CA2012934A1 - Enzymatic assay kit and method applicable to whole cells - Google Patents
Enzymatic assay kit and method applicable to whole cellsInfo
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- CA2012934A1 CA2012934A1 CA002012934A CA2012934A CA2012934A1 CA 2012934 A1 CA2012934 A1 CA 2012934A1 CA 002012934 A CA002012934 A CA 002012934A CA 2012934 A CA2012934 A CA 2012934A CA 2012934 A1 CA2012934 A1 CA 2012934A1
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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
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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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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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
- G01N33/53—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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Abstract
IN THE CANADIAN PATENT AND TRADEMARK OFFICE
PATENT APPLICATION
entitled: Enzymatic 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 assay-ing the endogenous cell enzymes 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) one or more solutions providing the reagents (substrate and chromogen) necessary for developing the activity of the endogenous enzyme to be assayed It further relates to an enzymatic method of assaying the endogenous enzymes of a cell population or subpopulation.
The assay kit and the enzymatic method according to the invention can be used for assaying the myelo-peroxidase in the granulocytes of blood or urine.
PATENT APPLICATION
entitled: Enzymatic 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 assay-ing the endogenous cell enzymes 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) one or more solutions providing the reagents (substrate and chromogen) necessary for developing the activity of the endogenous enzyme to be assayed It further relates to an enzymatic method of assaying the endogenous enzymes of a cell population or subpopulation.
The assay kit and the enzymatic method according to the invention can be used for assaying the myelo-peroxidase in the granulocytes of blood or urine.
Description
2012~3~
ENZYM~IC ASSAY KIT A~ E~Q~L~EEI~c~BLE T--Q WHOI.E ~EI~
The pre~ent invention relates to a kit and to an enzymatic a~ay method u~ing thi~ kit for a~saying endo-genous enzymes characteri~tic of cell popuIations or sub-05 population~. The en~ymatic a~ay method and the corres-ponding kit are al o intended for the assay o the cell~
them3elves via a~say of their endogenou~ enzy~es. These as~ays are applicable to diagnosis The method according t;o the invention con~ists in specifically immobilizing a cell population by immunocap~
ture on a solid support and then in a~aying an endo-genous enzyme ~acteristic of this p~ation or of a o~l subpopulation by u~ing a ~ubstrate appropriate for th~o enzyme and, if appropriate, the nece~sary cofactors or auxiliary ~ubstance~
According to the pre~ent invsntion, in the de~-cription and the claim~ which follow, the term "endo-genou~ enzyme" is under~tood a~ meaning an enzyme which i~ contained in the cytopl~m of the cell and whose ~ub-~trate is a low-molecular molecule capable of penetratin~
inside the cell, where it will be tran~formed by the enzyme, thereby releasing into the medium a colored or fluorescent reaction product which permit~ the final mea~urement of a signal proportional to the acti~ity of the enzyme.
Cell immunocapture i~ a proce s which con~ist~ in retaining the cells on a ~olid support by binding between one or more surface antigens of the~e cell~ and one or more monoclonal antibodies specific for thi~ antigen or these antigens.
Knowledge of cell ~urface antigen~ or marker~ has made enormou~ ad~nce~ with the development o~ lymphocyte hybridization and the discov~ry of monoclonal antibodies by KOEHLER and MI~STEIN (Nature, 1975, 2~, 495~497)-particular, monoclonal antibodie~ have made it possible ` 2~2~3~
to reveal and analyze surface markers or membrane anti-gens of cells of the widest possible variety of origins.
These markers (or antigens) can be of different kinds:
proteins, glycoproteins or glycolipids. The characteri-05 zations sought therefore apply mainly to tissue or organmarkers, to markers of states o~ 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 hemopoiesis (erythrocyte, megakaryocyte, granu-locyte, monocyte, lymphocyte).
Thus, for example, monoclonal antibodies have made it possible to specify the respective surface characteristics of T and B lymphocyte~. The correspon~
ding markers, by themselves or in combination, identify stages of differentiation and functional specialization of the lymphocyte~. 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, 1~84, and described in Bulletin of the World Health Organization, 1984, 62 (5), 813-815.
Monoclonal antibodies are now irreplaceable tools of clinical biology applied to cell analyses.
Cell counting methods exist which utilize the labeling of their surface antigens, but these methods are often lengthy, laborious and dificult to carry out and their results are sometimes random.
One group of methods for the measurement of anti-gens is based on quantitati~e evalua-tion o the surface markers of the overall cell population These methods make it possible to measure the antigens either by direct labeling or by indirect labPling, the latter most fre-quently being carried out in two, thr-ee or four steps.
In all cases, the reagent employed in the last labeling 2~ 2~
step carries a probe which is either of isotopic charac-ter, for example iodine 125~ for an assay of the radio-immunometric type (BROW et al., J Immunol. Methods, 1979, 31, 201; STOCKER and HEVSSER, J. Immunol. Methods, 05 1979, 26, 87-95), or an enzyme for an assay of the enzyme immunometric type, most frequently peroxidase, alkaline phosphatase or beta-galactosidase (VAN LEUVEN et al., J~
Immunol. Methods, 19789 23, 109-116; MORRIS, Transplan-tation, l9B3, 36(6), 719; BAUMGARTEN, J. Immunol.
Methods, 1906, 94, 91-98).
It ~ill be noted that the enzymes used in such methods are analytical reagents introduced during the assay in the form of a conjugate with an antibody which recognizes an antigen exposed on the outside of the cell membrane. These enzymes cannot therefore be confused with the endogenous enzymes of the cell, which form part of its natural enzymatic equipment.
These methods are rather inconvenient, laboriouæ
and risky to apply because of the need to wash and cen-trifuge the cell material many times, it is sometimesnecessary to take a sample of the colored medium resul-ting from the enzyme reaction in order to carry out the final spectrophotometric measurement; lastly, chemical fixation of the cells, which is used in most cases, causes irreversible destruction of certain antigens which are particularly sen~itive to the customary chemical fixative~ such as glutaraldehyde or methanol (DROVER et al., J. Immunol. Methods, 1986, 90, 275-281).
Cell i~munocapture on a solid 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 requires that the antibody used be fixed to the solid support by a complex macromolecular struc-~0129~
ture, called a network-relay, which i~ capable of en-suring a preferential orientation of the antibody rela-tive to that of the corresponding cell antigen. Said patent application gives no indication of an a~plication ~ of the technique to the quantitative assay of a cell enzyme.
Cell immunocapture i8 also de~cribed in patent application W0 84/03151 for an analytical application.
In said patent application, the object i~ 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 antibodie~ arranged accor-ding to a particular geometry on very specialized sup-port~ (microscope 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 analytical applications permit-ting the quantitative determination of a marker charac~ *ic of the cell, and in particular a constituent enzyme of the cell.
The assay method forming the subject of the present invention has considerable advantages over all the tPchniques known and used in the prior art, since it permits quantitative mea3urement of any endogenous enzyme of a cell population. Thi~ assay is performed on cells which have not undergone any chemical or physical inter-vention at the moment of their specific capture and which are therefore in their state of physiological integrity.
Furthermore, the assay method according to the invention ha~ the characteristics of very high specificity which are inherent in the double recognition systems involving two different specific markers carried by the sa~e cell, one being an antigen selected for immunocapture of the 3~ cells which it is desired to analyze, and the other being ~ 2~3~
an endogenous enzyme present in the cells which have been captured. 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 05 purposes in clinical biology laboratories handlin8 these large numbers.
The present invention thus relates to a kit -for assaying at leas~ one endogenous enzyme characteristic of a cell population or subpopulation7 said kit comprising the following components:
a) a solid support to which one or more mono-clonal antibodies are fixed by covalent bonding or physi-cal adsorption, said monoclonal antibodieR being directed against surface antigens of the cell population examined, and being intended for immobilization, on the support, o~
the cells which include those of the subpopulation pos-sessing the enzyme to be assayed;
b) a developer for the endogenous enzyme, namely one or more solutions providing the rea~ent~ (substrate and, if appropriate, chromogen) necessary for developin~
the activity of the enzyme, and, if appropriate, a reagent which improves the permeability of the cell mem-brane; and c) a ~uffer solution intended for washing of the 2S solid support.
The term "cell" as used in the present descrip~
tion and in the claims which follow encompasses human cells or animal cells and especially blood cell3~ in-cluding the platelets.
The assay method according to the invention applies to whole cells, i.e. non-lyzed cells.
These cells have not undergone any physical or chemical intervention at the moment of their immuno-capture and they are used in a state of complete physio-logical inte~rity. This situation constitutes the be~t ~01%~3~
guarantee of integrity of the antigen used for capture and of the endogenous enzyme chosen as the assay target.
As the solid support it is possible to u~e any device suitable for handling cell suspensions, and pre-05 ferably tubes, particulate magnetic supports or rigid orflexible microtiter plates made of polyethylene, poly-styrene, polyvinyl chloride or nitrocellulose, which con-tain microwells. The monoclonal antibodies intended for immobilization of the cells can be fixed to the solid supports either by covalent chemical bonding or by physical adsorption according to the classical techni~ueæ
well known to those skilled in the art, such as the tech-nigues described by STOCKER and HEUSSER, J. Immunol.
Methods, 1979, vol. 26, 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 cPll population which includes the cell population or populations containing the enzyme to be assayed. When this population consists of human cells, the preferred monoclonal antibodies for immuno-capture are the anti-class I HLA antibodies which are specific for the common part of the HLA-A, -B and -C
antigens present on the leukocytes and numerous other cell lines of the organism. Likewise~ monoclonal anti-bodies specific for the antigens of the differentiation classes which ha~e been determined on the leukocytes are preferred. Of these antibodies, the one called S-class I, marketed by BIOSYS, is particularly preferred.
In other cases where the cells ~xamined 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 substrate for the endogenous enzyme to be 2~:l2~3~
assayed and the reagents are chosen so that the final product of the reaction or reaction qe~uence caused by the enzyme, involving these substances, is:
- either a colored or fluorescent s~bstance which dif-05 fuses into the liquid medium surrounding the cells andwhich is the ob~ect of the final spectrophotometric or, respectively, fluorimetric measurement, - or an insoluble colored substance which deposits on the cells and the walls to which they are fixed, and which can ke the object either of photometric measure-ment by reflection or of visual evaluation, if approp-riate against a scale of standard shades.
Thus it is not necessary to use a chromogen if the substrate alone makes it possible to obtain a colored or fluorescent substance.
A~ an additional component, the assay kit con-tains a buffer solution intended ~or washing of the solid support after immobilization of the cell~.
As other additional components, the assay kit can also contain the samples necessary for standardization and ~uality con-trol of the assay The present invention further relates to a method of assaying the endogenous enzymes of a cell population or subpopulation, said method consiæting in:
- immobilizing the cell population, which includes the subpopulation containing the endogenous enzyme, 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;
- observing an incubation period to allow immunocapture;
- washing the solid support to remove the non-immobilized cells, - adding the reagent or reagents (substrate and, if appropriate, chromogen) necessary for developing the %~ ~ 2~
activity of the endogenous enzyMe; and - reading the results by measuring the light signals (coloration or fluorescence) with reference, if approp-riate, to a standard scale.
05 Thus the developing of the endogenous enzyme to be assayed, which belongs to the immobilized cell Population or to one of its subpopulation~s, is effected direct by means of a substrate specific for this endog~nous enzyme, after which the actuaI assay of the endogenous enzyme i~
performed by photometric measurement by transmi~sion or reflection, or measurement of the fluorescencs emission.
The assay kit and method according to the inven-tion are particularly simple because, when preprepared immunocapture supports are available 7 they involve only the solution or solutions containing the specific re-agents necessary for ~easuring the activity of the enzyme (substrate and, if appropriate, chromogen).
The assay kit and the enzymatic method according to the invention are preferably applied to the assay of the endogenous enzyme~ of the for~ed elem~nt~ of human blood, especially the leukocytes and more particularly the granulocytæs.
The assay kit and the enzymatic method according to the invention make it possible to measure signals (absorbed or emitted light) which depend both on the number of cells present in the cell population examined and on the concentratlon of the endogenous enzyme measured in these cells. Measurement of the e signals permits quantitative evaluakion of the activity of the molecules of this enzyme which are contained in the cells of the cell population or subpopulation examined.
One application of the invention becomes apparent if a microtiter plate is chosen as the solid support.
The assay kit and the enzymatic method according to the invention can then advantageously be used for the assay, 2~2~
on a single plate, of a series of endo~enous enzymes characteristic of various sub,populations making up the cell population examined. For thi~ application, it i8 possible on the one hand to take ready-to-u~e microtiter 05 plates to which one or more monoclonal antibodie~ capable of retaining all the cells of the population examined have been fixed beforehand, and on the other hand to have a series of substrates which are each specific for an endogenous enzyme characteristic of one of the subpopu-lations to be evaluated. Thus, on one and the samesupport, it is possible to perform the ~uantitative a~say of all the endogenous enzymes necessary for characteriza~
tion of the chosen subpopulations.
A oase which ma~ be mentioned as an application of the invention is that of the human leukocytes, for which there are in particular the subpopulation of cell~
called polynuclear leukocytes or granulocytes.
The presence of human granulocytes can be evalua-ted in blood or in urine. Thus, in the case of urinary infections such a~ cystitis or pyelonephritis7 it i~ par-ticularly valuable to have a simple method of assaying the granulocytes in urine.
The assay kit and the enzymatic method accordin~
to the invention can be u~ed for assaying granulocyte~.
In this case, specific immobilization of the granulocytes in the sample examined i~ effected on a solid support and the assay of an endogenous enzyme characteristic of the granulocytes is performed using an appropriate substrate.
Myeloperoxidase is an endogenous enzyme of granulocyte~
and its assay is performed u~ing hydrogen peroxide as the substrate for the peroxidase.
Specific immobilization of the granulscytes is effected using anti-CD1~ monoclonal antibodies fixed to a solid support such as, for example, tha wall~ of the microwells of microtiter plates 2~.2~3~
The plates prepared in this way can be lyophi-lized and ~tored, preferably at 4~C. This step can be carried out on the industrial scale and i-t is thus possible to have ready-to-use plates for the assay kits 05 which can be applied to the granulocytes in the ~ample of blood or urine examined.
The samples containing the cell~ to be aæsayed, which originate from blood or from any appropriate bio-logical fluid - normal or pathological - in par-ticular urine, can be used as such or after preparation, especi-ally after concentration.
Aliquots of the appropriate cell suspen~ion are brought into contact with the solid support, for example in tubes containing the microparticulate immunocapture support or in the microwells of a microtiter plate pre-pared beforehand. After washing, the solution forming part of the assay kit and containing the substrate specific for the endogenous enzyme characteristic of the target cell population is added.
The time re~uired for immobilization of the cells is preferably less than or equal to 15 minute~ During this time, the solid support can be centrifuged to improve the immobilization of the cells. The solid support, for example the tube or microtiter plate, is then washed to remove the unfixed cells The appearance of a colored or fluorescent pro-duct is brought about by adding, to the ~olid support to which the cell population carrying the endogenous enzyme to be a~sayed has been fixed, a solution containing the substrate for the enzyme and, if necessary, one or more auxiliary reagents such that the reaction product which is finally obtained is either a colored product ~oluble in the medium, or an insoluble colored product, or a soluble fluore~cent product, a~ explained earlier. The light signal coming ~rom the sample~ treated in this way ~0~2~3~
is then measured with the equipment appropriate to each case, i.e. a tranæmi.ssion or reflection photometer or, respectively, a fluorimeter~ When the solid support is a microtiter plate, the light æignal can be read æe~uen-05 tially in all the wells of one and the æame plate bymeans of automated readeræ commonly used in biology laboratorie~, æuch asl fvr example, the Titertek plate reader or the Fluoro~can plate reader for the spectro-photometric or, respectively, fluorometric reading~.
The reagentæ used to develop the endogenous peroxidase or the endogenous myeloperoxidase 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 i9 colored and soluble in the medium, or elæe 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole or 4-chloro-alpha-naphthol to give an inæoluble final reaction pro-duct, or else parahydroxyphenylpropionic acid to give a fluorescent reaction product which i3 soluble in the medium.
In a preferred form, the kit according to the invention for assaying myeloperoxidase characteristic of the granulocytes compriseæ:
a) a microtiter plate in whose well~ one or more anti-granulocyte monoclonal antibodies have been fixed;
bl) a solution containing hydrogen peroxide, which is the substrate for the enzyme~ in an appropriate buffer; and b2) a solut.ion containing the chromo~en used to develop the expression of the activity of the enzyme and, if appropriate, a reagent which improves the permeability of the cell membrane.
In another preferred form, the kit according to the invention for asæaying myeloperoxidaæe characteristic 2~2~3~
of the granulocytes comprises, as the solid support, tubes or alternatively particu:Late magnetic supports to which one or more anti-granulocyte monoclonal antibodies have been fixed.
05 The results of the assay of the endogenous en-zymes according to the invention can be expressed accor-ding to any procedure appropriate to the examination carried out. More particularly, these results can he expressed as the total activity of a particular endo-genous enzyme present in a given volume of the sample examined (for example per microliter of blood).
The activity of a particular endogenous en~yme in the sample examined will preferably be determined u~ing a standard scale consisting of appropriate cells or prepa-rations containing the endogenous enzyme to be assayed,which will have been calibrated beforehand by a known reference method. These standards will preferably consist either of cells identical in their o~igin to the cells which are to form the subject of the assay, or of cells of established cell lines containing the de~ired endogenous enzyme, or of cell-free preparations contain-ing the chosen enzyme.
These standards are then treated in exactly the same way as the samples ~o be examined. The resulting signals are used to build up a standard scale against which the signal~ measured with the samples to be examined are compared. The subse~uent calculations are conventional.
The enzymatic assay method according to the invention is simple, rapid and reproducible. Its use is totally suitable for the analysis of a large number of samples. For an understanding o~ its advantages compared with the other methods described, the various steps should be analyzed.
Immobilization of the cells on the solid ~upport 2~12~
is the stage of the assay which usually present~ the mo~t difficulties or which is the most critical to carry out.
The means often used i8 chemical fixation of the cells with glutaraldehyde or methanol in cups which may or may 05 not have been treated with poly-L-lysine (VAN LEUVEN F.
et al , J. Immunol. Methods, 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 is a very serious disadvantage (DROVER and MARSHALL, J.
Immunol. Methods, 1986, 90, 275-281).
Furthermore, the chemical fi~ation method has to be carried out in several steps: 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 antigens which might be useful forimmunocapture of the cellss as well as the endogenous enzymes to be assayed.
Furthermore, the reproducibility of this method is doubtful; in fact, the settling of the cell~ in the assay microwells and the drying of the cells can vary from one experiment to the next. Finally, this assay is lengthy to perform because the cell drying st2p alone takes more than 2 hours.
The immobilization of lymphocyte populations ha~
also been achieved by using polyclonal antibodies adsor-bed in microwells (STOCKER and HEUSSER, J. Immunol.
Methods, 1979, 26, 87-95). This method makes it possible to immobilize cells foreign to the single population which it is desired to analyze; this also represents a certain disadvantage.
2~ 3~
The use of highly specific and related monoclonal antibodies adsorbed on or fixed to the ~olid support, and especially in the assay wells, in the tubes or on the particulate support, permits exclusive capture of the 05 desired cells, the other non-retained cell population~
being removed in the course of the washes carried out.
Furthermore, no chemical or physical agent modifies the characteristics of the antigens in this ~tep because the various operations for chemical or phy~ical 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 method which makes it possible to simpli-fy the step for immobilizing the cells carrying the enzyme to be assayed, while at the ~ame time making the results more reliable.
The method according to the invention, which com prises the use of a procedure for immunocapture of whole cells without physical or chemical intervention on the cells, and the measurement, in all or some of these cells, of the activity of an endogenous enzyme by using its specific substrate, is the first method to permit the quantitative assay of the chosen endogenous enzymes on the cells themselves.
According to the invention, the direct labeling of immunologically immobilized cells permits:
- a saving of reagents, - an improved reliability through a reduction in the number of steps and manipulation3;
- a time saving; and - the possibility of treating large numbers of samplss at the same time, exclusively with the use of conventional equipment and apparatuses.
The time required to immobilize the cell popula-tion or cell subpopulation to be assayed is short. It is 20:~2~
less than or equal to 15 minutes in the case of the assay of granulocytes in the blood or urine. Likewisel the period for assay of the activity of the endogenous enzyme is less than or equal to 1~ minutes.
05 After the solid support has been wa~hed, the actual assay is performed by using conventional appara-tuses to observe a signal which is precise and simple to measure: light absorptlon or emission.
Thus, overall, the method according to the inven-tion has numerous advantages: it is rapid, reliable, economic and simple.
It has been verified that the signals recorded (photometric measurements) make it possible to obtain satisfactory uniform ~tandard curves as a function of the number of cells used, under the customary handling con-ditions.
In the E~amples below~ the following termq or their abbreviations will be used indiscriminately:
BSA: bovine serum albumin PBS: phosphate buffered saline at pH 7.4 26 LEUKOCYTES OE HUM~N ~LOO~: ASSAY PERFORM~D QN A
Myeloperoxidase is an enzyme of glycoprotein character compri~ing a heme structure. This enzyme, which is abundant in the polynuclear leukocytes of human blood, is involved ln the bactericidal and antimicrobial functions of calls (KLEBANOFF S.J., J. Bacteriol., 1968, 95, 2131; DIAMOND et al., J. Clin. Invest., 1580, ~, 908-917).
The assay of myeloperoxidase in the polynuclear 3~ leukocytes, according to the mæthod of the invention, 3~
comprises three steps carried out in ~ucce~sion:
1. separation of the polynuclear leukocytes from whole blood, 2. capture and selective immobilization of the cella by 05 means of a monoclonal antibody adsorbed beforehand in the microwells of a microtiter plate, and 3. developing of the activity of the cell enzyme.
a) Preparation of the plate The plate used i~ a plastic microtiter plate con-taining 96 microwells, marketed by NUNC (reference 64394). Each microwell receives 200 ~1 of a solution containing the purified anti-CD15 monoclonal antibody (called SMY 15a) used to immobilize the polynuclear leukocytes, i.e. to effect their immunocapture. Thi~
antibody~ marketed by BIOSYS, Compiègne, France~ under the reference SMY 15a, i5 used at a concentration of 5 .g/ml in a phosphate buffered saline (PBS) at pH 7.4.
The adsorption of the monoclonal antibody i~
effected at 4C for 12 hours. The excess antibody is removed by turning the plate over.
A solution containing 0.1% of gelatin and 0.3% of BSA in a phosphate buffered saline is prepared. 250 ~1 of this solution are introduced into each microwell ~o 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 stored at 4C.
b) Separation of the polynuclear leukocytes The blood sample is taken on an anticoagulant ~heparin). 2 ml of MONO-POLY separating medium (FLOW
ref. 16.980-49) are introduced into a 5 ml hemolysis tube and 2 ml of blood are deposited on top. The tube is then centrifuged at 400 x g for 40 minutes at laboratory tem-perature. Two cell suspension rings are formed; the lower ring contains the polynuclear leukocytes, which are 2~ 2~3~
recovered with a micropipette c~ Cell immunocapture Cell capture is effected by means of the adsorbed anti-CD15 monoclonal antibody 05 100 ~l of the cell suspension, adjusted to 5 x 10~ cells per ml of PBS, are introduced into the micro-wells of the plate. To improve the fixation of tha cells on the support, the plate is centrifuged for 3 minutes at 150 x g after a wait of 10 minutes at room temperature.
d) Developing and measurement of the myeloperoxi-dase The microwells are emptied by turning the plate over. TheY are washed twice with 200 ~l of PBS, making it possible to remove the undesirable cell populations, such as the monocytes or erythrocytes, which can cause a ~purious absorbance signal. After the second wash, 100 ~l of the developing reagent~ prepared for immediate use~
are added to each well.
The developing reagent is obtained in the fol-lowin~ manner: a 0.1 M citrate buffer i~ prepared by dis-solving citric acid monohydrate in water to give a 2%
solution and adjusting the pH to ~ by the addition of 7 N
sodium hydroxide solution. A 2% solution of hexadecyl-trimethylammonium bromide (= CETAB, TOUZART and MATIGNON
T 5650) in the buffer is then prepared. The reagent renders the cell membranes permeable and improves the activity of the myeloperoxidase on the ~ub~trate. 30 mg of orthophenylenediamine dihydrochloride and then 40 ~l of hydrogen peroxide (substrate for the enzyme) are added to 20 ml of this solution before use After incubation for 10 minutes, the absorbance is measured on a spectrophotometer at 4~0 nm (type 310 C
Titertek Multiskan apparatus - Flow Laboratories).
~or one experiment 7 the absorbances obtained with 50,000 cells are given in Table 1 below:
3 ~
~hE~
CELLS ONLY CELLS CELLS
+ reagents ~ reagents 05 r _ without CETAB with CETAB
¦ Absorbance0.003 0.370 1.545 The presence of CETAB in the developing medium produces a 4-fold increa~e in the optical denslty due to the reaction catalyzed by myeloperoxidase, making it possible to reduce the number of cells required for the assay.
ASSAY OF MYELQPERQXIDASE IN THE POLYN~lCLEAR
l,EUKOCYTES OF BLOOD AND URINE: A~SAY PERFORME~ ON
This Example is intended to show that the time required to assay an endogenous enzyme can be reduced, compared with the conditions described in Example 1, by modifying the cell immunocapture process. The support used to capture the cells consists of magnetic beads known as DYNABEADS. The beads (ref. DYN-llOOl, marketed by BIOSYS) carry anti-mouso immunoglobulin antibodies on their surface. Before use, the beads are treated with a solution of anti-CD15 monoclonal antibodies (ref. SMY
15a - BIOSYS, France) for 12 hours at 4C. This is done by mixing 25 ~l of the suspension of beads with 0.5 ml of antibody solution containing 100 ~g~ml of PBS. The beads are subsequently washed 3 times with PBS and then satu-rated for 12 hours at 4C with a 0.3% solution of bovine serum albumin (BSA). The read~-to-use beads are kept at 4~C.
The myeloperoxidase is assayed in a 5 ml hemo-2012~3 !~
lysis tube, 300 ~1 of PBS, 25 ~1 of the suspension of beads and 100 ~1 of whole blood taken on lithium hepari-nate (VACUTAINER tube ref. 606 484) are introduced into the tube. After 5 minutes of gentle shaking, the beads 05 are separated off by means of a magnet and the cells fixed to the beads are washed with PBS (5 washes).
The myeloperoxidase is developed with a mixed reagent containing the substrate (hydrogen peroxide) and the chromogen (orthophenylenediamine), prepared as in Example 1, with or without the incorporation o~ CETAB.
The absorbance is measured under conditions identical to those of Ex~mple 1. The results are given in Table 2.
Measurement of the myeloperoxidases in the granulocytes present in urine during urinary infections is an important diagnostic tool for pyelonephritis and pyuria. It was shown in the second part of the Example that magnetic particles carrying the same anti-CD15 monoclonal antibody are capable of capturing the poly-nuclear leukocytes in the urinary fluid. The assay is performed on 400 ~1 of urinary fluid containing about 2 x 105 granulocytes per ml. The cells are captured with 25 ~1 of the suspension of beads and then treated under the same conditions as in the previous Example. The results are given in Table 2 below.
~LEæ
ABSORBANCE BEADS + BEADS + BEADS -~ BEADS
developing CELLS CELLS -~ CELLS ~
reagentonly developing developing reagent reagent without with CETAB CETAB
Whole n_ _ _ blood 0.0250.011 0.0323 1.324 . __ ... __ _ _._.
Urine 0.0250.008 0.286 1.115 2~12~3~
According to the method described in thi~
E~ample, the assay was performed direct on the blood sample and then on the urine sample without the need for prior separation of the cell population to be analyzed.
05 Furthermore, the incubation period is only 5 minutes.
Thus, by using magnetic bead~ as the solid support, the assay operations taken as a whole are particularly simple and rapid.
ENZYM~IC ASSAY KIT A~ E~Q~L~EEI~c~BLE T--Q WHOI.E ~EI~
The pre~ent invention relates to a kit and to an enzymatic a~ay method u~ing thi~ kit for a~saying endo-genous enzymes characteri~tic of cell popuIations or sub-05 population~. The en~ymatic a~ay method and the corres-ponding kit are al o intended for the assay o the cell~
them3elves via a~say of their endogenou~ enzy~es. These as~ays are applicable to diagnosis The method according t;o the invention con~ists in specifically immobilizing a cell population by immunocap~
ture on a solid support and then in a~aying an endo-genous enzyme ~acteristic of this p~ation or of a o~l subpopulation by u~ing a ~ubstrate appropriate for th~o enzyme and, if appropriate, the nece~sary cofactors or auxiliary ~ubstance~
According to the pre~ent invsntion, in the de~-cription and the claim~ which follow, the term "endo-genou~ enzyme" is under~tood a~ meaning an enzyme which i~ contained in the cytopl~m of the cell and whose ~ub-~trate is a low-molecular molecule capable of penetratin~
inside the cell, where it will be tran~formed by the enzyme, thereby releasing into the medium a colored or fluorescent reaction product which permit~ the final mea~urement of a signal proportional to the acti~ity of the enzyme.
Cell immunocapture i~ a proce s which con~ist~ in retaining the cells on a ~olid support by binding between one or more surface antigens of the~e cell~ and one or more monoclonal antibodies specific for thi~ antigen or these antigens.
Knowledge of cell ~urface antigen~ or marker~ has made enormou~ ad~nce~ with the development o~ lymphocyte hybridization and the discov~ry of monoclonal antibodies by KOEHLER and MI~STEIN (Nature, 1975, 2~, 495~497)-particular, monoclonal antibodie~ have made it possible ` 2~2~3~
to reveal and analyze surface markers or membrane anti-gens of cells of the widest possible variety of origins.
These markers (or antigens) can be of different kinds:
proteins, glycoproteins or glycolipids. The characteri-05 zations sought therefore apply mainly to tissue or organmarkers, to markers of states o~ 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 hemopoiesis (erythrocyte, megakaryocyte, granu-locyte, monocyte, lymphocyte).
Thus, for example, monoclonal antibodies have made it possible to specify the respective surface characteristics of T and B lymphocyte~. The correspon~
ding markers, by themselves or in combination, identify stages of differentiation and functional specialization of the lymphocyte~. 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, 1~84, and described in Bulletin of the World Health Organization, 1984, 62 (5), 813-815.
Monoclonal antibodies are now irreplaceable tools of clinical biology applied to cell analyses.
Cell counting methods exist which utilize the labeling of their surface antigens, but these methods are often lengthy, laborious and dificult to carry out and their results are sometimes random.
One group of methods for the measurement of anti-gens is based on quantitati~e evalua-tion o the surface markers of the overall cell population These methods make it possible to measure the antigens either by direct labeling or by indirect labPling, the latter most fre-quently being carried out in two, thr-ee or four steps.
In all cases, the reagent employed in the last labeling 2~ 2~
step carries a probe which is either of isotopic charac-ter, for example iodine 125~ for an assay of the radio-immunometric type (BROW et al., J Immunol. Methods, 1979, 31, 201; STOCKER and HEVSSER, J. Immunol. Methods, 05 1979, 26, 87-95), or an enzyme for an assay of the enzyme immunometric type, most frequently peroxidase, alkaline phosphatase or beta-galactosidase (VAN LEUVEN et al., J~
Immunol. Methods, 19789 23, 109-116; MORRIS, Transplan-tation, l9B3, 36(6), 719; BAUMGARTEN, J. Immunol.
Methods, 1906, 94, 91-98).
It ~ill be noted that the enzymes used in such methods are analytical reagents introduced during the assay in the form of a conjugate with an antibody which recognizes an antigen exposed on the outside of the cell membrane. These enzymes cannot therefore be confused with the endogenous enzymes of the cell, which form part of its natural enzymatic equipment.
These methods are rather inconvenient, laboriouæ
and risky to apply because of the need to wash and cen-trifuge the cell material many times, it is sometimesnecessary to take a sample of the colored medium resul-ting from the enzyme reaction in order to carry out the final spectrophotometric measurement; lastly, chemical fixation of the cells, which is used in most cases, causes irreversible destruction of certain antigens which are particularly sen~itive to the customary chemical fixative~ such as glutaraldehyde or methanol (DROVER et al., J. Immunol. Methods, 1986, 90, 275-281).
Cell i~munocapture on a solid 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 requires that the antibody used be fixed to the solid support by a complex macromolecular struc-~0129~
ture, called a network-relay, which i~ capable of en-suring a preferential orientation of the antibody rela-tive to that of the corresponding cell antigen. Said patent application gives no indication of an a~plication ~ of the technique to the quantitative assay of a cell enzyme.
Cell immunocapture i8 also de~cribed in patent application W0 84/03151 for an analytical application.
In said patent application, the object i~ 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 antibodie~ arranged accor-ding to a particular geometry on very specialized sup-port~ (microscope 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 analytical applications permit-ting the quantitative determination of a marker charac~ *ic of the cell, and in particular a constituent enzyme of the cell.
The assay method forming the subject of the present invention has considerable advantages over all the tPchniques known and used in the prior art, since it permits quantitative mea3urement of any endogenous enzyme of a cell population. Thi~ assay is performed on cells which have not undergone any chemical or physical inter-vention at the moment of their specific capture and which are therefore in their state of physiological integrity.
Furthermore, the assay method according to the invention ha~ the characteristics of very high specificity which are inherent in the double recognition systems involving two different specific markers carried by the sa~e cell, one being an antigen selected for immunocapture of the 3~ cells which it is desired to analyze, and the other being ~ 2~3~
an endogenous enzyme present in the cells which have been captured. 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 05 purposes in clinical biology laboratories handlin8 these large numbers.
The present invention thus relates to a kit -for assaying at leas~ one endogenous enzyme characteristic of a cell population or subpopulation7 said kit comprising the following components:
a) a solid support to which one or more mono-clonal antibodies are fixed by covalent bonding or physi-cal adsorption, said monoclonal antibodieR being directed against surface antigens of the cell population examined, and being intended for immobilization, on the support, o~
the cells which include those of the subpopulation pos-sessing the enzyme to be assayed;
b) a developer for the endogenous enzyme, namely one or more solutions providing the rea~ent~ (substrate and, if appropriate, chromogen) necessary for developin~
the activity of the enzyme, and, if appropriate, a reagent which improves the permeability of the cell mem-brane; and c) a ~uffer solution intended for washing of the 2S solid support.
The term "cell" as used in the present descrip~
tion and in the claims which follow encompasses human cells or animal cells and especially blood cell3~ in-cluding the platelets.
The assay method according to the invention applies to whole cells, i.e. non-lyzed cells.
These cells have not undergone any physical or chemical intervention at the moment of their immuno-capture and they are used in a state of complete physio-logical inte~rity. This situation constitutes the be~t ~01%~3~
guarantee of integrity of the antigen used for capture and of the endogenous enzyme chosen as the assay target.
As the solid support it is possible to u~e any device suitable for handling cell suspensions, and pre-05 ferably tubes, particulate magnetic supports or rigid orflexible microtiter plates made of polyethylene, poly-styrene, polyvinyl chloride or nitrocellulose, which con-tain microwells. The monoclonal antibodies intended for immobilization of the cells can be fixed to the solid supports either by covalent chemical bonding or by physical adsorption according to the classical techni~ueæ
well known to those skilled in the art, such as the tech-nigues described by STOCKER and HEUSSER, J. Immunol.
Methods, 1979, vol. 26, 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 cPll population which includes the cell population or populations containing the enzyme to be assayed. When this population consists of human cells, the preferred monoclonal antibodies for immuno-capture are the anti-class I HLA antibodies which are specific for the common part of the HLA-A, -B and -C
antigens present on the leukocytes and numerous other cell lines of the organism. Likewise~ monoclonal anti-bodies specific for the antigens of the differentiation classes which ha~e been determined on the leukocytes are preferred. Of these antibodies, the one called S-class I, marketed by BIOSYS, is particularly preferred.
In other cases where the cells ~xamined 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 substrate for the endogenous enzyme to be 2~:l2~3~
assayed and the reagents are chosen so that the final product of the reaction or reaction qe~uence caused by the enzyme, involving these substances, is:
- either a colored or fluorescent s~bstance which dif-05 fuses into the liquid medium surrounding the cells andwhich is the ob~ect of the final spectrophotometric or, respectively, fluorimetric measurement, - or an insoluble colored substance which deposits on the cells and the walls to which they are fixed, and which can ke the object either of photometric measure-ment by reflection or of visual evaluation, if approp-riate against a scale of standard shades.
Thus it is not necessary to use a chromogen if the substrate alone makes it possible to obtain a colored or fluorescent substance.
A~ an additional component, the assay kit con-tains a buffer solution intended ~or washing of the solid support after immobilization of the cell~.
As other additional components, the assay kit can also contain the samples necessary for standardization and ~uality con-trol of the assay The present invention further relates to a method of assaying the endogenous enzymes of a cell population or subpopulation, said method consiæting in:
- immobilizing the cell population, which includes the subpopulation containing the endogenous enzyme, 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;
- observing an incubation period to allow immunocapture;
- washing the solid support to remove the non-immobilized cells, - adding the reagent or reagents (substrate and, if appropriate, chromogen) necessary for developing the %~ ~ 2~
activity of the endogenous enzyMe; and - reading the results by measuring the light signals (coloration or fluorescence) with reference, if approp-riate, to a standard scale.
05 Thus the developing of the endogenous enzyme to be assayed, which belongs to the immobilized cell Population or to one of its subpopulation~s, is effected direct by means of a substrate specific for this endog~nous enzyme, after which the actuaI assay of the endogenous enzyme i~
performed by photometric measurement by transmi~sion or reflection, or measurement of the fluorescencs emission.
The assay kit and method according to the inven-tion are particularly simple because, when preprepared immunocapture supports are available 7 they involve only the solution or solutions containing the specific re-agents necessary for ~easuring the activity of the enzyme (substrate and, if appropriate, chromogen).
The assay kit and the enzymatic method according to the invention are preferably applied to the assay of the endogenous enzyme~ of the for~ed elem~nt~ of human blood, especially the leukocytes and more particularly the granulocytæs.
The assay kit and the enzymatic method according to the invention make it possible to measure signals (absorbed or emitted light) which depend both on the number of cells present in the cell population examined and on the concentratlon of the endogenous enzyme measured in these cells. Measurement of the e signals permits quantitative evaluakion of the activity of the molecules of this enzyme which are contained in the cells of the cell population or subpopulation examined.
One application of the invention becomes apparent if a microtiter plate is chosen as the solid support.
The assay kit and the enzymatic method according to the invention can then advantageously be used for the assay, 2~2~
on a single plate, of a series of endo~enous enzymes characteristic of various sub,populations making up the cell population examined. For thi~ application, it i8 possible on the one hand to take ready-to-u~e microtiter 05 plates to which one or more monoclonal antibodie~ capable of retaining all the cells of the population examined have been fixed beforehand, and on the other hand to have a series of substrates which are each specific for an endogenous enzyme characteristic of one of the subpopu-lations to be evaluated. Thus, on one and the samesupport, it is possible to perform the ~uantitative a~say of all the endogenous enzymes necessary for characteriza~
tion of the chosen subpopulations.
A oase which ma~ be mentioned as an application of the invention is that of the human leukocytes, for which there are in particular the subpopulation of cell~
called polynuclear leukocytes or granulocytes.
The presence of human granulocytes can be evalua-ted in blood or in urine. Thus, in the case of urinary infections such a~ cystitis or pyelonephritis7 it i~ par-ticularly valuable to have a simple method of assaying the granulocytes in urine.
The assay kit and the enzymatic method accordin~
to the invention can be u~ed for assaying granulocyte~.
In this case, specific immobilization of the granulocytes in the sample examined i~ effected on a solid support and the assay of an endogenous enzyme characteristic of the granulocytes is performed using an appropriate substrate.
Myeloperoxidase is an endogenous enzyme of granulocyte~
and its assay is performed u~ing hydrogen peroxide as the substrate for the peroxidase.
Specific immobilization of the granulscytes is effected using anti-CD1~ monoclonal antibodies fixed to a solid support such as, for example, tha wall~ of the microwells of microtiter plates 2~.2~3~
The plates prepared in this way can be lyophi-lized and ~tored, preferably at 4~C. This step can be carried out on the industrial scale and i-t is thus possible to have ready-to-use plates for the assay kits 05 which can be applied to the granulocytes in the ~ample of blood or urine examined.
The samples containing the cell~ to be aæsayed, which originate from blood or from any appropriate bio-logical fluid - normal or pathological - in par-ticular urine, can be used as such or after preparation, especi-ally after concentration.
Aliquots of the appropriate cell suspen~ion are brought into contact with the solid support, for example in tubes containing the microparticulate immunocapture support or in the microwells of a microtiter plate pre-pared beforehand. After washing, the solution forming part of the assay kit and containing the substrate specific for the endogenous enzyme characteristic of the target cell population is added.
The time re~uired for immobilization of the cells is preferably less than or equal to 15 minute~ During this time, the solid support can be centrifuged to improve the immobilization of the cells. The solid support, for example the tube or microtiter plate, is then washed to remove the unfixed cells The appearance of a colored or fluorescent pro-duct is brought about by adding, to the ~olid support to which the cell population carrying the endogenous enzyme to be a~sayed has been fixed, a solution containing the substrate for the enzyme and, if necessary, one or more auxiliary reagents such that the reaction product which is finally obtained is either a colored product ~oluble in the medium, or an insoluble colored product, or a soluble fluore~cent product, a~ explained earlier. The light signal coming ~rom the sample~ treated in this way ~0~2~3~
is then measured with the equipment appropriate to each case, i.e. a tranæmi.ssion or reflection photometer or, respectively, a fluorimeter~ When the solid support is a microtiter plate, the light æignal can be read æe~uen-05 tially in all the wells of one and the æame plate bymeans of automated readeræ commonly used in biology laboratorie~, æuch asl fvr example, the Titertek plate reader or the Fluoro~can plate reader for the spectro-photometric or, respectively, fluorometric reading~.
The reagentæ used to develop the endogenous peroxidase or the endogenous myeloperoxidase 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 i9 colored and soluble in the medium, or elæe 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole or 4-chloro-alpha-naphthol to give an inæoluble final reaction pro-duct, or else parahydroxyphenylpropionic acid to give a fluorescent reaction product which i3 soluble in the medium.
In a preferred form, the kit according to the invention for assaying myeloperoxidase characteristic of the granulocytes compriseæ:
a) a microtiter plate in whose well~ one or more anti-granulocyte monoclonal antibodies have been fixed;
bl) a solution containing hydrogen peroxide, which is the substrate for the enzyme~ in an appropriate buffer; and b2) a solut.ion containing the chromo~en used to develop the expression of the activity of the enzyme and, if appropriate, a reagent which improves the permeability of the cell membrane.
In another preferred form, the kit according to the invention for asæaying myeloperoxidaæe characteristic 2~2~3~
of the granulocytes comprises, as the solid support, tubes or alternatively particu:Late magnetic supports to which one or more anti-granulocyte monoclonal antibodies have been fixed.
05 The results of the assay of the endogenous en-zymes according to the invention can be expressed accor-ding to any procedure appropriate to the examination carried out. More particularly, these results can he expressed as the total activity of a particular endo-genous enzyme present in a given volume of the sample examined (for example per microliter of blood).
The activity of a particular endogenous en~yme in the sample examined will preferably be determined u~ing a standard scale consisting of appropriate cells or prepa-rations containing the endogenous enzyme to be assayed,which will have been calibrated beforehand by a known reference method. These standards will preferably consist either of cells identical in their o~igin to the cells which are to form the subject of the assay, or of cells of established cell lines containing the de~ired endogenous enzyme, or of cell-free preparations contain-ing the chosen enzyme.
These standards are then treated in exactly the same way as the samples ~o be examined. The resulting signals are used to build up a standard scale against which the signal~ measured with the samples to be examined are compared. The subse~uent calculations are conventional.
The enzymatic assay method according to the invention is simple, rapid and reproducible. Its use is totally suitable for the analysis of a large number of samples. For an understanding o~ its advantages compared with the other methods described, the various steps should be analyzed.
Immobilization of the cells on the solid ~upport 2~12~
is the stage of the assay which usually present~ the mo~t difficulties or which is the most critical to carry out.
The means often used i8 chemical fixation of the cells with glutaraldehyde or methanol in cups which may or may 05 not have been treated with poly-L-lysine (VAN LEUVEN F.
et al , J. Immunol. Methods, 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 is a very serious disadvantage (DROVER and MARSHALL, J.
Immunol. Methods, 1986, 90, 275-281).
Furthermore, the chemical fi~ation method has to be carried out in several steps: 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 antigens which might be useful forimmunocapture of the cellss as well as the endogenous enzymes to be assayed.
Furthermore, the reproducibility of this method is doubtful; in fact, the settling of the cell~ in the assay microwells and the drying of the cells can vary from one experiment to the next. Finally, this assay is lengthy to perform because the cell drying st2p alone takes more than 2 hours.
The immobilization of lymphocyte populations ha~
also been achieved by using polyclonal antibodies adsor-bed in microwells (STOCKER and HEUSSER, J. Immunol.
Methods, 1979, 26, 87-95). This method makes it possible to immobilize cells foreign to the single population which it is desired to analyze; this also represents a certain disadvantage.
2~ 3~
The use of highly specific and related monoclonal antibodies adsorbed on or fixed to the ~olid support, and especially in the assay wells, in the tubes or on the particulate support, permits exclusive capture of the 05 desired cells, the other non-retained cell population~
being removed in the course of the washes carried out.
Furthermore, no chemical or physical agent modifies the characteristics of the antigens in this ~tep because the various operations for chemical or phy~ical 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 method which makes it possible to simpli-fy the step for immobilizing the cells carrying the enzyme to be assayed, while at the ~ame time making the results more reliable.
The method according to the invention, which com prises the use of a procedure for immunocapture of whole cells without physical or chemical intervention on the cells, and the measurement, in all or some of these cells, of the activity of an endogenous enzyme by using its specific substrate, is the first method to permit the quantitative assay of the chosen endogenous enzymes on the cells themselves.
According to the invention, the direct labeling of immunologically immobilized cells permits:
- a saving of reagents, - an improved reliability through a reduction in the number of steps and manipulation3;
- a time saving; and - the possibility of treating large numbers of samplss at the same time, exclusively with the use of conventional equipment and apparatuses.
The time required to immobilize the cell popula-tion or cell subpopulation to be assayed is short. It is 20:~2~
less than or equal to 15 minutes in the case of the assay of granulocytes in the blood or urine. Likewisel the period for assay of the activity of the endogenous enzyme is less than or equal to 1~ minutes.
05 After the solid support has been wa~hed, the actual assay is performed by using conventional appara-tuses to observe a signal which is precise and simple to measure: light absorptlon or emission.
Thus, overall, the method according to the inven-tion has numerous advantages: it is rapid, reliable, economic and simple.
It has been verified that the signals recorded (photometric measurements) make it possible to obtain satisfactory uniform ~tandard curves as a function of the number of cells used, under the customary handling con-ditions.
In the E~amples below~ the following termq or their abbreviations will be used indiscriminately:
BSA: bovine serum albumin PBS: phosphate buffered saline at pH 7.4 26 LEUKOCYTES OE HUM~N ~LOO~: ASSAY PERFORM~D QN A
Myeloperoxidase is an enzyme of glycoprotein character compri~ing a heme structure. This enzyme, which is abundant in the polynuclear leukocytes of human blood, is involved ln the bactericidal and antimicrobial functions of calls (KLEBANOFF S.J., J. Bacteriol., 1968, 95, 2131; DIAMOND et al., J. Clin. Invest., 1580, ~, 908-917).
The assay of myeloperoxidase in the polynuclear 3~ leukocytes, according to the mæthod of the invention, 3~
comprises three steps carried out in ~ucce~sion:
1. separation of the polynuclear leukocytes from whole blood, 2. capture and selective immobilization of the cella by 05 means of a monoclonal antibody adsorbed beforehand in the microwells of a microtiter plate, and 3. developing of the activity of the cell enzyme.
a) Preparation of the plate The plate used i~ a plastic microtiter plate con-taining 96 microwells, marketed by NUNC (reference 64394). Each microwell receives 200 ~1 of a solution containing the purified anti-CD15 monoclonal antibody (called SMY 15a) used to immobilize the polynuclear leukocytes, i.e. to effect their immunocapture. Thi~
antibody~ marketed by BIOSYS, Compiègne, France~ under the reference SMY 15a, i5 used at a concentration of 5 .g/ml in a phosphate buffered saline (PBS) at pH 7.4.
The adsorption of the monoclonal antibody i~
effected at 4C for 12 hours. The excess antibody is removed by turning the plate over.
A solution containing 0.1% of gelatin and 0.3% of BSA in a phosphate buffered saline is prepared. 250 ~1 of this solution are introduced into each microwell ~o 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 stored at 4C.
b) Separation of the polynuclear leukocytes The blood sample is taken on an anticoagulant ~heparin). 2 ml of MONO-POLY separating medium (FLOW
ref. 16.980-49) are introduced into a 5 ml hemolysis tube and 2 ml of blood are deposited on top. The tube is then centrifuged at 400 x g for 40 minutes at laboratory tem-perature. Two cell suspension rings are formed; the lower ring contains the polynuclear leukocytes, which are 2~ 2~3~
recovered with a micropipette c~ Cell immunocapture Cell capture is effected by means of the adsorbed anti-CD15 monoclonal antibody 05 100 ~l of the cell suspension, adjusted to 5 x 10~ cells per ml of PBS, are introduced into the micro-wells of the plate. To improve the fixation of tha cells on the support, the plate is centrifuged for 3 minutes at 150 x g after a wait of 10 minutes at room temperature.
d) Developing and measurement of the myeloperoxi-dase The microwells are emptied by turning the plate over. TheY are washed twice with 200 ~l of PBS, making it possible to remove the undesirable cell populations, such as the monocytes or erythrocytes, which can cause a ~purious absorbance signal. After the second wash, 100 ~l of the developing reagent~ prepared for immediate use~
are added to each well.
The developing reagent is obtained in the fol-lowin~ manner: a 0.1 M citrate buffer i~ prepared by dis-solving citric acid monohydrate in water to give a 2%
solution and adjusting the pH to ~ by the addition of 7 N
sodium hydroxide solution. A 2% solution of hexadecyl-trimethylammonium bromide (= CETAB, TOUZART and MATIGNON
T 5650) in the buffer is then prepared. The reagent renders the cell membranes permeable and improves the activity of the myeloperoxidase on the ~ub~trate. 30 mg of orthophenylenediamine dihydrochloride and then 40 ~l of hydrogen peroxide (substrate for the enzyme) are added to 20 ml of this solution before use After incubation for 10 minutes, the absorbance is measured on a spectrophotometer at 4~0 nm (type 310 C
Titertek Multiskan apparatus - Flow Laboratories).
~or one experiment 7 the absorbances obtained with 50,000 cells are given in Table 1 below:
3 ~
~hE~
CELLS ONLY CELLS CELLS
+ reagents ~ reagents 05 r _ without CETAB with CETAB
¦ Absorbance0.003 0.370 1.545 The presence of CETAB in the developing medium produces a 4-fold increa~e in the optical denslty due to the reaction catalyzed by myeloperoxidase, making it possible to reduce the number of cells required for the assay.
ASSAY OF MYELQPERQXIDASE IN THE POLYN~lCLEAR
l,EUKOCYTES OF BLOOD AND URINE: A~SAY PERFORME~ ON
This Example is intended to show that the time required to assay an endogenous enzyme can be reduced, compared with the conditions described in Example 1, by modifying the cell immunocapture process. The support used to capture the cells consists of magnetic beads known as DYNABEADS. The beads (ref. DYN-llOOl, marketed by BIOSYS) carry anti-mouso immunoglobulin antibodies on their surface. Before use, the beads are treated with a solution of anti-CD15 monoclonal antibodies (ref. SMY
15a - BIOSYS, France) for 12 hours at 4C. This is done by mixing 25 ~l of the suspension of beads with 0.5 ml of antibody solution containing 100 ~g~ml of PBS. The beads are subsequently washed 3 times with PBS and then satu-rated for 12 hours at 4C with a 0.3% solution of bovine serum albumin (BSA). The read~-to-use beads are kept at 4~C.
The myeloperoxidase is assayed in a 5 ml hemo-2012~3 !~
lysis tube, 300 ~1 of PBS, 25 ~1 of the suspension of beads and 100 ~1 of whole blood taken on lithium hepari-nate (VACUTAINER tube ref. 606 484) are introduced into the tube. After 5 minutes of gentle shaking, the beads 05 are separated off by means of a magnet and the cells fixed to the beads are washed with PBS (5 washes).
The myeloperoxidase is developed with a mixed reagent containing the substrate (hydrogen peroxide) and the chromogen (orthophenylenediamine), prepared as in Example 1, with or without the incorporation o~ CETAB.
The absorbance is measured under conditions identical to those of Ex~mple 1. The results are given in Table 2.
Measurement of the myeloperoxidases in the granulocytes present in urine during urinary infections is an important diagnostic tool for pyelonephritis and pyuria. It was shown in the second part of the Example that magnetic particles carrying the same anti-CD15 monoclonal antibody are capable of capturing the poly-nuclear leukocytes in the urinary fluid. The assay is performed on 400 ~1 of urinary fluid containing about 2 x 105 granulocytes per ml. The cells are captured with 25 ~1 of the suspension of beads and then treated under the same conditions as in the previous Example. The results are given in Table 2 below.
~LEæ
ABSORBANCE BEADS + BEADS + BEADS -~ BEADS
developing CELLS CELLS -~ CELLS ~
reagentonly developing developing reagent reagent without with CETAB CETAB
Whole n_ _ _ blood 0.0250.011 0.0323 1.324 . __ ... __ _ _._.
Urine 0.0250.008 0.286 1.115 2~12~3~
According to the method described in thi~
E~ample, the assay was performed direct on the blood sample and then on the urine sample without the need for prior separation of the cell population to be analyzed.
05 Furthermore, the incubation period is only 5 minutes.
Thus, by using magnetic bead~ as the solid support, the assay operations taken as a whole are particularly simple and rapid.
Claims (15)
1. A kit for assaying an endogenous enzyme characteristic of a cell population or subpopulation, said kit compri-sing 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;
b) one or more solutions providing the reagents (substrate and, if appropriate, chromogen) necessary for developing the activity of the enzyme; and c) an additional component consisting of a buffered washing solution and, if appropriate, 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;
b) one or more solutions providing the reagents (substrate and, if appropriate, chromogen) necessary for developing the activity of the enzyme; and c) an additional component consisting of a buffered washing solution and, if appropriate, samples permitting standardization and quality control of the assay
2. A kit according to claim 1, also comprising a reagent which improves the permeability of the cell membrane.
3. A kit according to claim 1 or claim 2 in which com-ponent (a) is a solid support consisting of a microtiter plate in whose wells the antibody or antibodies intended for immobilization of the cells, including those of the population or subpopulation containing the enzyme to be assayed, are fixed.
4. A kit according to claim 1 or claim 2 in which com-ponent (a) is a particulate magnetic support.
5. A kit according to any one of claims 1 to 3 in which component (a) is a tube.
6. A kit according to any one of claims 1 to 5 in which component (a) consists of a solid support to which one or more anti-class I HLA monoclonal antibodies are fixed.
7. A kit according to claim 6 in which the monoclonal antibody fixed is the antibody called S-class I.
8. A kit according to any one of claims 1 to 7 in which component (b) comprises essentially hydrogen peroxide and a chromogen selected from orthophenylenediamine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid, 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole or one of their water-soluble salts.
9. A method of assaying the endogenous enzymes of a cell population or subpopulation, said method consisting in:
a) immobilizing the cell population containing the enzyme to be assayed, or a cell population including the subpopulation containing the endogenous enzyme 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 recog-nizing an antigen present on the surface of the cells;
b) observing an incubation period;
c) washing the support to remove the non-immobilized cells, d) adding the reagent or reagents (substrate and chromogen) necessary for developing the activity of the endogenous enzyme; and e) reading the results by measuring the light signals (coloration or fluorescence) with reference, if appropriate, to a standard scale.
a) immobilizing the cell population containing the enzyme to be assayed, or a cell population including the subpopulation containing the endogenous enzyme 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 recog-nizing an antigen present on the surface of the cells;
b) observing an incubation period;
c) washing the support to remove the non-immobilized cells, d) adding the reagent or reagents (substrate and chromogen) necessary for developing the activity of the endogenous enzyme; and e) reading the results by measuring the light signals (coloration or fluorescence) with reference, if appropriate, to a standard scale.
10. An assay method according to claim 3 in which the solid support is as defined in any one of claims 3, 4 and 5.
11 An assay method according to claim 9 or claim 10 in which the activity of the enzyme is developed by hydrogen peroxide and a chromogen selected from orthophenylene-diamine, 2,2'-azino-bis(3-ethylbenzothiazoline-8-sulfonic) acid, 3,3'-diaminobenzidine, 3-amino-9-ethyl-carbazole or one of their water-soluble salts.
12. A method according to any one of claims 9, 10 and 11 in which the total time required to perform the assay is less than or equal to 30 minutes.
13. A method according to claim 9 in which the monoclonal antibody or antibodies fixed to the solid support are anti-class I HLA antibodies.
14. A method according to claim 9 in which the monoclonal antibody fixed to the solid support is the antibody called S-class I.
15. A method according to claim 9 for assaying the endo-genous myeloperoxidase in the granulocytes of human blood or of urine.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8903943A FR2644894B1 (en) | 1989-03-24 | 1989-03-24 | ENZYMATIC DOSAGE KIT AND METHOD APPLICABLE TO WHOLE CELLS |
| FR8903943 | 1989-03-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2012934A1 true CA2012934A1 (en) | 1990-09-24 |
Family
ID=9380065
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002012934A Abandoned CA2012934A1 (en) | 1989-03-24 | 1990-03-23 | Enzymatic assay kit and method applicable to whole cells |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0389381B1 (en) |
| JP (1) | JPH02298866A (en) |
| KR (1) | KR900014887A (en) |
| AT (1) | ATE113725T1 (en) |
| CA (1) | CA2012934A1 (en) |
| DE (1) | DE69013730T2 (en) |
| FI (1) | FI96723C (en) |
| FR (1) | FR2644894B1 (en) |
| IE (1) | IE66587B1 (en) |
| IL (1) | IL93862A0 (en) |
| PT (1) | PT93548A (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 |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19952155A1 (en) * | 1999-10-29 | 2001-05-03 | Volkswagen Ag | Cable, pipe or wire fastening clip for use in road vehicle has L-shaped bracket holding U-section clip member with curved arm defining space for cable |
| DK1353666T3 (en) | 2001-01-02 | 2013-10-14 | Cleveland Clinic Foundation | Myeloperoxidase, a risk indicator for cardiovascular disease |
| US7780950B2 (en) | 2002-01-02 | 2010-08-24 | The Cleveland Clinic Foundation | Systemic marker for monitoring anti-inflammatory and antioxidant actions of therapeutic agents |
| US7459286B1 (en) | 2003-10-22 | 2008-12-02 | The Cleveland Clinic Foundation | Assessing the risk of a major adverse cardiac event in patients with chest pain |
| SI2433138T1 (en) * | 2009-05-18 | 2017-03-31 | Technische Universitaet | Method for detecting a wound infection |
| CN104950111A (en) * | 2015-05-22 | 2015-09-30 | 北京协和洛克生物技术有限责任公司 | Liquid chip kit for quantitatively detecting concentration of myeloperoxidase (MPO) in sample and preparation method of liquid chip kit |
Family Cites Families (8)
| 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 |
| DE3438683A1 (en) * | 1984-10-22 | 1986-04-24 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR DETERMINING PEROXIDASE |
| 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 FR8903943A patent/FR2644894B1/en not_active Expired - Fee Related
-
1990
- 1990-03-22 PT PT93548A patent/PT93548A/en not_active Application Discontinuation
- 1990-03-23 EP EP90400805A patent/EP0389381B1/en not_active Expired - Lifetime
- 1990-03-23 IE IE108190A patent/IE66587B1/en not_active IP Right Cessation
- 1990-03-23 CA CA002012934A patent/CA2012934A1/en not_active Abandoned
- 1990-03-23 FI FI901468A patent/FI96723C/en not_active IP Right Cessation
- 1990-03-23 DE DE69013730T patent/DE69013730T2/en not_active Expired - Fee Related
- 1990-03-23 IL IL93862A patent/IL93862A0/en unknown
- 1990-03-23 AT AT90400805T patent/ATE113725T1/en not_active IP Right Cessation
- 1990-03-24 KR KR1019900004001A patent/KR900014887A/en not_active Application Discontinuation
- 1990-03-26 JP JP2076562A patent/JPH02298866A/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 |
|---|---|
| KR900014887A (en) | 1990-10-25 |
| IE901081L (en) | 1990-09-24 |
| JPH02298866A (en) | 1990-12-11 |
| FR2644894A1 (en) | 1990-09-28 |
| IL93862A0 (en) | 1990-12-23 |
| PT93548A (en) | 1990-11-07 |
| FI96723B (en) | 1996-04-30 |
| FI901468A0 (en) | 1990-03-23 |
| EP0389381A1 (en) | 1990-09-26 |
| FR2644894B1 (en) | 1994-05-13 |
| IE66587B1 (en) | 1996-01-24 |
| EP0389381B1 (en) | 1994-11-02 |
| ATE113725T1 (en) | 1994-11-15 |
| DE69013730T2 (en) | 1995-06-01 |
| FI96723C (en) | 1996-08-12 |
| DE69013730D1 (en) | 1994-12-08 |
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Legal Events
| Date | Code | Title | Description |
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| FZDE | Discontinued |