CH675309A5 - - Google Patents

Description

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CH 675 309 A5

Description

The present invention relates to reagents, kits and methods for immunoassay.

The present invention relates more particularly to a novel reagent for binding and detecting antibodies, as well as kits and methods incorporating this reagent. Assay methods for the binding of specific antibodies, also called immunoassay methods, constitute a rapid growth analytical technique, "used more widely for diagnosis and research, because of their high specificity and sensitivity.

The most recent versions of this technique which use radioisotopic markers have been increasingly replaced in recent years by enzyme immunoassay (EIA) techniques using enzyme markers, which techniques are also sensitive but safer, simpler and less expensive.

Among the EIA techniques, so-called “heterogeneous” test methods are commonly used using antibodies or antigens labeled with enzymes and fixed to (“immobilized on”) sensitized surfaces of solid supports, such as test tubes, beads. polystyrene or plates with hollow "wells". These techniques are generally designated by the abbreviation “ELISA” which means “enzyme-linked-immunosorbent assay” (enzyme linked immunosorbent assay). According to a modification, this technique is combined with the known use of a “second antibody” thus designated, namely an antiserum with the specific “first” antibody. The second antibody serves as a non-specific detector for any "first antibody" either in the free state or linked to its specific antigen in an antigen-antibody complex.

According to a version of the above method, which also reacts to the known technique of the "coated tube", an antigen is fixed to the inner wall surface of a test tube in which the binding reaction is then carried out. , after which the free specific antibody of said antigen present in the test sample will be fixed to the surface of the tube via the antigen immobilized thereon. Then, the liquid reaction mixture is removed from the test tube, the latter is washed and a second solution containing a second antibody labeled with enzymes is introduced into the tube. This second labeled antibody will also be immobilized by attaching it to any antibody that is bound to the surface of the tube via the antigen. The test tube is again emptied, rinsed and filled with a suitable substrate sensitive to the enzyme label of the second antibody and the enzyme activity is measured. This activity is directly proportional to the concentration of the antibody in the test sample.

In recent years, it has been found that the "second antibody" in immunoassay techniques can be replaced by a certain protein, namely the so-called "protein A" which is a main component of the cell wall of many Staphylococcus aureus strains. This protein A is covalently linked to the cell wall of staphyiococci but can be solubilized and is capable of binding to many classes of immuloblobulin molecules with a high affinity. It thus also binds to antibody-antigen complexes. Solubilized Protein A may further be labeled, for example, with radioactive iodine or enzymes, and enzyme labeled Protein A preparations are now becoming an increasingly common component in test kits immunological, in which they serve as a detector.

More recently, LARS BJORCK et AL. described in The Journal of Immunology, vol. 133 No. 2 August

1984, pages 969-974, their findings regarding the purification and properties of Streptococcus G protein as a new IgG binding receptor.

As described in this article, protein G, which is a protein of the wall of bacterial cells with affinity for immunoglobulin G (IgG), was isolated from a strain of human group G streptococci (G148) and it has been found to bind to all subclasses of human IgG and also to rabbit, mouse and goat IgG.

In another article by Bo Akerstrom et al, in The Journal of Immunology, vol. 135 no 4, October

1985, pages 2589 to 2592, we study the avidity of the G protein with regard to various monoclonal and polyclonal G-class Ig as well as the use of the radiolabelled G protein for the binding and detection of antibodies .

The present invention constitutes another important improvement of the ELISA, protein A and protein G techniques mentioned above and is based on the inventive concept according to which the enzyme-labeled protein A and the radiolabelled protein G can be replaced by non-viable and intact stabilized bacterial cells having an active receptor for immunoglobulin and an active labeling enzyme which has undergone appropriate pretreatment. So, for example, staphylococcus cells can effectively and quickly bind to immunoglobulin (as with any specific "first antibody") due to protein A present in their cell walls, and streptococcus G cells can similarly bind to the immunoglobulin due to the G protein present in their cell walls. At the same time, the enzyme marker is provided by enzymes which are naturally present in cells (i.e. endogenous or native enzymes), the activity of which can be easily measured, or alternatively in embodiments Particularly preferred of the present invention, as described below, the enzyme marker is the result of a gene for said enzyme which has been inserted into one of its parental cells.

Thus, the present invention provides an immunoassay reagent for binding and detection

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antibodies comprising stabilized non-viable bacterial cells having an active receptor for immunoglobulin and an active labeling enzyme.

In a first preferred embodiment of the present invention, this reagent comprises non-viable stabilized staphylococcus cells.

In another preferred embodiment of the present invention, this reagent comprises non-viable stabilized Streptococcus cells.

The above reagent proposed in accordance with the present invention, namely a suspension of properly treated bacterial cells, has considerable advantages as a "detector suspension" compared to all known detectors for antibodies or their complexes, in that it avoids the labor, time and costs involved in:

(a) isolating the binding protein or preparing the anti-immunoglobulins,

(b) the preparation of the enzyme to be used as a marker,

(c) the binding of the enzyme marker to the binding protein which is known to be a waste operation, and / or

(d) radiolabelling of proteins.

In light of the special properties of the reagent presently proposed, the invention also provides a test kit for an enzyme immunoassay, comprising one or more containers containing an aqueous suspension of stabilized non-viable bacterial cells having an active receptor for immunoglobulin and an active labeling enzyme, or a dry stabilized preparation of cells suitable for in situ reconstitution.

In accordance with another aspect of the invention, there is provided a test kit for carrying out an immunoassay comprising, in a conditioned combination, one or more solid supports on at least a portion of the surface of which is fixed a capture antibody or a hapten or antigen, the specific antibody of which is to be tested as well as one or more receptacles containing an aqueous suspension of the stabilized bacterial cells of the invention or a dry stabilized preparation of these cells suitable for an in situ reconstitution to obtain an aqueous suspension of cells.

On the basis of the above concept, the invention also provides, according to one of its aspects, a method of heterogeneous specific binding tests for determining an unbound antibody in a liquid medium, this method comprising the steps consisting in:

i) incubating a sample of said liquid medium in contact with a solid support on the surface of which is fixed an antigen or hapten specific for said antibody;

ii) separating the solid support of said sample from the liquid medium and rinsing it with an aqueous solution to remove all traces of said sample;

iii) incubating the solid support in contact with an aqueous suspension of non-viable stabilized bacterial cells having an active receptor for immunoglobulin and an active labeling enzyme;

iv) separating the solid support from said suspension and washing it with an aqueous solution; and v) incubating this solid support in contact with a suitable substrate and determining the enzymatic activity of the labeling enzyme on cells retained on the surface of the solid support.

The specific binding test method according to the invention can be applied to the determination of a hapten or antigen in a liquid medium by adding, to a sample of said liquid medium, an antibody specific for the hapten or antigen which is to be determined and by incubating the reaction mixture in contact with a solid support on the surface of which is fixed the same hapten or antigen which is to be determined, process in which, in step (i) is added to the test sample an antibody specific for said hapten or antigen, and, as a result of binding of said hapten or antigen in said sample, the amount of antibody available for binding to hapten or antigen attached to the solid surface is proportionally decreased. Steps ii) to v) described above are then carried out and the enzymatic activity which is determined in step v) is inversely proportional to the concentration of the hapten or antigen in the sample. This concentration can be calculated by methods well known in the immunoassay techniques, for example by comparison to a series of reference samples having known standard concentrations of the hapten or antigen.

The cell suspension of the detector of the present invention can similarly be used in immunological assays of the “sandwich” type, that is to say in assays in which a “capture antibody” is immobilized on a solid surface to capture antigen present in the test sample, and a "tracer" antibody is then applied to said antigen to form a "sandwich". The binding of the "tracer" antibody is revealed in a conventional manner by a marker incorporated into this antibody molecule.

In the present method, the detector suspension can be used to eliminate the need to label the second antibody. The only requirement is that the detector cells must not bind to the capture antibody. This requirement can be met by using antibodies which do not bind to

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protein A (eg IgY from chicken serum or egg yolk) or a modified antibody in which the Fc segment is missing (eg Fab or F (ab ') 2).

Selective binding to the second antibody can often be seen with standard antibody preparations showing that the capture antibody is fixed in a configuration which renders the immunoglobulin protein A binding fragment (Fc segment) unavailable for the connection of the detector cell.

A test kit suitable for carrying out the test method according to the invention for determining a hapten or antigen in a liquid medium can also comprise one or more containers containing standardized solutions of an antibody specific to said hapten or antigen which is to be determined. The test kits in accordance with the invention also optionally comprise reagents or auxiliary solutions conditioned to carry out the test process, such as buffer solutions, standardized reference solutions of the analyte to be determined, solutions of substrate and detector reagents for measuring the activity of the endogenous enzyme serving as a marker, etc. Such test kits which comprise a stabilized lyophilized preparation of staphylococcus aureus cells may also further comprise an aqueous medium conditioned for the formation of the required aqueous suspension of said cells in situ.

In preferred embodiments of the present invention there is also provided a test kit further comprising an absorbent reagent carrier having incorporated therein a reagent specific for said labeling enzyme.

As noted above, the staphylococcal cells of the present invention are properly treated before use so as to ensure that the cell suspension is healthy (i.e. non-pathogenic), standardized and stable, while retaining its capacity binding and the activity of the labeling enzyme which is chosen to serve as a marker. It has been found that these requirements can be met, for example by fixing the staph cells by treatment with a 0.5% formaldehyde solution at room temperature for 2 hours, followed by heating for about 5 to 15 min to about 50 ° -70 ° C. The treated cells can be stored, for example in the form of a 10% suspension with a preservative such as sodium azide at 4 ° C. or in the form of a lyophilized granule of staphylococcal cells stored in sealed ampoules. . Alternatively, a healthy and stable reagent can be obtained by lyophilizing a suspension of staphylococcal cells and storing the product in sealed ampoules.

The lyophilized cells can be reconstituted in situ for use by suspending them in a suitable liquid medium.

It should be noted that, although stabilized staphylococcus cells are commercially available, standard treatment for their stabilization normally inactivates any endogenous enzymes found thereon and thus non-viable stabilized staphylococcus cells having an active receptor for immunoglobulin and an active labeling enzyme have not been available to date and have not been suggested for use as reagents for immunoassays.

In one embodiment of the present invention, the labeling enzyme is an endogenous catalase and determines its catalase activity in staphylococcus cells.

In particularly preferred embodiments of the present invention, non-viable stabilized staphylococcus cells having an active receptor for immunoglobulin and an active labeling enzyme are used as reagents, the gene for said enzyme having been inserted into a parental cell. of the latter by genetic engineering techniques known per se.

Thus, for use in the present invention, a strain obtained by genetic engineering of Cowan Staphylococcus is prepared which differs from the original strain in that it contains a gene for the formation of β-lactamase.

The general advantage of using this approach to inserting a new gene lies in the fact that there is no need to use enzyme markers which happen to be present in the original strain. Thus, one can add to the existing repertoire of endogenous enzymes a new hereditary marker which is more suitable for an immunoassay.

The specific advantages of the gene for ß-lactamase are that it fulfills all of the following requirements:

1. The enzyme is largely linked to the cell.

2. The enzyme is completely accessible to the substrate.

3. The enzyme is very stable and its activity can be well preserved in the treatment of cells.

4. The enzyme has a very high rate of return.

5. The substrate is stable, inexpensive, and its use does not imply any health hazard or safety precautions.

6. The test for the ß-lactamase reaction is simple, rapid and very sensitive.

7. The test does not require any instrumentation and one can keep a permanent recording of the results without other additional manipulations.

As previously indicated, several types of Streptococcus pyogenes are known to produce

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re cell wall proteins that can serve as receptors for the Fc portion of immunoglobulins. The specificity of the receptor for such proteins can vary with the type of production strain. Of particular interest are Streptococci types C and G which produce powerful receptors of broad specificity. Such bacterial cells can be used as reagents in a manner analogous to that described for Cowan I cells. However, the following differences should be noted:

1. The broader specificity of streptococcus Fc receptors allows the extension of the present methodology to the detection of immunoglobulins which do not bind adequately to protein A.

2. Streptococcus cells do not contain catalase, but have a rich variety of other cell-binding enzymes, including hemolysins, which can serve as endogenous indicators of binding to the immunoglobulin molecule.

3. In analogy to the genetic modification of the Cowan strain, as described above, the insertion of suitable genes provides additional enzyme markers which may be advantageous in diagnostic kits for immunoassays.

From what is mentioned above, it is evident that streptococcal cells possessing Fc receptors can in themselves serve as a reagent source for detecting a variety of immunoglobulins. If preferable, such cells can be used in combination with the Cowan reagent described above. The combined use of the two types of cells can take one of several forms:

1. The reagent can consist of a mixture of cells of both types. Each type can carry its own distinctive enzyme marker or a single assay can be used if similar enzyme activity is chosen as the marker for both types of cells.

2. The mixed cells are linked together (for example with glutaraldehyde) to form a combined reagent, which now carries a single enzyme marker (for example ß-lactamase) but two kinds of Fc receptors.

3. A combined reagent is formed by interaction with the immunoglobulins present in the test or is deliberately added for this purpose.

Although the invention will now be described in conjunction with certain preferred embodiments in the following examples, so that certain aspects of the invention can be more fully understood and appreciated, the invention should not be limited to these forms of particular realization. On the contrary, it should be understood that it covers other variants, modifications and equivalents which may be included within the scope of the invention as defined by the appended claims. Thus, the following examples which include preferred embodiments serve to illustrate the practice of this invention, it being understood that the features shown are exemplary and only for the purpose of an illustrated discussion of the embodiments preferred of the present invention and are presented for the purpose of providing what is considered to be the most useful and easily understandable description of the formulation methods as well as aspects of principle and concepts of the invention.

EXAMPLE 1

Preparation of a stabilized suspension of Staphylococcus aureus cells

Staphylococcus aureus (Cowan I strain, ATCC No 125928) is grown in TSB (Trypticase Soybean Culture Broth, Difco), in 500 ml Erlenmeyer flasks, on an alternative shaker for 18 hours at 37 ° C. The cells are then centrifuged (10 min at 10,000 rpm), washed twice with phosphate buffered saline (PBS), at pH 7, and suspended in 10 volumes of PBS (pH 7.3) containing 0 , 5% formaldehyde. After 2 hours at room temperature, the cells are centrifuged and washed as above, and then suspended in 10 volumes of PBS (pH 7.3). The suspension is incubated for 10 min at 60 ° C with moderate shaking, centrifuged as above, and resuspended in 10 volumes of PBS (pH 7.3). Samples of this suspension, streaked on AB3 plates (Difco) did not show growth after 24 h at 37 ° C. After 12 months storage at 4 ° C, the suspension shows no sign of change in its binding and catalytic properties. Before use, the suspension is diluted 1:10 in PBS.

EXAMPLE 2

Preparation of a lyophilized stabilized staphylococcus aureus reagent

Staphylococcus (Cowan I strain, ATCC) is cultivated and collected as described in Example 1 below.

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above. The cells are washed with PBS, resuspended in 10 volumes of fresh TSB, incubated for 10 min at 60 ° C. and distributed in 0.5 ml portions in lyophilization vials. After lyophilization, the sealed ampoules are stored at room temperature (22-25 ° C). For use, the contents of one ampoule are suspended in 5.0 ml of PBS containing 0.01% sea thiolate.

EXAMPLE 3

Detection of rabbit anti-BSA

Microtitration plates (type U, Nunc) are activated by filling the wells with 100 μl of a 0.2% solution of glutaraldehyde in a borate buffer, at pH 9.0 and by incubation for 3 h at 56 °. vs. The plates are then washed 10 times with twice distilled water and coated with antigen by filling the wells with 100 μl of bovine serum albumin (BSA) (50 g / ml) by incubating for 2 h at 37 ° C and storing at 4 ° C for 18 h. After 3 washes with PBS, the unoccupied surfaces are blocked by filling the wells with 100 μl of ovalbumin (1.0 mg / ml) and by incubating for 2 h at 37 ° C., followed by 3 washes with PBS.

Control wells are prepared as above, omitting BSA.

An antibody to BSA (Anti-BSA) is prepared in rabbits. Normal rabbit serum (NRS) serves as a non-specific control. Serial dilutions are made in PBS and 100 µl of each is added to the antigen coated wells. After incubation for 30 min at 37 ° C, the wells are washed 3 times with PBS. For the detection of the antibody retained by the antigen, an aliquot (100 μl) of a cell suspension of staphylococci (prepared in accordance with examples 1 and 2 above) is added to each well. After incubation for 20 min. at 37 ° C, the wells are washed once with 1% Tween 20 and twice with PBS. The retention of cells by the bound antibody is detected by the activity of acid phosphatase or catalysis, in accordance with the method described in the specification of Israeli Patent No. 36,496.

RESULTS

The test system described above was used to compare the sensitivity of the detectors, namely the suspensions of staphylococcal cells prepared according to Examples 1 and 2 above, to the sensitivity of a protein A preparation. radioiodine, conventionally used in sensitive solid phase radioimmunoassays. The results are summarized in Table I.

Table 1

Detector prepared

Anti-BSA

Anti-BSA

Anti-BSA

NRS

in accordance with

1: 1000

1: 3000

1:10 000

1: 3000

Example 1

Pos.

Pos.

Pos.

Neg.

Example 2 *

Pos.

Pos.

Pos.

Neg.

Protein A

Pos.

Pos.

Neg.

Neg.

radioiodine

* Based on a catalase activity manifested 5 minutes after the addition of the substrate, namely 50 μl of a 6% solution of hydrogen peroxide.

EXAMPLE 4

A gene for the formation of constitutive penicillinase (pen 1) is inserted into the Cowan I strain of staphylococcus aureus. The gene is originally found in a plasmid (PI258, pen 1443) carried by the strain (RN 453) of staphylococcus aureus (Novick, RP, and Brodsky, R., 1972), Studies on replication of Plasmids ; Replication of Plasmids not established in S Aureus. J. Mol. Bio) 68: 285-302), and is transduced with phage o11. Transduction is as described by Novick (Novick, R.P., 1967), properties of a high frequency cryptic transduction phage in staphylococcus aureus; Viro-logy 33: 155-166. A stabilized suspension of these cells comprising β-lactamase as a labeling enzyme is prepared according to the procedure of Example I.

EXAMPLES

Detection of brucella antibodies in bovine sera

We activate a Petri dish made of polystyrene (standard size, miniplate) in 8 equidistant pre-selected places by placing 50 nl of a 25% solution of glutaraldehyde (Merck) at each place.

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After 150 sec. at 24 ° C., the dish is rinsed with water and 10 μl of the antigen suspension are added at each location. The antigen suspension consists of cells killed by heat (90 min at 72 ° C) of Brucella abortus. suspended in PS (0.5% phenol and 0.85% NaCl in distilled water) at an optical density corresponding to 200 Klett units. The places are rinsed thoroughly and the Petri dish is covered with 5 ml of a blocking solution consisting of BSA (20 mg / ml) in PBS. After 2 h at 37 ° and 16 h at 4 °, the blocking solution is removed and the Petri dish is rinsed twice with PBS, then twice with 0.05% Tween 20 in PBS and again with PBS. The dish is then air dried and 20 µl of bovine sera samples, diluted as shown in Table II, are added to the marked locations. After 60 min at 37 °, the dish is rinsed and washed as above, and each place receives 20 μl of a detector suspension. The detector suspension consists of the genetically modified staphylococcus cells of example 4 suspended in PBS to OD corresponding to 200 KU. After rinsing and drying in air, the β-lactamase activity of the detector cells retained at the locations is tested by placing a developer strip on each location. The developer strip is a section (12 × 15 mm) of Whatman No. 3 filter paper impregnated with a reagent solution containing iodine (12 mM), potassium iodide (63 mM) and soluble starch (1 , 0% w / w) and penicilin (50 mM) in PBS. The activity of the labeling enzyme of the detector cells, namely β-lactamase, is obtained by determining the time required for the appearance of a white circle in the center of the dark blue detector band. The white circle indicates that the iodine has been absorbed by pe-nicilloic acid and thus removed from the complex which gives the dark color to the detector band. Pe-nicilloic acid is the product of hydrolysis of a ß-lactam (here a penicillin) catalyzed by ß-lactamase. Therefore the discoloration rate of the circular area around the marked location is inversely proportional to the amount of ß-lactamase retained there.

Table 2

Serum n °

Agglutination title *

Supplement fixation title *

Title of this essay **

2

1:10

1: 5

1: 200

6

1:20

1: 5

1: 200

32

Neg.

Neg.

1: 1000

53

1:80

1: 5

1: 1000

77

Neg.

Neg.

Neg.

26

1:40

1:50

1: 2000

111

1: 1250

-

1: 16000

Normal

Neg.

Neg.

Neg.

Bovine serum

* As determined at the Kimron Veterinary Institute, Beth Dagan, Israel.

** The highest dilution (in PBS) which shows discoloration in less than 10 min.

It will be obvious to a person skilled in the art that the invention is not limited to the details of the preceding illustrative examples and that the present invention can be implemented in other specific forms without departing from its attributes essential and it is therefore desired that the present embodiments and examples are considered, in all respects, as illustrative and not limiting, reference being made to the appended claims, rather than to the preceding description, and all changes which fall within the meaning of equivalence of claims are therefore considered to be presently included.

Claims (17)

Claims 1. Immunoassay reagent for binding and detecting antibodies, comprising stabilized non-viable bacterial cells having an active receptor for immunoglobulin and an active labeling enzyme. 2. An immunoassay reagent for binding and detecting antibodies according to claim 1, comprising stabilized non-viable staphylococcus cells. 3. A reagent for antibody binding and antibody detection tests according to claim 1, comprising non-viable stabilized Streptococcus cells. 4. Kit for enzyme immunoassays comprising one or more containers containing an assay reagent according to claim 1 in the form of an aqueous suspension of stabilized non-viable bacterial cells having an active receptor immunoglobulin and an active labeling enzyme or of a dry stabilized preparation of such cells, suitable for an in situ reconstitution. 7 5 10 15 20 25 30 35 40 45 50 55 CH 675 309 A5 5. An assay kit according to claim 4, in which said cells are a Cowan I strain of staphylococcus aureus cells containing a gene for the formation of β-lactamase, the gene for said enzyme having been inserted into one of the parental cells. 6. An assay kit according to claim 4, wherein said cells are G148 group G streptococcus strain cells. 7. Test kit according to claim 4, further comprising one or more solid supports on at least a part of the surface of which is fixed a capture antibody or a hapten or antigen, the specific antibody of which is to be determined. 8. Test kit according to claim 4, which further comprises one or more containers containing standardized solutions of a specific antibody against a hapten or antigen to be determined. 9. Test kit according to claim 4, which further comprises an absorbent reactive support incorporating thereon a specific reagent of said labeling enzyme. 10. A heterogeneous specific binding assay method for determining an unbound antibody in a liquid medium, using a reagent according to claim 1 comprising the steps consisting in: i) incubating a sample of said liquid medium in contact with a solid support on the surface of which is fixed an antigen or hapten specific for said antibody; ii) separating the solid support from said sample of liquid medium and rinsing it with an aqueous solution to remove all traces of said sample; iii) incubating the solid support in contact with the reagent in the form of an aqueous suspension of non-viable stabilized bacterial cells having an active receptor for immunoglobulin and an active labeling enzyme; iv) separating the solid support from said suspension and washing it with an aqueous solution; and v) incubating said solid support in contact with a suitable substrate and determining the enzymatic activity of the labeling enzyme on cells retained on the surface of solid support. 11. Method for determining a hapten or antigen in a liquid medium by means of a reagent according to claim 1, comprising the steps consisting in: i) incubating a sample of said liquid medium in contact with a solid support on the surface of which is fixed an antigen or hapten specific for said antibody; ii) separating the solid support from said sample of liquid medium and rinsing it with an aqueous solution to remove all traces of said sample; iii) incubating the solid support in contact with an aqueous suspension of stabilized non-viable bacterial cells having an active receptor for immunoglobulin and an active labeling enzyme; iv) separating the solid support from said suspension and washing it with an aqueous solution; and v) incubating said solid support in contact with a suitable substrate and determining the enzymatic activity of the labeling enzyme on cells retained on the surface of solid support. 12. The method of claim 10, wherein said aqueous suspension contains staphylococcus cells stabilized by heat treatment and formaldehyde to maintain their binding capacity and the activity of the labeling enzyme. 13. The method of claim 12, wherein said labeling enzyme is an endogenous catalase and the catalase activity of the staphylococcus cells is determined. 14. The method of claim 10, comprising incubating said solid support with an aqueous solution of non-viable stabilized staphylococcal cells having an active receptor for immunoglobulin and an activating labeling enzyme, the gene for said enzyme having been inserted into one of the parental cells. 15. The method of claim 10, wherein said aqueous suspension contains stabilized Streptococcus cells. 16. The method of claim 15, wherein said labeling enzyme is endogenous hemolysin and the hemolysin activity of Streptococcus cells is determined. 17. The method of claim 10, wherein said aqueous suspension contains a Cowan I strain of staphylococcus aureus containing a gene for the formation of β-lactamase, the gene for said enzyme having been inserted into a parental cell thereof. 8