CA2012939A1 - Assay kit and method applicable to whole cells - Google Patents
Assay kit and method applicable to whole cellsInfo
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- CA2012939A1 CA2012939A1 CA002012939A CA2012939A CA2012939A1 CA 2012939 A1 CA2012939 A1 CA 2012939A1 CA 002012939 A CA002012939 A CA 002012939A CA 2012939 A CA2012939 A CA 2012939A CA 2012939 A1 CA2012939 A1 CA 2012939A1
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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
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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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
- 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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- 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
- G01N33/56977—HLA or MHC typing
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- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
IN THE CANADIAN PATENT AND TRADEMARK OFFICE
PATENT APPLICATION
entitled: Assay kit and method applicable to whole cells in the name of: Dominique CARRIERE
Assignee: SANOFI
ABSTRACT OF THE DISCLOSURE
The present invention relates to a kit for assaying the surface receptors characteristic of a cell population or subpopulation, said kit comprising the following components:
a) a solid support to which one or more mono-clonal antibodies are fixed by covalent bonding or by physical adsorption, said monoclonal antibodies being directed against surface antigens of the cell population to be assayed; and b) a solution containing the ligand specific for the membrane receptor of the cell population or sub-population to be assayed, said ligand being labeled with a radioisotopic probe.
It further relates to a method of assaying the surface receptors of a cell population or subpopulation.
The assay kit and the method according to the invention can be used for assaying the subpopulation of activated T lymphocytes carrying the interleukin-2 receptor.
PATENT APPLICATION
entitled: Assay kit and method applicable to whole cells in the name of: Dominique CARRIERE
Assignee: SANOFI
ABSTRACT OF THE DISCLOSURE
The present invention relates to a kit for assaying the surface receptors characteristic of a cell population or subpopulation, said kit comprising the following components:
a) a solid support to which one or more mono-clonal antibodies are fixed by covalent bonding or by physical adsorption, said monoclonal antibodies being directed against surface antigens of the cell population to be assayed; and b) a solution containing the ligand specific for the membrane receptor of the cell population or sub-population to be assayed, said ligand being labeled with a radioisotopic probe.
It further relates to a method of assaying the surface receptors of a cell population or subpopulation.
The assay kit and the method according to the invention can be used for assaying the subpopulation of activated T lymphocytes carrying the interleukin-2 receptor.
Description
2 ~ 3 9 A~say kit a~_meth~ g~}lis~h1Q to whol~ell$
The pre~ent invention relate~ to a ~it and to an assay method u~ing thi~ kit for assaying membrane re-ceptors characteri3tic of cell population~ or sub-05 population~. The assay method and ths corresponding kitare also intended for assaying the cells themselves via the assay of their surface receptor~. These a~says are applicable to diagno i9.
Surface receptors or membrane receptor~ form par~
of the cell surface marker~ in the ~ame way as ~urface antigens. A membrane receptor of a cell i~ a macro-molecular structure inserted in the membrane of a cell and capable of binding to a specific ligand characteris-tic of this receptor. A membrane receptor can also be considered as an antigen and a~ such~ it can bind to a specific antibody.
The membrane receptor~ of a cell are ~acteristic of a cell population either because they are present for this population under norm~l condition~ of viability of the cell or because they have been expressed on the surface of the cell through the intervention of an endo-genous or exogenous factor and therefore label either a state of differentiation or a physiological ~tate of ths cell in question.
Knowledge of cell surface antigens or marker~ h~
made enormous advances with the development o~ lymphocyte hybridization and the discovery of monoclonal antibodie~
by KOEHLER and MILSTEIN (Nature) 1975 2~6 495-497). In particular monoclonal antibodie~ have made it possible to reveal and analyze surface markers or membrane anti-~ens of cell~ of the widest pos~ible variety of ori~ins.
The characterizations ~ought apply mainly to ti~su~ or organ markers~ to marker~ of states of differentiation or activation of normal cells and to the identification or typing of normal or cancerous cells. A particularlY
2~2~a~
important field of application is the study of the cell lines of hemopoiesis (erythrocyte, megakaryocyte, granu-locyte, monocyte, lymphocyte).
Thus, for example, monoclonal antibodies have 0~ made it possible to specify the respective surface characteristics of T and B ly~nphocytes. The correspon-ding markers, by themselves o:r in combination, identify stages of differentiation and functional specialization - o~ the lymphocytes By international convention, the surface markers of human leukocytes have been classified in differentiation groups or differentiation classes (CD) defined by the IUIS-WHO subcommittee, 1984, and de3cribed in Bulletin of the World Health Organization, 1984, 62 (~), 813-81~. As research progresses, an increa3ing pro-portion of th~se antigens are proving to be functionalmolecules and~ in particular, receptors for specific ligands.
The receptors located on the surface of cells are conventionally detected by binding of the correspondin~
radiolabeled ligands. These receptors are frequently hormone receptors, for which the ligand3 are hormones.
Radiolabeled ligands are commercially available or are prepared according to known methods (F.C. ~REEN-WOOD, W.M. HUNTER et al., Biochem. J., 1963, 89~ 114).
Cell immunocapture on a ~olid support is des-cribed in patent application WO 86/02091, in which the object is to remove undesirable cells from samples of bone marrow intended for transplants. In said patent application, cell capture is effected on floating microbeads and re~uires that the antibody used be fixed to the solid support by a complex macromolecular struc-ture, called a network-relay, which is capable of en-suring a preferential orientation of the antibody rela-tive -to that of the correspondin~ cell antigen. Said patent application gives no indication of an application 2 0 ~
of the techni~ue to the ~uantitative a~say of a membrane receptor.
Cell immunocapture is also described in patent application W0 84/03151 for an analytical application.
05 In said patent application, the object is to identify the tissue groups to which the examined cells belong (this operation generally being called HLA typing). Cell cap-ture is effected by means of antibodies arranged accor-ding to a particular geometry on very specialized sup-ports (microscope cover glasses). The results areobtained simply by visual observation of the support and produce "all or nothing" re~ponses. Thus the cell im-munocapture systems described hitherto do not lead to analytical applications permitting the quantitative determination of a receptor expres~ed at the membrane of certain cells The assay method forming the subJect of the present invention has considerable advan~ages over all the techniques known and used in the prior art, since it permits quantitative measurement of an~ membrane receptor of a cell population in a single analytical step. This assay is performed on cells which have not undergone any chemical or physical intervention and which are in their siate of physiological integrity. Furthermore, the a~say method according to the invention has the characteristics of very high specificity which are inherent in the double recognition systems involving 2 different specific markers carried by the same cell, one being an antigen selected for immunocapture of the cells which it is desired to analyze, and the other being the membrane receptor to be assayed, which is recognized very speci-fically by the corresponding ligand. This method i3 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 2~2~3~
biology laboratories handling these large numbers.
The present invention thus relates to a kit Ior assaying at least one membrane receptor characteristic of a cell population or subpopulation, said kit comprising 05 the following components:
a) a solid support to which one or more mono-clonal antibodies are fixed by covalent bonding or physical adsorption, said monoclonal antibodies being directed against surface antigens of the cell population examined, other than said receptor, and being intended for immobilization, on the support, of the cells which include those of the subpopulation carrying the receptor to be assayed; and b) a solution containing, in radiolabeled form, the ligand specific for the membrane receptor to be assayed.
The term "cell" as used in the present descrip-tion and in the claims which follow encompasse~ human cells or animal cells and especially blood cells, in-cluding the platelets.
The assay method according to the inventionapplies to whole cell~, i.e non-lyzed cells, carrying the receptor to be assayed.
These cells have not undergone any physical or chemical intervention and they are used in a state of complete physiological integrity. This situation con-stitutes the best guarantee of integrity of the chosen membrane markers, the immunocapture antigen and the receptor to be assayed.
As the solid support it is possible to use any device suitable for handling cell suspensions, and pre-ferably tubes, particulate magnetic supports or rigid or flexible microtiter plates m~de of polyethylene, poly-styrene, polyvinyl chloride or nitrocellulose, which con-tain microwells. The monoclonal antibodies intended for 2~2~3~
immobilization of the cells can be fixed to the solid supports either by covalent chemical bonding or by physical adsorption according to the classical techniques well known to those skilled in the art, such as the tech-05 niques described by STOCKER and HEUSSER, J. Immunol.Methods, 1979, vol. 20, p 87-95 Advantageously, the support can be saturated with a protein beforehand According to the invention, the monoclonal anti-body or antibodies fixed to the solid support must permit immunocapture of the cell population which includes the cell population or populations carrying the membrane receptors to be assayed When this population consists of human cells, the preferred monoclonal antibodies for immunocapture are the anti-class I HLA antibodies which are specific for the common part of the ~LA-A, -B and -C
antigens present on the leukocytes and numerous other cell lines of the organism. Of these antibodies, the one called S-class I, marketed by BIOSYS, is particularly 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 immunocapture accor-ding to the invention.
The expression "a ligand in radiolabeled form for the receptor" or "a ligand labeled with a radioisotopic probe" means that this ligand carries~ on a component inherent in its structure, a radioactive isotope which enables it to be assayed by counting the radioactivity associated therewith.
As an additional component, the assay kit can contain a buffer solution intended for washing of the solid support after immobilization and labeling of the membrane receptors with the radiolabeled ligand for the receptor 2~2~3~
As additional components, the as~ay kit can also contain the samples necessar~ for standardization and quality control of the assay..
The present inventiorl further relates to a method 05 of assaying the surface receptors of a cell population or subpopulation, said method consisting in:
a) immobi.lizing the cell population carrying the receptor to be assayed, or a subpopulation carrying the receptor to be assayed, on a ~olid support using one or more monoclonal antibodies fixed to said support before-hand by covalent bonding or by physical ad~orption and capable of recognizing an antigen present on the surface of the cells, other than the receptor to be assayed, and, in the same step, directly labeling certain cell3 belon-ging to the immobilized cell population or subpopulationby means of at leaqt one ligand specific for this recep-tor -to be assayed, said ligand carrying a radioisotopic probe;
b) observing an incubation period to allow sin~ul-taneous immunocapture and labeling, c) washing the solid support to remove the non-immobilized cells and the excess ligand carrying the radioisotopic probe; and d~ reading the results by countin~ the fixed radioactivity with reference, if appropriate, to a standard scale.
The assay kit and the method accordin~ to the invention are preferably applied to assay of the surface receptors of the formed elements of human blood, espec-ially the leukocytes and more particularly the lympho-cytes.
The assay kit and the method according to the invention make it possible to mea~ure radioactivity signals which depend both on the number of cells present 3~ in the cell population examined and on the density of the ~.2~c-1~
receptor measured on the surface of these cells. Meas-urement of these signals permits quantitative evaluation of the total number of molecules of this receptor which are carried by the cell population or subpapulation 05 examined.
A case which may be mentioned as an illustration of an application of the invention is that of the human T
lymphocytes, for which there are in particular a sub-population of cells: the lymphocyte~ characterized by the presence of the interleukin-2 receptor.
This receptor is expressed with a greatly in-creased density on the surface of the lymphocytes in the case where the immune system is stressed, for exampls during infectious pathological conditions, in certain dysimmune diseases or in organ graft~.
The assay kit and the method according to the invention can be u~ed to measure the number of activated lymphocytes in a sample of blood taken from a patient and to relate it, if appropriate, to the number of total lym-phocytes.
Likewise, it i~ possible to mea~ure the ratio o~the quiescent lymphocytes to the activated lymphocyte~
and the variation in this ratio during the course of the disease as a function of the therapy administered to the patient.
Specific immobilization of the T lymphocytes of the sample is preferably effected using one or more monoclonal antibodies which are capable, by themselve~ or in combination, of recognizing all the T cells of the sample, this being the case of the anti-common leukocyte (or anti-CD~15) antibodies or antibodies which recognize the whole of the T population (called "pan-T" anti-bodies), such as the anti-CD2 (or anti-T11), anti-CD5 (or anti-T1) or anti-CD7 (or anti-T2) antibodies or other pan-T antibodies which have not yet been assigned to a 2~
differentiation class according to the WH0 criteria For axample, 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 th~
05 walls of microwells. These monoclonal antibodies will enable the whole population of T cell~ of the sample examined to be immobilized in the microwell~ at a later stage. The plate~ prepared in this way can be lyophi lized and stored, preferably at 4C. Thi~ step can be carried out on the indu~trial scale and it is thus possible to have ready-to-use plates for the assay kits which can be applied to any subpopulation of T lympho-cytes characterized by the pre~qence of the receptor for any identified ligand which can be obtained in radio-labeled form.
The samples containing the cells to be assayed,which originate from blood or from any appropriate bio-logical fluid - normal or pathological - can be used a~
such or after preparation, especially by density gradient centrifugation according to the methods already known, and in particular in a high-density medium such as, for example, FICOLL-PAQUE marketed by Pharmacia. To assay the blood lymphocytes, the blood sample to be a~sayed can also be treated with a so-called lysis buffer ~olution, which lyzes the erythrocyte~.
Aliquots of the appropriate cell quqpension are brought into contact with the solid support, for example in the microwells of a microtiter plate prepared before-hand, at the same time a~ the qolution forming part of the assay kitl which contain~ the ligand specific for the receptor of the target cell population and carrying a radioisotopic probe. Ligand~ ~pecific for membrane receptors are generally available commercially; other-wise, they are prepared for example by labeling the ligand with iodine 125 or iodine 131, for example in the 2~2~3~
presence of chloramine T, according to a known method (F.C. GREENWOOD, W.M. HUNTER et al., Biochem. J., 1963, ~9, 114).
The incubation period, i e. the time required for 05 immobilization and ~imultaneous labeling of -the cell~, is preferably less than or equal to 1 hour. During this time, the solid support can be centrifuged, if necessary, to improve the immobilization of the cells. The solid support, for example the microtiter plate, is then wa~hed to remove the unfixed cells and at the same time the excess ligand carrying the radioisotopic probe~
The radioactivity associated with the cells is counted in a gamma counter according to any appropriate procedure and, for example, after solubilization of the cell~ with an alkaline solu~ion (for example a sodium hydroxide solution) and recovery of the solution con-taining the radioactivity by means of an absorbent buffer.
The results of the assay of the membrane recep-tors according to the invention can be expres~ed accor-ding to any procedure appropriate to the examination carried out. More particularly, these results can be expressed as the total number of molecules of a parti-cular receptor which are pre~ent in a given volume of the sample examined (for example per microliter of blood).
The number of molecules of a particular receptor in the sample examined will preferably be determined using a ~tandard scale con~isting of appropriate cells or cell preparations carrying the receptor to be assayed, which will have been calibrated beforehand by a known reference method. These standards will preferably con-~ist either of cells identical in their origin to the cells which are to form the subject of the assay, or of cells of established cell line~ carrying the desired receptor, or of preparations, for example membrane pre-2~:~2~3~
parations, originatin~ from these cells.
These standards are then treated in exactly the same way as the samples to be examined. The resulting signals are used to build up a standard scale against 0~ which the signals measured with the samples to be examined are compared. The subsequent calculations are conventional The assay method according to the invention is simple, rapid and reproducible. Its use i~ 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 should be analyzed.
Immobilization of the cells on the solid support is the stage of the assay which usually presents the most difficulties or which is the most critical to carry out.
The means often used is chemical fixation of the cells with glutaraldehyde or methanol in cups which may or may not have been treated with poly-L-lysine (VAN LE~VEN F.
et al., J. Immunol. Method~, 1978, 23, 109). However, chemical fixations performed in this way can reduce or even suppress the desired specific detection or, conver-sely, can induce false-positive labeling of cells, which i~ a very serious disadvantage (DROVER and MARSHALL, J.
Immunol. Methods, 1986, 90, 275-281).
Furthermore, the chemical fixation method has to be carried out in several step~: centrifugation of the cells, preparation of the fixative mixture, fixation and then washing of the fixed cells several times.
Drying of the cells at 37C, optionally followed by fixation with methanol in the microwells, has also been proposed (BAUMGARTEN, J. Immunol. Methods, 1986, 94, 91-98). Actually, drying of the cells at 37C can de-grade certain fragile membrane constituents and render the pericellular plasma membrane ~ermeable, thereby 2 ~ 3 ~
facilitatin~ access of the radiolabeled ligand to intra-cellular structure~ which are not the intended targets.
Furthermore, the reproducibility of this method is doubtful; in fact, the settling of the cells in the 05 assay microwells and the dry;;ng of the cells can vary from one experiment to the next. Finally, this as3ay is lengthy to perform because the cell drying 3tep alone takes more than 2 hours.
The immobilization of lymphocyte populations has also been achieved by using polyclonal antibodies adsor-bed in microwells ~STOCKER and HEUSSER, J Immunol Method~, 1979, 26, 87-95). This method makes it possible to immobilize cells foreign to the single population which it is desired to analyze.
The use of highly specific and related monoclonal antibodies adsorbed on or fixed to the solid support, and especially in the assay wells, permits exclusive capture of the desired cells, the other non-retained cell popula-tions being removed in the cour~e of the washes carried out when labeling of the antigen3 to be assayed is com-plete. Furthermore, no chemical or phy~ical agent modifies the characteristics of the antigens in this step becau~e the various operations for chemical or physical fixation of the cell~ to the support are omitted.
Thus, according to the present invention, it ha~
been found that the immobilization of cells by monoclonal antibodies is a method which makes it po~sible to simpli-fy the step for immobilizing the cell carrying the re-ceptor to be assayed~ while at the same time making the results more reliable.
The method according to the invention, which com-prises the simultaneous use, in a ~ingle step~ of a pro-cedure for immunocapture of whole cells without physical or chemical intervention on the cell~, and the labeling of all or some of these cells with one or more ligands 2~X~
directly carrying a radioisotopic probe, is the fir~t method to permit the ~uantitative a~say of chosen mem-brane receptors on the cells themselves.
According to the invention, the direct labeling 05 of immunologically immobilized cells permits:
- simplification of the as3ay method by elimination of the intermediate manipulations repeated between the successive steps of the labeling process in the case o~
indirect labeling: centrifugation of the cells several times, removal of the labeling reagents and resuspen-sion;
- a saving of reagents, - an improved reliability 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 conventional equipment and apparatuses The incubation period for immobilization of the cell population and simultaneous direct labeling of the receptors of the cell subpopulation to be assayed is short. It is less than or equal to 1 hour in the case of the assay of activated T lymphocytes carrying the inter-leukin-2 receptor.
After the solid support has been washed, the actual assay is performed by using conventional appara-tuses to observe a signal which is precise and simple to meaæure: radioactivity.
Thus, overall, the method according to the in-vention ha~ numerous advantage~: it is rapid, reliable, economic and simple.
The method according to the invention makes it possible to assay surface receptor~ of a cell population over a wide range o cell concentrations.
3~ The sensitivity of the method in respect of the 20~2g3~
num~er of cells depend~ on the receptor density of the cell population assayed. For each receptor it is pos-sible, if desired, to define the minimum molar concentra-tion of receptor which can be measured by the method 05 according to the invention.
It has been verified that the æignals recorded (radioactivity count) make i1; possible to obtain satis-factory uniform standard curves as a function of the number of cells used, under the customary handling con-ditions In the Examples below, the following terms ortheir abbreviations will be used indiscriminately:
BSA: bovine serum albumin PBS: phosphate buffered saline at pH 7.4 cpm: counts per minute dpm: disintegrations per minute AS~;AY OF INTE~LEUKIN-2 ~ECEPTORS E~Y MEANS OF A
LABELE:D PHY~;I OLOGI CAL LI GAl~D
Interleukin-2 receptors appear on the m~smbrane of activated T lymphocytes when the immune system is stressed.
In the laboratory, it is pos~ible to work on a non-pathological blood sample provided activated lympho-cytes are obtained from the quiescent lymphocytes by the intervention of an exogenous agent such as phytohemagglu-tinin (PHA), concanavalin A or other lectins. Assay of the interleukin-2 receptors from human blood uses inter-leukin-2 labeled with iodine 125, which is commercially available (NEX-229, DUPONT).
A) Separation of the lymphocytes from whole blood and activation with PHA
0.5 ml of the blood sample to be assayed is taken 2~)~.2~3~
and mixed with 1.5 ml of phosphate buffered saline. 1.5 ml of Ficoll-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 05 Ficoll. After centrifugation at 900 x g for 5 minutes at room temperature, the suspension ring containing the mononucleate cells is removecl under sterile conditions in a volu~e of O.5 ml. 1.5 ml of culture medium are added to this sample. The cells are then counted and cultured at a rate of 5 106 cells per ml of RPMI 1640 medium (Flow) containing 10% of fetal calf serum (Flow).
The T lymphocytes are activated with PHA (HA 15, WELLCOME), introduced at a final concentration of 50 ~g/
ml of culture medium, for 3 days.
B) Preparation o~ the plate The plate used is a plastic microtiter plate con-taining 96 microwells, marketed by NUNC (reference 64394). Each microwell receives 200 ~l of a solution containing the purified anti-CD2 monoclonal antibody (called ST 11) used to immobilize the total T lympho-cytes, i.e. to effect their immunocapture~ This anti-body, marketed by BIOSYS, Compiègne, France, under the reference ST 11, is u~ed at a concentration of 10 ~g/ml in a phosphate bu~fered ~aline (PBS) at pH 7.4.
The adsorption of the monoclonal antibody iæ
effected at 4C 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 ~l of this solution are introduced into each microwell so as to saturate the surface of the wells with protein, which takes 1 hour at 37C; the plates are washed 3 times with phosphate buffered saline. The plates prepared in this way are lyophilized and stored at 4C in a sealed plastic bag.
~12~3~
C) Incubation of the cells The assay is performed by introducing into the microwells of the plate a volume of 100 ~l of cell 9US-pension (2.5.106 cells per ml) containing the cell~
05 treated with P~ for 3 days. 100 ~l of a solution of interleukin-2 labeled with iodine 125 (product ref. NEX-229 DUPONT, i.e. 120,000 dpm in 100 ~l of PBS containing 5% of calf serum) are then added. After 1 hour at room temperature, the microwells are emptied by turning the plate over The microwells are washed 4 times with 200 ~l of PBS per well. 75 ~l of 1 M sodium hydroxide solution are then introduced into each well. After 10 minutes, the contents of each well are recovered with an absorbent buffer before the radioactivity is counted with a ~ counter (LKB multiwell counter).
The results ars calculated and expressed in dpm in the Table below.
.
Number of cell~ Blank without in the sample cells 2.5 x 10~ cells Interleukin-2 435 dpm 185 dpm receptor __
The pre~ent invention relate~ to a ~it and to an assay method u~ing thi~ kit for assaying membrane re-ceptors characteri3tic of cell population~ or sub-05 population~. The assay method and ths corresponding kitare also intended for assaying the cells themselves via the assay of their surface receptor~. These a~says are applicable to diagno i9.
Surface receptors or membrane receptor~ form par~
of the cell surface marker~ in the ~ame way as ~urface antigens. A membrane receptor of a cell i~ a macro-molecular structure inserted in the membrane of a cell and capable of binding to a specific ligand characteris-tic of this receptor. A membrane receptor can also be considered as an antigen and a~ such~ it can bind to a specific antibody.
The membrane receptor~ of a cell are ~acteristic of a cell population either because they are present for this population under norm~l condition~ of viability of the cell or because they have been expressed on the surface of the cell through the intervention of an endo-genous or exogenous factor and therefore label either a state of differentiation or a physiological ~tate of ths cell in question.
Knowledge of cell surface antigens or marker~ h~
made enormous advances with the development o~ lymphocyte hybridization and the discovery of monoclonal antibodie~
by KOEHLER and MILSTEIN (Nature) 1975 2~6 495-497). In particular monoclonal antibodie~ have made it possible to reveal and analyze surface markers or membrane anti-~ens of cell~ of the widest pos~ible variety of ori~ins.
The characterizations ~ought apply mainly to ti~su~ or organ markers~ to marker~ of states of differentiation or activation of normal cells and to the identification or typing of normal or cancerous cells. A particularlY
2~2~a~
important field of application is the study of the cell lines of hemopoiesis (erythrocyte, megakaryocyte, granu-locyte, monocyte, lymphocyte).
Thus, for example, monoclonal antibodies have 0~ made it possible to specify the respective surface characteristics of T and B ly~nphocytes. The correspon-ding markers, by themselves o:r in combination, identify stages of differentiation and functional specialization - o~ the lymphocytes By international convention, the surface markers of human leukocytes have been classified in differentiation groups or differentiation classes (CD) defined by the IUIS-WHO subcommittee, 1984, and de3cribed in Bulletin of the World Health Organization, 1984, 62 (~), 813-81~. As research progresses, an increa3ing pro-portion of th~se antigens are proving to be functionalmolecules and~ in particular, receptors for specific ligands.
The receptors located on the surface of cells are conventionally detected by binding of the correspondin~
radiolabeled ligands. These receptors are frequently hormone receptors, for which the ligand3 are hormones.
Radiolabeled ligands are commercially available or are prepared according to known methods (F.C. ~REEN-WOOD, W.M. HUNTER et al., Biochem. J., 1963, 89~ 114).
Cell immunocapture on a ~olid support is des-cribed in patent application WO 86/02091, in which the object is to remove undesirable cells from samples of bone marrow intended for transplants. In said patent application, cell capture is effected on floating microbeads and re~uires that the antibody used be fixed to the solid support by a complex macromolecular struc-ture, called a network-relay, which is capable of en-suring a preferential orientation of the antibody rela-tive -to that of the correspondin~ cell antigen. Said patent application gives no indication of an application 2 0 ~
of the techni~ue to the ~uantitative a~say of a membrane receptor.
Cell immunocapture is also described in patent application W0 84/03151 for an analytical application.
05 In said patent application, the object is to identify the tissue groups to which the examined cells belong (this operation generally being called HLA typing). Cell cap-ture is effected by means of antibodies arranged accor-ding to a particular geometry on very specialized sup-ports (microscope cover glasses). The results areobtained simply by visual observation of the support and produce "all or nothing" re~ponses. Thus the cell im-munocapture systems described hitherto do not lead to analytical applications permitting the quantitative determination of a receptor expres~ed at the membrane of certain cells The assay method forming the subJect of the present invention has considerable advan~ages over all the techniques known and used in the prior art, since it permits quantitative measurement of an~ membrane receptor of a cell population in a single analytical step. This assay is performed on cells which have not undergone any chemical or physical intervention and which are in their siate of physiological integrity. Furthermore, the a~say method according to the invention has the characteristics of very high specificity which are inherent in the double recognition systems involving 2 different specific markers carried by the same cell, one being an antigen selected for immunocapture of the cells which it is desired to analyze, and the other being the membrane receptor to be assayed, which is recognized very speci-fically by the corresponding ligand. This method i3 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 2~2~3~
biology laboratories handling these large numbers.
The present invention thus relates to a kit Ior assaying at least one membrane receptor characteristic of a cell population or subpopulation, said kit comprising 05 the following components:
a) a solid support to which one or more mono-clonal antibodies are fixed by covalent bonding or physical adsorption, said monoclonal antibodies being directed against surface antigens of the cell population examined, other than said receptor, and being intended for immobilization, on the support, of the cells which include those of the subpopulation carrying the receptor to be assayed; and b) a solution containing, in radiolabeled form, the ligand specific for the membrane receptor to be assayed.
The term "cell" as used in the present descrip-tion and in the claims which follow encompasse~ human cells or animal cells and especially blood cells, in-cluding the platelets.
The assay method according to the inventionapplies to whole cell~, i.e non-lyzed cells, carrying the receptor to be assayed.
These cells have not undergone any physical or chemical intervention and they are used in a state of complete physiological integrity. This situation con-stitutes the best guarantee of integrity of the chosen membrane markers, the immunocapture antigen and the receptor to be assayed.
As the solid support it is possible to use any device suitable for handling cell suspensions, and pre-ferably tubes, particulate magnetic supports or rigid or flexible microtiter plates m~de of polyethylene, poly-styrene, polyvinyl chloride or nitrocellulose, which con-tain microwells. The monoclonal antibodies intended for 2~2~3~
immobilization of the cells can be fixed to the solid supports either by covalent chemical bonding or by physical adsorption according to the classical techniques well known to those skilled in the art, such as the tech-05 niques described by STOCKER and HEUSSER, J. Immunol.Methods, 1979, vol. 20, p 87-95 Advantageously, the support can be saturated with a protein beforehand According to the invention, the monoclonal anti-body or antibodies fixed to the solid support must permit immunocapture of the cell population which includes the cell population or populations carrying the membrane receptors to be assayed When this population consists of human cells, the preferred monoclonal antibodies for immunocapture are the anti-class I HLA antibodies which are specific for the common part of the ~LA-A, -B and -C
antigens present on the leukocytes and numerous other cell lines of the organism. Of these antibodies, the one called S-class I, marketed by BIOSYS, is particularly 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 immunocapture accor-ding to the invention.
The expression "a ligand in radiolabeled form for the receptor" or "a ligand labeled with a radioisotopic probe" means that this ligand carries~ on a component inherent in its structure, a radioactive isotope which enables it to be assayed by counting the radioactivity associated therewith.
As an additional component, the assay kit can contain a buffer solution intended for washing of the solid support after immobilization and labeling of the membrane receptors with the radiolabeled ligand for the receptor 2~2~3~
As additional components, the as~ay kit can also contain the samples necessar~ for standardization and quality control of the assay..
The present inventiorl further relates to a method 05 of assaying the surface receptors of a cell population or subpopulation, said method consisting in:
a) immobi.lizing the cell population carrying the receptor to be assayed, or a subpopulation carrying the receptor to be assayed, on a ~olid support using one or more monoclonal antibodies fixed to said support before-hand by covalent bonding or by physical ad~orption and capable of recognizing an antigen present on the surface of the cells, other than the receptor to be assayed, and, in the same step, directly labeling certain cell3 belon-ging to the immobilized cell population or subpopulationby means of at leaqt one ligand specific for this recep-tor -to be assayed, said ligand carrying a radioisotopic probe;
b) observing an incubation period to allow sin~ul-taneous immunocapture and labeling, c) washing the solid support to remove the non-immobilized cells and the excess ligand carrying the radioisotopic probe; and d~ reading the results by countin~ the fixed radioactivity with reference, if appropriate, to a standard scale.
The assay kit and the method accordin~ to the invention are preferably applied to assay of the surface receptors of the formed elements of human blood, espec-ially the leukocytes and more particularly the lympho-cytes.
The assay kit and the method according to the invention make it possible to mea~ure radioactivity signals which depend both on the number of cells present 3~ in the cell population examined and on the density of the ~.2~c-1~
receptor measured on the surface of these cells. Meas-urement of these signals permits quantitative evaluation of the total number of molecules of this receptor which are carried by the cell population or subpapulation 05 examined.
A case which may be mentioned as an illustration of an application of the invention is that of the human T
lymphocytes, for which there are in particular a sub-population of cells: the lymphocyte~ characterized by the presence of the interleukin-2 receptor.
This receptor is expressed with a greatly in-creased density on the surface of the lymphocytes in the case where the immune system is stressed, for exampls during infectious pathological conditions, in certain dysimmune diseases or in organ graft~.
The assay kit and the method according to the invention can be u~ed to measure the number of activated lymphocytes in a sample of blood taken from a patient and to relate it, if appropriate, to the number of total lym-phocytes.
Likewise, it i~ possible to mea~ure the ratio o~the quiescent lymphocytes to the activated lymphocyte~
and the variation in this ratio during the course of the disease as a function of the therapy administered to the patient.
Specific immobilization of the T lymphocytes of the sample is preferably effected using one or more monoclonal antibodies which are capable, by themselve~ or in combination, of recognizing all the T cells of the sample, this being the case of the anti-common leukocyte (or anti-CD~15) antibodies or antibodies which recognize the whole of the T population (called "pan-T" anti-bodies), such as the anti-CD2 (or anti-T11), anti-CD5 (or anti-T1) or anti-CD7 (or anti-T2) antibodies or other pan-T antibodies which have not yet been assigned to a 2~
differentiation class according to the WH0 criteria For axample, 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 th~
05 walls of microwells. These monoclonal antibodies will enable the whole population of T cell~ of the sample examined to be immobilized in the microwell~ at a later stage. The plate~ prepared in this way can be lyophi lized and stored, preferably at 4C. Thi~ step can be carried out on the indu~trial scale and it is thus possible to have ready-to-use plates for the assay kits which can be applied to any subpopulation of T lympho-cytes characterized by the pre~qence of the receptor for any identified ligand which can be obtained in radio-labeled form.
The samples containing the cells to be assayed,which originate from blood or from any appropriate bio-logical fluid - normal or pathological - can be used a~
such or after preparation, especially by density gradient centrifugation according to the methods already known, and in particular in a high-density medium such as, for example, FICOLL-PAQUE marketed by Pharmacia. To assay the blood lymphocytes, the blood sample to be a~sayed can also be treated with a so-called lysis buffer ~olution, which lyzes the erythrocyte~.
Aliquots of the appropriate cell quqpension are brought into contact with the solid support, for example in the microwells of a microtiter plate prepared before-hand, at the same time a~ the qolution forming part of the assay kitl which contain~ the ligand specific for the receptor of the target cell population and carrying a radioisotopic probe. Ligand~ ~pecific for membrane receptors are generally available commercially; other-wise, they are prepared for example by labeling the ligand with iodine 125 or iodine 131, for example in the 2~2~3~
presence of chloramine T, according to a known method (F.C. GREENWOOD, W.M. HUNTER et al., Biochem. J., 1963, ~9, 114).
The incubation period, i e. the time required for 05 immobilization and ~imultaneous labeling of -the cell~, is preferably less than or equal to 1 hour. During this time, the solid support can be centrifuged, if necessary, to improve the immobilization of the cells. The solid support, for example the microtiter plate, is then wa~hed to remove the unfixed cells and at the same time the excess ligand carrying the radioisotopic probe~
The radioactivity associated with the cells is counted in a gamma counter according to any appropriate procedure and, for example, after solubilization of the cell~ with an alkaline solu~ion (for example a sodium hydroxide solution) and recovery of the solution con-taining the radioactivity by means of an absorbent buffer.
The results of the assay of the membrane recep-tors according to the invention can be expres~ed accor-ding to any procedure appropriate to the examination carried out. More particularly, these results can be expressed as the total number of molecules of a parti-cular receptor which are pre~ent in a given volume of the sample examined (for example per microliter of blood).
The number of molecules of a particular receptor in the sample examined will preferably be determined using a ~tandard scale con~isting of appropriate cells or cell preparations carrying the receptor to be assayed, which will have been calibrated beforehand by a known reference method. These standards will preferably con-~ist either of cells identical in their origin to the cells which are to form the subject of the assay, or of cells of established cell line~ carrying the desired receptor, or of preparations, for example membrane pre-2~:~2~3~
parations, originatin~ from these cells.
These standards are then treated in exactly the same way as the samples to be examined. The resulting signals are used to build up a standard scale against 0~ which the signals measured with the samples to be examined are compared. The subsequent calculations are conventional The assay method according to the invention is simple, rapid and reproducible. Its use i~ 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 should be analyzed.
Immobilization of the cells on the solid support is the stage of the assay which usually presents the most difficulties or which is the most critical to carry out.
The means often used is chemical fixation of the cells with glutaraldehyde or methanol in cups which may or may not have been treated with poly-L-lysine (VAN LE~VEN F.
et al., J. Immunol. Method~, 1978, 23, 109). However, chemical fixations performed in this way can reduce or even suppress the desired specific detection or, conver-sely, can induce false-positive labeling of cells, which i~ a very serious disadvantage (DROVER and MARSHALL, J.
Immunol. Methods, 1986, 90, 275-281).
Furthermore, the chemical fixation method has to be carried out in several step~: centrifugation of the cells, preparation of the fixative mixture, fixation and then washing of the fixed cells several times.
Drying of the cells at 37C, optionally followed by fixation with methanol in the microwells, has also been proposed (BAUMGARTEN, J. Immunol. Methods, 1986, 94, 91-98). Actually, drying of the cells at 37C can de-grade certain fragile membrane constituents and render the pericellular plasma membrane ~ermeable, thereby 2 ~ 3 ~
facilitatin~ access of the radiolabeled ligand to intra-cellular structure~ which are not the intended targets.
Furthermore, the reproducibility of this method is doubtful; in fact, the settling of the cells in the 05 assay microwells and the dry;;ng of the cells can vary from one experiment to the next. Finally, this as3ay is lengthy to perform because the cell drying 3tep alone takes more than 2 hours.
The immobilization of lymphocyte populations has also been achieved by using polyclonal antibodies adsor-bed in microwells ~STOCKER and HEUSSER, J Immunol Method~, 1979, 26, 87-95). This method makes it possible to immobilize cells foreign to the single population which it is desired to analyze.
The use of highly specific and related monoclonal antibodies adsorbed on or fixed to the solid support, and especially in the assay wells, permits exclusive capture of the desired cells, the other non-retained cell popula-tions being removed in the cour~e of the washes carried out when labeling of the antigen3 to be assayed is com-plete. Furthermore, no chemical or phy~ical agent modifies the characteristics of the antigens in this step becau~e the various operations for chemical or physical fixation of the cell~ to the support are omitted.
Thus, according to the present invention, it ha~
been found that the immobilization of cells by monoclonal antibodies is a method which makes it po~sible to simpli-fy the step for immobilizing the cell carrying the re-ceptor to be assayed~ while at the same time making the results more reliable.
The method according to the invention, which com-prises the simultaneous use, in a ~ingle step~ of a pro-cedure for immunocapture of whole cells without physical or chemical intervention on the cell~, and the labeling of all or some of these cells with one or more ligands 2~X~
directly carrying a radioisotopic probe, is the fir~t method to permit the ~uantitative a~say of chosen mem-brane receptors on the cells themselves.
According to the invention, the direct labeling 05 of immunologically immobilized cells permits:
- simplification of the as3ay method by elimination of the intermediate manipulations repeated between the successive steps of the labeling process in the case o~
indirect labeling: centrifugation of the cells several times, removal of the labeling reagents and resuspen-sion;
- a saving of reagents, - an improved reliability 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 conventional equipment and apparatuses The incubation period for immobilization of the cell population and simultaneous direct labeling of the receptors of the cell subpopulation to be assayed is short. It is less than or equal to 1 hour in the case of the assay of activated T lymphocytes carrying the inter-leukin-2 receptor.
After the solid support has been washed, the actual assay is performed by using conventional appara-tuses to observe a signal which is precise and simple to meaæure: radioactivity.
Thus, overall, the method according to the in-vention ha~ numerous advantage~: it is rapid, reliable, economic and simple.
The method according to the invention makes it possible to assay surface receptor~ of a cell population over a wide range o cell concentrations.
3~ The sensitivity of the method in respect of the 20~2g3~
num~er of cells depend~ on the receptor density of the cell population assayed. For each receptor it is pos-sible, if desired, to define the minimum molar concentra-tion of receptor which can be measured by the method 05 according to the invention.
It has been verified that the æignals recorded (radioactivity count) make i1; possible to obtain satis-factory uniform standard curves as a function of the number of cells used, under the customary handling con-ditions In the Examples below, the following terms ortheir abbreviations will be used indiscriminately:
BSA: bovine serum albumin PBS: phosphate buffered saline at pH 7.4 cpm: counts per minute dpm: disintegrations per minute AS~;AY OF INTE~LEUKIN-2 ~ECEPTORS E~Y MEANS OF A
LABELE:D PHY~;I OLOGI CAL LI GAl~D
Interleukin-2 receptors appear on the m~smbrane of activated T lymphocytes when the immune system is stressed.
In the laboratory, it is pos~ible to work on a non-pathological blood sample provided activated lympho-cytes are obtained from the quiescent lymphocytes by the intervention of an exogenous agent such as phytohemagglu-tinin (PHA), concanavalin A or other lectins. Assay of the interleukin-2 receptors from human blood uses inter-leukin-2 labeled with iodine 125, which is commercially available (NEX-229, DUPONT).
A) Separation of the lymphocytes from whole blood and activation with PHA
0.5 ml of the blood sample to be assayed is taken 2~)~.2~3~
and mixed with 1.5 ml of phosphate buffered saline. 1.5 ml of Ficoll-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 05 Ficoll. After centrifugation at 900 x g for 5 minutes at room temperature, the suspension ring containing the mononucleate cells is removecl under sterile conditions in a volu~e of O.5 ml. 1.5 ml of culture medium are added to this sample. The cells are then counted and cultured at a rate of 5 106 cells per ml of RPMI 1640 medium (Flow) containing 10% of fetal calf serum (Flow).
The T lymphocytes are activated with PHA (HA 15, WELLCOME), introduced at a final concentration of 50 ~g/
ml of culture medium, for 3 days.
B) Preparation o~ the plate The plate used is a plastic microtiter plate con-taining 96 microwells, marketed by NUNC (reference 64394). Each microwell receives 200 ~l of a solution containing the purified anti-CD2 monoclonal antibody (called ST 11) used to immobilize the total T lympho-cytes, i.e. to effect their immunocapture~ This anti-body, marketed by BIOSYS, Compiègne, France, under the reference ST 11, is u~ed at a concentration of 10 ~g/ml in a phosphate bu~fered ~aline (PBS) at pH 7.4.
The adsorption of the monoclonal antibody iæ
effected at 4C 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 ~l of this solution are introduced into each microwell so as to saturate the surface of the wells with protein, which takes 1 hour at 37C; the plates are washed 3 times with phosphate buffered saline. The plates prepared in this way are lyophilized and stored at 4C in a sealed plastic bag.
~12~3~
C) Incubation of the cells The assay is performed by introducing into the microwells of the plate a volume of 100 ~l of cell 9US-pension (2.5.106 cells per ml) containing the cell~
05 treated with P~ for 3 days. 100 ~l of a solution of interleukin-2 labeled with iodine 125 (product ref. NEX-229 DUPONT, i.e. 120,000 dpm in 100 ~l of PBS containing 5% of calf serum) are then added. After 1 hour at room temperature, the microwells are emptied by turning the plate over The microwells are washed 4 times with 200 ~l of PBS per well. 75 ~l of 1 M sodium hydroxide solution are then introduced into each well. After 10 minutes, the contents of each well are recovered with an absorbent buffer before the radioactivity is counted with a ~ counter (LKB multiwell counter).
The results ars calculated and expressed in dpm in the Table below.
.
Number of cell~ Blank without in the sample cells 2.5 x 10~ cells Interleukin-2 435 dpm 185 dpm receptor __
Claims (12)
1. A kit for assaying a surface receptor characteristic of a cell population or subpopulation, said kit com-prising the following components:
a) a solid support to which one or more mono-clonal antibodies are fixed by covalent bonding or by physical adsorption, said monoclonal antibodies being directed against surface antigens of the cell population to be assayed, other than said receptor;
b) a solution containing the ligand specific for the membrane receptor to be assayed, said ligand carrying a radioisotopic probe; and c) if appropriate, an additional component con-sisting of a buffered washing solution and/or samples permitting standardization and quality control of the assay.
a) a solid support to which one or more mono-clonal antibodies are fixed by covalent bonding or by physical adsorption, said monoclonal antibodies being directed against surface antigens of the cell population to be assayed, other than said receptor;
b) a solution containing the ligand specific for the membrane receptor to be assayed, said ligand carrying a radioisotopic probe; and c) if appropriate, an additional component con-sisting of a buffered washing solution and/or samples permitting standardization and quality control of the assay.
2. A kit according to claim 1 in which component (a) is a solid support consisting of a microtiter plate in whose wells the antibody or antibodies intended for immobili-zation of the cells, including those of the population or subpopulation carrying the receptor to be assayed, are fixed.
3. A kit according to claim 1 in which component (a) is a particulate magnetic support.
4. A kit according to any one of claims 1 to 3 in which component (a) consists of a solid support to which one or more class I HLA monoclonal antibodies are fixed.
5. A kit according to claim 4 in which the fixed mono-clonal antibody is the antibody called S-class I.
6. A kit according to any one of claims 1 to 5 in which the specific ligand is interleukin-2 labeled with iodine 125.
7. A method of assaying the surface receptors of a cell population or subpopulation, said method consisting in:
a) immobilizing the cell population carrying the receptor to be assayed, or a cell population including the subpopulation carrying the receptor to be assayed, on a solid support using one or more monoclonal antibodies fixed to 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 receptor to be assayed, and, in the same step, directly labeling certain cells of the immobilized cell population or subpopulation by means of at least one ligand specific for the membrane receptor to be assayed, said ligand carrying a radioisotopic probe;
b) observing an incubation period;
c) washing the support to remove the non-immobilized cells and the excess ligand; and d) reading the results by counting the radio-activity with reference, if appropriate, to a standard scale.
a) immobilizing the cell population carrying the receptor to be assayed, or a cell population including the subpopulation carrying the receptor to be assayed, on a solid support using one or more monoclonal antibodies fixed to 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 receptor to be assayed, and, in the same step, directly labeling certain cells of the immobilized cell population or subpopulation by means of at least one ligand specific for the membrane receptor to be assayed, said ligand carrying a radioisotopic probe;
b) observing an incubation period;
c) washing the support to remove the non-immobilized cells and the excess ligand; and d) reading the results by counting the radio-activity with reference, if appropriate, to a standard scale.
8. An assay method according to claim 7 in which the solid support is as defined in claim 2 or claim 3.
9. An assay method according to claim 7 in which the ligands carry an iodine 125 or iodine 131 radioisotopic probe.
10. A method according to any one of claims 7, 8 and 9 in which the total time required to perform the assay is less than or equal to 1 hour.
11. A method according to claim 7 in which the monoclonal antibody or antibodies fixed to the solid support are anti-class I HLA antibodies.
12. A method according to claim 7 in which the monoclonal antibody fixed to the solid support is the antibody called S-class I.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8903942 | 1989-03-24 | ||
| FR8903942A FR2644893B1 (en) | 1989-03-24 | 1989-03-24 | DOSAGE KIT AND METHOD APPLICABLE TO WHOLE CELLS |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2012939A1 true CA2012939A1 (en) | 1990-09-24 |
Family
ID=9380064
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002012939A Abandoned CA2012939A1 (en) | 1989-03-24 | 1990-03-23 | Assay kit and method applicable to whole cells |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0389380A1 (en) |
| JP (1) | JPH02291966A (en) |
| KR (1) | KR900014885A (en) |
| CA (1) | CA2012939A1 (en) |
| FI (1) | FI901467A0 (en) |
| FR (1) | FR2644893B1 (en) |
| IL (1) | IL93861A0 (en) |
| PT (1) | PT93547A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3047860A1 (en) * | 1980-12-18 | 1982-07-15 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR DETERMINING HLA ANTIGENS |
| US4591570A (en) * | 1983-02-02 | 1986-05-27 | Centocor, Inc. | Matrix of antibody-coated spots for determination of antigens |
| FR2571498B1 (en) * | 1984-10-04 | 1988-04-08 | Immunotech Sa | METHOD FOR SEPARATING CELLS USING LOW DENSITY ANTIBODIES AND BALLS |
| US4770995A (en) * | 1985-08-29 | 1988-09-13 | New York Blood Center, Inc | Detection of the sensitivity of cells to the effects of tumor necrosis factor and lymphotoxin |
| DE3541033A1 (en) * | 1985-11-19 | 1987-05-21 | Boehringer Mannheim Gmbh | METHOD FOR QUANTIFYING CELL POPULATIONS OR SUBPOPULATIONS AND REAGENT SUITABLE FOR THIS |
| CA1326435C (en) * | 1987-03-13 | 1994-01-25 | Wallace H. Coulter | Method and apparatus for rapid mixing of small volumes for enhancing biological reactions |
| US5100777A (en) * | 1987-04-27 | 1992-03-31 | Tanox Biosystems, Inc. | Antibody matrix device and method for evaluating immune status |
-
1989
- 1989-03-24 FR FR8903942A patent/FR2644893B1/en not_active Expired - Fee Related
-
1990
- 1990-03-22 PT PT93547A patent/PT93547A/en not_active Application Discontinuation
- 1990-03-23 CA CA002012939A patent/CA2012939A1/en not_active Abandoned
- 1990-03-23 EP EP90400804A patent/EP0389380A1/en not_active Withdrawn
- 1990-03-23 FI FI901467A patent/FI901467A0/en not_active Application Discontinuation
- 1990-03-23 IL IL93861A patent/IL93861A0/en unknown
- 1990-03-24 KR KR1019900004000A patent/KR900014885A/en not_active Application Discontinuation
- 1990-03-26 JP JP2076561A patent/JPH02291966A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0389380A1 (en) | 1990-09-26 |
| JPH02291966A (en) | 1990-12-03 |
| FR2644893A1 (en) | 1990-09-28 |
| FR2644893B1 (en) | 1991-07-12 |
| PT93547A (en) | 1990-11-07 |
| IL93861A0 (en) | 1990-12-23 |
| FI901467A0 (en) | 1990-03-23 |
| KR900014885A (en) | 1990-10-25 |
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