CA1340973C - Immunometric assay kit and method applicable to whole cells - Google Patents
Immunometric assay kit and method applicable to whole cellsInfo
- Publication number
- CA1340973C CA1340973C CA000578730A CA578730A CA1340973C CA 1340973 C CA1340973 C CA 1340973C CA 000578730 A CA000578730 A CA 000578730A CA 578730 A CA578730 A CA 578730A CA 1340973 C CA1340973 C CA 1340973C
- Authority
- CA
- Canada
- Prior art keywords
- cells
- antigen
- enzyme
- antibody
- lymphocytes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
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- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
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- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Zoology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a kit for the determination of a surface antigen characteristic of a cell population or subpopulation, which comprises the following components, namely a) a solid support to which one or more monoclonal antibodies are fixed by covalent bonding or physical adsorption, the said monoclonal antibodies being directed against surface antigens of the cell to he determined, other than the said characteristic antigen; b) one or more solutions each containing a monoclonal antibody specific for the said antigen characterstic of the cell population or subpopulation to be determined, which is labeled with a radioactive probe or an enzyme probe; c) in the case of antibodies labeled with an enzyme probe, one or more solutions providing the reagents necessary for developing the activity of the enzyme; and d) if appropriate, an additional component consisting of a washing buffer solution and/or samples permitting standardization and quality control of the determination. Also disclosed is a process for immunometric assay of antigens characteristic of a cell population of subpopulation, particularly for the determination of lymphocytes T or subpopulations T4 and T8.
Description
~340g73 Immunometri<: assa;~ kit and method applicable to whole cells.
The present invention relates to a kit and to an immunometric assay method using this kit for the determina-tion of surface antigens characteristic of populations or subpopulations of cells.T~'=e immunometric assay method and the corresponding kit are also intended for the ~7eter-ruination of the <.el.ls themselves via the determination of their surface antigens. These determinations are applicable to diagnosis.
Knowledge of the antigens or markers on the cell surface has made enormous advances with the development of lymphocyte hybridization and the discovery of monoclonal antibodies by KOEHLER and MILSTEIN (Nature, 1975, 256, 495-497). In particular, monoclonal antibodies have made it possible to reveal and analyze membrane antigens or surface markers of cells of the widest: possible variety of origins. These markers (or antigens) can be of different kinds:
proteins, glycoproteins or glycolipids. The characteriza-tions sought therefore apply mainly to tissue or organ markers, to markers of states of differentiation or activation of normal cells and to the identification or typing of normal or cancerous cells. A particularly important field of application is the study of the cell lines of hernopoiesis (erythrocyte, rnegakaryocyte, granulocyte, monocyte, Lymphocyte).
Thus, for example, rnonoc onal antibodies have made it possible to specify the respective surface characteristics of T and B lymphocytes. The corres-ponding markers, by themselves or in combination, identify stages of differentiation and functional specialization of the lymphocytes. By international convention, the surface markers of human leukocytes have been classified in differentiation groups or differentiation classes (CD) defined by the ItIIS-WHO
~340g73 subcommittee, 1984, and described in Bulletin of the World Health Organization, 1.984, 62(5), 813-815.
The identification of these markers which has been made possible by monoclonal antibodies has provided access to their structure and their biological functions.
For example, the m~.olecules of the CD4 and CD8 markers participate in leukocyte adhesion functions and are present on the surface of T lymphocytes with an auxiliary and inductive function (CD4 marker) or, respectively, with a cytotoxic and suppressive function (CD8 marker).
With this knowledge established, it has been possible to use these markers, by virtue of the anti-bodies which recognize them, for diagnosing and following up a variety of pathologies including, in particular, malignant hemopathies (leukemias, lymphomas, etc.) and states of dysfunction of the immune system (autoimmune diseases, congenital or acquired immune deficiencies such as AIDS, etc.) (BRETON-GORILJS and VAINCHENKER, Le Riologiste, 1987, XXI, no. 167, 63-70; SHAW, Immunology Today, 1987, 8(1), 1-3).
Monoclonal antibodies are now irreplaceable tools of clinical biology applied to cell analyses.
Cell counting methods exist which use the marking of their surface antigens, but these methods are often lengthy, laborious and difficult to carry out and their results are sometimes random.
The known methods used for measuring the normal or modified expression of antigens on the surface of the cell can be separated into two groups. In the first group, the antigens are measured with the aid of complex and specialized laboratory equipment based on flux cytometry (sf~e, in particular, PONCELET et al., J.
Immunol. Methods, 1985, 85, 65-74) or quantitative microscopy techniques (POULTER et al., J. Immunol.
Methods, 198?, 98, 227-234). These methods for the evaluation of cell. antigens are based on the measurement of signals provided by anticell antibodies coupled directly or indirectly to a. reagent labeled with fluorescent substances (or flu~orochromes) such as fluorescein iso-thiocyanate or rhodamine isothiocyanate, or with enzymes such as peroxidase or alkaline phosphatase. The use of these fluorescent or enzyme reagents in association with appropriate washing steps then leads to the appearance of fluorescences or colorations which are strictly limited to the cell membranes and do not diffuse into the surrounding medium. Common use of these methods in the laboratory is still restricted by the need for specialized and expensive equipment (a fluorescence microscope which may or may not be associated with an image analyzer, a cryostat and a flux cytomet~=_r). Moreover, the analysis and interpreta-tion of the immunolabeling of cells by these processes demand the competence of a specialist in cytology.
A second group of methods for the measurement of antigens .is based on the quantitative evaluation of the markers of the overall cell population. These methods make it possible to measure antigens either by direct labeling or by indirect labeling, the latter most frequently be=ing carried out in two, three or four steps. In al_1 case's, the reagent employed in the last labeling steF~ carr=ies a probe which is either of iso-topic character, for example iodine 125, for a deter-mination of the ra,d.i.oimmunomet;ric typ,~~ (BROW et al.,,~.
Immunol_. Methods, 1.979, 31, 201; STOCKER and HEUSSER, J. Immunol. Methods, 1979, 26, 87-95), or an enzyme for a determination of the enzyme immunometric -type, most frequently peroxida.se, alkaline phosphatase or beta-galactosi'dasei(VAN LEUVEN et al., J. Immunol. Methods, 1978, 23, 109-116; MORRIS, Transplantation, 1983, 36(6), 719; AAIIMGARTEN, J. Immunol. Methods, 1986, 94, 91-98).
The present invention relates to a kit and to an immunometric assay method using this kit for the determina-tion of surface antigens characteristic of populations or subpopulations of cells.T~'=e immunometric assay method and the corresponding kit are also intended for the ~7eter-ruination of the <.el.ls themselves via the determination of their surface antigens. These determinations are applicable to diagnosis.
Knowledge of the antigens or markers on the cell surface has made enormous advances with the development of lymphocyte hybridization and the discovery of monoclonal antibodies by KOEHLER and MILSTEIN (Nature, 1975, 256, 495-497). In particular, monoclonal antibodies have made it possible to reveal and analyze membrane antigens or surface markers of cells of the widest: possible variety of origins. These markers (or antigens) can be of different kinds:
proteins, glycoproteins or glycolipids. The characteriza-tions sought therefore apply mainly to tissue or organ markers, to markers of states of differentiation or activation of normal cells and to the identification or typing of normal or cancerous cells. A particularly important field of application is the study of the cell lines of hernopoiesis (erythrocyte, rnegakaryocyte, granulocyte, monocyte, Lymphocyte).
Thus, for example, rnonoc onal antibodies have made it possible to specify the respective surface characteristics of T and B lymphocytes. The corres-ponding markers, by themselves or in combination, identify stages of differentiation and functional specialization of the lymphocytes. By international convention, the surface markers of human leukocytes have been classified in differentiation groups or differentiation classes (CD) defined by the ItIIS-WHO
~340g73 subcommittee, 1984, and described in Bulletin of the World Health Organization, 1.984, 62(5), 813-815.
The identification of these markers which has been made possible by monoclonal antibodies has provided access to their structure and their biological functions.
For example, the m~.olecules of the CD4 and CD8 markers participate in leukocyte adhesion functions and are present on the surface of T lymphocytes with an auxiliary and inductive function (CD4 marker) or, respectively, with a cytotoxic and suppressive function (CD8 marker).
With this knowledge established, it has been possible to use these markers, by virtue of the anti-bodies which recognize them, for diagnosing and following up a variety of pathologies including, in particular, malignant hemopathies (leukemias, lymphomas, etc.) and states of dysfunction of the immune system (autoimmune diseases, congenital or acquired immune deficiencies such as AIDS, etc.) (BRETON-GORILJS and VAINCHENKER, Le Riologiste, 1987, XXI, no. 167, 63-70; SHAW, Immunology Today, 1987, 8(1), 1-3).
Monoclonal antibodies are now irreplaceable tools of clinical biology applied to cell analyses.
Cell counting methods exist which use the marking of their surface antigens, but these methods are often lengthy, laborious and difficult to carry out and their results are sometimes random.
The known methods used for measuring the normal or modified expression of antigens on the surface of the cell can be separated into two groups. In the first group, the antigens are measured with the aid of complex and specialized laboratory equipment based on flux cytometry (sf~e, in particular, PONCELET et al., J.
Immunol. Methods, 1985, 85, 65-74) or quantitative microscopy techniques (POULTER et al., J. Immunol.
Methods, 198?, 98, 227-234). These methods for the evaluation of cell. antigens are based on the measurement of signals provided by anticell antibodies coupled directly or indirectly to a. reagent labeled with fluorescent substances (or flu~orochromes) such as fluorescein iso-thiocyanate or rhodamine isothiocyanate, or with enzymes such as peroxidase or alkaline phosphatase. The use of these fluorescent or enzyme reagents in association with appropriate washing steps then leads to the appearance of fluorescences or colorations which are strictly limited to the cell membranes and do not diffuse into the surrounding medium. Common use of these methods in the laboratory is still restricted by the need for specialized and expensive equipment (a fluorescence microscope which may or may not be associated with an image analyzer, a cryostat and a flux cytomet~=_r). Moreover, the analysis and interpreta-tion of the immunolabeling of cells by these processes demand the competence of a specialist in cytology.
A second group of methods for the measurement of antigens .is based on the quantitative evaluation of the markers of the overall cell population. These methods make it possible to measure antigens either by direct labeling or by indirect labeling, the latter most frequently be=ing carried out in two, three or four steps. In al_1 case's, the reagent employed in the last labeling steF~ carr=ies a probe which is either of iso-topic character, for example iodine 125, for a deter-mination of the ra,d.i.oimmunomet;ric typ,~~ (BROW et al.,,~.
Immunol_. Methods, 1.979, 31, 201; STOCKER and HEUSSER, J. Immunol. Methods, 1979, 26, 87-95), or an enzyme for a determination of the enzyme immunometric -type, most frequently peroxida.se, alkaline phosphatase or beta-galactosi'dasei(VAN LEUVEN et al., J. Immunol. Methods, 1978, 23, 109-116; MORRIS, Transplantation, 1983, 36(6), 719; AAIIMGARTEN, J. Immunol. Methods, 1986, 94, 91-98).
The methods in this last group are rather inconvenient., laborious and risky to apply because of the need to wash and centrifuge the cell material many times; i.t is sometimes necessary to take a sample of the colored medium resulting from the enzyme reaction in order to carry out the final spectrophoto-metric measu.rement:; finally, chemical fixation of the cells, which. is used in most cases, causes the irrever-sible destruction of certain antigens which are particularly sensitive to the customary chemical binding agents such as glutaraldehyde or methanol (DROVER
et al., .1. Immunol.. Methods, 1986, 90, 275-281).
It is known in the literature that an antigen carrying several antigenic determinants, i.e. several epitopes, can be determined by fixing this antigen via one of its epit:opes using an antibody immobilized on a solid support, and by binding, to another epitope of the antigen, another antibody carrying an enzyme or radioisotopic marker enabling the determination to be carried out.
This kind of technique, which is often referred to as the sandwich technique, is described especially in French Patent 2 487 983, French Patent 2 500 166 and European Patent Application 119 736. None of these documents describes the application of this technique to whole cells, even though the word "cell"
is sometimes included in the list of antigens to which the process applies.
In the above patents, the various antigens forming the :subjects of the Examples described are in all cases so_Lely protein molecules soluble in water and physiological liquids, such as tumoral markers, enzymes or hormones in the bloodstream. On the other hand, it is clear that a cell is not a molecule and differs therefrom at least by being considerably larger and by the fact that it is not soluble in physiological media. Thus, the sandwich technique has so far never been applied to whole cells.
Furtliermors~, the immunocapture of cells on a solid support: is described in International Patent Application ~>6/020~a1, in which the object is to remove undesirable cells from samples of bone marrow intended for transplantations. In the said patent application, capture of tt-~e cel:Ls is effected on floating microbeads and requires that t:he antibody used be bound to the solid support: by a complex macromolecular structure, called a network-relay, which is capable of ensuring a preferential orient:ati.on of the antibody relative to that of the corresponding cell antigen. The said patent application gives no indication of an application of the technique to the quantitative determination of an antigen.
The immunoc:apture of cells is also described in International. Patent Application 84/03151 for an analytical application. In the said patent application, the object is to identify the tissue groups to which the examined cells belong (this operation generally being called HLA typing). The cells are captured by means of anti-bodies arranged according to a particular geometry on very specialized supports (m:icroscope cover glasses).
The results are obtained simply by visual observation of the support and produce "all or nothing" responses.
Thus the cell immunocapture systems described hitherto do not lead to ana7_ytical applications permitting the quantitative determination of an antigen expressed at the membrane of certain cells. Furthermore, all these systems may lack specificity because they are based on recognition of the cells by a single antibody.
The method of determination forming the subject of the present invention has considerable advantages compared with all the techniques known and used in the prior art, since it permits the quantitative measure-ment of any antigen of a cell population in a single analysis step. 'This determination is carried out on S cells which have not undergone any chemical or physical intervention and which are i.n their state of physio-logical integrity. Furthermore, the method of deter-mination according to the invention has the characteris-tics of very high specificity which are inherent in the double immunological recognition systems involving 2 different antibodies specific for 2 different antigens carried by the same cell. This method is simple, rapid and reproducible. It is totally suitable for the analysis of a large number of samples, which enables it to be used for diagnostic purposes in clinical biology laboratories handling these large numbers.
The ;resent invention thus relates to a kit for the determin~3tion of at least one surface antigen charac-teristic of ~~ cell population or subpopulation, which comprises the following components:
a) a solid support to which one or more mono-clonal antibodies are fixed by covalent bonding or physical. adsorption, the said monoclonal antibodies being directed against surface antigens of the cell population eacamined, other than the said characteristic antigen, and being intended to immobilize, on the support, the cells which inr.lude those of the subpopula-tion carrying the antigen to be det:ermined;
b) at: leasl_ one solution comprising a monoclonal antibody spec.:ific .or the said antigen characteristic of the cell population or subpopulation carrying the antigen to be deterrni.ned, which is labeled with a radioisotopic probe or an enzyme probe; and c) in the case of manoclonal antibodies labeled with an enzyme probe, a developer for the enzyme, namely one or more solutions containing the substrate for the enzyme and, if necessary, one or more reagents necessary for developing the activity o1.-" the enzyme.
The term "ce:ll" as used in the present specification and in the claims which follow includes human cells, animal cells, the cells of s protozoans and i:he cells of microorganisms (bacteria or fungi). As regards blood cells, the :present ;invention includes the nucleate formed elements, such as the leukocytes, and the anucleate formed elements, such as the erythrocytes or platelets.
The method o~f determination according to the invention applies to to whole cells, i.e~. non-ly;aed cells.
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 best guarantee of integrity of the membrane markers chosen as targets for determination.
t s As thc~ solid rapport it is possible to use any device suitable for the handling of cell suspensions, and preferably tubes, particulate magnetic supports or rigid or flexible microtiter plates made of polyethylene, polystyrene, polyvinyl chloride or nitrocellulose, which contain microwells.
The monoclonal antibodies intended for immobilization of the cells can be 2o fixed to the solid supports either by covalent chemical bonding or by physical adsorption according to conventional methods such as those disclosed by STOCKER a.nd HEUSSER J. Immunol. Methods, 1979, vol.
26, p. 87-95. Acivantage~ously, the support is saturated with a protein beforehand.
2s A .cording to the invention, the monoclonal antibody or antibodies fixed to t.a.e solid support must permit the irnmunocapt~re os the cell population which includes the cell population or populations carrying the antigens to be determined. When this population consists of human cells, the preferred monoclonal antibodies for immunocapture are the anti-class I
3o HLA antibodies 'which are specific for the common part of HLA-A, -B
- g _ and -C antigens Fresent on the leu'.tocytes and numerous other cell lines of the organism. Of these antibodies, the one called S-class I, marketed by BIOSYS, is par-ticularly preferred.
In other cases where the cells examined are human cells and in all cases where these cells are not human cells, monoclonal antibodies appropriate to the type of cells examined can also be used for the immuno-capture according to the invention.
The expression "a monoclonal antibody labeled with a radioisotapic probe" means that the monoclonal antibody carries, either on a component inherent in its structure, for example the constituent tyrosine residues, or on an app:ropri.ate radical which has been attached thereto, a radioactive isotope which enables it to be determined b_~ counting the radioactivity associated therewith.
The expression "a monoclonal antibody labeled with an enzyme probe" means that the monoclonal antibody is coupled to an enzyme which, when associated with the use of appropriate reagents, permits quan-titative measurement of this monoclonal antibody.
The ~~ubstr~~te and the reagents are chosen so that the final product of the reaction or reaction sequence cau__=;ed by the enzyme, involving these sub-stances, is:
- either a colored or fluorescent substance which diffuses into the liquid medium surrounding the cells and which is the object of the final spectrophoto-metric or, respectively, f:luorimetric measurement, - or an insoluble colored substance which deposits on the cells and the walls to which they are fixed, and which can be the object either of photometric measurement by reflection or of visual evaluation, if appropriate against a range of standard shades.
As an additional component, the assay kit can contain a buffer solution intended for washing the solid support: after immobilization and labeling of the cells with the antibody or antibodies carrying the chosen probe"
As additional components, the assay kit can also contain the samples necessary for standardization and quality control of the determination.
The presewt invention further relates to a process for the immunometric assay of the surface anti-gens of a cell population or subpopulation, the said process comprising .
a single step fog:- the specific immobilization or immunocapture of a cell population on the solid support using onE~ or more monoclonal antibodies fixed to the said support beforehand by covalent bonding or by physical adsorption and capable of recognizing an antigen present on the surface of the cells~other than the antigen to be determined, and, simultaneously, the direct labeling of the surface antigen to be determined, belonging to the immobilized cell popula-tion or to one o:E its subpopulations, with a mono-clonal antibody specific for this antigen to be determined, the acid monoclonal antibody carrying a radioisotopic or, alternately, enzyme: probe;
- an incubation peg°iod to allow the simultaneous immuno-capture anci labeling to take place;
- the washin~; of the solid support to remove the non-immobilized. undesirable cells and the excess of mono-clonal antibody carrying the radioisotopic or enzyme probe; and - the actual determination of the antigen to be deter-mined in the labeled cell population or subpopulation by counting the fixed radioactivity or, alternately, after treatment of the medium with the substrate for the enzyme and, if necessary, one or more appropriate auxiliary reagents, by photometric measurement by transmission or reflection, or measurement of the fluorescence emission.
The assay kit and the immunometric assay process according to the invention are preferably applied to the determination of the surface antigens of the formed elements of human i5lood, especially the leukocytes and more particularly !the lymphocytes, the T lymphocytes, the T'4 lymphocytes, the TS lymphocytes and the B lympho-cytes, as we~_1 as the granulocytes, the monocytes and the blood platelet;.
Another preferred application is the determina-tion of the :surface antigens of pathogenic micro-organisms, for example Candida a:lbicans.
The assay lcit and the immunometric assay process according to the invention are also particularly useful for the determination of the surface antigens of. tumoral cells, especially t=hose of cancers of the urinary system and those of malignant hemopathies.
The assay k:it and the immunometric assay process according to the invention make it possible to measure signals (absorbed or emitted light or radioactivity) which depend both on the number of cells present in the cell population examined and on the density of the antigen measured on the surface of these cells. l~leasure-ment of these signals permits quantitative evaluation of the total number of molecules of this antigen which are carried by the cell population or subpopulation examined, whether this antigen has a structural or functional role.
For example, in the case of the leukocyte markers of particular importance in hematology, it is known that, in the majority of situations in healthy subjects, the mean value of the antigen density does not vary substantially between samples for one and the same cell population, ~;o there is a good correlation between the cytological count of the cells carrying the antigen in question and the signal measured according to the invention, which i.s proportional to the total number of antigen molecules present in the sample examined.
Conversely, in some pathological states, the density of the surface antigens may vary for one and the same cell population without. a notable variation in the number or proportion of the positive cells. Such pathological immuno-metric assa;~ process according to the invention or by using the kit of the aresent invention than they would be if a kit of the present. invention than they would be if a conventional cytological counting procedure were used.
Another application of the invention becomes apparent if a mic:rotiter plate is chosen as the solid support. The assay kit and the immunometric assay process according to the invention can then advantageously be used for the determination, on a single plate, of_ a series of surface antigens characteristic of various subpopulations mak:i_ng up the cell population examined.
For this application, it is possible on the one hand to take ready-to-use microtit:er plates to which one or more monoclonal antibodies capable of retaining all the cells of the population examined have been fixed before-hand, and on the other hand t:o have a series of mono-clonal antibodies coupled to an appropriate probe and each specific for an antigen characteristic of one of the subpopulations to be evaluated. 'thus, in a single manipulation and on one and the same support, it is possible to carry out the quantitative determination of all the antigens necessary for characterization of the chosen subpopulations.
This application of the present invention is illustrated by the characterization of the antigenic - 12 - ~3~0973 equipment of cell~~ of interest in clinical biology. A
first case is represented by the determination of the tissue groups characterizing a given individual, which is conventionally known as HLA typing.
A second ease is represented by the typing of tumoral cells, in particular for patients afflicted by malignant hemopathi.es such as leukemias or lymphomas.
This diagnostic examination, which is practiced systematically, consists in characterizing the type and origin of the pati.ent's tumoral cells by the presence or absence on the:>e cells of a series of suitably chosen surface antigens.
Use o.f the kit according to the invention, which contains a microtiter plate on which one or more monoclonal antibodies capable of fixing all the cells of the population examined have been fixed beforehand, and solutions of different monoclonal antibodies labeled with an enzyme or radioisotopic probe and each specific for an antigen present on the tumoral cells, permits identification and quantitative evaluation of the antigens characteristic of the patient's tumoral cell population anal thus makes it possible to associate them with one of the major groups of clinically characterized cancers, especially malignant hemopathies.
Application of the process according to the invention thus makes it possible to carry out the qualitative and quantitative examination of the antigenic equipment of tumoral cel-is rapidly and on a sing~_e support.
A case which may be mentioned as an illustration of another application of the invention is that of the human T lymphocytes, for which there are in particular 2 subpopulations of cells: the lymphocytes characterized by the presence of the CD4 marker, which will be called positive T4 lymphocytes or, more simply, T4 lymphocytes, and the lymphocytes characterized by the presence of the _ 13 _ ~3'~0973 CD8 marker, which will be called positive T8 lymphocytes (or T8 lymphocyte:o).
Measurement of the numerical ratio T4/T8 is of great diagnostic interest in clinical biology. In fact, it is known that modifications of the T4/T8 ratio appear in various complaints of the immune system, such as dysimmune diseases, chronic infectious diseases, viral infections and, in particular, HIV complaints (AIDS virus).
The assay kit and the immunometric assay process accor-ding to the invention can be used for the determination of antigens universally characteristic of the T lymphocyte population and/or antigens characteristic of the T4 and T8 lymphocyte subpopulations. In this case, the T lympho-cytes of the sample examined are specifically immobilized on a solid support anti, simultaneously, the surface antigens of the T4 lymphocytes are labeled directly with an anti-CD4 monoclonal antibody carrying a radioisotopic or enzyme probe; in the same way, the surface antigens of the T8 lymphocytes are labeled directly with an anti-CD8 monoclonal antibody carrying an appropriate probe.
The total. T lymphocytes are preferably determined using an anti-CD7 monoclonal antibody (also called anti-T2 monoclonal antibody) carrying an appropriate probe.
Specific immobilization of the T lymphocytes of the sample is preferably carried out using one or more monoclonal antibodies which are capable, by themselves or in combination, of recognizing all the T cells of the sample, this being the case of the anti-common leukocyr_e (or anti-CD4.~) antibodies or antibodies which recognize the whole of the T population (called "pan-T" antibodies), such as the anti-CD2 (or anti-T11), anti-CD5 (or anti-T1 or anti-CD7 (or anti-T2) antibodies or other pan-T anti-bodies which have not yet been assigned to a differentiation class according to the WHO criteria.
The immur,.ometric assay method of the invention can advantageou:~ly be used for the determination of antigens characteristic of the population of T lymphocytes and T4 and T8 lymphoc!rtes on several parts of the same solid support. Measurement of the signals by radioactivity counting, photomel:ric measurement by transmission or reflection or fluorescence measurement enables the numerical ratio CI)4/CD8 to be calculated easily and directly.
In the same way, it is possible to determine, on one and the same solid support, the subpopulations called T lymphocytes and B lymphocytes which make up the whole of the lymphocyte line.
For example, a monoclonal antibody or a mixture of monoclonal antibodies specific for all the surface antigens of the T cells can be fixed by adsorption to the walls of the microwells. These monoclonal anti-bodies will enable the whole population of T cells of the sample examined to be immobilized in the microwells at a later stage. The plates prepared in this way can be lyophilized and stored, preferably at 4°C. This step can be carried out on the industrial scale and it will thus be possible to have ready-to-use plates for the assay kits which can be applied either to the total T
lymphocytes or to any subpopulation of T lymphocytes.
The samples containing the cells to be determined, which origin,3te from the blood or from any appropriate biological liquid - normal or pathological - can be used as such or a:Eter preparation, especially by density gradient centrifugation according to the methods already known, and in particular in a high-density medium such as, for example, F:ICOLL-PAQUE* marketed by Pharmacia. To determine thf~ blood lymphocytes, the blood sample to be determined can also be treated with a so-called lysis buffer solut~_on, which lyzes the erythrocytes.
* -Trademark ~t1 Aliquots of the appropriate cell suspension are brought into ~~ontact with the solid support, for example in the microwells of a microtiter plate prepared beforehand, at the same time as the solution forming part of the assay kit, which contains the monoclonal antibody specific for the target cell population and carrying an appropriate probe, i.e. a radioisotopic or enzyme tracer. Thus a radioisotopic probe can be prepared for example by labeling the monoclonal antibody with iodine 125 or iodine 131, for example in the presence of chloramine T, by a known process (F. C.
GREENWOOD, W.M. HUNTER et al., Biochem. J., 1963, 89, 114); alternately, an enzyme probe can be prepared by conjugating the monoclonal antibody with an enzyme such as alkaline phosphatase, peroxidase, beta-galac-tosidase or a~~etyl.cholinesterase, by a described method (see, for example, M. 0'SULLIVAN, Methods in Enzymology, 1981, 73, 147) or by a method based thereon. In some cases, in order to avoid certain disadvantages associated with the handling of radioactive substances and the limited shelf life of the reagents, enzymes will be used in preference to radioisotopic probes.
The incubation period, i.e. the time required f.or immobilization and simultaneous labeling of the cells, is preferably less than or equal to 1 hour.
During this tame, the solid support can be centrifuged, if necessary, in order to improve the immobilization of the cells. T'he solid support, for example the micro-titer plate, is then washed to remove the non-fixed cells and at nhe same time the excess of monoclonal antibody carr:~ing an enzyme or radioisotopic probe.
When a radioisotopic probe is used, for example iodine 125, tile radioactivity associated with the cells is counted in a gamma counter according to any appropriate procedure and" for example, after solubilization of the -lr~- 1340973 cells with an alkaline solution (for example a sodium hydroxide solution) and recovery of the solution con-taining the radioactivity by means of an absorbent buffer.
When an enzyme probe is used on the mono-clonal. antibody, the appearance of a colored or fluorescent product is brought about by adding, to the solid support to which the cell population carrying the antigen to be determined has been fixed, a solution containing the substrate for the enzyme and one or more auxiliary reagents such that the reaction product which is finally obtained is either a colored product soluble in the medium, or an insoluble colored product, or a soluble fluorescent product, as explained earlier. The light signal coming from the samples treated in this way is then measured with the equipment appropriate to each case, i.e. a transmission or reflection photometer or, respectively, a fluori.meter. When the solid support is a microtiter plate, the light signal can be read sequentially in all the wells of one and the same plate by means of automated readers commonly used in biology laboratories, such as the Titertek plate reader or the Fluoroscan plate reader for the spectrophotometric or, respectively, fluorometric readings.
When alkaline phosphatase is used as the enzyme probe, this enzyme is coupled to the monoclonal antibody acc~~rding to the method proposed by F3oehringer Mannheim - Biochemica. 'The preferred substrates for this enzyme are paranitrophenyl phosphate for a spectrophoto-metric final reading, 4-methylumbelliferyl phosphate for a fl.uorimetr:ic reading or 5-bromo-4-chloroindol-3-yl phosphate for obtaining an insoluble colored reaction product. Likewise, beta-galactosidase can be used as the enzyme probe, in which case the preferred sub-strates will be orthonitrophenyl beta-D-galactopyranoside or 4-methylumbelliferyl beta-D-galactopyranoside.
The monoclonal antibodies can preferably be coupled to peroxid.ase. In this case, the coupling process is derivef. from that described by M.B. WILSON
S and P.K. NAKANE in Immunofluorescence and Related Staining Techniques, edited by W. Knapp, K. Kolubar and G. Wicks, Elsevier/North Holland, Amsterdam, 1978, p. 215-224. The modifications which have been introduced by comparison with the initial protocol for preparation of the enzyme conjugate concern the following points:
- the molar ratio peroxidase/antibody is equal to 3 as opposed to 2 in the protocol, and - oxidation of the carbohydrate units of the peroxidase is less harsh ciu.e to a 33~ reduction in the proposed concentration of sodium periodate.
The reagents used to develop the peroxidase conjugated with th.e monoclonal antibodies contain hydrogen peroxide, which is the substrate for the enzyme, and an appropriate chromogen, for example orthophenylene-diamine or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid, or ABTS, to give a final reaction product which is colored a.nd soluble in the medium, or else 3,3'-diaminobenzidi.ne, 3-amino-9-ethylcarbazole or 4-chloro-alpha-naphthol to give an insoluble final reaction product, or else parahydroxyphenylpropionic acid to give a fluorescent reaction product which i.s soluble in the medium.
In a preferred form, the kit according to the invention for the determination of antigens characteristic of the T, T4 and T'8 lymphocytes comprises:
a) a microtiter plate in whose wells one or more monoclonal antibodies directed against T lymphocytes have been fixed, bl) a solution containing at least one peroxidase-labeled monoclonal antibody directed against T lymphocytes, b2) a solution containing at least one peroxidase-1<ibeled monoclonal antibody directed against CD4 antigen, b3) a solution containing at least one peroxidase-l~3beled monoclonal antibody directed against CD8 antigen, cl) ~3 solution containing hydrogen peroxide, which is the substrate for the enzyme, in an appropriate buffer, and c2) <3 solution containing the chromogen used to develop the expression of the enzyme's activity.
Another preferred embodiment of the invention is the use of monoclonal antibodies coupled to acetyl-cholinestera:~e.
The acetyl~~holinesterase is preferably coupled to the antibody using a process derived from that described in French Patent no. 2 550 799 or a process which schemat:icall:y involves the preparation of fragments of the antibody by a known technique, the modification of the enzyme by reaction with an appropriate hetero-bifun~.~'ional agent and, finally, the coupling of the resulting products. Other known processes for constructing immunoenzyme conjugates can also be used in this case.
Devel.opmen~~ of_ the enzyme activity specifically associated with the cell antigen recognized by the acetylcholinesteraae conjugate is preferably carried out by the known techn=ique which employs acetylthiocholine as the substrate for the enzyme and Ellman's reagent, or 5,5'-dithio-~.-nitrobenzoic acid, as the chromogen, in accordance with any variant suitable for the case under examination, for example the one described by Pradelles et al., Anal. Chem", 57 (1985) 1170-1173.
The chromo~;ens cited are used as such or in the form of. water-soluble salts.
The result: of the determination of antigens according to the invention can be expressed in any form appropriate to the examination carried out. More par-ticularly, these results can be expressed either as the total number of molecules of a particular antigen (for example the CD4 antigen) present in a given volume of the sample examined (for example per microliter of blood), or as the ratio of the number of molecules of one antigen to the number of molecules of another antigen in the sample examined (for example the ratio of the CD4 anti;~ens to the CD8 antigens - or CD4/CD8 ratio -in the blood sample examined).
The number of molecules of a particular antigen in the sample examined will preferably be determined using a standard range consisting of appropriate cells or cell preparations carrying the antigen to be determined, which will hive been calibrated beforehand by a known reference mei=hod. These standards will preferably con-sist either of cells identical in their origin to the cells which are to form the subject of the determination, or of cells of established cell lines carrying the desired antif;en, or of preparations, for example membrane preparations., originating from these cells.
These standards are then treated in exactly the same way as t:he samples to be examined. The resulting signals are used to build up a standard range against.
which the signals measured with the samples to be examined are compared. The subsequent calculations are conventional.
To determine the ratio of the numbers of molecules of two antigens in the sample to be examined, it is possible to use the standard system described above and finally to ca.lculat:e the desired ratio. A simpler possibility in many cases is directly to calculate the ratio of the specific signals obtained with each of the desired antigens and to correct it, if necessary, by means of known faci~ors such as the ratio of the dimen-sions of the samples used; this gives the desired ratio directly.
The inrmunomet;ric assay method according to the inven-tion is simp7.e, rapid and reproducible. Its use is totally suitable for the analysis of a large number of samples. For an understanding of its advantages compared with the other methods described, the various steps of the method according to the invention should be analyzed.
The immobilization of the cells on the solid support is the pha:~e of the determination which usually presents the most difficulties or which is the most critical to carry out. The means often used is chemical fixation of t:he ce_Lls with glutaraldehyde or methanol in cups which may or may not have been treated with poly-L-lysine (F. 'JAN LEUVEN et al., J. Immunol. Methods, 1978, 23, 1050. However, chemical fixations performed in this way c:an reduce or even suppress the desired specific detection or conversely can induce the false-positive labeling of cells, which is a very serious disadvantage (DROVhR and MARSHALL, J. Immunol. Methods, 1986, 90, 27~~-281)"
Furthermore, the chemical fixation process has to be carried. out ~_n several. steps: centrifugation of the cells, preparat=ion of the fixative mixture, fixation and then wasa.ing o1- the fixed 'cells several times.
Drying of t:he cells at 37°C, optionally followed by fixation with methanol in the microwells, has also been proposed (BALJ~1G.'~RTE1, J. lmmunol. .'Methods, 1986, 9'~, 91-98).
Actually, in addition to the fact that it can degrade certain fragile antigens, drying of the cells at 37°C
renders the pericel_lular plasmic membrane permeable, thereby facilitating the immunolabeling of intracyto p.lasmic antigens a~; well as .Labeling of the surface antigens, which leads to troublesome backgrounds or to false-positive results when it is desired to restrict a measurement to the surface antigens.
Furthermore, the reproducibility of this process is doubtful; in fact, the settling of the cells in the assay microwells and the drying of the cells can vary between experiments. Finally, this determination is long to perform because the cell drying step alone takes more than 2 hours.
The immobilization of lymphocytic populations has also been achieved by the use of_ polyclonal antibodies adsorbed in inicrowells (STOCKER and HEUSSER, J. Immunol.
Methods, 197'x, 26, 87-95). Apart from permitting the immobilization of cells foreign to the single population which it is desired to analyze, polyclonal antibodies also have the disadvantage of reacting with the antigens which are to be measured by means of the labeled anti-body, leading to a corresponding reduction in the signal which is fin;~lly measured.
The use of highly specific and related monoclonal antibodies adsorbed on or fixed to the solid support, and especial:iy in the assay wells, permits exclusive capture of the desired cells, the other non-retained cell populations being removed in the course of the washing operations carried out on completion of the labeling of i=he antigens to be determined. Furthermore, no chemical or physical agent modifies the characteris-tics of the antigens in this step because the various operations for chemical or physical. fixation of the cells to the support are omitted.
Thus;. according to the present invention, it has been found that the immobilization of cells by monoclonal antibodies is a process which makes it possible to simpli:Ey the step for immobilizing the cells carrying the antigen to be determined, while at the same ~ 3 40 97 3 _ 22 _ time making the results more reliable.
Tmmunometric: assays applied to cell populations are generally carried out by labeling the cells according to an indirect method in 2 or 3 successive steps, the S probe which provides the specific signal being fixed to the cells during the last step of the labeling process. In the labe?.ing of cells in two steps, the main reagents involved use anti-immunoglobulin (anti-Ig) antibodies coupled to beta-galactosidase (COBBOLD
et al., J. Immunol. :Methods, 1971, 44, 125-133) or to alkaline phosphatase (HESSIAN, .1. Immunol. Methods, 1986, 91, 29-34) or labeled with iodine 125 (SAVION, J. Immunol. Methods, 1987, 97, 49-56). The technique of labeling with the peroxidase-antiperoxidase reagent 1S is carried out in 3 steps (VAN LEUVEN, 1978).
Another process, also in 3 steps, uses a mono-clonal antibody specific for the antigenic marker to be analyzed, then anti-mouse Ig antibodies carrying biotin, and finally a streptavi.dine-peroxi.dase conjugate (BAUMGARTEN, 1986) or streptavidine-alkaline phosphatase conjugate (IGIETSEME et al., J. Immunol. Methods, 1987, 97, 123-131).
The process according to the invention, which comprises the simultaneous use, in a single step, of a procedure for immunocapture of whole cells without physical or chemi.ca7 intervention on the cells, and of the labelinf; of a:~ll. ar some of these cells with one or more monoclonal antibodies directly carrying a radioiso-topic or enzyme probe, is the first process to permit the quantitative determination of chosen membrane anti-~;ens on the cells themselves.
According to the invention, the direct labeling of immunologicall_y immobilized cells permits:
- simplification of the method of determination by elimination of the intermediate manipulations repeated ~ 3 4p g7 3 between the successive steps of the labeling process in the case of indirect labeling: centrifugation of the cells several times, removal of_ the labeling reagents and resuspension;
- a saving of reagents;
- an improved reliab:i:l_ity through a reduction in the number of steps and manipulations;
- a time saving; and - the possibility of treating large numbers of samples at the same time, exclusively with the use of con-ventional equipment and apparatuses.
The incubation period for immobilization of the cell population and simultaneous direct labeling of the antigens of the cell subpopulation to be determined is short. It i;s less than or equal to 1 hour in the case of the determination of T lymphocytes and subpopulations of T4 and T8 lymphocytes.
After the solid support has been washed, the actual determination is carried out by using conventional apparatuses 1.0 observe a signal which is precise and simple to measure: radioactivity or light absorption or emission.
Thus" overall, the process according to the invention ha:~ nume:rous advantages: it is rapid, reliable, economic and simply=_.
The proces:~ according to the invention makes it possible to determine surface antigens of a cell population over a wade range of ce7_1 concentrations.
The ;;ensit=ivity of the method in respect of the number of cells depends on the antigen density of the cell population determined. For each antigen it is possible, if desired, to define the minimum molar con-centration of antigen which can be measured by the process according to the invention.
Thus, for example, when the chosen solid support is a microtiter plate and 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 about 200 000 per 200 ~.rl microwell, the lower limit being imposed by the density of the measured antigen on the cells examined and the sensitivity of the chosen detection technique, while the upper limit depends essentially on the size and geometry of the solid support. The same applies in the case where the solid support consists of tubes.
It has been verified that the signals recorded (radioactivity count or photometric measurements) make it possible to obtain satisfactory uniform calibration curves as a function of the number of cells used, under the customary handling conditions.
Furthermare, the sensitivity of the method can be improved, if necessary, by the simultaneous fixation, to one and the same surface antigen, of several different monoclonal antibadies specific for several different epitopes of the same antigen. This has been verified by determination of the CD4 antigens on cells of the Ichikawa line (human T line), where the OKT4 and ST4 antibodies labeled with iodine 125 were used simul-taneously. The measured signal increased by about 500 relative to the signals obtavined with each of the anti-CD4 antihodi~s used by itselfi.
In the Rxamples which follow, the following forms or their abbreviations will be used indiscriminately:
FiS~~ . bovine serum albumin PBS . phosphate buffered saline of pH 7.4 POD . peroxidase IgG . immunoglobulin G
IgM . immunoglobulin M
anti-T IgG o:r anti-T . antibody directed against T
Lymphocytes anti-CD4 IgG or anti-CD4 . antibody directed against CD4 antigen anti-CD8 IgG or anti-CD8 , antibody directed against CD8 antigen cpm , counts per minute dpm . disintegrations per minute Enzyme immurrometric assay of the molecular concentration of the CD4 and CD8 antigens in samples of human blood, in which the antibodies are labeled with peroxidase A) Preparation of the assay kit a) Preparation of the plate The plate used is a plastic microtiter plate containing 9p microwells, marketed by NUNC (reference 64394). Eaclz mic.rowell receives 200 trl of a solution containing the purified anti-CD2 monoclonal antibody (called STlI) used to immobilize the total T lympho-cytes, i.e. to e.f.f.ect their immunocapture. This anti-body, marketed by BIOSYS, Compiegne, France, under the reference ST11, is used at a concentration of 10 ~g/ml in a phosphate buffered saline (PBS) of,pH 7.4. The adsorption o:E the ,monoclonal antibody is effected at 4°C for 12 hours. The excess antibody is removed by turning the plate over.
A solution containing 0.1% of gelatin and 0.5~
of BSA in a phosphate buffered saline is prepared.
250 Yl of this solution are introduced into each microwell so as to saturate the surface of the wells with protein, which takes J. hour at 37°C; the plates are washed 3 times with phosphavte buffered saline. The plates prepared in this way are lyophilized and stored at 4°C in a sealed plastic sachet.
b) Preparalwion of. the solution of monoclonal antibod~r-peroxidase conjugate The r~eroxidase (POD) marketed by Boehringer Mannheim Biochemica (reference 814 393) is used.
The process for coupling the antibody to the peroxidase is the one described by M.B. WILSON and P.K. NAKANE in Immunofluorescence and Related Techniques (edited by ~J. KNAPP, K. KOLLJBAR and G. WICK, 1978, Elsevier/North Holland, Amsterdam - p. 215-224), except that the oxidation of the peroxidase is carried out using 1.5 mg of PCD in 0.36 ml of distilled water and that 50 ul of a 0.2 M solution of sodium periodate are added. /The resulting product is coupled to 2 mg of anti-CD4 IgG contained in 500 ~l of carbonate buffer.
After treatment with sodium borohydride and dialysis against PBS, the IgG-POD conjugate is sterilized by filtration on a 0.22 ~m membrane and kept under sterile conditions at a concentration of 0.5 mg of IgG per ml, at 4°C, in glass tubes. The reagent is stable for at least 1 year.
The anti-CD8 IgG-POD conjugate is prepared in the same way. IgM-POD conjugates can be prepared by the same method.
c) Preparation of the developing reagent The developing reagent is obtained in the following manner: A 0.1 M citrate buffer is prepared by dissolving citric acid monohydrate in water at a con-centration of 2.1~ and adjusting the pH to 5 by the addition of 7 N sodium hydroxide solution. 30 mg of orthophenylenediamine dihydrochloride are then added to 20 ml of the citrate buffer obtained, after which 40 ~1 of 30~ hydrogen peroxide (substrate f_or the enzyme) are added, at the last moment, per 20 ml of citrate buffer containing orthophenylenediamine.
B) Immunometric assay process a) Separation of the cells 2 ml are taken from the blood sample to be determined. This is mixed with 2 ml of phosphate ~3~~9~3 _ 27 _ buffered saline and the resulting mixture is deposited on top of 3 ;ml of FICOLL-PAQUE (marketed by Pharmacia).
On centrifugation at 400 x g for 30 minutes at room temperature, a suspension ring containing the mono-nucleate cells is formed. The whole of this suspension is recovered in a volume of 1 ml and 6 x 100 ~l of the resulting suspension are distributed into 6 microwells of the preprepared plate.
b) Incubation of the cells The previously obtained POD-antibody conjugate is diluted 100-fold with PBS containing 1~ of a protein substance such as BSA or skimmed milk powder, and 100 rl of this solution are deposited in each microwell as follows:
- 2 microwel:Ls are each filled with 100 ~1 of the solution o:E the POD-anti-CD4 conjugate;
- 2 microwel:Ls are each filled with 100 ~1 of the solution off= the POD-anti-CD8 conjugate; and - 2 control rnicrow.ells are each filled with 100 ~1 of a 1~ solution of protein substance (reaction blank).
To improve the fixation of the cells to the support, the plate is centrifuged for 3 minutes at 150 x g after a wait of 1 hour at room temperature.
c) Development of the fixed enzyme and measurement The microwells are emptied by turning the plate over. They are waahed 4 times with 200 Y1 of PBS.
200 ul of then developing reagent, prepared for immediate use,Iare added to each well. Incubation is carried out for 20 minutes at room temperature in subdued light.
The optical density is measured on a spectrophotometer at 492 nm (type 310 C* Titertek Multiskan apparatus -Flow Laboratories)..
d) Standardization and expression of the results To standardize these experiments, a preparation of total human lymphocytes was used which had been * - Trademar?;
A
calibrated beforehand in respect of CD4 and CD8 antigens by the cytofl_uorom~=_tric technique of PONCELET et al.
(,T. Immunol. Methods, 1985, 85, 65-74). Known aliquots of this reference preparation were used to make up the 2 standard ranges ~celating to the CD4 antigen and CD8 antigen respective:Ly, against which the numbers of antigens corresponding to each sample are calculated.
By way of :indication, Table 1 below shows, for 20 samples of human blood taken from healthy donors, the optical density va=lues obtained, the corresponding numbers of moleculE~s of CD4 and CD8 antigens and the resulting ratios oj~ these numbers of antigens (CD4/CD8 ratios).
~ 3 40 97 3 CD4 anti;~en CD8 antigen Donor Molar number 0ptica7_ Number of Optical Number of ratio density antigen moleculesdensity antigen moleculesCD4/
(in m:illi.ons) (in millions) CD8 per ~yl of blood per ~1 o.f blood 1 0.64 '~3 1.29 92 0.25 2 0.71 27 0.75 37 0.73 3 0.82 32 1.10 81 0.39 4 0.49 17 0.55 23 0.74 5 0.74 'Z9 0.89 52 0.56 6 0.88 38 0.92 55 0.69 7 0.74 29 0.72 36 0.80 8 0.75 29 0.69 33 0.88 9 0.48 16 0.80 41 0.39 10 1.00 50 1.08 80 0.62 11 0.61 22 0.69 33 0.67 12 0.73 28 0.85 46 0.61 13 0,51 18 0.66 30 0.60 14 0.65 24 1.06 76 0.31 15 0.92 42 0.87 48 0.87 16 0.77 31 0.98 63 0.49 17 0.68 25 1.01 67 0.37 18 0.85 36 0.96 61 0.59 19 0.91 41 0.90 52 0.79 20 1.00 50 1.02 69 0.72 Enzyme immunometric assay of the molecularconcentra-tion of the C;D4 and CD8 antigens in samples of human blood, in which the antibodies are labeled with alkaline phosphatase This determination is carried out in the same way as that of Example 1. The monoclonal antibody-alkaline phosphate:>e conjugate is prepared according to the method indicated by Roehringer Mannheim - Biochemica (ref. 567 744). After the cells to be determined have been incubated for one hour with this conjugate, the fixed enzyme is developed by the addition of a solution containing 1 mg/ml of paranitrophenyl phosphate in a buffer containing J..2~ of diethanolamine 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 on a spectrophotometer at 405 nm.
The results are calculated and expressed as in Example 1.
Radio:Lmmunometric assay of the molecular concentration of the CD4 and CD8 antigens in samples of human blood, in which the antibodies are :Labeled with iodine 125 The antibody labeled with iodine 125 is prepared according to the technique described by F.C. Greenwood, V.M. Hunter et al., Biochem. J., 1963, 89, 114.
50 ~g of anti-CD4 or anti-CD8 monoclonal antibody contained in 50 rrl of phosphate buffered saline o.f pH
7.2 are mixed with 37 MBq of iodine 125 in the form of sodium iodide and ''.0 trl of a solution containing 0.33 mg of chloramine T per m1l in a phosphate buffered saline.
After 1 minute of shaking, t:he reaction for labeling the monoclonal antibody is stopped by the addition of 100 ul of a solution containing 2.5 mg/ml of_ sodium 1340973'' metabisulfite" The solution prepared in this way is passed through a PD 10 column (Pharmacia - Sephadex G25M) and the fraction containing the radiolabeled antibody is recovered in the effluent. The determination is carried oul: by introducing into 4 microwells a volume of 100 ul of cell suspension containing mononucleate human bllood cf~lls prepared as in Example 1. 100 ul of dilute solution of radioactive antibody in a PBS 'buffer containing 5% of BSA are then deposited so as to introduce 150 000 cpm into each well; 2 microwells are each filled with 100 ~l of the solution of the anti-CD4-125I conjugate. and 2 microwells are each filled with 100 ul of the solution of the anti-CD8-125I conjugate.
The /fixation of the cells is improved by centrifugation of the plate :nor 3 minutes at 150 x g after incubation for 1 hour at room temperature. The microwells are emptied by turning the plate over. The microwells are washed 4 timer with 200 rl of PBS per well. 75 ~l of 1 M sodium hydroxide solution are then introduced into each well. A:Eter 10 minutes, the contents of each well are recovered with an absorbent buffer before the radio-activity is counted in a X counter (LKB multiwell counter).
The results are calculated and expressed as in Example l, the optical density values being replaced with the count results in dpm.
Checking the validity of the method On 20 samples of human blood, the CD4 and CD8 antigens of the human T cells were measured by following the peroxidase immunometric assay method described in Example 1.
The ratio of l:he number of positive T4 cells to the number of positive 'T8 cells was also determined on the same blood samples 'using the conventional cytological counting technique (W. W. ERBER et al., Lancet, 1984, (8385 ) , 1042--1045 ) .
The correlation coefficient r between the ratio CD4/CD8 obtained by enzyme immunornetric assay and the ratio T4/T8 obtained by cytological counting is 0.72.
Likewise, on 7 other samples of human blood, the ratio CD~E/CD8 determined by the radioimmunometric assay method using labeling with iodine 125 by the process of the present ~:nvent:ion was compared with the cell ratio T4/T8 determined b:y cell counting. The correlation coef.fici.ent r is 0.87.
In both cases, the correlation coefficients obtained show that, despite a satisfactory overall agreement bet=ween the results of the 2 techniques, the information provided by the 2 ratios is not equivalent;
this is obvious since the determination of antigens according to the invention allows not only for the number of positive cells, as is the case for the cyto-logical method, but also for the density of the antigen in question on the positive cells of each sample. The information given by the technique described in the present invention is therefore more complete than that given by the conventional reference technique (W. W.
FRBFR et al., Lancet, 1984, (8385), 1.042-1045).
Enzym<~ immunometric assay of the molecular concentration of the CD4 and CD8 antigens of the T lymphocytes in samples of human blood, in which the antibodies are labeled with peroxidase The purpose of this Example is to show that the time required for the determination can be reduced, relative to 'she conditions described in Example 1, by modifying on the one hand the cell separation process and on the o~=her hand the incubation time required by the step for immunocapture and labeling of the cells.
A) Influence of the process for the separation of cells from whole blood a) Technique involving brief centrifugation:
0.5 ml is taken from the blood sample to be determined and mixed with 1.5 ml of phosphate buffered saline. 1.5 ml of Fi.coll-Paque, marketed by Pharmacia, are introduced into a 5 ml hemolysis tube and the blood sample, diluted in PBS, is deposited on the surface of the layer of Ficoll. Centrifugation at 900 x g for 5 minutes at room temperature is followed by removal of the suspension ring contai.ni.ng the mononucleate cells in a volume of 0.5 ml. 1.5 ml of PBS are added to this sample.
b) Technique involving lysis of the erythrocytes:
Another rapid method for the separation of cells from blood is to use a buffer which lyzes the erythro-cytes. The lysis buffer, used by way of a non-limiting example, has the following composition:
ammonium chloride: 8.29 g potassium bicarbonate: 1 g disodium salt of ethylenediaminetetraacetic acid:
0.0307 g per liter of distilled water, the pH being adjusted to 7.3.
5 ml of lysis buffer are mixed with 0.250 ml of blood. After 10 minutes of shaking, the mixture is centrifuged at 6t:)C~ x g for L0 minutes. The cell residue formed is taken up in 1 ml of PBS buffer.
By subsequent determination of the CD4.and CD8 antigens under the conditions of Example l, and by comparison with the reference protocol described in Example 1 for isolation of t:he mononucleate cells from blood, it was verified that both these variants of the method for the separation of the mononucleate cells from whole blood did indeed enable all the lymphocytes to be collected from the blood samples examined.
B) Influence of the incubation time for immunocapture and labeling of the cells In order to check whether the period of 1 hour used in Example 1 might be reduced in order to accelerate the determination, the results obtained for identical samples each containing 20 000 mononucleate cells per well were compared when the time allowed for capture and labeling of t:he cells was varied from 10 minutes to 1 hour. The results obtained are shown in Table 2, all other operating conditions being the same as in Example 1.
Incubation Optical density time (minutes) CD4 antigen CD8 antigen Test Blank'~~ 'Test Blank 10 0.322 0.037 0.329 0.023 0.438 0.049 0.401 0.025 0.620 0.055 0.498 0.027 15 ~ These optical density values correspond to the reagent blank obtainE~d in 1_he absence of cells. The control values obtaiu:ed in the presence of cells with either the conjugate or the substrate being omitted are not greater than the values indicated.
20 These resu_Lts show that:
- The non-specific signal (reagent blank) always remains at low values which become lower as the time for which the conjugate is present becomes shorter.
- After 10 minutes of contact, the specific signal reaches a value which can be processed analytically with precision and reproducibility. This indicates that it is possible, relative to the conditions of Example l, signifi~antl.y to reduce the time taken to carry out the determination without a substantial loss of precision in the results.
In practice, and if the time savings which can be achieved in the preparation of the blood sample and in immunocapture are added together, the kit and the process of t'he invention make it possible to determine the CD4 and CD8 antigens (by way of non-limiting examples) of samples of whole blood, at a rate of 10 or 20 samplers per plate of 96 wells, in a total time (taken from reception of the samples in the laboratory) of no more tinan 1 hour. This level of productivity is greater than that of all the alternate techniques known to date.
Immur~ometric assay of the molecular concentration of the CD5 antiF;ens of the human T lymphocytes in samples of human blood, in which the antibody is labeled with acetylcholin~~sterase A) Preparation of the assay kit a) Solid support A micrati.ter plate prepared in the manner des-cribed in Example 1 is used.
b) Manoclo:nal antibody-acetylcholinesterase conjugate The acetylr_holinesterase of. Electrophorus electricus i;~ used, which is obtained by the technique described in French Patent no. 2 550 799.
This enzyme is coupled to the anti-CD5 antibody called ST1 and may°keted by BIOSYS. Coupling is carried out in accordance ~~rith the process described by YOSHITAKE
et al., Eur. J. Biochem., 101 (1979), 395-399.
c) Developing reagent This reagent comprises both the substrate for the enzyme (acetylt:hiocholine) and Ellman's reagent and has the following composition:
- acetylthiocholine 7.5 x 10-4 M
- 5,5'-dithio-2-nit:robenzoic acid (DTNB) 5 x 10 4 M
in 1 M sodium phosphate buffer of pH 7.4.
This solution is diluted 100-fold in distilled water before use.
B) Immunometric assay process a) The mononucleate cells are separated off using the method described in Example 1.
b) Incubation of the cells for the immunocapture and labeling step takes 20 minutes, as in Example 5.
c) Development of the fixed enzyme and measure-ment.
100 ~l of developing reagent are added to each microwell. T'he ab:;orbance is measured at 412 nm after 45 minutes of incubation.
The measured optical densities for 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.
Number of T introduced lymphocytes into each well and of number CD5 of antigen molecules present in each millions well, in (32) (65) (130) (260) (520) (1040) Optical density0.030 0.060 0.138 0,260 0.470 0.850 These results reveal the extreme sensitivity of the technique developed in this way. In fact, the optical density of 0.030 obtained in the well containing 540 T lymphocytes (i.e. 32 million molecules of CD5 antigen), which is measurable with precision and equal to 10 times the background, corresponds to about 5~10 17 mol of CD5 antigen per assay well. Such sensitivity is of the same order as that of the best immunoassays known, which use the most sensitive radioactive tracers.
Determination of different antigens expressed on activated human T lymphocytes Activation of the T lymphocytes is a physio-logical process which takes place prematurely every time the immune system is stressed, for example as in infectious pathologies, organ transplantations and certain dysimmune diseases. This natural process is also commonly used in vitro in the laboratory in tests such as, in particular, mixed lymphocytic reactions or lymphoblastic transformation tests. In the latter case, polyclonal activation is obtained by the use of agents such as phyt:ohemagglutinin (PHA), concanavalin A or other lectins. Activation of the T lymphocytes is accompanied by a considerable increase in the expression of several membrane markers, and especially the CD25 antigen (receptor of i.nterleukin '?) c>r the CD2 antigen.
These antigens therefore constitute excellent markers of the activation state and their determination is of great interest both in clinical biology and for laboratory work, as indicated above, the use of radioactive reagents being avoided in all cases.
These antigens are measured using the method described in Example 1 with the specifications detailed in Table 4.
Measuremento:E the surface antigensof activated T lymphocytes Antigen Antibody used for Antibody labeled with measured immunocapture, peroxidase, marketed by marketed by CD2 STl BIOSYS ST11 BIOSYS
CD2.5 ST11 BIOSYS I0T14 IMMUNOTECH
The optical density values measured at 450 nm for the 25~103 mononucleate cells used are reported in S Table 5 below and show the comparison between the cells without stimulation with PHA and the same cells after 3 days of stimulation.
Optical density Antigen Non--activated Cells activated Reagent blanf<
cel=Ls for 3 days CD25 0.064 0.529 0.044 CD 2 (),129 0. 768 0. 027 These results show that the specific signals of the 2 antigens examined increase considerably when activation takes place.
Determination of the CD22 and HLA-Dr (or class II
HL,A) antigens present on B lymphocytes The present Example describes the determination of these antigens on cells of the RAJI line, which is a human B lymphoid line described by PULVERTAFT in J.
Clin. Pathol., 1965, 18, 261-274.
39 ~ (~ O ~ 7 3 For immunocapture of the cells, either the S-class II antibody (BIOSYS) for determination of the CD22 antigens, or the SB4 antibody (BIOSYS) for determination of the class II HLA (or HLA-Dr) antigens, was adsorbed in the assay wells as indicated in Example 1.
s a) Determination of the CD22 antigen The S;B22 antibody (BIOSYS) was labeled with iodine 125 by the method described in F;xample 3. The CD22 antigens are determined in the manner indicated in Example 3 for the CD4 and CD8 antigens, the following being introduced successively into the microwells : 5.104 cells of ~o the RAJI line and then 105 cpm of the labeled SB22 antibody. After a period of one hour at 4°C, the microwells are emptied by turning the plate over and then washed 4 times with 200 ~1 of PBS per well for each wash.
The radioactivity fixed to the cells is recovered and counted as in Example 3. This gives a count of 7Ei0 dpm for the well containing 5 x 104 RAJI cells is with a reagent blank (without cells) of 50 dpm.
b) DetE~rmination of the HLA-Dr antigen 105 cell'.s are introduced into the wells, followed by the conjugate of S-class II antibody labeled with peroxidase by the process described in Example 1. The dntennina~tion is carried out according to the same protocol 2o as in Example 1. ~Chis gives an optical density of 1.840 and a reagent blank, without cells, of 0.105.
EXAMfPLE 9 Use of the process according to the invention for the 2s measurement of antigens carried by the T lymphocytes and B lymphocytes of human blood A) Preparation of the plates The plates used are microtiter plates prepared as in Example 1 except that monoclonal antibodies capable of fixing at least all the T lymphocytes (S-class I from BIOSYS) and the B lymphocytes (S-class II
from BIOSYS), each one being used at a concentration of 5 ug/ml, are simultaneously adsorbed in each well of the milcroplate.
B) Immunometric assay .
The mononucleate cells are separated from the whole blood with the aid of Fico:ll under the conditions of Example 1 or Example 5, after which 80 000 mono-nucleate cells in 75 ul of PBS buffer are introduced into each well. The l'C lymphocytes are measured in some of the wells with the ST11 (anti-CD2) antibody from BIOSYS; the B lymphocytes are measured in other wells with the SB3 (anti.-CD37) antibody from BIOSYS, which recognizes all the peripheral B lymphocytes. These antibodies are first coupled to peroxidase according to the protoc~~l described in Example 1. The actual determination is carried out as in Example 1.
C) Results The al5solute numbers of T lymphocytes and B
lymphocytes contained in the sample examined are deter-mined by reference to calibration curves established by carrying out an identical treatment on mononucleate cells in which the 'r and B lymphocytes have been counted by a reference technique.
Alternately, by means of appropriate standards, the results can also be expressed as molar concentrations of the CD2 anc~ CD37 antigens in the sample examined.
Determination of the GpIIb-IIIa and CD9 antigens of human blood platelets This determ:W ation is carried out according to Example 3 using monoclonal antibodies radiolabeled with iodine 125.
The platelets are isolated from human blood by centrifugation in the presence of PLASMION perfusion liquid (Laboratoire R. Bellon). 5 ml of blood plus ml of phosphate huffered saline (PBS) and 10 mI of 5 PLASMION are mixed in a tube. The tube is then centrifuged for 10 minutes at 1500 rpm. The platelets which have collected in the supernatant are removed by suction with a pipette. They are washed once in PBS by mixing 1 volume of the platelet suspension with 10 ml of PBS, and then centrifuged for 10 minutes at 1000 x g.
Finally, the plate_Let residue is collected and resus-pended in PB~> (2 m_L). The platelets are counted and their concentration adjusted to 2.5 million/ml of PBS.
The C~pIIb-=LIIa antigen is determined by immuno-capture of the platelets by the monoclonal antibody I0B2 (Immunot:ech) and labeling of the GpIIb-IIIa antigen with the monoclona~L antibody P2 (Immunotech).
The C;D9 antigen is determined by immunocapture by the monoclonal antibody P2 and labeling with the monoclonal antibody IOB2. The number of dpm is measured for a sample compr~'_sing 200 000 platelets.
The r~esult:~ are shown in Table 6.
Antigen Sample Control without measured measured cells (dpm) (dpm) GpIIb-IIIa 2891 238 Determination of the CD15 antigens carried by human granulocytes The CD15 antigen is a good specific marker of human granulocytes (also called polynuclear leukocytes).
These cells can be evaluated by means of this antigen, in the blood, in the same way as for any other leuko-cytic subpopulation. This evaluation is also useful for assessing the presence of granulocytes in the urine, for example in cases of urinary infections such as cystitis or pyelonephritis. In the present Example, the determination was carried out on granulocytes originating from whole blood and separated off as indicated below.
The granulocytes, as well as some of the mono-nucleate cells, are separated from the erythrocytes by a process analogous to that described in Example 10 for the platelets, except that t:he tube containing the cells is left to stand for 45 minutes at room temperature and the suspension of leukocytes is recovered in a volume of 500 til taken from the surface of the liquid.
The granulocytes are immobilized in the wells with a monoclonal antibody specific for the CD45 antigen present on the cell membrane of all the leukocytes:
the anti-LCA antibody (BIOSYS) is used and is adsorbed in the microwells of the titer plate, as indicated in Example 1.
The determination is carried out using the anti-CD15 monoclonal antibody SMY-15a (BIOSYS) labeled with peroxidase by the technique of Example 1.
'Thus, w:it:h a total of 12 500 cells introduced into each well, the specific signal of the CD15 antigen of the granulocytes is 0.616 after correction of the crude optical density for a reagent blank of 0.100, obtained in the presence of the cells and in the absence of enzyme conjugate and due to the endogenous peroxidases of the granulocytes.
~ 3 4o g~ 3 EXAMPLE. 12 Evaluation of the phenotype of a leukemia by the enzyme immunometric assay process The phenotyping of the tumoral cells of a leukemia patient is a systematic diagnostic examination performed in order to characterize the type and origin of the patient's leukemia cells (T, B, granulocytes, myeloblasts, etc.). This examination is conventionally performed by the i_mmunofluorescence technique by using a set of monoclonal antibodies and by observing and counting the positive cells under a microscope. Flux cytometry is also used to analyze the labeling of the cells.
The use of monoclonal antibodies in the process according to the invention makes it possible to identify the major clinically important groups of acute or chronic leukemias .and also provides quantitative infor-mation on the relative densities of the different antigens examined.
6 peroxidase-coupled monoclonal antibodies specific for antigens present on the leukemia cells were prepared in this Example:
- STl and ST:Il (BI~OSYS): anti-CD5 and anti-CD2 - SB3 (BIOSYS): anti-CD37 - S-CALLA (SANOFI): anti-CALLA (or anti-CD10) - S-class II (BIOSYS): anti-HLA-Dr (or anti-class II HLA) - SMY15 (BIO;;YS): anti-CD15 The rnicrotiter plate is prepared by the prior adsorption oil a mixture of anti-CD45 monoclonal anti bodies (S-LCA, BIOSYS) and anti-class I HLA monoclonal antibodies (;>-class I, BIOSYS).
The phenotype of chronic lymphoid leukemia B
(CLL-B) was evaluated using the 6 monoclonal antibodies selected above; it was also characterized by the con-ventional method.
The optical densities measured at 492 nm are reported in 'table 7 below .for a total of 80 000 mono-nucleate cells introduced into each well or for the control wells without cells. For the control well con-s taming the cells but no peroxidase-labeled antibody, the optical density is 0.110.
Monoclonal Optical density antibody and corresponding Sample with Control antigen cells without cells ST11 (CD2) 0.270 0.030 ST1 (CD5) 1.350 0.014 SB3 (CD37) 0.850 0.005 S-class II
(HLA-Dr) 1.640 0 .
SMY15 (CD15) 0.235 0.007 CALLA (CD10) 0.160 0.002 Thus the leukemia cells studied carry an abundance of the CDS, CD37 anal class II HLA antigens, but they carry no CD15, CALL,A and CD2 antigens, or very few, which is characteristic of a ChL-B.
Determination of antigens associated with cancers of the urinary system The kits and processes of the present invention are very particularly suitable for the detection of tumoral cells, especially in the case of tumors of the urinary system, and are applicable to the mass screening of high-risk :populations, for example workers in the 1 3 4~ 97 3 chemical industry of other very exposed groups.
The present Example shows the application of the invention to the determination of an antigen associated with cancer of the bladder, on cells of the RT4 line originating from a human urinary papilloma (C.C. Rigby and L.M. Franks, Brit. J. Cancer, 1970, 24, 746-754).
The plates intended for immunocapture of the cells are prepared as in Example 1, the monoclonal antibody S-class I (BIOSYS), which recognizes a class I
HLA antigen .and i.s capable of retaining all the epi-thelial cells, being adsorbed in the wells.
The labeled conjugates are obtained by the processes of Example 1 (for enzym a labeling) or Example 3 (for radioisotopic labeling) with the mono-clonal antibody 12F6 (SANOFI), which recognizes an antigen associated with cancer of the bladder.
For the determination, 75 ul of the cell suspension are distributed into 4 Imicrowells (i.e.
50~103 cells per well). The conjugate of the monoclonal antibody 12F6 (SANOFI) coupled with peroxidase is introduced into 2 wells and the antibody 12F6 labeled with iodine 125 is introduced into the other 2 wells (100 000 cpm/well). The results obtained by applying the enzyme immunometric assay process or the radioimmuno-metric assay process are reported in Table 8.
Labeled Optical density Ratio of (at. 492 nm) or cpm monoclona-L positive antibody Sample to be Control signal to non-determined specific signal 12F6- 0.782 0.078 10.0 peroxidase 12F6- 1612 350 4.6 iodine 125 Enzyme immunometric assay of a membraned antigen of the yeast Candida albicans The (~andid,3 albicans cells (serotype 17) originate from the Institut Pasteur collection, Paris.
The yeasts are cultivated for 18 hours in the usual way on a solid synthetic medium. The cells are counted and a cell suspension containing 105 cells per ml of PBS
buffer solution is prepared.
The anti-Candida albicans monoclonal antibodies used were prepared by lymphocytic hybridization as described in the thesis of T. Chardes, Faculty of Pharmacy, Iln:iversity of Montpellier I, 1988.
The ,antibody CA4 is used for immunocapture while the antibody CA12 labeled with peroxidase is used for the determination under the conditions of Example 1.
The ~~ptical density values measured at 492 nm for the well containing the introduced cells and for the control aample without cells are 1.025 and 0.080 respectively.
Enzyme immuriometri.c assay carried out on magnetic beads determinatioiz of the CD5 antigen of human T lymphocytes with a conju;~ate of antibody labeled with acetyl-cholinesterase and a particulate support formed of magnetic beads The support used to capture the cells consists of magnetic beads known as DYNABEADS*. The beads, marketed by~ BIOS~'S under ref. DfV-11101, carr; on their surface an anti-CD2 antibody capable of firing all the T lymphocytes in the sample examined. These beads are treated again with a solution of anti-CD2 antibody before use in order to improve the performance characteristics of the determination.
This is done by mixing 10 Y1 of the suspension of beads with 1 ml of antibody solution containing 25 ~g/
ml of PRS buffer, for 1 hour, with gentle shaking. The beads are subsequently washed 4 times with PBS buffer and then kept in 4 m:1 of PBS buffer.
For the determination, 200 ul of the suspension of beads and 80 ~I. of a suspension lof mononucleate cells separated from whole blood with the aid of Ficoll-Paque (PHARMACIA) are introduced successively into a 5 ml tube and 280 ~1 a~f a conjugate of antibody STl labeled with acetylcholin.esterase, identical to that used in Example 6, are then added..
After one hour of gentle shaking, the beads are separated off by means of a magnet and the cells fixed to the bead; are washed with PBS buffer (5 washes).
The acety7_cholinesterase fixed to the cells is developed ire the same way as in Example 6. The optical density signal obtained is 0.168 in the presence of the labeled cells; the' blank obtained without the cells gives a value of 0.062.
* - Tradema:rk ,F., ,4:. -~~ -
et al., .1. Immunol.. Methods, 1986, 90, 275-281).
It is known in the literature that an antigen carrying several antigenic determinants, i.e. several epitopes, can be determined by fixing this antigen via one of its epit:opes using an antibody immobilized on a solid support, and by binding, to another epitope of the antigen, another antibody carrying an enzyme or radioisotopic marker enabling the determination to be carried out.
This kind of technique, which is often referred to as the sandwich technique, is described especially in French Patent 2 487 983, French Patent 2 500 166 and European Patent Application 119 736. None of these documents describes the application of this technique to whole cells, even though the word "cell"
is sometimes included in the list of antigens to which the process applies.
In the above patents, the various antigens forming the :subjects of the Examples described are in all cases so_Lely protein molecules soluble in water and physiological liquids, such as tumoral markers, enzymes or hormones in the bloodstream. On the other hand, it is clear that a cell is not a molecule and differs therefrom at least by being considerably larger and by the fact that it is not soluble in physiological media. Thus, the sandwich technique has so far never been applied to whole cells.
Furtliermors~, the immunocapture of cells on a solid support: is described in International Patent Application ~>6/020~a1, in which the object is to remove undesirable cells from samples of bone marrow intended for transplantations. In the said patent application, capture of tt-~e cel:Ls is effected on floating microbeads and requires that t:he antibody used be bound to the solid support: by a complex macromolecular structure, called a network-relay, which is capable of ensuring a preferential orient:ati.on of the antibody relative to that of the corresponding cell antigen. The said patent application gives no indication of an application of the technique to the quantitative determination of an antigen.
The immunoc:apture of cells is also described in International. Patent Application 84/03151 for an analytical application. In the said patent application, the object is to identify the tissue groups to which the examined cells belong (this operation generally being called HLA typing). The cells are captured by means of anti-bodies arranged according to a particular geometry on very specialized supports (m:icroscope cover glasses).
The results are obtained simply by visual observation of the support and produce "all or nothing" responses.
Thus the cell immunocapture systems described hitherto do not lead to ana7_ytical applications permitting the quantitative determination of an antigen expressed at the membrane of certain cells. Furthermore, all these systems may lack specificity because they are based on recognition of the cells by a single antibody.
The method of determination forming the subject of the present invention has considerable advantages compared with all the techniques known and used in the prior art, since it permits the quantitative measure-ment of any antigen of a cell population in a single analysis step. 'This determination is carried out on S cells which have not undergone any chemical or physical intervention and which are i.n their state of physio-logical integrity. Furthermore, the method of deter-mination according to the invention has the characteris-tics of very high specificity which are inherent in the double immunological recognition systems involving 2 different antibodies specific for 2 different antigens carried by the same cell. This method is simple, rapid and reproducible. It is totally suitable for the analysis of a large number of samples, which enables it to be used for diagnostic purposes in clinical biology laboratories handling these large numbers.
The ;resent invention thus relates to a kit for the determin~3tion of at least one surface antigen charac-teristic of ~~ cell population or subpopulation, which comprises the following components:
a) a solid support to which one or more mono-clonal antibodies are fixed by covalent bonding or physical. adsorption, the said monoclonal antibodies being directed against surface antigens of the cell population eacamined, other than the said characteristic antigen, and being intended to immobilize, on the support, the cells which inr.lude those of the subpopula-tion carrying the antigen to be det:ermined;
b) at: leasl_ one solution comprising a monoclonal antibody spec.:ific .or the said antigen characteristic of the cell population or subpopulation carrying the antigen to be deterrni.ned, which is labeled with a radioisotopic probe or an enzyme probe; and c) in the case of manoclonal antibodies labeled with an enzyme probe, a developer for the enzyme, namely one or more solutions containing the substrate for the enzyme and, if necessary, one or more reagents necessary for developing the activity o1.-" the enzyme.
The term "ce:ll" as used in the present specification and in the claims which follow includes human cells, animal cells, the cells of s protozoans and i:he cells of microorganisms (bacteria or fungi). As regards blood cells, the :present ;invention includes the nucleate formed elements, such as the leukocytes, and the anucleate formed elements, such as the erythrocytes or platelets.
The method o~f determination according to the invention applies to to whole cells, i.e~. non-ly;aed cells.
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 best guarantee of integrity of the membrane markers chosen as targets for determination.
t s As thc~ solid rapport it is possible to use any device suitable for the handling of cell suspensions, and preferably tubes, particulate magnetic supports or rigid or flexible microtiter plates made of polyethylene, polystyrene, polyvinyl chloride or nitrocellulose, which contain microwells.
The monoclonal antibodies intended for immobilization of the cells can be 2o fixed to the solid supports either by covalent chemical bonding or by physical adsorption according to conventional methods such as those disclosed by STOCKER a.nd HEUSSER J. Immunol. Methods, 1979, vol.
26, p. 87-95. Acivantage~ously, the support is saturated with a protein beforehand.
2s A .cording to the invention, the monoclonal antibody or antibodies fixed to t.a.e solid support must permit the irnmunocapt~re os the cell population which includes the cell population or populations carrying the antigens to be determined. When this population consists of human cells, the preferred monoclonal antibodies for immunocapture are the anti-class I
3o HLA antibodies 'which are specific for the common part of HLA-A, -B
- g _ and -C antigens Fresent on the leu'.tocytes and numerous other cell lines of the organism. Of these antibodies, the one called S-class I, marketed by BIOSYS, is par-ticularly preferred.
In other cases where the cells examined are human cells and in all cases where these cells are not human cells, monoclonal antibodies appropriate to the type of cells examined can also be used for the immuno-capture according to the invention.
The expression "a monoclonal antibody labeled with a radioisotapic probe" means that the monoclonal antibody carries, either on a component inherent in its structure, for example the constituent tyrosine residues, or on an app:ropri.ate radical which has been attached thereto, a radioactive isotope which enables it to be determined b_~ counting the radioactivity associated therewith.
The expression "a monoclonal antibody labeled with an enzyme probe" means that the monoclonal antibody is coupled to an enzyme which, when associated with the use of appropriate reagents, permits quan-titative measurement of this monoclonal antibody.
The ~~ubstr~~te and the reagents are chosen so that the final product of the reaction or reaction sequence cau__=;ed by the enzyme, involving these sub-stances, is:
- either a colored or fluorescent substance which diffuses into the liquid medium surrounding the cells and which is the object of the final spectrophoto-metric or, respectively, f:luorimetric measurement, - or an insoluble colored substance which deposits on the cells and the walls to which they are fixed, and which can be the object either of photometric measurement by reflection or of visual evaluation, if appropriate against a range of standard shades.
As an additional component, the assay kit can contain a buffer solution intended for washing the solid support: after immobilization and labeling of the cells with the antibody or antibodies carrying the chosen probe"
As additional components, the assay kit can also contain the samples necessary for standardization and quality control of the determination.
The presewt invention further relates to a process for the immunometric assay of the surface anti-gens of a cell population or subpopulation, the said process comprising .
a single step fog:- the specific immobilization or immunocapture of a cell population on the solid support using onE~ or more monoclonal antibodies fixed to the said support beforehand by covalent bonding or by physical adsorption and capable of recognizing an antigen present on the surface of the cells~other than the antigen to be determined, and, simultaneously, the direct labeling of the surface antigen to be determined, belonging to the immobilized cell popula-tion or to one o:E its subpopulations, with a mono-clonal antibody specific for this antigen to be determined, the acid monoclonal antibody carrying a radioisotopic or, alternately, enzyme: probe;
- an incubation peg°iod to allow the simultaneous immuno-capture anci labeling to take place;
- the washin~; of the solid support to remove the non-immobilized. undesirable cells and the excess of mono-clonal antibody carrying the radioisotopic or enzyme probe; and - the actual determination of the antigen to be deter-mined in the labeled cell population or subpopulation by counting the fixed radioactivity or, alternately, after treatment of the medium with the substrate for the enzyme and, if necessary, one or more appropriate auxiliary reagents, by photometric measurement by transmission or reflection, or measurement of the fluorescence emission.
The assay kit and the immunometric assay process according to the invention are preferably applied to the determination of the surface antigens of the formed elements of human i5lood, especially the leukocytes and more particularly !the lymphocytes, the T lymphocytes, the T'4 lymphocytes, the TS lymphocytes and the B lympho-cytes, as we~_1 as the granulocytes, the monocytes and the blood platelet;.
Another preferred application is the determina-tion of the :surface antigens of pathogenic micro-organisms, for example Candida a:lbicans.
The assay lcit and the immunometric assay process according to the invention are also particularly useful for the determination of the surface antigens of. tumoral cells, especially t=hose of cancers of the urinary system and those of malignant hemopathies.
The assay k:it and the immunometric assay process according to the invention make it possible to measure signals (absorbed or emitted light or radioactivity) which depend both on the number of cells present in the cell population examined and on the density of the antigen measured on the surface of these cells. l~leasure-ment of these signals permits quantitative evaluation of the total number of molecules of this antigen which are carried by the cell population or subpopulation examined, whether this antigen has a structural or functional role.
For example, in the case of the leukocyte markers of particular importance in hematology, it is known that, in the majority of situations in healthy subjects, the mean value of the antigen density does not vary substantially between samples for one and the same cell population, ~;o there is a good correlation between the cytological count of the cells carrying the antigen in question and the signal measured according to the invention, which i.s proportional to the total number of antigen molecules present in the sample examined.
Conversely, in some pathological states, the density of the surface antigens may vary for one and the same cell population without. a notable variation in the number or proportion of the positive cells. Such pathological immuno-metric assa;~ process according to the invention or by using the kit of the aresent invention than they would be if a kit of the present. invention than they would be if a conventional cytological counting procedure were used.
Another application of the invention becomes apparent if a mic:rotiter plate is chosen as the solid support. The assay kit and the immunometric assay process according to the invention can then advantageously be used for the determination, on a single plate, of_ a series of surface antigens characteristic of various subpopulations mak:i_ng up the cell population examined.
For this application, it is possible on the one hand to take ready-to-use microtit:er plates to which one or more monoclonal antibodies capable of retaining all the cells of the population examined have been fixed before-hand, and on the other hand t:o have a series of mono-clonal antibodies coupled to an appropriate probe and each specific for an antigen characteristic of one of the subpopulations to be evaluated. 'thus, in a single manipulation and on one and the same support, it is possible to carry out the quantitative determination of all the antigens necessary for characterization of the chosen subpopulations.
This application of the present invention is illustrated by the characterization of the antigenic - 12 - ~3~0973 equipment of cell~~ of interest in clinical biology. A
first case is represented by the determination of the tissue groups characterizing a given individual, which is conventionally known as HLA typing.
A second ease is represented by the typing of tumoral cells, in particular for patients afflicted by malignant hemopathi.es such as leukemias or lymphomas.
This diagnostic examination, which is practiced systematically, consists in characterizing the type and origin of the pati.ent's tumoral cells by the presence or absence on the:>e cells of a series of suitably chosen surface antigens.
Use o.f the kit according to the invention, which contains a microtiter plate on which one or more monoclonal antibodies capable of fixing all the cells of the population examined have been fixed beforehand, and solutions of different monoclonal antibodies labeled with an enzyme or radioisotopic probe and each specific for an antigen present on the tumoral cells, permits identification and quantitative evaluation of the antigens characteristic of the patient's tumoral cell population anal thus makes it possible to associate them with one of the major groups of clinically characterized cancers, especially malignant hemopathies.
Application of the process according to the invention thus makes it possible to carry out the qualitative and quantitative examination of the antigenic equipment of tumoral cel-is rapidly and on a sing~_e support.
A case which may be mentioned as an illustration of another application of the invention is that of the human T lymphocytes, for which there are in particular 2 subpopulations of cells: the lymphocytes characterized by the presence of the CD4 marker, which will be called positive T4 lymphocytes or, more simply, T4 lymphocytes, and the lymphocytes characterized by the presence of the _ 13 _ ~3'~0973 CD8 marker, which will be called positive T8 lymphocytes (or T8 lymphocyte:o).
Measurement of the numerical ratio T4/T8 is of great diagnostic interest in clinical biology. In fact, it is known that modifications of the T4/T8 ratio appear in various complaints of the immune system, such as dysimmune diseases, chronic infectious diseases, viral infections and, in particular, HIV complaints (AIDS virus).
The assay kit and the immunometric assay process accor-ding to the invention can be used for the determination of antigens universally characteristic of the T lymphocyte population and/or antigens characteristic of the T4 and T8 lymphocyte subpopulations. In this case, the T lympho-cytes of the sample examined are specifically immobilized on a solid support anti, simultaneously, the surface antigens of the T4 lymphocytes are labeled directly with an anti-CD4 monoclonal antibody carrying a radioisotopic or enzyme probe; in the same way, the surface antigens of the T8 lymphocytes are labeled directly with an anti-CD8 monoclonal antibody carrying an appropriate probe.
The total. T lymphocytes are preferably determined using an anti-CD7 monoclonal antibody (also called anti-T2 monoclonal antibody) carrying an appropriate probe.
Specific immobilization of the T lymphocytes of the sample is preferably carried out using one or more monoclonal antibodies which are capable, by themselves or in combination, of recognizing all the T cells of the sample, this being the case of the anti-common leukocyr_e (or anti-CD4.~) antibodies or antibodies which recognize the whole of the T population (called "pan-T" antibodies), such as the anti-CD2 (or anti-T11), anti-CD5 (or anti-T1 or anti-CD7 (or anti-T2) antibodies or other pan-T anti-bodies which have not yet been assigned to a differentiation class according to the WHO criteria.
The immur,.ometric assay method of the invention can advantageou:~ly be used for the determination of antigens characteristic of the population of T lymphocytes and T4 and T8 lymphoc!rtes on several parts of the same solid support. Measurement of the signals by radioactivity counting, photomel:ric measurement by transmission or reflection or fluorescence measurement enables the numerical ratio CI)4/CD8 to be calculated easily and directly.
In the same way, it is possible to determine, on one and the same solid support, the subpopulations called T lymphocytes and B lymphocytes which make up the whole of the lymphocyte line.
For example, a monoclonal antibody or a mixture of monoclonal antibodies specific for all the surface antigens of the T cells can be fixed by adsorption to the walls of the microwells. These monoclonal anti-bodies will enable the whole population of T cells of the sample examined to be immobilized in the microwells at a later stage. The plates prepared in this way can be lyophilized and stored, preferably at 4°C. This step can be carried out on the industrial scale and it will thus be possible to have ready-to-use plates for the assay kits which can be applied either to the total T
lymphocytes or to any subpopulation of T lymphocytes.
The samples containing the cells to be determined, which origin,3te from the blood or from any appropriate biological liquid - normal or pathological - can be used as such or a:Eter preparation, especially by density gradient centrifugation according to the methods already known, and in particular in a high-density medium such as, for example, F:ICOLL-PAQUE* marketed by Pharmacia. To determine thf~ blood lymphocytes, the blood sample to be determined can also be treated with a so-called lysis buffer solut~_on, which lyzes the erythrocytes.
* -Trademark ~t1 Aliquots of the appropriate cell suspension are brought into ~~ontact with the solid support, for example in the microwells of a microtiter plate prepared beforehand, at the same time as the solution forming part of the assay kit, which contains the monoclonal antibody specific for the target cell population and carrying an appropriate probe, i.e. a radioisotopic or enzyme tracer. Thus a radioisotopic probe can be prepared for example by labeling the monoclonal antibody with iodine 125 or iodine 131, for example in the presence of chloramine T, by a known process (F. C.
GREENWOOD, W.M. HUNTER et al., Biochem. J., 1963, 89, 114); alternately, an enzyme probe can be prepared by conjugating the monoclonal antibody with an enzyme such as alkaline phosphatase, peroxidase, beta-galac-tosidase or a~~etyl.cholinesterase, by a described method (see, for example, M. 0'SULLIVAN, Methods in Enzymology, 1981, 73, 147) or by a method based thereon. In some cases, in order to avoid certain disadvantages associated with the handling of radioactive substances and the limited shelf life of the reagents, enzymes will be used in preference to radioisotopic probes.
The incubation period, i.e. the time required f.or immobilization and simultaneous labeling of the cells, is preferably less than or equal to 1 hour.
During this tame, the solid support can be centrifuged, if necessary, in order to improve the immobilization of the cells. T'he solid support, for example the micro-titer plate, is then washed to remove the non-fixed cells and at nhe same time the excess of monoclonal antibody carr:~ing an enzyme or radioisotopic probe.
When a radioisotopic probe is used, for example iodine 125, tile radioactivity associated with the cells is counted in a gamma counter according to any appropriate procedure and" for example, after solubilization of the -lr~- 1340973 cells with an alkaline solution (for example a sodium hydroxide solution) and recovery of the solution con-taining the radioactivity by means of an absorbent buffer.
When an enzyme probe is used on the mono-clonal. antibody, the appearance of a colored or fluorescent product is brought about by adding, to the solid support to which the cell population carrying the antigen to be determined has been fixed, a solution containing the substrate for the enzyme and one or more auxiliary reagents such that the reaction product which is finally obtained is either a colored product soluble in the medium, or an insoluble colored product, or a soluble fluorescent product, as explained earlier. The light signal coming from the samples treated in this way is then measured with the equipment appropriate to each case, i.e. a transmission or reflection photometer or, respectively, a fluori.meter. When the solid support is a microtiter plate, the light signal can be read sequentially in all the wells of one and the same plate by means of automated readers commonly used in biology laboratories, such as the Titertek plate reader or the Fluoroscan plate reader for the spectrophotometric or, respectively, fluorometric readings.
When alkaline phosphatase is used as the enzyme probe, this enzyme is coupled to the monoclonal antibody acc~~rding to the method proposed by F3oehringer Mannheim - Biochemica. 'The preferred substrates for this enzyme are paranitrophenyl phosphate for a spectrophoto-metric final reading, 4-methylumbelliferyl phosphate for a fl.uorimetr:ic reading or 5-bromo-4-chloroindol-3-yl phosphate for obtaining an insoluble colored reaction product. Likewise, beta-galactosidase can be used as the enzyme probe, in which case the preferred sub-strates will be orthonitrophenyl beta-D-galactopyranoside or 4-methylumbelliferyl beta-D-galactopyranoside.
The monoclonal antibodies can preferably be coupled to peroxid.ase. In this case, the coupling process is derivef. from that described by M.B. WILSON
S and P.K. NAKANE in Immunofluorescence and Related Staining Techniques, edited by W. Knapp, K. Kolubar and G. Wicks, Elsevier/North Holland, Amsterdam, 1978, p. 215-224. The modifications which have been introduced by comparison with the initial protocol for preparation of the enzyme conjugate concern the following points:
- the molar ratio peroxidase/antibody is equal to 3 as opposed to 2 in the protocol, and - oxidation of the carbohydrate units of the peroxidase is less harsh ciu.e to a 33~ reduction in the proposed concentration of sodium periodate.
The reagents used to develop the peroxidase conjugated with th.e monoclonal antibodies contain hydrogen peroxide, which is the substrate for the enzyme, and an appropriate chromogen, for example orthophenylene-diamine or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid, or ABTS, to give a final reaction product which is colored a.nd soluble in the medium, or else 3,3'-diaminobenzidi.ne, 3-amino-9-ethylcarbazole or 4-chloro-alpha-naphthol to give an insoluble final reaction product, or else parahydroxyphenylpropionic acid to give a fluorescent reaction product which i.s soluble in the medium.
In a preferred form, the kit according to the invention for the determination of antigens characteristic of the T, T4 and T'8 lymphocytes comprises:
a) a microtiter plate in whose wells one or more monoclonal antibodies directed against T lymphocytes have been fixed, bl) a solution containing at least one peroxidase-labeled monoclonal antibody directed against T lymphocytes, b2) a solution containing at least one peroxidase-1<ibeled monoclonal antibody directed against CD4 antigen, b3) a solution containing at least one peroxidase-l~3beled monoclonal antibody directed against CD8 antigen, cl) ~3 solution containing hydrogen peroxide, which is the substrate for the enzyme, in an appropriate buffer, and c2) <3 solution containing the chromogen used to develop the expression of the enzyme's activity.
Another preferred embodiment of the invention is the use of monoclonal antibodies coupled to acetyl-cholinestera:~e.
The acetyl~~holinesterase is preferably coupled to the antibody using a process derived from that described in French Patent no. 2 550 799 or a process which schemat:icall:y involves the preparation of fragments of the antibody by a known technique, the modification of the enzyme by reaction with an appropriate hetero-bifun~.~'ional agent and, finally, the coupling of the resulting products. Other known processes for constructing immunoenzyme conjugates can also be used in this case.
Devel.opmen~~ of_ the enzyme activity specifically associated with the cell antigen recognized by the acetylcholinesteraae conjugate is preferably carried out by the known techn=ique which employs acetylthiocholine as the substrate for the enzyme and Ellman's reagent, or 5,5'-dithio-~.-nitrobenzoic acid, as the chromogen, in accordance with any variant suitable for the case under examination, for example the one described by Pradelles et al., Anal. Chem", 57 (1985) 1170-1173.
The chromo~;ens cited are used as such or in the form of. water-soluble salts.
The result: of the determination of antigens according to the invention can be expressed in any form appropriate to the examination carried out. More par-ticularly, these results can be expressed either as the total number of molecules of a particular antigen (for example the CD4 antigen) present in a given volume of the sample examined (for example per microliter of blood), or as the ratio of the number of molecules of one antigen to the number of molecules of another antigen in the sample examined (for example the ratio of the CD4 anti;~ens to the CD8 antigens - or CD4/CD8 ratio -in the blood sample examined).
The number of molecules of a particular antigen in the sample examined will preferably be determined using a standard range consisting of appropriate cells or cell preparations carrying the antigen to be determined, which will hive been calibrated beforehand by a known reference mei=hod. These standards will preferably con-sist either of cells identical in their origin to the cells which are to form the subject of the determination, or of cells of established cell lines carrying the desired antif;en, or of preparations, for example membrane preparations., originating from these cells.
These standards are then treated in exactly the same way as t:he samples to be examined. The resulting signals are used to build up a standard range against.
which the signals measured with the samples to be examined are compared. The subsequent calculations are conventional.
To determine the ratio of the numbers of molecules of two antigens in the sample to be examined, it is possible to use the standard system described above and finally to ca.lculat:e the desired ratio. A simpler possibility in many cases is directly to calculate the ratio of the specific signals obtained with each of the desired antigens and to correct it, if necessary, by means of known faci~ors such as the ratio of the dimen-sions of the samples used; this gives the desired ratio directly.
The inrmunomet;ric assay method according to the inven-tion is simp7.e, rapid and reproducible. Its use is totally suitable for the analysis of a large number of samples. For an understanding of its advantages compared with the other methods described, the various steps of the method according to the invention should be analyzed.
The immobilization of the cells on the solid support is the pha:~e of the determination which usually presents the most difficulties or which is the most critical to carry out. The means often used is chemical fixation of t:he ce_Lls with glutaraldehyde or methanol in cups which may or may not have been treated with poly-L-lysine (F. 'JAN LEUVEN et al., J. Immunol. Methods, 1978, 23, 1050. However, chemical fixations performed in this way c:an reduce or even suppress the desired specific detection or conversely can induce the false-positive labeling of cells, which is a very serious disadvantage (DROVhR and MARSHALL, J. Immunol. Methods, 1986, 90, 27~~-281)"
Furthermore, the chemical fixation process has to be carried. out ~_n several. steps: centrifugation of the cells, preparat=ion of the fixative mixture, fixation and then wasa.ing o1- the fixed 'cells several times.
Drying of t:he cells at 37°C, optionally followed by fixation with methanol in the microwells, has also been proposed (BALJ~1G.'~RTE1, J. lmmunol. .'Methods, 1986, 9'~, 91-98).
Actually, in addition to the fact that it can degrade certain fragile antigens, drying of the cells at 37°C
renders the pericel_lular plasmic membrane permeable, thereby facilitating the immunolabeling of intracyto p.lasmic antigens a~; well as .Labeling of the surface antigens, which leads to troublesome backgrounds or to false-positive results when it is desired to restrict a measurement to the surface antigens.
Furthermore, the reproducibility of this process is doubtful; in fact, the settling of the cells in the assay microwells and the drying of the cells can vary between experiments. Finally, this determination is long to perform because the cell drying step alone takes more than 2 hours.
The immobilization of lymphocytic populations has also been achieved by the use of_ polyclonal antibodies adsorbed in inicrowells (STOCKER and HEUSSER, J. Immunol.
Methods, 197'x, 26, 87-95). Apart from permitting the immobilization of cells foreign to the single population which it is desired to analyze, polyclonal antibodies also have the disadvantage of reacting with the antigens which are to be measured by means of the labeled anti-body, leading to a corresponding reduction in the signal which is fin;~lly measured.
The use of highly specific and related monoclonal antibodies adsorbed on or fixed to the solid support, and especial:iy in the assay wells, permits exclusive capture of the desired cells, the other non-retained cell populations being removed in the course of the washing operations carried out on completion of the labeling of i=he antigens to be determined. Furthermore, no chemical or physical agent modifies the characteris-tics of the antigens in this step because the various operations for chemical or physical. fixation of the cells to the support are omitted.
Thus;. according to the present invention, it has been found that the immobilization of cells by monoclonal antibodies is a process which makes it possible to simpli:Ey the step for immobilizing the cells carrying the antigen to be determined, while at the same ~ 3 40 97 3 _ 22 _ time making the results more reliable.
Tmmunometric: assays applied to cell populations are generally carried out by labeling the cells according to an indirect method in 2 or 3 successive steps, the S probe which provides the specific signal being fixed to the cells during the last step of the labeling process. In the labe?.ing of cells in two steps, the main reagents involved use anti-immunoglobulin (anti-Ig) antibodies coupled to beta-galactosidase (COBBOLD
et al., J. Immunol. :Methods, 1971, 44, 125-133) or to alkaline phosphatase (HESSIAN, .1. Immunol. Methods, 1986, 91, 29-34) or labeled with iodine 125 (SAVION, J. Immunol. Methods, 1987, 97, 49-56). The technique of labeling with the peroxidase-antiperoxidase reagent 1S is carried out in 3 steps (VAN LEUVEN, 1978).
Another process, also in 3 steps, uses a mono-clonal antibody specific for the antigenic marker to be analyzed, then anti-mouse Ig antibodies carrying biotin, and finally a streptavi.dine-peroxi.dase conjugate (BAUMGARTEN, 1986) or streptavidine-alkaline phosphatase conjugate (IGIETSEME et al., J. Immunol. Methods, 1987, 97, 123-131).
The process according to the invention, which comprises the simultaneous use, in a single step, of a procedure for immunocapture of whole cells without physical or chemi.ca7 intervention on the cells, and of the labelinf; of a:~ll. ar some of these cells with one or more monoclonal antibodies directly carrying a radioiso-topic or enzyme probe, is the first process to permit the quantitative determination of chosen membrane anti-~;ens on the cells themselves.
According to the invention, the direct labeling of immunologicall_y immobilized cells permits:
- simplification of the method of determination by elimination of the intermediate manipulations repeated ~ 3 4p g7 3 between the successive steps of the labeling process in the case of indirect labeling: centrifugation of the cells several times, removal of_ the labeling reagents and resuspension;
- a saving of reagents;
- an improved reliab:i:l_ity through a reduction in the number of steps and manipulations;
- a time saving; and - the possibility of treating large numbers of samples at the same time, exclusively with the use of con-ventional equipment and apparatuses.
The incubation period for immobilization of the cell population and simultaneous direct labeling of the antigens of the cell subpopulation to be determined is short. It i;s less than or equal to 1 hour in the case of the determination of T lymphocytes and subpopulations of T4 and T8 lymphocytes.
After the solid support has been washed, the actual determination is carried out by using conventional apparatuses 1.0 observe a signal which is precise and simple to measure: radioactivity or light absorption or emission.
Thus" overall, the process according to the invention ha:~ nume:rous advantages: it is rapid, reliable, economic and simply=_.
The proces:~ according to the invention makes it possible to determine surface antigens of a cell population over a wade range of ce7_1 concentrations.
The ;;ensit=ivity of the method in respect of the number of cells depends on the antigen density of the cell population determined. For each antigen it is possible, if desired, to define the minimum molar con-centration of antigen which can be measured by the process according to the invention.
Thus, for example, when the chosen solid support is a microtiter plate and 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 about 200 000 per 200 ~.rl microwell, the lower limit being imposed by the density of the measured antigen on the cells examined and the sensitivity of the chosen detection technique, while the upper limit depends essentially on the size and geometry of the solid support. The same applies in the case where the solid support consists of tubes.
It has been verified that the signals recorded (radioactivity count or photometric measurements) make it possible to obtain satisfactory uniform calibration curves as a function of the number of cells used, under the customary handling conditions.
Furthermare, the sensitivity of the method can be improved, if necessary, by the simultaneous fixation, to one and the same surface antigen, of several different monoclonal antibadies specific for several different epitopes of the same antigen. This has been verified by determination of the CD4 antigens on cells of the Ichikawa line (human T line), where the OKT4 and ST4 antibodies labeled with iodine 125 were used simul-taneously. The measured signal increased by about 500 relative to the signals obtavined with each of the anti-CD4 antihodi~s used by itselfi.
In the Rxamples which follow, the following forms or their abbreviations will be used indiscriminately:
FiS~~ . bovine serum albumin PBS . phosphate buffered saline of pH 7.4 POD . peroxidase IgG . immunoglobulin G
IgM . immunoglobulin M
anti-T IgG o:r anti-T . antibody directed against T
Lymphocytes anti-CD4 IgG or anti-CD4 . antibody directed against CD4 antigen anti-CD8 IgG or anti-CD8 , antibody directed against CD8 antigen cpm , counts per minute dpm . disintegrations per minute Enzyme immurrometric assay of the molecular concentration of the CD4 and CD8 antigens in samples of human blood, in which the antibodies are labeled with peroxidase A) Preparation of the assay kit a) Preparation of the plate The plate used is a plastic microtiter plate containing 9p microwells, marketed by NUNC (reference 64394). Eaclz mic.rowell receives 200 trl of a solution containing the purified anti-CD2 monoclonal antibody (called STlI) used to immobilize the total T lympho-cytes, i.e. to e.f.f.ect their immunocapture. This anti-body, marketed by BIOSYS, Compiegne, France, under the reference ST11, is used at a concentration of 10 ~g/ml in a phosphate buffered saline (PBS) of,pH 7.4. The adsorption o:E the ,monoclonal antibody is effected at 4°C for 12 hours. The excess antibody is removed by turning the plate over.
A solution containing 0.1% of gelatin and 0.5~
of BSA in a phosphate buffered saline is prepared.
250 Yl of this solution are introduced into each microwell so as to saturate the surface of the wells with protein, which takes J. hour at 37°C; the plates are washed 3 times with phosphavte buffered saline. The plates prepared in this way are lyophilized and stored at 4°C in a sealed plastic sachet.
b) Preparalwion of. the solution of monoclonal antibod~r-peroxidase conjugate The r~eroxidase (POD) marketed by Boehringer Mannheim Biochemica (reference 814 393) is used.
The process for coupling the antibody to the peroxidase is the one described by M.B. WILSON and P.K. NAKANE in Immunofluorescence and Related Techniques (edited by ~J. KNAPP, K. KOLLJBAR and G. WICK, 1978, Elsevier/North Holland, Amsterdam - p. 215-224), except that the oxidation of the peroxidase is carried out using 1.5 mg of PCD in 0.36 ml of distilled water and that 50 ul of a 0.2 M solution of sodium periodate are added. /The resulting product is coupled to 2 mg of anti-CD4 IgG contained in 500 ~l of carbonate buffer.
After treatment with sodium borohydride and dialysis against PBS, the IgG-POD conjugate is sterilized by filtration on a 0.22 ~m membrane and kept under sterile conditions at a concentration of 0.5 mg of IgG per ml, at 4°C, in glass tubes. The reagent is stable for at least 1 year.
The anti-CD8 IgG-POD conjugate is prepared in the same way. IgM-POD conjugates can be prepared by the same method.
c) Preparation of the developing reagent The developing reagent is obtained in the following manner: A 0.1 M citrate buffer is prepared by dissolving citric acid monohydrate in water at a con-centration of 2.1~ and adjusting the pH to 5 by the addition of 7 N sodium hydroxide solution. 30 mg of orthophenylenediamine dihydrochloride are then added to 20 ml of the citrate buffer obtained, after which 40 ~1 of 30~ hydrogen peroxide (substrate f_or the enzyme) are added, at the last moment, per 20 ml of citrate buffer containing orthophenylenediamine.
B) Immunometric assay process a) Separation of the cells 2 ml are taken from the blood sample to be determined. This is mixed with 2 ml of phosphate ~3~~9~3 _ 27 _ buffered saline and the resulting mixture is deposited on top of 3 ;ml of FICOLL-PAQUE (marketed by Pharmacia).
On centrifugation at 400 x g for 30 minutes at room temperature, a suspension ring containing the mono-nucleate cells is formed. The whole of this suspension is recovered in a volume of 1 ml and 6 x 100 ~l of the resulting suspension are distributed into 6 microwells of the preprepared plate.
b) Incubation of the cells The previously obtained POD-antibody conjugate is diluted 100-fold with PBS containing 1~ of a protein substance such as BSA or skimmed milk powder, and 100 rl of this solution are deposited in each microwell as follows:
- 2 microwel:Ls are each filled with 100 ~1 of the solution o:E the POD-anti-CD4 conjugate;
- 2 microwel:Ls are each filled with 100 ~1 of the solution off= the POD-anti-CD8 conjugate; and - 2 control rnicrow.ells are each filled with 100 ~1 of a 1~ solution of protein substance (reaction blank).
To improve the fixation of the cells to the support, the plate is centrifuged for 3 minutes at 150 x g after a wait of 1 hour at room temperature.
c) Development of the fixed enzyme and measurement The microwells are emptied by turning the plate over. They are waahed 4 times with 200 Y1 of PBS.
200 ul of then developing reagent, prepared for immediate use,Iare added to each well. Incubation is carried out for 20 minutes at room temperature in subdued light.
The optical density is measured on a spectrophotometer at 492 nm (type 310 C* Titertek Multiskan apparatus -Flow Laboratories)..
d) Standardization and expression of the results To standardize these experiments, a preparation of total human lymphocytes was used which had been * - Trademar?;
A
calibrated beforehand in respect of CD4 and CD8 antigens by the cytofl_uorom~=_tric technique of PONCELET et al.
(,T. Immunol. Methods, 1985, 85, 65-74). Known aliquots of this reference preparation were used to make up the 2 standard ranges ~celating to the CD4 antigen and CD8 antigen respective:Ly, against which the numbers of antigens corresponding to each sample are calculated.
By way of :indication, Table 1 below shows, for 20 samples of human blood taken from healthy donors, the optical density va=lues obtained, the corresponding numbers of moleculE~s of CD4 and CD8 antigens and the resulting ratios oj~ these numbers of antigens (CD4/CD8 ratios).
~ 3 40 97 3 CD4 anti;~en CD8 antigen Donor Molar number 0ptica7_ Number of Optical Number of ratio density antigen moleculesdensity antigen moleculesCD4/
(in m:illi.ons) (in millions) CD8 per ~yl of blood per ~1 o.f blood 1 0.64 '~3 1.29 92 0.25 2 0.71 27 0.75 37 0.73 3 0.82 32 1.10 81 0.39 4 0.49 17 0.55 23 0.74 5 0.74 'Z9 0.89 52 0.56 6 0.88 38 0.92 55 0.69 7 0.74 29 0.72 36 0.80 8 0.75 29 0.69 33 0.88 9 0.48 16 0.80 41 0.39 10 1.00 50 1.08 80 0.62 11 0.61 22 0.69 33 0.67 12 0.73 28 0.85 46 0.61 13 0,51 18 0.66 30 0.60 14 0.65 24 1.06 76 0.31 15 0.92 42 0.87 48 0.87 16 0.77 31 0.98 63 0.49 17 0.68 25 1.01 67 0.37 18 0.85 36 0.96 61 0.59 19 0.91 41 0.90 52 0.79 20 1.00 50 1.02 69 0.72 Enzyme immunometric assay of the molecularconcentra-tion of the C;D4 and CD8 antigens in samples of human blood, in which the antibodies are labeled with alkaline phosphatase This determination is carried out in the same way as that of Example 1. The monoclonal antibody-alkaline phosphate:>e conjugate is prepared according to the method indicated by Roehringer Mannheim - Biochemica (ref. 567 744). After the cells to be determined have been incubated for one hour with this conjugate, the fixed enzyme is developed by the addition of a solution containing 1 mg/ml of paranitrophenyl phosphate in a buffer containing J..2~ of diethanolamine 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 on a spectrophotometer at 405 nm.
The results are calculated and expressed as in Example 1.
Radio:Lmmunometric assay of the molecular concentration of the CD4 and CD8 antigens in samples of human blood, in which the antibodies are :Labeled with iodine 125 The antibody labeled with iodine 125 is prepared according to the technique described by F.C. Greenwood, V.M. Hunter et al., Biochem. J., 1963, 89, 114.
50 ~g of anti-CD4 or anti-CD8 monoclonal antibody contained in 50 rrl of phosphate buffered saline o.f pH
7.2 are mixed with 37 MBq of iodine 125 in the form of sodium iodide and ''.0 trl of a solution containing 0.33 mg of chloramine T per m1l in a phosphate buffered saline.
After 1 minute of shaking, t:he reaction for labeling the monoclonal antibody is stopped by the addition of 100 ul of a solution containing 2.5 mg/ml of_ sodium 1340973'' metabisulfite" The solution prepared in this way is passed through a PD 10 column (Pharmacia - Sephadex G25M) and the fraction containing the radiolabeled antibody is recovered in the effluent. The determination is carried oul: by introducing into 4 microwells a volume of 100 ul of cell suspension containing mononucleate human bllood cf~lls prepared as in Example 1. 100 ul of dilute solution of radioactive antibody in a PBS 'buffer containing 5% of BSA are then deposited so as to introduce 150 000 cpm into each well; 2 microwells are each filled with 100 ~l of the solution of the anti-CD4-125I conjugate. and 2 microwells are each filled with 100 ul of the solution of the anti-CD8-125I conjugate.
The /fixation of the cells is improved by centrifugation of the plate :nor 3 minutes at 150 x g after incubation for 1 hour at room temperature. The microwells are emptied by turning the plate over. The microwells are washed 4 timer with 200 rl of PBS per well. 75 ~l of 1 M sodium hydroxide solution are then introduced into each well. A:Eter 10 minutes, the contents of each well are recovered with an absorbent buffer before the radio-activity is counted in a X counter (LKB multiwell counter).
The results are calculated and expressed as in Example l, the optical density values being replaced with the count results in dpm.
Checking the validity of the method On 20 samples of human blood, the CD4 and CD8 antigens of the human T cells were measured by following the peroxidase immunometric assay method described in Example 1.
The ratio of l:he number of positive T4 cells to the number of positive 'T8 cells was also determined on the same blood samples 'using the conventional cytological counting technique (W. W. ERBER et al., Lancet, 1984, (8385 ) , 1042--1045 ) .
The correlation coefficient r between the ratio CD4/CD8 obtained by enzyme immunornetric assay and the ratio T4/T8 obtained by cytological counting is 0.72.
Likewise, on 7 other samples of human blood, the ratio CD~E/CD8 determined by the radioimmunometric assay method using labeling with iodine 125 by the process of the present ~:nvent:ion was compared with the cell ratio T4/T8 determined b:y cell counting. The correlation coef.fici.ent r is 0.87.
In both cases, the correlation coefficients obtained show that, despite a satisfactory overall agreement bet=ween the results of the 2 techniques, the information provided by the 2 ratios is not equivalent;
this is obvious since the determination of antigens according to the invention allows not only for the number of positive cells, as is the case for the cyto-logical method, but also for the density of the antigen in question on the positive cells of each sample. The information given by the technique described in the present invention is therefore more complete than that given by the conventional reference technique (W. W.
FRBFR et al., Lancet, 1984, (8385), 1.042-1045).
Enzym<~ immunometric assay of the molecular concentration of the CD4 and CD8 antigens of the T lymphocytes in samples of human blood, in which the antibodies are labeled with peroxidase The purpose of this Example is to show that the time required for the determination can be reduced, relative to 'she conditions described in Example 1, by modifying on the one hand the cell separation process and on the o~=her hand the incubation time required by the step for immunocapture and labeling of the cells.
A) Influence of the process for the separation of cells from whole blood a) Technique involving brief centrifugation:
0.5 ml is taken from the blood sample to be determined and mixed with 1.5 ml of phosphate buffered saline. 1.5 ml of Fi.coll-Paque, marketed by Pharmacia, are introduced into a 5 ml hemolysis tube and the blood sample, diluted in PBS, is deposited on the surface of the layer of Ficoll. Centrifugation at 900 x g for 5 minutes at room temperature is followed by removal of the suspension ring contai.ni.ng the mononucleate cells in a volume of 0.5 ml. 1.5 ml of PBS are added to this sample.
b) Technique involving lysis of the erythrocytes:
Another rapid method for the separation of cells from blood is to use a buffer which lyzes the erythro-cytes. The lysis buffer, used by way of a non-limiting example, has the following composition:
ammonium chloride: 8.29 g potassium bicarbonate: 1 g disodium salt of ethylenediaminetetraacetic acid:
0.0307 g per liter of distilled water, the pH being adjusted to 7.3.
5 ml of lysis buffer are mixed with 0.250 ml of blood. After 10 minutes of shaking, the mixture is centrifuged at 6t:)C~ x g for L0 minutes. The cell residue formed is taken up in 1 ml of PBS buffer.
By subsequent determination of the CD4.and CD8 antigens under the conditions of Example l, and by comparison with the reference protocol described in Example 1 for isolation of t:he mononucleate cells from blood, it was verified that both these variants of the method for the separation of the mononucleate cells from whole blood did indeed enable all the lymphocytes to be collected from the blood samples examined.
B) Influence of the incubation time for immunocapture and labeling of the cells In order to check whether the period of 1 hour used in Example 1 might be reduced in order to accelerate the determination, the results obtained for identical samples each containing 20 000 mononucleate cells per well were compared when the time allowed for capture and labeling of t:he cells was varied from 10 minutes to 1 hour. The results obtained are shown in Table 2, all other operating conditions being the same as in Example 1.
Incubation Optical density time (minutes) CD4 antigen CD8 antigen Test Blank'~~ 'Test Blank 10 0.322 0.037 0.329 0.023 0.438 0.049 0.401 0.025 0.620 0.055 0.498 0.027 15 ~ These optical density values correspond to the reagent blank obtainE~d in 1_he absence of cells. The control values obtaiu:ed in the presence of cells with either the conjugate or the substrate being omitted are not greater than the values indicated.
20 These resu_Lts show that:
- The non-specific signal (reagent blank) always remains at low values which become lower as the time for which the conjugate is present becomes shorter.
- After 10 minutes of contact, the specific signal reaches a value which can be processed analytically with precision and reproducibility. This indicates that it is possible, relative to the conditions of Example l, signifi~antl.y to reduce the time taken to carry out the determination without a substantial loss of precision in the results.
In practice, and if the time savings which can be achieved in the preparation of the blood sample and in immunocapture are added together, the kit and the process of t'he invention make it possible to determine the CD4 and CD8 antigens (by way of non-limiting examples) of samples of whole blood, at a rate of 10 or 20 samplers per plate of 96 wells, in a total time (taken from reception of the samples in the laboratory) of no more tinan 1 hour. This level of productivity is greater than that of all the alternate techniques known to date.
Immur~ometric assay of the molecular concentration of the CD5 antiF;ens of the human T lymphocytes in samples of human blood, in which the antibody is labeled with acetylcholin~~sterase A) Preparation of the assay kit a) Solid support A micrati.ter plate prepared in the manner des-cribed in Example 1 is used.
b) Manoclo:nal antibody-acetylcholinesterase conjugate The acetylr_holinesterase of. Electrophorus electricus i;~ used, which is obtained by the technique described in French Patent no. 2 550 799.
This enzyme is coupled to the anti-CD5 antibody called ST1 and may°keted by BIOSYS. Coupling is carried out in accordance ~~rith the process described by YOSHITAKE
et al., Eur. J. Biochem., 101 (1979), 395-399.
c) Developing reagent This reagent comprises both the substrate for the enzyme (acetylt:hiocholine) and Ellman's reagent and has the following composition:
- acetylthiocholine 7.5 x 10-4 M
- 5,5'-dithio-2-nit:robenzoic acid (DTNB) 5 x 10 4 M
in 1 M sodium phosphate buffer of pH 7.4.
This solution is diluted 100-fold in distilled water before use.
B) Immunometric assay process a) The mononucleate cells are separated off using the method described in Example 1.
b) Incubation of the cells for the immunocapture and labeling step takes 20 minutes, as in Example 5.
c) Development of the fixed enzyme and measure-ment.
100 ~l of developing reagent are added to each microwell. T'he ab:;orbance is measured at 412 nm after 45 minutes of incubation.
The measured optical densities for 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.
Number of T introduced lymphocytes into each well and of number CD5 of antigen molecules present in each millions well, in (32) (65) (130) (260) (520) (1040) Optical density0.030 0.060 0.138 0,260 0.470 0.850 These results reveal the extreme sensitivity of the technique developed in this way. In fact, the optical density of 0.030 obtained in the well containing 540 T lymphocytes (i.e. 32 million molecules of CD5 antigen), which is measurable with precision and equal to 10 times the background, corresponds to about 5~10 17 mol of CD5 antigen per assay well. Such sensitivity is of the same order as that of the best immunoassays known, which use the most sensitive radioactive tracers.
Determination of different antigens expressed on activated human T lymphocytes Activation of the T lymphocytes is a physio-logical process which takes place prematurely every time the immune system is stressed, for example as in infectious pathologies, organ transplantations and certain dysimmune diseases. This natural process is also commonly used in vitro in the laboratory in tests such as, in particular, mixed lymphocytic reactions or lymphoblastic transformation tests. In the latter case, polyclonal activation is obtained by the use of agents such as phyt:ohemagglutinin (PHA), concanavalin A or other lectins. Activation of the T lymphocytes is accompanied by a considerable increase in the expression of several membrane markers, and especially the CD25 antigen (receptor of i.nterleukin '?) c>r the CD2 antigen.
These antigens therefore constitute excellent markers of the activation state and their determination is of great interest both in clinical biology and for laboratory work, as indicated above, the use of radioactive reagents being avoided in all cases.
These antigens are measured using the method described in Example 1 with the specifications detailed in Table 4.
Measuremento:E the surface antigensof activated T lymphocytes Antigen Antibody used for Antibody labeled with measured immunocapture, peroxidase, marketed by marketed by CD2 STl BIOSYS ST11 BIOSYS
CD2.5 ST11 BIOSYS I0T14 IMMUNOTECH
The optical density values measured at 450 nm for the 25~103 mononucleate cells used are reported in S Table 5 below and show the comparison between the cells without stimulation with PHA and the same cells after 3 days of stimulation.
Optical density Antigen Non--activated Cells activated Reagent blanf<
cel=Ls for 3 days CD25 0.064 0.529 0.044 CD 2 (),129 0. 768 0. 027 These results show that the specific signals of the 2 antigens examined increase considerably when activation takes place.
Determination of the CD22 and HLA-Dr (or class II
HL,A) antigens present on B lymphocytes The present Example describes the determination of these antigens on cells of the RAJI line, which is a human B lymphoid line described by PULVERTAFT in J.
Clin. Pathol., 1965, 18, 261-274.
39 ~ (~ O ~ 7 3 For immunocapture of the cells, either the S-class II antibody (BIOSYS) for determination of the CD22 antigens, or the SB4 antibody (BIOSYS) for determination of the class II HLA (or HLA-Dr) antigens, was adsorbed in the assay wells as indicated in Example 1.
s a) Determination of the CD22 antigen The S;B22 antibody (BIOSYS) was labeled with iodine 125 by the method described in F;xample 3. The CD22 antigens are determined in the manner indicated in Example 3 for the CD4 and CD8 antigens, the following being introduced successively into the microwells : 5.104 cells of ~o the RAJI line and then 105 cpm of the labeled SB22 antibody. After a period of one hour at 4°C, the microwells are emptied by turning the plate over and then washed 4 times with 200 ~1 of PBS per well for each wash.
The radioactivity fixed to the cells is recovered and counted as in Example 3. This gives a count of 7Ei0 dpm for the well containing 5 x 104 RAJI cells is with a reagent blank (without cells) of 50 dpm.
b) DetE~rmination of the HLA-Dr antigen 105 cell'.s are introduced into the wells, followed by the conjugate of S-class II antibody labeled with peroxidase by the process described in Example 1. The dntennina~tion is carried out according to the same protocol 2o as in Example 1. ~Chis gives an optical density of 1.840 and a reagent blank, without cells, of 0.105.
EXAMfPLE 9 Use of the process according to the invention for the 2s measurement of antigens carried by the T lymphocytes and B lymphocytes of human blood A) Preparation of the plates The plates used are microtiter plates prepared as in Example 1 except that monoclonal antibodies capable of fixing at least all the T lymphocytes (S-class I from BIOSYS) and the B lymphocytes (S-class II
from BIOSYS), each one being used at a concentration of 5 ug/ml, are simultaneously adsorbed in each well of the milcroplate.
B) Immunometric assay .
The mononucleate cells are separated from the whole blood with the aid of Fico:ll under the conditions of Example 1 or Example 5, after which 80 000 mono-nucleate cells in 75 ul of PBS buffer are introduced into each well. The l'C lymphocytes are measured in some of the wells with the ST11 (anti-CD2) antibody from BIOSYS; the B lymphocytes are measured in other wells with the SB3 (anti.-CD37) antibody from BIOSYS, which recognizes all the peripheral B lymphocytes. These antibodies are first coupled to peroxidase according to the protoc~~l described in Example 1. The actual determination is carried out as in Example 1.
C) Results The al5solute numbers of T lymphocytes and B
lymphocytes contained in the sample examined are deter-mined by reference to calibration curves established by carrying out an identical treatment on mononucleate cells in which the 'r and B lymphocytes have been counted by a reference technique.
Alternately, by means of appropriate standards, the results can also be expressed as molar concentrations of the CD2 anc~ CD37 antigens in the sample examined.
Determination of the GpIIb-IIIa and CD9 antigens of human blood platelets This determ:W ation is carried out according to Example 3 using monoclonal antibodies radiolabeled with iodine 125.
The platelets are isolated from human blood by centrifugation in the presence of PLASMION perfusion liquid (Laboratoire R. Bellon). 5 ml of blood plus ml of phosphate huffered saline (PBS) and 10 mI of 5 PLASMION are mixed in a tube. The tube is then centrifuged for 10 minutes at 1500 rpm. The platelets which have collected in the supernatant are removed by suction with a pipette. They are washed once in PBS by mixing 1 volume of the platelet suspension with 10 ml of PBS, and then centrifuged for 10 minutes at 1000 x g.
Finally, the plate_Let residue is collected and resus-pended in PB~> (2 m_L). The platelets are counted and their concentration adjusted to 2.5 million/ml of PBS.
The C~pIIb-=LIIa antigen is determined by immuno-capture of the platelets by the monoclonal antibody I0B2 (Immunot:ech) and labeling of the GpIIb-IIIa antigen with the monoclona~L antibody P2 (Immunotech).
The C;D9 antigen is determined by immunocapture by the monoclonal antibody P2 and labeling with the monoclonal antibody IOB2. The number of dpm is measured for a sample compr~'_sing 200 000 platelets.
The r~esult:~ are shown in Table 6.
Antigen Sample Control without measured measured cells (dpm) (dpm) GpIIb-IIIa 2891 238 Determination of the CD15 antigens carried by human granulocytes The CD15 antigen is a good specific marker of human granulocytes (also called polynuclear leukocytes).
These cells can be evaluated by means of this antigen, in the blood, in the same way as for any other leuko-cytic subpopulation. This evaluation is also useful for assessing the presence of granulocytes in the urine, for example in cases of urinary infections such as cystitis or pyelonephritis. In the present Example, the determination was carried out on granulocytes originating from whole blood and separated off as indicated below.
The granulocytes, as well as some of the mono-nucleate cells, are separated from the erythrocytes by a process analogous to that described in Example 10 for the platelets, except that t:he tube containing the cells is left to stand for 45 minutes at room temperature and the suspension of leukocytes is recovered in a volume of 500 til taken from the surface of the liquid.
The granulocytes are immobilized in the wells with a monoclonal antibody specific for the CD45 antigen present on the cell membrane of all the leukocytes:
the anti-LCA antibody (BIOSYS) is used and is adsorbed in the microwells of the titer plate, as indicated in Example 1.
The determination is carried out using the anti-CD15 monoclonal antibody SMY-15a (BIOSYS) labeled with peroxidase by the technique of Example 1.
'Thus, w:it:h a total of 12 500 cells introduced into each well, the specific signal of the CD15 antigen of the granulocytes is 0.616 after correction of the crude optical density for a reagent blank of 0.100, obtained in the presence of the cells and in the absence of enzyme conjugate and due to the endogenous peroxidases of the granulocytes.
~ 3 4o g~ 3 EXAMPLE. 12 Evaluation of the phenotype of a leukemia by the enzyme immunometric assay process The phenotyping of the tumoral cells of a leukemia patient is a systematic diagnostic examination performed in order to characterize the type and origin of the patient's leukemia cells (T, B, granulocytes, myeloblasts, etc.). This examination is conventionally performed by the i_mmunofluorescence technique by using a set of monoclonal antibodies and by observing and counting the positive cells under a microscope. Flux cytometry is also used to analyze the labeling of the cells.
The use of monoclonal antibodies in the process according to the invention makes it possible to identify the major clinically important groups of acute or chronic leukemias .and also provides quantitative infor-mation on the relative densities of the different antigens examined.
6 peroxidase-coupled monoclonal antibodies specific for antigens present on the leukemia cells were prepared in this Example:
- STl and ST:Il (BI~OSYS): anti-CD5 and anti-CD2 - SB3 (BIOSYS): anti-CD37 - S-CALLA (SANOFI): anti-CALLA (or anti-CD10) - S-class II (BIOSYS): anti-HLA-Dr (or anti-class II HLA) - SMY15 (BIO;;YS): anti-CD15 The rnicrotiter plate is prepared by the prior adsorption oil a mixture of anti-CD45 monoclonal anti bodies (S-LCA, BIOSYS) and anti-class I HLA monoclonal antibodies (;>-class I, BIOSYS).
The phenotype of chronic lymphoid leukemia B
(CLL-B) was evaluated using the 6 monoclonal antibodies selected above; it was also characterized by the con-ventional method.
The optical densities measured at 492 nm are reported in 'table 7 below .for a total of 80 000 mono-nucleate cells introduced into each well or for the control wells without cells. For the control well con-s taming the cells but no peroxidase-labeled antibody, the optical density is 0.110.
Monoclonal Optical density antibody and corresponding Sample with Control antigen cells without cells ST11 (CD2) 0.270 0.030 ST1 (CD5) 1.350 0.014 SB3 (CD37) 0.850 0.005 S-class II
(HLA-Dr) 1.640 0 .
SMY15 (CD15) 0.235 0.007 CALLA (CD10) 0.160 0.002 Thus the leukemia cells studied carry an abundance of the CDS, CD37 anal class II HLA antigens, but they carry no CD15, CALL,A and CD2 antigens, or very few, which is characteristic of a ChL-B.
Determination of antigens associated with cancers of the urinary system The kits and processes of the present invention are very particularly suitable for the detection of tumoral cells, especially in the case of tumors of the urinary system, and are applicable to the mass screening of high-risk :populations, for example workers in the 1 3 4~ 97 3 chemical industry of other very exposed groups.
The present Example shows the application of the invention to the determination of an antigen associated with cancer of the bladder, on cells of the RT4 line originating from a human urinary papilloma (C.C. Rigby and L.M. Franks, Brit. J. Cancer, 1970, 24, 746-754).
The plates intended for immunocapture of the cells are prepared as in Example 1, the monoclonal antibody S-class I (BIOSYS), which recognizes a class I
HLA antigen .and i.s capable of retaining all the epi-thelial cells, being adsorbed in the wells.
The labeled conjugates are obtained by the processes of Example 1 (for enzym a labeling) or Example 3 (for radioisotopic labeling) with the mono-clonal antibody 12F6 (SANOFI), which recognizes an antigen associated with cancer of the bladder.
For the determination, 75 ul of the cell suspension are distributed into 4 Imicrowells (i.e.
50~103 cells per well). The conjugate of the monoclonal antibody 12F6 (SANOFI) coupled with peroxidase is introduced into 2 wells and the antibody 12F6 labeled with iodine 125 is introduced into the other 2 wells (100 000 cpm/well). The results obtained by applying the enzyme immunometric assay process or the radioimmuno-metric assay process are reported in Table 8.
Labeled Optical density Ratio of (at. 492 nm) or cpm monoclona-L positive antibody Sample to be Control signal to non-determined specific signal 12F6- 0.782 0.078 10.0 peroxidase 12F6- 1612 350 4.6 iodine 125 Enzyme immunometric assay of a membraned antigen of the yeast Candida albicans The (~andid,3 albicans cells (serotype 17) originate from the Institut Pasteur collection, Paris.
The yeasts are cultivated for 18 hours in the usual way on a solid synthetic medium. The cells are counted and a cell suspension containing 105 cells per ml of PBS
buffer solution is prepared.
The anti-Candida albicans monoclonal antibodies used were prepared by lymphocytic hybridization as described in the thesis of T. Chardes, Faculty of Pharmacy, Iln:iversity of Montpellier I, 1988.
The ,antibody CA4 is used for immunocapture while the antibody CA12 labeled with peroxidase is used for the determination under the conditions of Example 1.
The ~~ptical density values measured at 492 nm for the well containing the introduced cells and for the control aample without cells are 1.025 and 0.080 respectively.
Enzyme immuriometri.c assay carried out on magnetic beads determinatioiz of the CD5 antigen of human T lymphocytes with a conju;~ate of antibody labeled with acetyl-cholinesterase and a particulate support formed of magnetic beads The support used to capture the cells consists of magnetic beads known as DYNABEADS*. The beads, marketed by~ BIOS~'S under ref. DfV-11101, carr; on their surface an anti-CD2 antibody capable of firing all the T lymphocytes in the sample examined. These beads are treated again with a solution of anti-CD2 antibody before use in order to improve the performance characteristics of the determination.
This is done by mixing 10 Y1 of the suspension of beads with 1 ml of antibody solution containing 25 ~g/
ml of PRS buffer, for 1 hour, with gentle shaking. The beads are subsequently washed 4 times with PBS buffer and then kept in 4 m:1 of PBS buffer.
For the determination, 200 ul of the suspension of beads and 80 ~I. of a suspension lof mononucleate cells separated from whole blood with the aid of Ficoll-Paque (PHARMACIA) are introduced successively into a 5 ml tube and 280 ~1 a~f a conjugate of antibody STl labeled with acetylcholin.esterase, identical to that used in Example 6, are then added..
After one hour of gentle shaking, the beads are separated off by means of a magnet and the cells fixed to the bead; are washed with PBS buffer (5 washes).
The acety7_cholinesterase fixed to the cells is developed ire the same way as in Example 6. The optical density signal obtained is 0.168 in the presence of the labeled cells; the' blank obtained without the cells gives a value of 0.062.
* - Tradema:rk ,F., ,4:. -~~ -
Claims (33)
1. A kit for the determination of at least one surface antigen characteristic of a cell population or subpopulation selected from the group consisting of leukocytes, lymphocytes, granulocytes and tumoral cells, which comprises the following components:
a) a solid support to which one or more monoclonal antibodies are fixed by covalent bonding or physical adsorption, the said monoclonal antibodies being directed against surface antigens of the cell population to be determined, other than the said characteristic antigen;
b) one or more solutions each containing a monoclonal antibody specific for the said antigen characteristic of the cell population or subpopulation to be determined, which is labeled with a radioactive probe or an enzyme probe;
c) in the case of antibodies labeled with an enzyme probe, one or more solutions providing the reagents necessary for developing the activity of the enzyme.
a) a solid support to which one or more monoclonal antibodies are fixed by covalent bonding or physical adsorption, the said monoclonal antibodies being directed against surface antigens of the cell population to be determined, other than the said characteristic antigen;
b) one or more solutions each containing a monoclonal antibody specific for the said antigen characteristic of the cell population or subpopulation to be determined, which is labeled with a radioactive probe or an enzyme probe;
c) in the case of antibodies labeled with an enzyme probe, one or more solutions providing the reagents necessary for developing the activity of the enzyme.
2. The kit according to claim 1 which further comprises at least one member selected from the group consisting of a washing buffer solution and samples permitting standardization and quality control of the determination.
3. The kit according to claim 2 in which component (a) is a solid support consisting of a microtiter plate in whose wells are fixed the antibody or antibodies for immobilization of the cells, including those of the population or subpopulation carrying the antigen to be determined.
4. The kit according to claim 2 in which component (a) is a particulate magnetic support.
5. The kit according to any one of claims 1, 2, 3 or 4 in which component (a) consists of a solid support to which one or more anti-class I HLA monoclonal antibodies are fixed.
6. The kit according to claim 5 in which the fixed monoclonal antibody(ies) is the antibody called S-class I.
7. The kit according to any one of claims 1, 2, 3 or 4 in which the monoclonal antibodies of component (b) are labeled with an iodine 125 or iodine 131 radioisotopic probe.
8. The kit according to any one of claims 1, 2, 3 or 4 in which the monoclonal antibodies of component (b) are labeled with an enzyme probe.
9. The kit according to claim 8 in which the monoclonal antibodies of component (b) are labeled with peroxidase and in which component (c) essentially comprises hydrogen peroxide and a chromogen selected from the group comprising orthophenylenediamine, 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic) acid, 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole or one of their water-soluble salts.
10. The kit according to claim 8 in which the monoclonal antibodies of component (b) are labeled with alkaline phosphatase and in which component (c) consists of paranitrophenyl phosphate, 4-methylumbelliferyl phosphate or 5-bromo-4-chloroindol-3-yl phosphate.
11. The kit according to claim 7 in which the monoclonal antibodies of component (b) are labeled with beta-galactosidase and in which component (c) consists of orthonitrophenyl beta-D-galactopyranoside or 4-methyl-umbelliferyl beta-D-galactopyranoside.
12. The kit according to claim 8 in which the monoclonal antibodies of component (b) are labeled with acetyl cholinesterase and in which component (c) essentially comprises acetylthiocholine and 5,5'-dithio-2-nitro-benzoic acid or one of its water-soluble salts.
13. The kit according to claim 1 for the determination of the CD4 or CD8 antigen of the human T lymphocytes carrying this antigen, which are called T4 lymphocytes or T8 lymphocytes.
14. The kit according to claim 1 in which the tumoral cells are those of cancers of the urinary system or those of malignant hemopathies.
15. The kit according to any one of claims 1, 2 or 3 in which component (a) is a microtiter plate in whose wells one or more monoclonal antibodies specific for surface antigens of a cell population have been fixed, and component (b) consists of several solutions each containing a monoclonal antibody specific for a surface antigen of a cell subpopulation forming part of the fixed cell population.
16. The kit according to claim 15 for the determination of specific antigens of T, T4, T8 and B lymphocytic subpopulations.
17. The kit according to claim 15 for the characterization and determination of different antigens constituting the surface antigenic equipment of the tumoral cells of malignant hemopathies.
18. A process for the immunometric assay of at least one surface antigen characteristic of a cell population or subpopulation selected from the group consisting of leukocytes, lymphocytes, granulocytes and tumoral cells, which consists in:
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 by using one or more monoclonal antibodies fixed to the said support beforehand by covalent bonding or by physical adsorption and capable of recognizing an antigen present on the surface of the cells, other than the antigen to be determined, and, in the same strap, directly labeling the cell population or subpopulation to be determined with at least one monoclonal antibody specific for the antigen to be determined, the said antibody carrying a radioisotopic or enzyme probe;
b) observing an incubation period;
c) wishing the support to remove the non-immobilized cells and the excess antibody;
d) in the case where the antibody carries an enzyme probe, adding the reagent or reagents (substrate and chromogen) necessary for developing the activity of the enzyme; and e) reading the results either by counting the radioactivity or by measuring the light signals (coloration or fluorescence) with reference to a standard range.
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 by using one or more monoclonal antibodies fixed to the said support beforehand by covalent bonding or by physical adsorption and capable of recognizing an antigen present on the surface of the cells, other than the antigen to be determined, and, in the same strap, directly labeling the cell population or subpopulation to be determined with at least one monoclonal antibody specific for the antigen to be determined, the said antibody carrying a radioisotopic or enzyme probe;
b) observing an incubation period;
c) wishing the support to remove the non-immobilized cells and the excess antibody;
d) in the case where the antibody carries an enzyme probe, adding the reagent or reagents (substrate and chromogen) necessary for developing the activity of the enzyme; and e) reading the results either by counting the radioactivity or by measuring the light signals (coloration or fluorescence) with reference to a standard range.
19. The process according to claim 18 wherein the solid support consists of a microtiter plate in whose cells are fixed the antibody or antibodies for immobilization of the cells including those of the population or subpopulation carrying the antigen to be determined, or is a particulate magnetic support.
20. The process according to claim 18 or 19 in which the monoclonal antibodies intended for labeling carry an enzyme probe.
21. The process according to claim 20 in which the enzyme probe is peroxidase and in which the activity of the enzyme is developed by hydrogen peroxide and a chromogen selected from the group comprising ortho-phenylenediamine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid, 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole or one of their water-soluble salts.
22. The process according to claim 20 in which the enzyme probe is alkaline phosphatase and in which the activity of the enzyme is developed by paranitrophenyl phosphate, 4-methylumbelliferyl phosphate or 5-bromo-4-chloroindol-3-yl phosphate.
23. The process according to claim 20 in which the enzyme probe is beta-galactosidase and in which the activity of the enzyme is developed by orthonitrophenyl beta-D-galacaopyranoside or 4-methylumbelliferyl beta-D-galactopyranoside.
24. The process according to claim 20 in which the enzyme probe is acetylcholinesterase and in which the activity of the enzyme is developed by acetylthiocholine and, as the chromogen, 5,5'-dithio-2-nitrobenzoic acid or one of its water-soluble salts.
25. The process according to claim 18 or 19 in which the monoclonal antibodies intended for labeling carry an iodine 125 or iodine 131 radioisotopic probe.
26. The process according to claim 18 or 19 in which the total time required to carry out the determination is less than or equal to 1 hour.
27. The process according to claim 26 in which the monoclonal antibody or antibodies fixed to the solid support are anti-class I HLA antibodies.
28. The process according to claim 27 in which the mono-clonal antibody(ies) fixed to the solid support is the antibody called S-class I.
29. The process according to claim 18, for the assay of the T lymphocytes carrying the CD4 antigen, called T4 lymphocytes, and of the T lymphocytes carrying the CD8 antigen, called T8 lymphocytes.
30. The process according to claim 18 in which the tumoral cells are those of cancers of the urinary system or those of malignant hemopathies.
31. The process according to claim 18 or 19 which consists in immobilizing a cell population on a micro-titer plate with one or more monoclonal antibodies specific for surface antigens of this population, and, in the same step, directly labeling cell subpopulations making up the fixed population with several solutions each containing a monoclonal antibody each specific for a surface antigen of one of the cell subpopulations to be determined.
32. The process according to claim 31 for the assay of specific antigens of T, T4, T8 and B lymphocytic subpopulations.
33. The process according to claim 31 for the characterization and determination of different antigens constituting the surface antigenic equipment of the tumoral cells of malignant hemopathies.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8713537A FR2621128B1 (en) | 1987-09-30 | 1987-09-30 | IMMUNOMETRIC ASSAY KIT AND METHOD FOR WHOLE CELLS |
| FR8713537 | 1987-09-30 |
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| CA000578730A Expired - Fee Related CA1340973C (en) | 1987-09-30 | 1988-09-28 | Immunometric assay kit and method applicable to whole cells |
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| EP (1) | EP0311492B1 (en) |
| JP (1) | JPH01121755A (en) |
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| AT (1) | ATE103711T1 (en) |
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| CA (1) | CA1340973C (en) |
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| DK (1) | DK174032B1 (en) |
| ES (1) | ES2053785T3 (en) |
| FI (1) | FI95752C (en) |
| FR (1) | FR2621128B1 (en) |
| HK (1) | HK1001570A1 (en) |
| IE (1) | IE63426B1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5256532A (en) * | 1988-05-02 | 1993-10-26 | Zynaxis Technologies, Inc. | Methods, reagents and test kits for determination of subpopulations of biological entities |
| US5385822A (en) * | 1988-05-02 | 1995-01-31 | Zynaxis, Inc. | Methods for detection and quantification of cell subsets within subpopulations of a mixed cell population |
| US4953561A (en) * | 1989-09-18 | 1990-09-04 | Cancer Diagnostics, Inc. | Urine testing module and method of collecting urine antigen |
| US5466574A (en) * | 1991-03-25 | 1995-11-14 | Immunivest Corporation | Apparatus and methods for magnetic separation featuring external magnetic means |
| US5795470A (en) * | 1991-03-25 | 1998-08-18 | Immunivest Corporation | Magnetic separation apparatus |
| US5646001A (en) * | 1991-03-25 | 1997-07-08 | Immunivest Corporation | Affinity-binding separation and release of one or more selected subset of biological entities from a mixed population thereof |
| FR2684186B1 (en) * | 1991-11-25 | 1994-02-25 | Pasteur Sanofi Diagnostics | KIT FOR THE RAPID ENUMERATION OF GRANULOCYTES, AND METHOD USING THE SAME. |
| TW378213B (en) * | 1992-11-04 | 2000-01-01 | Shionogy Seiyaku Kk | Basophil-binding monoclonal antibody, method for separation of basophils, method for chemical mediator released from basophils, and testing method for release of basophil-derived chemical mediators |
| US5374531A (en) * | 1993-03-22 | 1994-12-20 | Zynaxis, Inc. | Immunoassay for determination of cells |
| ES2066722B1 (en) * | 1993-05-20 | 1995-11-01 | Univ Pais Vasco | PROCEDURE FOR THE DIAGNOSIS OF INVASIVE CANDIDIASIS. |
| EP0772778B1 (en) | 1994-07-23 | 1999-10-27 | Roche Diagnostics GmbH | Method for the determination of the prethrombotic state |
| US5658745A (en) * | 1995-02-17 | 1997-08-19 | E. I. Du Pont De Nemours And Company | Cell enumeration immunoassay |
| GB9912631D0 (en) * | 1999-05-28 | 1999-07-28 | Nat Blood Authority The | Cell counter |
| KR20020032751A (en) * | 2000-10-27 | 2002-05-04 | 김희태 | A micro well plate protein chip using nitrocellulose membrane as a support and a method for the preparation thereof |
| GB0129776D0 (en) * | 2001-12-13 | 2002-01-30 | Sec Dep For Environment Food & | Assay device and method |
| EP1599730A2 (en) * | 2003-03-03 | 2005-11-30 | Kouyama, Yoshihisa | Methods and apparatus for use in detection and quantitation of various cell types and use of optical bio-disc for performing same |
| JP5144950B2 (en) * | 2007-03-29 | 2013-02-13 | 株式会社バイオマーカーサイエンス | Evaluation method of natural killer activity |
| WO2019084458A1 (en) * | 2017-10-26 | 2019-05-02 | The University Of Houston System | Highly-specific assays |
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| EP0101724A1 (en) * | 1982-02-23 | 1984-03-07 | Ventrex Laboratories Inc. | Multipurpose supports for immunological and biological use |
| US4471056A (en) * | 1982-04-02 | 1984-09-11 | The Board Of Trustees Of The Leland Stanford Junior University | Method for HLA-DR typing of total human lymphocyte sample |
| US4606855A (en) * | 1982-07-26 | 1986-08-19 | Mex Research Associates C/O Leon Reimer | Monoclonal antibody to digoxin |
| US4503143A (en) * | 1982-08-20 | 1985-03-05 | Btc Diagnostics Limited Partnership | Enzyme immunoassay with two-part solution of tetramethylbenzidine as chromogen |
| US4591570A (en) * | 1983-02-02 | 1986-05-27 | Centocor, Inc. | Matrix of antibody-coated spots for determination of antigens |
| US4474892A (en) * | 1983-02-16 | 1984-10-02 | Board Of Trustees Of The Leland Stanford Junior University | Two-site immunoassays using monoclonal antibodies of different classes or subclasses and test kits for performing same |
| US4599304A (en) * | 1983-10-07 | 1986-07-08 | Becton, Dickinson And Company | Method for monitoring activated cell subpopulations |
| FR2571498B1 (en) * | 1984-10-04 | 1988-04-08 | Immunotech Sa | METHOD FOR SEPARATING CELLS USING LOW DENSITY ANTIBODIES AND BALLS |
| US4677061A (en) * | 1984-10-19 | 1987-06-30 | Genetic Systems Corporation | T-cell lymphocyte subset monitoring of immunologic disease |
| WO1986002733A1 (en) * | 1984-10-31 | 1986-05-09 | Alcon Laboratories, Incorporated | Sandwich immunoassay of antigens involved in ophthalmic disorders |
| US4661446A (en) * | 1985-02-19 | 1987-04-28 | Dana-Farber Cancer Institute, Inc. | Monoclonal antibodies and method of immunizing therewith |
| EP0204522A3 (en) * | 1985-05-30 | 1987-09-16 | Genetic Systems Corporation | Monoclonal antibody panel for histocompatibility typing |
| WO1986007633A1 (en) * | 1985-06-17 | 1986-12-31 | Electro-Nucleonics, Inc. | Test for determining false positive reactions in a testing procedure to detect the presence of antibody to microorganisms. |
| AU590405B2 (en) * | 1986-02-26 | 1989-11-02 | University Of Melbourne, The | Hla-b27 testing |
| AU7357687A (en) * | 1986-04-29 | 1987-11-24 | Murex Corp. | Diagnostic kit for sexually transmitted diseases |
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- 1987-09-30 FR FR8713537A patent/FR2621128B1/en not_active Expired - Fee Related
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1988
- 1988-09-28 PT PT88615A patent/PT88615B/en not_active IP Right Cessation
- 1988-09-28 CA CA000578730A patent/CA1340973C/en not_active Expired - Fee Related
- 1988-09-29 NO NO884327A patent/NO177444C/en unknown
- 1988-09-29 IL IL87882A patent/IL87882A/en not_active IP Right Cessation
- 1988-09-29 AU AU22948/88A patent/AU621310B2/en not_active Ceased
- 1988-09-29 FI FI884472A patent/FI95752C/en not_active IP Right Cessation
- 1988-09-29 IE IE293988A patent/IE63426B1/en not_active IP Right Cessation
- 1988-09-30 KR KR1019880012838A patent/KR0126236B1/en not_active Expired - Fee Related
- 1988-09-30 DE DE3888779T patent/DE3888779T2/en not_active Expired - Fee Related
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- 1988-09-30 JP JP63247289A patent/JPH01121755A/en active Pending
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- 1988-09-30 EP EP88402475A patent/EP0311492B1/en not_active Expired - Lifetime
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1997
- 1997-11-26 HK HK97102252A patent/HK1001570A1/en not_active IP Right Cessation
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| FI884472A0 (en) | 1988-09-29 |
| ATE103711T1 (en) | 1994-04-15 |
| FR2621128B1 (en) | 1994-05-06 |
| KR890005518A (en) | 1989-05-15 |
| FR2621128A1 (en) | 1989-03-31 |
| FI95752C (en) | 1996-03-11 |
| ES2053785T3 (en) | 1994-08-01 |
| KR0126236B1 (en) | 1997-12-24 |
| IE882939L (en) | 1989-03-30 |
| EP0311492B1 (en) | 1994-03-30 |
| AU2294888A (en) | 1989-04-06 |
| IL87882A (en) | 1993-01-14 |
| HK1001570A1 (en) | 1998-06-26 |
| NO177444C (en) | 1995-09-13 |
| DK550288D0 (en) | 1988-09-30 |
| NO884327D0 (en) | 1988-09-29 |
| NO177444B (en) | 1995-06-06 |
| EP0311492A2 (en) | 1989-04-12 |
| DE3888779D1 (en) | 1994-05-05 |
| PT88615B (en) | 1993-07-30 |
| EP0311492A3 (en) | 1989-05-31 |
| DK550288A (en) | 1989-03-31 |
| FI95752B (en) | 1995-11-30 |
| IL87882A0 (en) | 1989-03-31 |
| AU621310B2 (en) | 1992-03-12 |
| DE3888779T2 (en) | 1994-09-08 |
| JPH01121755A (en) | 1989-05-15 |
| FI884472L (en) | 1989-03-31 |
| DK174032B1 (en) | 2002-04-29 |
| IE63426B1 (en) | 1995-04-19 |
| PT88615A (en) | 1989-07-31 |
| NO884327L (en) | 1989-03-31 |
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