg of monoclonal antibody anti-CD4 or anti-CD8 contained in 50 µl of phosphate-buffered saline solution at pH 7.2 is mixed with

37 MBq of iodine 125 in the form of sodium iodide in 30 µl of a solution of chloramine T in a phosphate-buffered saline solution containing 0.33 mg of chloramine T per ml. After one minute under stirring, the labeling reaction of the monoclonal antibody is stopped by adding 100 µl of a solution of sodium metabisulphite dosed to 2.5 mg/ml. The thus prepared solution is placed on a PD column 10 (Pharmacia - Sephadex G25M), and the fraction containing the radiolabeled antibody is recovered in the effluent. The dosage is carried out by adding to 4 microwells a volume of 100 µl of the cellular suspension containing the mononuclear cells of human blood prepared as in example 1. Then one adds 100 µl of the diluted solution of radioactive antibody in a PVS buffer which contains 5% BSA, by adding 150,000 cpm per well; 2 microwells are each filled with 100 µl of the solution of conjugate anti-CD4-125-I, 2 microwells are each filled with 100 µl of the solution of conjugate anti-CD8 125 I. The fixation of the cells is improved by centrifuging the plate for 3 minutes at 150 x g after 1 hour incubation at room temperature. The microwells are emptied by rotating the plate. The microwells are rinsed 4 times with 200 µl PVS per well. 75 µl of 1 M sodium hydroxide solution is then added to each well. After 10 minutes, the contents of each well are set up with an absorbent tampon and the radioactivity is counted in a gamma counter (multiwell counter LKB).

The results are calculated and expressed as in example l, with the values of the optical densities being replaced with the results from counting in dpm.

EXAMPLE 4

Verification of the validity of the method.

Anti-genes CD4 and CD8 in human T cells are measured on 20 human blood samples taken in advance by following the immunometric method with peroxidase as described in example 1. On the same blood samples it was also determined by using the conventional technique (W.W. ERBER et Coll. Lancet, 1984, (8385), 1042-1045) for cytological count the ratio between the number of T4 positive cells and the number of T8 positive cells.

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

In the same way, on 7 other previously drawn human blood samples, the CD4/CD8 ratio determined by the immunoradiometric method using labeling with iodine 125 in the method according to the present invention and the ratio between cells T4/T8 determined by counting cells were compared . The correlation coefficient is r=0.87.

In the 2 cases, the obtained correlation coefficients show that despite a satisfactory overall agreement between the results in the 2 techniques, the information presented in the 2 reports is not equivalent, which is clear since the dosage of antigens according to the invention takes into account in the calculation not only the number of positive cells as is the case for the cytological method, but also the density of the considered antigen on the positive cells in each sample. The information provided by the described technique according to the present invention is thus more complete than that provided by the conventional reference technique (W. W. ERBER et coil. Lancet, 1984, (8385), 1042-1045).

EXAMPLE 5

Immunoenzymometric measurement of the molecular concentration of the antigen CD4 and CD8 to T-lymphocytes from human blood samples which includes labeling of antibodies with peroxidase.

This example is intended to show that the necessary duration for dosing can be reduced, in relation to the conditions described in example 1, by changing the procedure for separating cells on the one hand and the incubation time necessary for it on the other hand immunological capture steps and for labeling the cells. A) The influence of the separation procedure on the cells in whole blood.

a) Technique with short centrifugation:

0.5 ml of the required blood sample is taken beforehand

is dosed, which is mixed with 1.5 ml of phosphate-buffered saline solution. In a 5 ml hemolysis tube, add 1.5 ml of Ficoll-Pague, sold by Pharmacia, and deposit the blood sample diluted in PBS on the surface of the Ficoll layer. After 5 minutes of centrifugation at 900 x g at room temperature, the ring of suspension containing the mononuclear cells in

a volume of 0.5 ml. 1.5 ml of PBS is added to this sample.

b) Technique for lysis of erythrocytes:

Another quick method for separating blood cells is the use of a buffer that lyses the red blood cells. The light buffer, used as an example, with the following composition:

ammonium chloride: 8.29 g

potassium bicarbonate: 1 g

disodium salt of ethylenediaminetetraacetic acid: 0.0307 g for 1 liter of distilled water, the pH being adjusted to 7.3.

5 ml of lysis buffer and 0.250 ml of blood are mixed. After 10 minutes with stirring, centrifuge at 600 x g for 10 minutes. The precipitate of the formed cells is taken up in 1 ml of PBS buffer.

By further carrying out the dosage of antigens CD4 and CD8, under the conditions of example 1, and by comparison with the reference protocol described in example 1 for the isolation of mononuclear cells from blood, it has been verified that both of these two variants of the method for separating mononuclear cells from total blood well has enabled the collection of the entire amount of lymphocytes in the examined blood samples. B) Influence of incubation time for the immunological capture and labeling of the cells.

In order to verify whether the time period of 1 hour used in example 1 cannot be reduced in order to accelerate the course of the dosing, the results obtained for identical samples, each containing 20,000 mononuclear cells per well, when varying this time limit from 10 minutes to 1 hour in accordance with the step for capturing and labeling the cells. As the operating conditions apart from this are the same as in example 1, the results obtained are shown in table 2.

* These optical density values correspond to the reagent blank obtained in the absence of cells. The sample values obtained in the presence of cells and by omitting either the conjugate or the substrate are not greater than the indicated values.

These results show that:

the non-specific signal (blank test on reagents) remains at always weak values, as much lower as the time for

presence of conjugate is shorter.

the specific signal has, after 10 minutes of contact, a value that can be used analytically with precision and reproducibility. This indicates that it is possible, in relation to the conditions in example 1, in a significant way to reduce the duration for carrying out dosing without an important loss in the precision of the results.

In practice and if one adds up the gains in time that can be achieved at the level of preparation for the blood sample and at the level of immunological capture, it is possible with the equipment package and the method according to the invention to dose the antigen CD4 and CD8 in the samples from whole blood against a background of 10 or 20 samples per plate with 96 wells, in a total time period (after receipt of the samples in the laboratory) that does not exceed 1 hour. This level of productivity is greater than the level of all the alternative techniques known to date.

EXAMPLE 6

Immunometric dosing of the molecular concentration of antigen CD5 from human T-lymphocytes from human blood samples that includes labeling of antibodies with acetylcholinesterase.

A) Preparation of the equipment package for dosing.

a) Fixed carrier.

A microtitration plate manufactured as

described in example 1.

b) Conjugate monoclonal antibody-acetylcholinesterase.

Acetylcholinesterase from Electrophorus is used

electricus obtained according to the technique described in French patent No. 2,550,799.

This enzyme is bound to antibody anti-CD5 called ST 1 and marketed by BIOSYS. The binding is carried out according to the method described by YOSHITAKE et al. Eur. J. Biochem. 101 (1979), 395-399.

c) Revelation reagent.

This reagent also includes the substrate of the enzyme

(acetylthiocholine) and the Ellman reagent and with the following composition:

Acetylthiocholine 7.5 x 10"*M

5,5'-dithio-2-nitrobenzoic acid (DTNB) 5 x 10~<*>M

a sodium phosphate buffer pH 7.4

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

B) Immunometric procedure.

a) The mononuclear cells are separated using the method described in example 1. b) Incubation of the cells for the step of immunological capture and labeling lasts 20 minutes as in example 5.

c) Disclosure of the fixed and measured enzyme.

100 µl of disclosure reagent is added to each microwell.

The absorbance is measured at 412 nm after 45 minutes of incubation.

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

These results reveal the extreme sensitivity of the technique thus performed. In fact, the optical density of 0.030 obtained in the well containing 540 T-lymphocytes (i.e. 32 million molecules of antigen CD5) which is measurable with precision and equal to 10 times the background noise, corresponds to approximately 5.10"<17> mol of antigen CD5 per dosing well. A such sensitivity is of the order of the best known immunodosages, which use the most sensitive radioactive markers.

EXAMPLE 7

Dosing of different antigens expressed on human activated T lymphocytes.

Activation of T-lymphocytes is a physiological process that occurs early whenever the immune system is affected, such as in infectious pathological conditions, in organ transplants, in certain dysimmune diseases.

This natural process is also easily initiated in vitro in the laboratory in such experiments as, in particular, the mixed lymphocytic reactions or experiments with lymphoplast transformation. In the latter case, polyclonal activation is achieved by using substances such as phytohemagglutinin (PHA), concanavalin A or other lectins. Activation of T-lymphocytes is followed by a strong increase in expression of several membrane markers and in particular antigen CD25 (receptor for interleukin 2) or antigen CD2. These antigens contain excellent markers in the activation state, and their dosage is at the same time of great interest in clinical biology and for laboratory work as indicated above, in that in all cases the use of radioactive reagents is avoided.

These antigens are measured by using the method described in example 1 with the specifications specified in table 4.

The values of the optical densities measured at 450 nm for 25.10<3 >used mononuclear cells are reproduced in Table 5 below and compare the same cells without stimulation by PHA and after 3 days of stimulation.

These results show the considerable rise of the specific signals for the 2 investigated antigens, when the activation occurs.

EXAMPLE 8

Dosage aV antigens CD22 and HLA-Dr (or HLA from class II) present on B-lymphocytes.

The present example describes the dosing of antigens on cells from line RAJI which is a B-human lymphoid line described by PULVERTAFT in J. Clin. Pathol., 1965, 18, 261-274.

For the immunological capture of cells, either antibody S class II (BIOSYS) for dosing antigens CD22, or antibody SB4 (BIOSYS) for dosing antigens HLA in class II (or HLA -Dr.).

a) Dosage of antigen CD22:

Antibody SB22 (BIOSYS) was labeled with iodine 125 according to the method described in example 3. The dosage of the antigen CD22 is carried out as indicated in example 3 for antigens CD4 and CD8, by successively adding to the microwells: 5.10'' cells from the line RAJI, then 10<5> cpm of the labeled antibody SB 22. After a time period of one hour at 4°C, the microwells are emptied by rotating the plate, then rinsed 4 times with 200 μl of PBS per well for each rinse. The radioactivity fixed on the cells is collected and counted as in example 3. Thus, a count of 760 dpm is obtained for the well containing 5 x 10<*> cells RAJI with a blank sample

for reagents (without cells) of 50 dpm.

b) Dosage of antigen HLA-Dr:

One successively adds IO<5> cells to the wells and that

conjugated antibody S-class II labeled with peroxidase according to the method described in example 1. The dosage is carried out according to the same method as in example 1. Thus, an optical density of 1.840 is obtained and a blank test on reagents without cells of 0.105.

EXAMPLE 9

Application of the method according to the invention for measuring antigens carried by T-lymphocytes and B-lymphocytes from human blood.

A) Production of plates:

Microtitration plates prepared in example 1 are used, with the exception that in each well of the microplate, monoclonal antibodies capable of fixing at least the total amount of T-lymphocytes (S class I from BIOSYS) and B-lymphocytes (S class II from BIOSYS), each being used in a concentration of 5 µg/ml.

B) Immunometric dosing:

The mononuclear cells are separated from whole blood using Ficoll under conditions as in example 1 or example 5, then 80,000 mononuclear cells are added per well in 75 µl PBS buffer. The T lymphocytes are measured in these wells with antibody ST 11 (anti-CD 2) from BIOSYS; The B-lymphocytes are measured in other wells with antibody SB 3 (anti-CD 37) from BIOSYS, which recognizes all the peripheral B-lymphocytes. These antibodies are previously bound to peroxidase according to the method described in example 1. The dosage in the true sense is carried out as in example 1.

C) Results.

The absolute number of T-lymphocytes and B-lymphocytes contained in the examined sample is determined by referring to standardization curves obtained by identically treating the mononuclear cells in which the T- and B-lymphocytes were counted by a reference technique.

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

EXAMPLE 10

Dosage of the antigens GpIIb-IIIa and CD 9 in human platelets.

This dosage is carried out according to example 3 by using the monoclonal antibodies radioactively labeled with iodine 125.

Platelets are isolated from human blood by centrifugation in the presence of perfusion fluid PLASMION (laboratory R. Bellon). 5 ml of blood plus 5 ml of phosphate-buffered saline (PBS) and 10 ml of PLASMION are mixed in a tube. The tube is then centrifuged for 10 minutes at 1500 rpm. The platelets collected in the supernatant are taken up by aspiration with a pipette. They are rinsed once in PBS by mixing a volume of the platelet suspension with 10 ml of PBS, and then centrifuged for 10 minutes at 1000 x g. Finally, the precipitate is collected with the platelets, which are resuspended in PBS (2 ml). The platelets are counted and the concentration is adjusted to 2.5 million/ml PBS.

To dose antigen GpIIb-IIIa, immunological capture of blood platelets is carried out using monoclonal antibody I0B2 (Immunotech) and labeling of antigen GpIIb-IIIa and monoclonal antibody P2 (Immunotech).

To dose antigen CD9, immunological capture is carried out using monoclonal antibody P2 and labeling using monoclonal antibody IOB2. The number of dpm is measured for a sample that includes 200,000 platelets.

The results are shown in table 6.

EXAMPLE 11

Dosage of the antigen CD15 carried by human granulocytes.

Antigen CD15 is a good specific marker of human granulocytes (also called polynuclear). Evaluation of these cells using this antigen can be practiced in blood, as for any other leukocyte subpopulation. It is similarly applicable to detect the presence of granulocytes in urine, for example in cases of urinary tract infections such as cystitis or pyelonephritis. In the present example, the determination was performed on granulocytes derived from whole blood and separated as indicated later.

The granulocytes, together with certain mononuclear cells, are separated from red blood cells according to a procedure analogous to that described in Example 10 for the platelets, with the exception that the tube containing the cells is allowed to stand for 45 minutes at room temperature and that the suspension is recovered of leukocytes of 500 /il taken up in the liquid surface.

The granulocytes are immobilized in wells with a monoclonal antibody specific for antigen CD45 present on the cellular membrane of all leukocytes: antibody anti-LCA (BIOSYS) is used absorbed in the microwells of the titration plate, as indicated in example 1.

For dosing, the monoclonal antibody anti-CD 15 SMY-15a (BIOSYS) labeled with peroxidase is used according to the technique in example 1.

Thus, with 12,500 total cells added per well, the specific signal for antigen CD15 from granulocytes is 0.616 after correction for the gross optical density for a reagent blank of 0.100 obtained in the presence of cells and in the absence of enzymatic conjugate, and caused by endogenous peroxidases from granulocytes.

EXAMPLE 12

Evaluation of the phenotype from a leukemia using the immunoenzymometric method.

Phenotyping of tumor cells in a leukemic patient is a systematic diagnostic examination carried out to characterize the type and origin of leukemic cells in the patient (T, B, granulocytes, myeloblasts, etc). This examination is carried out in a classical way using the immunofluorescence technique by using a collection of monoclonal antibodies and by observing and counting positive cells under a microscope. It is used in the same way

.flow cytometry to analyze the labeling of cells.

Use of monoclonal antibodies in the method according to the invention enables the identification of large, clinically important groups of acute or chronic leukemias by producing quantitative information on the relative densities of the various investigated antigens.

In this example, 6 monoclonal antibodies were produced, bound to peroxidase, specific for antigens present on the leukemia cells:

ST 1 and ST 11 (BIOSYS): anti-CD 5 and anti-CD 2

SB 3 (BIOSYS): anti-CD 37

S-CALLA (SANOFI): anti-CALLA (or anti-CD 10)

S-class II (BIOSYS): anti-HLA-Dr (or anti-HLA from

class II)

SMY 15 (BIOSYS): anti-CD 15

The microtiter plate is prepared by absorbing in advance a mixture of monoclonal antibodies anti-CD 45 (S-LKA, BIOSYS) and anti-HLA from class I (S-class I, BIOSYS).

By using the 6 monoclonal antibodies selected above, the phenotype of a chronic B-lymphoid leukemia (LLC-B) which has also been characterized by conventional methods was evaluated.

For 80,000 total mononuclear cells added per well or for the control wells without cells, the optical densities measured at 492 nm are reproduced in Table 7 below. For the control well, which includes cells but no antibodies labeled with peroxidase, the optical density is 0.110.

Thus, the studied leukemic cells carry abundant antigens CD5, CD37 and HLA from class II, but they do not carry or very little antigens CD 15, CALLA and CD 2 which are characteristic of an LLC-B.

EXAMPLE 13

Dosing of antigens associated with cancer in the urinary system.

The equipment packages and the methods according to the present invention are all specially adapted to the detection of tumor cells, particularly in types of tumors in the urinary system and are often used for mass detection in populations with increased risk, for example workers in the chemical industry or other highly exposed groups.

The present example shows the application of the invention to the determination of an antigen associated with cancer in the urinary bladder on cells from line RT4 originating a human urinary papilloma (CC. Rigby et L.M. Franks, Brit. J. Cancer, 1970, 24, 746-754) .

The plates intended for the immunological capture of cells are prepared as in example 1, absorbing in each well the monoclonal antibody S class I (BIOSYS) which recognizes an HLA antigen in class I and is able to retain all the epithelial cells .

The conjugates from the labeling are obtained using the methods in example 1 (for the enzymatic labeling) or from example 3 (for the radioisotopic labeling) with the monoclonal antibody 12F6 (Sanofi) which recognizes an antigen associated with bladder cancer.

For dosing, distribute 75 µl of the cellular suspension in 4 microwells (i.e. 50.10<3> cells per well).

In 2 wells, the conjugated monoclonal antibody is added

12 F 6 (SANOFI) bound to peroxidase, in 2 other wells, antibody 12 F 6 labeled with iodine 125 (100,000 cpm/well). The results obtained by using the immunoenzymometric or therein radioimmunometric method are reproduced in table 8.

EXAMPLE 14

Immunoenzymometric dosing of a membrane antigen from the fungus Candida albicans.

The cells from Candida albicans (serotype 17) come from the collection from the Pasteur Institute in Paris. The fungi are cultivated for 18 hours in the usual way in a fixed synthetic environment. The cells are counted and a cell suspension containing 10<5> cells per ml buffer solution PBS.

The anti-Candida albicans monoclonal antibodies used were prepared by lymphocytic hybridization as described in the thesis of T. Chardes, Faculté de Pharmacie, Université de Montpellier I, 1988.

Antibody CA4 is used for immunological capture, while antibody CA12 labeled with peroxidase is used for dosing under the conditions in example 1.

The values for the measured optical densities at 492 nm for the well containing the added cells and for the control sample without cells are 1.025 and 0.080, respectively.

EXAMPLE 15

Immunoenzymometric dosing performed on magnetic beads: dosing of antigen CD5 from human T-lymphocytes with a conjugated antibody labeled with acetylcholinesterase and a special carrier formed by magnetic beads.

The carrier used to trap the cells consists of DYNABEADS magnetic balls. The referred beads DYN-11101, marketed by BIOSYS, carry an antibody anti-CD 2 on their surface capable of fixing all T-lymphocytes in the examined sample. These beads are then treated with a solution of antibody anti-CD2 prior to use to improve dosing performance. For this, 10 µl of the bead suspension is mixed with 1 ml of a solution of antibody in 25 µg/ml PBS buffer for 1 hour with gentle stirring. The beads are then washed 4 times with PBS buffer, then preserved in 4 ml of PBS buffer.

For dosing, successively add in a 5 ml tube:

200 µl of the bead suspension, 80 µl of the suspension of mononuclear cells separated from whole blood using Ficoll-Paque (PHARMACIA), then add 280 µl of conjugate antibody STI labeled with acetylcholinesterase, identical to that used in example 6.

After one hour under gentle stirring, the spheres are separated using a magnet, and the cells fixed on the spheres are rinsed with PBS buffer (5 rinses).

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

Claims (31)

1. Procedure for immunometric determination of surface antigens from a cellular population or subpopulation selected from among tumor cells and blood cells, characterized in that it consists of: a) immobilization of the cell population carrying the antigen to be determined or a cell population comprising the sub-population carrying the antigen to be determined, on a solid support, by using one or more monoclonal antibodies previously fixed by covalent binding or by physical absorption on said carrier and capable of recognizing an antigen present on the surface of cells different from the antigen to be determined, and, in the same step, direct labeling of the cellular population or subpopulation to be determined, by means of at least one monoclonal antibody specific for the antigen to be determined, said antibody carrying a radioisotopic or enzymatic marker; b) observation of an incubation period; c) washing the support to eliminate unimmobilized cells and excess antibody; d) in the case where the antibody carries an enzymatic marker, adding the necessary reagent(s) (substrate and chromogen) to reveal the activity of the enzyme, e) reading the results, either by counting radioactivity or by measuring light signals (staining or fluorescence) referring to a standard form if necessary. 2. Method for determination according to claim 1, characterized in that the solid support consists of a microtiter plate in whose wells the antibody(ies) are fixed which are intended to immobilize the cells among which there are cells from the population or subpopulation which carries the antigen to be determined. 3. Method according to claim 1, characterized in that the solid carrier is a particulate magnetic carrier. 4. Method according to one of claims 1 to 3, characterized in that the monoclonal antibodies intended for labeling carry an enzymatic marker. 5. Method according to claim 4, characterized in that the enzymatic marker is peroxidase and in that the enzyme's activity is revealed using oxygenated water and a chromogen selected from orthophenylenediamine, 2,2'-azino-bis (3-ethyl-benzothiazoline- 6-sulfonic acid) 3,3'-diamino-benzidine, 3-amino-9-ethyl-carbazole or one of their water-soluble salts. 6. Method according to claim 4, characterized in that the enzymatic marker is alkaline phosphatase and in that the activity of the enzyme is revealed by means of paranitrophenyl phosphate, 4-methyl-umbelliferyl phosphate or by 5-bromo-4-chloro-3-indolyl phosphate. 7. Method according to claim 4, characterized in that the enzymatic marker is beta-galactosidase and in that the enzyme's activity is revealed by means of orthonitrophenyl-beta-D-galactopyranoside or 4-methyl-umbel1i fery1-beta-D-galactopyranoside id. 8. Method according to claim 4, characterized in that the enzymatic marker is acetylcholinesterase and in that the enzyme's activity is revealed by means of acetylthiocholine and as chromogen 5,5'-dithio-2-nitro-benzoic acid or their water-soluble salts. 9. Method according to one of claims 1 to 3, characterized in that the monoclonal antibodies intended for labeling carry a radioisotopic marker with iodine 125 or iodine 131. 10. Method according to any one of claims 1 to 3, characterized in that the total duration for carrying out the determination is less than or equal to 1 hour. 11. Method according to claim 1, characterized in that the monoclonal antibody(s) fixed on the solid carrier are HLA antibodies from class I. 12. Method according to claim 11, characterized in that the monoclonal antibody which is fixed on the solid support is the antibody called S-class I. 13. Method according to claim 1, characterized in that it is for the determination of one or more surface antigens from blood cells from human blood. 14. Method according to claim 13, characterized in that blood cells from human blood are leukocytes, lymphocytes, T-lymphocytes, which carry the marker CD4, called T4-lymphocytes, T-lymphocytes which carry the marker CD8, called T8-lymphocytes, B- lymphocytes, granulocytes, platelets. 15. Method according to claim 1, characterized in that it is for the determination of any one or more of the membrane antigens from tumor cells. 16. Method according to claim 15, characterized in that the tumor cells are cancer cells from the urinary system or cells from malignant hemopathies. 17. Method according to claims 1 to 3, characterized in that it consists in immobilizing a cell population on a microtiter plate using one or more monoclonal antibodies that are specific for surface antigens from this population, and in the same step direct labeling of the cellular subpopulations that make up the fixed population, using several solutions each containing a monoclonal antibody each specific for a surface antigen from one of the cellular subpopulations to be determined. 18. Method according to claim 17, characterized in that it is for the determination of antigens that are specific for the lymphocytic subpopulations T, T4, T8 and B. 19. Method according to claim 17, characterized in that it is for the characterization and determination of different antigens that make up the antigenic equipment on the surface of tumor cells from malignant hemopathies. 20. Equipment package for use in the method according to any one of claims 1 to 19, characterized in that it comprises the following components: a) a solid support on which one or more monoclonal antibodies are fixed by covalent binding or by physical adsorption substances directed against surface antigens from the cell population to be determined different from the characteristic antigen to be determined b) one or more solutions each containing a monoclonal antibody specific for said antigen characteristic of the cellular population or subpopulation to be determined, labeled with a radioactive marker or an enzymatic marker; c) in the case of antibody labeled with an enzymatic marker, one or more solutions containing the reagents necessary to reveal the activity of the enzyme; d) possibly an additional component consisting of a buffer solution for washing and/or samples that enables standardization and quality control of the determination. 21. Equipment package according to claim 20, characterized in that component (a) is a solid carrier consisting of a microtitration plate in whose wells the antibody(s) is fixed which is intended to immobilize the cells among which there are cells from the population or subpopulation carrying the antigen to be determined. 22. Equipment package according to claim 20, characterized in that component (a) is a particle-shaped magnetic carrier. 23. Equipment package according to any one of claims 20 to 22, characterized in that component (a) consists of a solid support on which one or more monoclonal antibodies HLA from class I are fixed. 24. Equipment package according to claim 23, characterized in that the fixed monoclonal antibody is the antibody with the designation S-class I. 25. Equipment package according to any one of claims 20 to 22, characterized in that the monoclonal antibodies in component (b) are labeled with a radioisotope marker with iodine 125 or iodine 131. 26. Equipment package according to any one of claims 20 to 22, characterized in that the monoclonal antibodies in component (b) are labeled with an enzymatic marker. 27. Equipment package according to claim 26, characterized in that the monoclonal antibodies in component (b) are labeled with peroxidase and in that component (c) essentially consists of oxygenated water and a chromogen selected from orthophenylenediamine, 2,2'- azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, 3,3'-diamino-benzidine, 3-amino-9-ethyl-carbazole or one of their water-soluble salts. 28. Equipment package according to claim 26, characterized in that the monoclonal antibodies in component (b) are labeled with alkaline phosphatase and in that component (c) consists of paranitrophenyl phosphate, of 4-methyl-umbelliferyl phosphate or of 5-bromo-4- chloro-3-indolyl phosphate. 29. Equipment package according to claim 26, characterized in that the monoclonal antibodies in component (b) are labeled with beta-galactosidase and in that component (c) consists of /3-orthonitrophenyl-D-galactopyranoside or 4-methyl-jØ- umbelliferyl-D-galactopyranoside. 30. Equipment package according to claim 26, characterized in that the monoclonal antibodies in component (b) are labeled with acetylcholinesterase and in that component (c) essentially comprises acetylthiocholine and 5,5'-dithio-2-nitro-benzoic acid or one of their water-soluble salts. 31. Equipment package according to one of claims 20 to 22, characterized in that component (a) is a microtiter plate in whose wells one or more monoclonal antibodies specific for surface antigens from a cellular population have been fixed and component (b) consists of several solutions each containing a monoclonal antibody specific for a surface antigen from a cellular subpopulation comprising the fixed cell population.