IE901081L - Enzymatic assay kit and method applicable to whole cells - Google Patents

Abstract

The invention relates to a kit for determination of the endogenous cell enzymes characteristic of a cell population or subpopulation, comprising, as components: a) a solid support to which one or more monoclonal antibodies directed towards surface antigens of the cell population to be determined are bound by covalent bonding or by physical adsorption; b) one or more solutions supplying the necessary reagents (substrate and chromogen) for visualising the activity of the endogenous enzyme to be determined. The invention also relates to a method for enzymatic determination of the endogenous enzymes of a cell population or subpopulation. The kit for determination and the enzymatic method according to the invention may be used for the determination of myeloperoxidase in the granulocytes in the blood or urine.

Description

_ !_ 66587 EMZIMAIIC^ASSAY KIT AND METHOD APPLICABT.F.

TO WHOLF. CF.T.T.S The present invention relates to a kit and to an enzymatic assay method using this kit for assaying endogenous enzymes characteristic of cell populations or subpopulations. The enzymatic assay method and the corresponding kit are also intended for the assay of the cells themselves via assay of their endogenous enzymes. These assays are applicable to diagnosis.

The method according to the invention consists in specifically immobilizing a cell population by immunocapture on a solid support and then in assaying an endogenous enzyme specific for this population or for a cell subpopulation by using a substrate appropriate for this enzyme and, if appropriate, the necessary cofactors or auxiliary substances.

According to the present invention, in the description and the claims which follow, the term "endogenous enzyme" is understood as meaning an enzyme which is contained in the cytoplasm of the cell and whose substrate is a lovv-molecular molecule capable of penetrating inside the cell, where it will be transformed by the enzyme, thereby releasing into the medium a colored or fluorescent reaction product which permits the final measurement of a signal proportional to the activity of the enzyme.

Cell immunocapture is a process which consists in retaining the cells on a solid support by binding between one or more surface antigens of these cells and one or more monoclonal antibodies specific for this antigen or these antigens.

Knowledge of cell surface antigens or markers has made enormous advances with the development of lymphocyte hybridization and the discovery of monoclonal antibodies by KOEHLER and MILSTEIN (Nature, 1975, 256. 495-497). In particular, monoclonal antibodies have made it possible to reveal and analyze surface markers or membrane antigens of cells of the widest possible variety of origins. These markers (or antigens) can be of different kinds: proteins, glycoproteins or glvcolipids. The characterizations sought therefore apply mainly to tissue or organ markers, to markers of states of differentiation or activation of normal cells and to the identification or typing of normal or cancerous cells. A particularly important field of application is the study of the cell lines of hemopoiesis (erythrocyte, megakaryocyte, granulocyte, monocyte, lymphocyte).

Thus, for example, monoclonal antibodies have made it possible to specify the respective surface characteristics of T and B lymphocytes. 6 6 5 8 7 The corresponding markers, by themselves or in combination, identify stages of differentiation and functional specialization of the lymphocytes. By international convention, the surface markers of human leukocytes have been classified in differentiation groups or differentiation classes (DC) 5 defined by the IUIS-WHO subcommittee, 1984, 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 10 surface antigens, but these methods are often lengthy, laborious and difficult to carry out and their results are sometimes random.

One group of methods for the measurement of antigens is based on quantitative evaluation of the surface markers of the overall cell population. These methods make it possible to measure the antigens either 15 by direct labeling or by indirect labeling, the latter most frequently being carried out in two, three or four steps. In all cases, the reagent employed in the last labeling step carries a probe which is either of isotopic character, for example iodine 125, for an assay of the radioimmunometric type (BROW et al., J. Immunol. Methods, 1979, 31, 201, - STOCKER et HEUSSER J. 20 Immunol. Methods, 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, 1978, 23, 109-113 - MORRIS, Transplantation, 1983, 36(6), 719 - BAUMGARTEN, J. Immunol. Methods, 1986, 94, 91-98). 25 It will 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 30 equipment.

These methods are rather inconvenient, laborious and risky to apply because of the need to wash and centrifuge the cell material many times; it is sometimes necessary to take a sample of the colored medium resulting from the enzyme reaction in order to carry out the final 35 spectrophotometry measurement; lastly, chemical fixation of the cells, which is used in most cases, causes irreversible destruction of certain antigens which are particularly sensitive to the customary chemical fixatives such as glutaraldehyde or methanol (DROVER et al., J. Immunol. Methods, 1986,90,275-281).

Cell immunocapture on a solid support is described 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 structure, called a network-relay, which is capable of ensuring a preferential orientation of the antibody relative to that of the corresponding cell antigen. Said application gives no indication of an application of the technique to the quantitative assay of a cell enzyme.

Cell immunocapture is also described in patent application WO 84/03151 for an analytical application. In said application, the object is to identify the tissue groups to which the examined cells belong (this operation generally being called HLA typing). Cell capture is effected by means of antibodies arranged according to a particular geometry on very specialized supports (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 permitting the quantitative determination of a marker specific for 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 techniques known and used in the prior art, since it permits quantitative measurement of any endogenous enzyme of a cell population. This assay is performed on cells which have not undergone any chemical or physical intervention 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 has the characteristics of very high specificity which are inherent in the double recognition systems involving two 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 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 purposes in clinical biology laboratories handling these large numbers.

The present invention thus relates to a kit for assaying at least one endogenous enzyme characteristic of a cell population or subpopulation comprising the following components: a) a solid support to which one or more monoclonal antibodies are fixed by covalent bonding or physical adsorption, said monoclonal antibodies being directed against surface antigens of the cell population examined, and being intended for immobilization, on the support, of the cells which include those of the subpopulation possessing the enzyme to be assayed; b) a solution containing the substrate for the enzyme for developing the activity of the enzyme, said substrate being a low-molecular molecule capable of penetrating inside said cell population; c) a buffer solution intended for washing of the solid support.

The kit may also comprise a solution containing a chromogenous reagent for developing the activity of the enzyme, and samples permitting standardization and quality control of the assay.

The term "cell" as used in the present description and in the claims which follow encompasses human cells or animal cells and especially blood cells, including 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 immunocapture and they are used in a state of complete physiological integrity. This situation constitutes the best 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 use any device suitable for handling cell suspensions, and preferably tubes, particulate magnetic supports or rigid or flexible microtiter plates made of polyethylene, polystyrene, polyvinyl chloride or nitrocellulose, which contain microwells. The monoclonal antibodies intended for immobilization of the cells can be 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 techniques 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 antibody or antibodies 5 fixed to the solid support must permit immunocapture of the cell 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 immunocapture are the anti-class I HLA antibodies which are specific for the common part of the HLA-A, -B and -C 10 antigens present on the leukocytes and numerous other cell lines of the organism. Likewise, monoclonal antibodies specific for the antigens of the differentiation classes which have 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 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 according to the invention.

The substrate for the endogenous enzyme to be assayed and the 20 reagents are chosen so that the final product of the reaction or reaction sequence caused by the enzyme, involving these substances, is: - either a colored or fluorescent substance which diffuses into the liquid medium surrounding the cells and which is the object of the final spectrophotometric or, respectively, fluorimetric measurement, 25 - or an insoluble colored substance which deposits on the cells and the walls to which they are fixed, and which can be the object either of photometric measurement by reflection or of visual evaluation, if appropriate against a scale of standard shades.

Thus it is not necessary to use a chromogen if the substrate 30 alone makes it possible to obtain a colored or fluorescent substance.

As an additional component, the assay kit contains a buffer solution intended for washing of the solid support after immobilization of the cells.

As other additional components, the assay kit can also contain 35 the samples necessary for standardization and quality control of the assay. 6 The present invention further relates to a meihod of assaying the endogenous enzymes of a cell population or subpopulation, characterized in that it consists 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 activity of the endogenous enzyme; - reading the results by measuring the light signals (coloration or fluorescence) with reference, if appropriate, to a standard scale.

Thus the developing of the endogenous enzyme to be assayed, which belongs to the immobilized cell population or to one of its subpopulations, is effected direct by means of a substrate specific for this endogenous enzyme, after which the actual assay of the endogenous enzyme is performed by photometric measurement by transmission or reflection, or measurement of the fluorescence emission.

The assay kit and method according to the invention are particularly simple because, when preprepared immunocapture supports are available, they involve only the solution or solutions containing the specific reagents necessary for measuring 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 enzymes of the formed elements of human blood, especially the leukocytes and more particularly the granulocytes.

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 concentration of the endogenous enzyme measured in these cells. Measurement of these signals permits quantitative evaluation 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, on a single plate, of a series of endogenous enzymes characteristic of various subpopulations making up the cell population examined. For this application, it is possible on the one hand to take ready-to-use microtiter plates to which one or more monoclonal antibodies 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 subpopulations to be evaluated. Thus, on one and the same support, it is possible to perform the quantitative assay of all the endogenous enzymes necessary for characterization of the chosen subpopulations.

A case which may be mentioned as an application of the invention is that of the human leukocytes, for which there are in particular the subpopulation of cells called polvnuclear leukocytes or granulocytes.

The presence of human granulocytes can be evaluated in blood or in urine. Thus, in the case of urinary infections such as cystitis or pyelonephritis, it is particularly valuable to have a simple method of assaying the granulocytes in urine.

The assay kit and the enzymatic method according to the invention can be used for assaying granulocytes. In this case, specific immobilization of the granulocytes in the sample examined is 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 granulocytes and its assay is performed using hydrogen peroxide as the substrate for the peroxidase.

Specific immobilization of the granulocytes is effected using anti-DC 15 monoclonal antibodies fixed to a solid support such as, for example, the walls of the microwells of microtiter plates.

The plates prepared in this way can be Ivophilized and stored, preferably at 4"C. This step can be carried out on the industrial scale and it is thus possible to have ready-io-use plates for the assay kits which can be applied to the granulocytes in the sample of blood or urine examined. 8 The samples containing the cells to be assayed, which originate from blood or from any appropriate biological fluid - normal or pathological - in particular urine, can be used as such or after preparation, especially after concentration.

Aliquot portions of the appropriate cell suspension 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 prepared 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 required for immobilization of the cells is preferably less than or equal to 15 minutes. 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 product is brought about by adding, to the solid support to which the cell population carrying the endogenous enzyme to be assayed 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 soluble in the medium, or an insoluble colored product, or a soluble fluorescent product, as explained earlier. The light signal coming from the samples treated in this way is then measured with the equipment appropriate to each case, i.e. a transmission or reflection photometer or, respectively, a fluorimeter. When the solid support is a microtiter plate, the light signal can be read sequentially in all the wells of one and the same plate by means of automated readers commonly used in biology laboratories, such as, for example, the Titertek plate reader or the Fluoroscan plate reader for the spectrophotometric or, respectively, fluorometric readings.

The reagents 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 orthophenylenediamine or 2,2'-azino-bis(3-ethylbenzothiazoline-S-sulfonic acid), or ABTS, to give a final reaction product which is colored and soluble in the medium, or else 3,3'-diaminobenzidine, 3-amino-9- ethvlcarbazole or 4-chloro-alpha-naphthol to give an insoluble final reaction product, or else parahvdroxvphenylpropionic acid to give a fluorescent reaction product which is soluble in the medium.

In a preferred form, the kit according to the invention for assaying myeloperoxidase characteristic of the granulocytes comprises: a) a microtiter plate in whose wells one or more anti-granulocvte monoclonal antibodies have been fixed; b1) a solution containing hydrogen peroxide, which is the substrate for the enzyme, in an appropriate buffer; b2) a solution containing the chromogen 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 embodiment, the kit according to the invention for assaying myeloperoxidase characteristic of the granulocytes comprises, as the solid support, tubes or alternatively particulate magnetic supports to which one or more anti-granulocvte monoclonal antibodies have been fixed.

The results of the assay of the endogenous enzymes according to the invention can be expressed according to any procedure appropriate to the examination carried out. More particularly, these results can be expressed as the total activity of a particular endogenous enzyme present in a given volume of the sample examined (for example per microliter of blood).

The activity of a particular endogenous enzyme in the sample examined will preferably be determined using a standard scale consisting of appropriate cells or preparations 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 origin to the cells which are to form the subject of the assay, or of cells of established cell lines containing the desired endogenous enzyme, or of cell-free preparations containing the chosen enzyme.

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 which the signals measured with the samples to be examined are compared. The subsequent 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 of its advantages compared with the other methods described, the various steps should be analyzed. 5 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 polv-L-lvsine (VAN LEUVEN F. et al., J. Immunol. Methods, 10 1978, 23, 109). However, chemical fixations performed in this way can reduce or even suppress the desired specific detection or, conversely, 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 fixation method has to be carried out 15 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 37*C, 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 37*C 20 can degrade certain fragile antigens which might be useful for immunocapture of the cells, as well as the endogenous enzymes to be assayed.

Furthermore, the reproducibility of this method is doubtful; in fact, the settling of the cells in the assay microwells and the drying of the cells 25 can vary from one experiment to the next. Finally, this assay is lengthy to perform because the cell drying step alone takes more than 2 hours.

The immobilization of lymphocyte populations has also been achieved by using polyclonal antibodies adsorbed in microwells (STOCKER and HEUSSER, J. Immunol. Methods, 1979, 26, 87-95). This method 30 makes it possible to immobilize cells foreign to the single population which it is desired to analyze; this also represents a certain disadvantage.

The use of highly specific and affine monoclonal antibodies adsorbed on or fixed to the solid support, and especially in the assay wells, in the tubes or on the particulate support, permits exclusive capture of the 35 desired cells, the other non-retained cell populations being removed in the course of the washes carried out. Furthermore, no chemical or physical 11 agent modifies the characteristics of the antigens in this step because the various operations for chemical or physical fixation of the cells to the support are omitted.

Thus, according to the present invention, it has been found that the immobilization of cells by monoclonal antibodies is a method which makes it possible to simplify the step for immobilizing the cells carrying the enzyme to be assayed, while at the same time making the results more reliable.

The method according to the invention, which comprises 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 manipulations; - a time saving; - the possibility of treating large numbers of samples at the same time, exclusively with the use of conventional equipment and apparatuses.

The time required to immobilize the cell population or cell subpopulation to be assayed is short. It is less than or equal to 15 minutes in the case of the assay of granulocytes in the blood or urine. Likewise, the period for assay of the activity of the endogenous enzyme is less than or equal to 15 minutes.

After the solid support has been washed, the actual assay is performed by using conventional apparatuses to observe a signal which is precise and simple to measure: light absorption or emission.

Thus, overall, the method according to the invention 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 standard curves as a function of the number of cells used, under the customary handling conditions.

In the Examples below, the following terms or their abbreviations will be used indiscriminately: BSA: bovine serum albumin PBS: phosphate buffered saline at pH 7.4 EXAMPLE 1 ASSAY OF MYELOPEROXIDASE IN THE POLYNUCLEAR LEUKOCYTES OF HUMAN BLOOD: ASSAY PERFORMED ON A MICROTITER PLATE Myeloperoxidase is an enzyme of glycoprotein character comprising a heme structure. This enzyme, which is abundant in the polvnuclear leukocytes of human blood, is involved in the bactericidal and antimicrobial functions of cells. (KLEBANOFF S.J., J. Bacterid., 1968, 95, 2131 - DIAMOND et al., J. Clin. Invest., 1980, 66, 908-917).

The assay of myeloperoxidase in the polvnuclear leukocytes, according to the method of the invention, comprises three steps carried out in succession: 1. separation of the polvnuclear leukocytes from whole blood, 2. capture and selective immobilization of the cells by means of a monoclonal antibody adsorbed beforehand in the microwells of a microtiter plate, 3. developing of the activity of the cell enzyme. a) Preparation of the plate The plate used is a plastic microtiter plate containing 96 microwells, marketed by NUNC (reference 64394). Each microwell receives 200 jul of a solution containing the purified anti-DCl5 monoclonal antibody (called SMY 15a) used to immobilize the polvnuclear leukocytes, i.e. to effect their immunocapture. This antibody, marketed by BIOSYS, Compiegne, France, under the reference SMY 15a, is used at a concentration of 5 /ig/ml in phosphate buffered saline (PBS) at pH 7.4.

The adsorption of the monoclonal antibody is effected at 4"C for 12 hours. The excess antibody is removed by turning the plate over.

A solution containing 0.1% of gelatin and 0.3% of BSA in phosphate buffered saline is prepared. 250 /j\ of this solution are introduced into each microvt/ell so as to saturate the surface of the wells with protein, 13 which takes 1 hour at 37*C; the plates are washed 3 times with phosphate buffered saline. The plates prepared in this way are stored at 4"C. b) Separation of the polynuclear leukocytes The blood 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 then 2 ml of blood are deposited therein. The tubs is then centrifuged a 400 x g for 40 minutes at laboratory temperature. Two cell suspension rings are formed; the lower ring contains the polynuclear leukocytes, which are recovered with a micropipette. c) Cell immunocapture Cell capture is effected by means of the adsorbed anti-DC15 monoclonal antibody. 100 fj\ of the cell suspension, adjusted to 5 x 105 cells per ml of PBS, are introduced into the microwells of the plate. To improve the fixation of the 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 myeloperoxidase The microwells are emptied by turning the plate over. They are washed twice with 200 /jI of PBS, making it possible to remove the undesirable cell populations, such as the monocytes or erythrocytes, which can cause a spurious absorbance signal. After the second v/ash, 100 fjtl of the developing reagent, prepared for immediate use, are added to each well.

The developing reagent is obtained in the following manner: a 0.1 M citrate buffer is prepared by dissolving citric acid monohydrate in water to give a 2% solution and adjusting the pH to 5 by the addition of a 7 N solution of sodium hydroxide. A 2% solution of hexadecvltri-methylammonium bromide (= CETAB, TOUZART and MATIGNON T 5350) in the buffer is then prepared. The reagent renders the cell membranes permeable and improves the activity of the myeloperoxidase on the substrate. 30 mg of orthophenvlenediamine dihvdrochloride and then 40 /Jl 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 450 nm (type 310 C Titertek iVfultiskan apparatus -Flow Laboratories). 14 For one experiment, the absorbances obtained with 50,000 cells are given in Table 1 below: TABLE 1 CELLS ONLY CELLS -r reagents without CETAB CELLS + reagents with CETAB Absorbance 0.003 0.370 1.545 The presence of CETAB in the developing medium produces a 4-fold increase in the optical density due to the reaction catalyzed by myeloperoxidase, making it possible to reduce the number of cells required for the assay.

EXAMPLE 2 ASSAY OF MYELOPEROXIDASE IN THE POLYNUCLEAR LEUKOCYTES OF BLOOD AND URINE: ASSAY PERFORMED ON MAGNETIC BEADS 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-11001, marketed by BIOSYS) carry anti-mouse immunoglobulin antibodies on their surface. Before use, the beads are treated with a solution of anti-DCI 5 monoclonal antibodies (ref. SMY 15a - BIOSYS, France) for 12 hours at 4*C. This is done by mixing 25 }Jt\ of the suspension of beads with 0.5 ml of antibody solution at a concentration of 100 jug/ml of PBS. The beads are subsequently washed 3 times with PBS and then saturated for 12 hours at 4*C with a 0.3% solution of bovine serum albumin (BSA). The ready-to-use beads are kept at 4*C.

The myeloperoxidase is assayed in a 5 ml hemolysis tube; 300 /Lil of PBS, 25 /Lil of the suspension of beads and 100 /jI of whole blood taken on lithium heparinate (VACUTAINER tube ref. 606 484) are introduced into the tube. After 5 minutes of gentle shaking, the beads 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 (orthophenvlenediamine), prepared as in Example 1, with or without the incorporation of CETAB. The absorbance is measured under conditions identical to those of Example 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-DC15 monoclonal antibody are capable of capturing the polynuclear leukocytes in the urinary fluid. The assay is performed on 400 /jI of urinary fluid containing about 2 x 10s granulocytes per ml. The cells are captured with 25 fiI 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.

TABLE 2 ABSORBANCE BEADS + BEADS-f BEADS+ BEADS+ developing CELLS only CELLS + CELLS + reagent developing developing reagent without reagent with CETAB CETAB Whole blood 0.025 0.011 0.0323 1.324 Urine 0.025 0.008 0.286 1.115 According to the method described in this Example, 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. Furthermore, the incubation period is only 5 minutes. Thus, by using magnetic beads as the solid support, the assay operations taken as a whole are particularly simple and rapid. 16

Claims (19)

CLAIMS;

1. Kit for assaying an endogenous enzyme characteristic of a cell population or subpopulation comprising the following components: a) a solid support to which one or more monoclonal 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) a solution containing the substrate for the enzyme for developing the activity of the enzyme, said substrate being a low-molecular weight molecule capable of penetrating inside said cell population; c) a buffer washing solution.

2. Kit according to claim 1 also comprising a chromogenous reagent for developing the activity of the enzyme.

3. Kit according to one of claims 1 and 2 also comprising samples permitting standardization and quality control of the assay.

4. Kit according to any one of claims 1 to 3 also comprising a reagent which improves the permeability of the cell membrane.

5. Kit according to any one of claims 1 to 4 in which component (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.

6. Kit according to any one of claims 1 to 4 in which component (a) is a particulate magnetic support.

7. Kit according to any one of claims 1 to 4 in which component (a) is a tube.

8. Kit according to any one of claims 1 to 7 in which component (a) consists of a solid support to which one or more anti-class I HLA monoclonal antibodies are fixed.

9. Kit according to any one of claims 2 to 8 in which component (b) comprises essentially hydrogen peroxide and a chromogen selected from orthophenvlenediamine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 3,3-diaminobenzidine, 3-amino-9-eihvlcarbazole or one of their Wetter-soluble salts. 17

10. Method of assaying the endogenous enzymes of a cell population or subpopulation, characterized in that it consists 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 recognizing 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 a solution containing the substrate for the enzyme as defined in claim 1 for developing the activity of the enzyme; e) reading the results by measuring the light signals (coloration or fluorescence).

11. Method according to claim 10, characterized in that a chromogen is added in step d).

12. Method according to claim 10 or claim 11, characterized in that the results are obtained with reference to a standard scale.

13. Assay method according to any one of claims 10 to 12 in which the solid support is as defined in any one of claims 5, 6 or 7.

14. Assay method according to any one of claims 11 to 13 in which the activity of the enzyme is developed by hydrogen peroxide and a chromogen selected from orthophenvlenediamine, 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid), 3,3'-diaminobenzidine, 3-amino-9-ethyl-carbazole or one of their water-soluble salts.

15. Method according to any one of claims 10 to 14 in which the total time required to perform the assay is less than or equal to 30 minutes.

16. 13. Method according to claim 10 in which the monoclonal antibody or antibodies fixed to the solid support are anti-class I HLA antibodies.

17. Method according to claim 11 for assaying the endogenous myeloperoxidase in the granulocytes of human blood or of urine. - 18 -

18. A kit for assaying an endogenous enzyme characteristic of a cell population or subpopulation according to claim 1 s substantially as herein described in the Examples,

19. A method of assaying the endogenous enzymes of a cell population or subpopulation according to claim 109 substantially as herein described in the Examples- MACLACHLAN & DONALDSON, Applicants® Agents, 47s Merrion Square. DUBLIN 2.