GB2197468A

GB2197468A - Immunoassay reagents, kits and methods

Info

Publication number: GB2197468A
Application number: GB08721276A
Authority: GB
Inventors: Nathan Citri
Original assignee: Yissum Research Development Co of Hebrew University of Jerusalem
Current assignee: Yissum Research Development Co of Hebrew University of Jerusalem
Priority date: 1986-09-19
Filing date: 1987-09-10
Publication date: 1988-05-18
Anticipated expiration: 2007-09-10
Also published as: NL8702235A; AU7862387A; FR2604259A1; AU594503B2; FR2604259B1; GB2197468B; CA1294871C; IL80313A0; IT8721950A0; DE3731227A1; CH675309A5; IT1223303B; GB8721276D0

Description

1 GB2197468A 1

SPECIFICATION

Immunoassay reagents, kits J and methods The present invention relates to immunoassay reagents, kits and methods, More particularly the present invention relates to a noval reagent for binding and detection of antibodies and to kits and methods incorporating the same.

Specific antibody binding assay methods, also called immunoassay methods, are a rapidly emerging analytical technique, most widely used in diagnosis and research, owing to their high specificity and sensitivity. The earlier versions of this technique which used radioisotopic labels 10 have in recent years been increasingly replaced with enzyme immunoassay(EIA) techniques using enzyme labels, which techniques are equally sensitive but safer, simpler and cheaper.

Among the EIA techniques there are commonly used the so-called "heterogenous" assay methods emplo-ing enzyme-labeled antibodies or antigens attached to ("immobilized on") sensi tized surfaces of solid carriers, such as test tubes, polystyrene beads or plates provided with recessed "wells". Such techniques are generally referred to by the abbreviation "ELISA" which stands for "enzyme-linked immunosorbent assay". In accordance with one modification, this technique is combined with the known use of a so-called "second antibody", namely an antiserum to the specific "first" antibody. The second antibody serves as a non-specific detector for any "first antibody" either in the free state or when bound to its specific antigen in an 20 antigen-antibody complex.

In accordance with one version of the above method, which reacts also to the known "coated tube" technique, an antigen is attached to the inner wall surface of a test tube in which the binding reaction is then carried out, whereupon the free specific antibody to said antigen present in the test sample will be attached to the surface of the tube via the antigen immobilized thereon. Thereafter the liquid reaction mixture is removed from the test tube, the latter is washed and a second solution containing an enzyme-labeled second antibody is introduced into the tube. This labeled second antibody will also be immobilized by attaching itself to any antibody which is bound to the surface of the tube via the antigen. The test tube is again emptied, rinsed and filled with a suitable substrate responsive to the enzyme-label of the second 30 antibody and the enzymatic activity is measured. This activity is directly proportional to the antibody concentration 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 major cell wall component of many strains of Staphylococcus aureus. This protein A is covalently linked to the 35 cell wall of the staphylococci but can be solubilized and is capable of binding many classes of immunoglobulin molecules, with high affinity. It thus also binds anti- body-antigen complexes. The solubilized protein A can, furthermore, be labeled, e.g. with radioiodine or with enzymes, and enzyme-labeled protein A preparations are now becoming an increasingly popular component of immunoassay kits, in which they serve as the detector.

More recently Lars Bjorck et aL have described in The Journal of Immunology Vol. 133 No. 2 Aug. 1984 pp. 969-974 their findings with regard to the purification and properties of Strepto coccal Protein G, as a novel IgG-binding receptor.

As described therein Protein G, a bacterial cell wall protein with affinity for immunoglobulin G(IgG) was isolated from a human group G streptococcal strain (G148) and found to bind all 45 human IgG subclasses and also rabbit, mouse and goat IgG.

In a further article by Bo Akerstrom et al, in The Journal of Immunology Vol. 135 No. 4 Oct.

1985 pp. 2589-2592 the avidity of protein G for various monoclonal and polyclonal Ig of the G class was studied as well as the use of radiolabeled protein G for binding and detection of antibodies.

The present invention constitutes a yet further important improvement of the abovementioned ELISA, protein A and protein G techniques and is based on the inventive concept that the enzyme-labeled protein A and radiolabeled protein G can be replaced by intact non-viable stabilized bacterial cells having an active receptor for immunoglobulin and an active marker enzyme, which have undergone an appropriate pre-treatment. Thus, e.g., staphylococcal cells can effectively and rapidly bind immunoglobulin (such as any specific "first antibody") by virtue of the protein A in ther cell walls and G streptococcal cells can similarly bind immunoglobulin by virtue of the protein G in their cell walls. At the same time the enzyme-label is provided by enzymes which are naturally present in the cells (i.e. the endogeneous or autochtonous enzymes), the activity of which can be readily measured, or in especially preferred embodiments of the present 60 invention, as described hereinafter, the enzyme label or marker is the result of a gene for said enzyme having been inserted ir#a parental cell thereof.

Thus the present invention provides an immuno-assay reagent for binding and detection of antibodies comprising non-viable stabilized bacterial cells having an active receptor for immuno- globulin and an active marker enzyme.

2 GB2197468A 2 In a first preferred embodiment of the present invention said reagent comprises non-viable stabilized staphylococcus cells.

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

The above reagent proposed in accordance with the present invention, namely a suspension of appropriately treated bacterial cells, has considerable advantages as a "detector suspension" over all known detectors of antibodies or their complexes, in that it obviates the work, time and costs involved in:

(a) isolating the binding protein or preparing anti-immunoglobulins; (b) preparing the enzyme to be used as label; (C) linking the enzyme-label to the binding protein-a notoriously wasteful step, and/or (d) radiolabeling of proteins.

In light of the special properties of the present proposed reagent the invention also provides a test kit for enzyme immunoassay comprising one or more containers holding an aqueous suspen- sion of non-viable stabilized bacterial cells having an active receptor for immunoglobulin and an 15 active marker enzyme, or a dry stabilized preparation of such cells, suitable for in-situ reconstitu tion.

In accordance with a further aspect of the invention there is provided a test kit for carrying out immunoassay, comprising in a packaged combination, one or more solid carriers having fixed on at least a portion of the surface thereon a capture antibody or a hapten or antigen the specific antibody to which is to be assayed and one or more containers holding an aqueous suspension of the stabilized bacterial cells of the invention or a dry stabilized preparation of such cells, suitable for in-situ reconstitution to obtain an aqueous suspension of cells.

Based on the above concept the invention also provides, in accordance with one aspect thereof, a heterogenous specific binding assay method for determining an unbound antibody in a 25 liquid medium, comprising the steps of:

i) incubating a sample of said liquid medium in contact with a solid carrier having fixed on its surface an antigen or hapten specific to said antibody; ii) separating the solid carrier from said liquid medium sample and rinsing it with aqueous solution to remove all traces of said sample; iii) incubating the solid carrier in contact with an aqueous suspension of non-viable stabilized bacterial cells having an active receptor for immunoglobulin and an active marker enzyme; iv) separating the solid carrier from said suspension and washing it with aqueous solution; and v) incubating said solid carrier in contact with a suitable substrate and assaying for enzymatic activity of the marker enzyme on cells retained on the solid carrier surface.

The specific binding assay method according to the invention can be applied to the determina tion of a hapten or antigen in a liquid medium by adding to a sample of said liquid medium an antibody specific to the hapten or antigen which is to be determined and incubating the reaction mixture in contact with a solid carrier having fixed on its surface the same hapten or antigen which is to be assayed wherein in step (i) a specific antibody to said hapten or antigen is added 40 to the test sample, and as the result of binding of said hapten or antigen in said sample, the amount of antibody available for binding to the hapten or antigen fixed on the solid surface is proportionately reduced. The above-described steps ii) to v) 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 immunoassay techniques, e.g. by comparison with a series reference samples having known standard concentrations of the hapten or antigen.

The detector cell suspension of the present invention can be similarly used in "sandwich" type immunoassays, namely in assays where a "capture antibody" is immobilized on a solid surface so as to capture antigen present in the test sample, and a "tracer" antibody to that antigen is then applied to form a "sanwich". The binding of the "tracer" antibody is convention ally revealed by a label incorporated in that antibody moledule.

In the present method the detector suspension can be used to eliminate the need for labeling the detector cells must not bind to the using antibodies which do not bind Protein A.

(e.g. IgY from chicken serum or from yolk) or modified antibody in which the F. segment is missing (e.g. Fab or F(ab'),).

Selective binding to the second antibody can often be observed with conventional antibody preparations, indicating that the capture antibody is fixed in a configuration which makes the Protein A binding moiety (Fc segment) of the immunoglobulin unavailable for binding of the detector cell.

A test kit adapted for carrying out the assay method according to the invention for determin ing a hapten or antigen in a liquid medium may additionally comprise one or more containers holding standardized solutions of a specific antibody to said hapten or antigen which is to be assayed. The test kits according to the invention optionally further comprise packaged auxiliary 65 the second antibody. The only requirement is that 55 capture antibody. This requirement can be met by 3 GB2197468A 3 1 reagents or solutions for carrying out the assay method, such as buffer solutions, standardized reference solutions of the analyte to be determined, substrate solutions and detector reagents for measuring the activity of the enclogeneous enzyme serving as label, etc. Such test kits which comprise a Iyophilized stabilized preparation of Staphylococcus aureus cells may also additionally comprise packaged aqueous medium for forming the required aqueous suspension of said cells in situ.

In preferred embodiments of the present invention there is also provided a test kit additionally comprising an absorbent reagent carrier incorporating thereon a reagent specific to said marker enzyme.

As stated above, the staphylococcal cells of the present invention are appropriately treated before use in order to ensure that the cell suspension is safe (i.e. non-pathogenic), standardized and stable whilst preserving its binding capacity and the activity of the marker enzyme which is selected to serve as the label. It has been found that these requirements can be met, e.g. by fixing the staphylococcal cells by treatment with 0.5% formaldehyde solution at room tempera- ture for 2 hours followed by heating for about 5-15 mins. at about 50'- 70'C. The treated cells can be preserved, e.g., as a 10% suspension with a preservative such as sodium axide at 40C or as a freeze-dried pellet of staphylococcal cells stored in sealed ampoules. Alternatively, a safe and stable reagent can be obtained by freeze-drying a suspension of staphylococcal cells and storing the product in sealed ampoules. The freez-dried cells can be reconstituted in situ for use by suspending them in a suitable liquid medium.

It is to be noted that while stabilized staphylococcus cells are commercially available the standard treatment for stabilization thereof normally inactives any endogenous enzymes found thereon and thus non-viable stabilized Staphylococcus cells having an active receptor for immu noglobulin and an active marker enzyme heretofor have not been available and also have not been suggested for use as immunoassay reagents.

In one embodiment of the present invention said marker enzyme is endogenous catalase and the catalase activity of the Staphylococcus cells is assayed.

In especially preferred embodiments of the present invention there are used as reagents non viable stabilized Staphylococcus cells having an active receptor for immunoglobulin and an active marker enzyme, the gene for said enzyme having been inserted into a parental cell thereof by 30 genetic engineering procedures known per se.

Thus for use in the present invention there was prepared a genetically engineered strain of the Cowan Staphylococcus which differs from the original strain in having a gene for the formation of-lactamase.

The general advantage of using this approach of inserting a new gene is in not having to rely 35 on enzyme markers which happen to be present in the original strain. Thus there can be added to the existing repertoire of the endogenous enzymes a new heritable marker which is more suitable for immunoassay.

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

1. The enzyme is largely cellbound.

2. The enzyme is fully accessible to the substrate.

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

4. The enzyme has a very high turnover rate.

5. The substrate is stable, inexpensive and its use involves no health hazards or safety precautions.

6. The assay of the -lactamase reaction is simple, rapid and very sensitive.

7. The assay requires no instrumentation and a permanent record of the results can be retained without any additional manipulations.

As indicated hereinbefore several types of Streptococcus pyogenes are known to produce cell wall proteins which can serve as receptors for the F., portion of immunoglobulins. The receptor - specificity of such proteins may vary with the type of the producing strain. Of special interest are streptococci of 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 the Cowan 1 55 cells. The following differences must be noted, however:

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

2. The streptococcal cells have no catalase, but have a rich variety of other cell bound enzymes including hemolysins, which can serve as endogenous indicators of binding to the 60 immunoglobulin molecule.

3. In analogy to the genetic modification of the Cowan strain, as described above, insertion of suitable genes will provide additional enzyme labels which may be more advantageous in immunoassay diagnostic kits.

From the aforementioned, it will be obvious that streptococcal cells possessing Fc receptors 65 4 GB2197468A 4 can by themselves serve as a source of reagent for detecting a variety of immunoglobulins. When preferable, such cells can be used in combination with the Cowan reagent described before. The combined use of both types of cells may take one of several forms:

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

2. The mixed cells are linked together (e.g. with glutaraldehyde) to form a combined reagent, which now carries a single enzyme label (e.g. lactamase) but two kinds of F. receptors.

3. A combined reagent is formed through interaction with immunoglobulins present in the assay or deliberately added for that purpose.

While the invention will now be described in connection with certain preferred embodiments in the following examples so that aspects thereof may be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims. Thus, the following examples which 15 include preferred embodiments will serve to illustrate the pratice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodimens of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of the invention.

EXAMPLE I

Preparation of stabilized suspension of Staphylococcus aureus cells Staphylococcus aureus (strain Cowan 1, ATCQ, was grown in TSB (Trypticase Soy Broth, Difco), in 500 ml ErIenmeyer flasks, on a reciprocal shaker, for 18 hours at 37'C. The cells were 25 then spun down (10 min at 10,000 RPM), washed twice with phosphate buffered saline (PBS), pH 7.3 and suspended in 10 volumes of PBS (pH 7.3) containing 0.5% formaldehyde. After 2 hours at room temperature the cells were spun down and washed as before, and then sus pended in 10 volumes of PBS (pH 7.3). The suspension was incubated for 10 minutes at 60'C under gentle shaking, spun as before and resuspended in 10 volumes of PBS (pH 7.3). Samples 30 of that suspension, streaked on AB3(Difco) plates, failed to show growth after 24 hours at 37'C. After 12 months storage at 4'C the suspension showed no signs of change in binding and catalytic properties. Before use the suspension was diluted 1:10 in PBS.

EXAMPLE 2 Preparation of freeze-dried stabilized Staphylococcus aureus reagent Staphylococcus aureus (strain Cowan 1, ATCQ was cultivated and harvested as desribed in Example 1 above. The cells were washed with PBS, resuspended in 10 volumes of fresh TSB, incubated for 10 minutes at 60'C and dispensed in 0.5 ml portions into Iyophilization-ampoules.

After freeze-drying the sealed ampoules were stored at room temperature (22-25'C). For use, the contents of one ampoule were suspended in 5.0 ml PBS containing 0.0 1 % merthiolate.

EXAMPLE 3

Detection of Rabbit anti-BSA Microtitre plates (U type, Nunc) wre activated by filling the wells with 100 1 of 0.2% glutaral- 45 dehyde solution in borate buffer, pH 9.0, and incubation for 3 hours at 56'C. The plates were then washed 10 times with twice-distilled water and coated with antigen by filling the wells with 1 of bovine serum albumin (BSA) (50 g/ml), incubating for 2 hours at 37'C and storing at 4-C for 18 hours. After 3 washes with PBS the unoccupied surfaces were blocked by filling the wells with 100 1 of ovalburnin (1.0 mg/ml) and incubating for 2 hours at 37'C, followed by 3 50 washes with PBS.

Control wells were prepared as above with the BSA being omitted.

Antibody to BSA (Anti-BSA) was prepared in rabbits. Normal rabbit serum (NRS) served as a non-specific control. Serial dilutions were made in PBS, and 100 1 of each were added to the antigen coated wells. After 30 minutes incubation at 37'C the wells were washed thrice with 55 PBS For detection of the antibody retained by the antigen, an aliquot (100 1) of a staphylococcal cell suspension (prepared in accordance with Examples 1 and 2 above) was added to each well.

After 20 minutes incubation at 37'C the wells were washed once with 1% Tween 20 and twice with PBS. The retention of the cells by the bound antibody was detected by acid phosphatase or catalase activity, in accordance with the method described in Israel Patent Specification No. 60 36496.

RESULTS The test system described above was used to compare the sensitivity of the detectors, i.e.

the staphylococcal cell suspensions prepared in accordance with Examples 1 and 2 above with 65 GB2197468A 5 the sensitivity of a radioiodinated protein A preparation, conventionally used in sensitive solidphase radioimmunoassays. The results are summarized in Table 1.

TABLE I

Detector prepared Anti-BSA Anti-BSA Anti-BSA NRS 10 according to 1:1000 1:3000 1:10000 1:1000 Example 1 Pos. Pos. Pos. Neg.

Example 2 Pos. Pos. Pos. Neg. 15 Radioiodinated Protein A Pos. Pos. Neg. Neg. 20 () Based on catalase activity displayed 5 minutes after the additon of the substrate, namely 50 1 of a 6% hydrogen peroxide solution.

1 EXAMPLE 4

A gene for constitutive penicillinase formation (pen I was inserted into the Cowan I strain of Staphylococcus aureus. The gene originated in a plasmid (PI... pen 1443) hosted by strain (RN 453) of Staphylococcus aureus (Novick, R.P., and Brodsky. R., 1972). Studies on plasmid 30 replication: 1. Replication of unestablished plasmids in S Aureus. J.Mol. Biol 68: 285-302), and was transduced with phase o 11. The transduction was as described by Novick (Novick, R.P., 1967), Properties of a cryptic high frequency transducing phage in Staphylococcus aureus; Virology 33:155-166. A stabilized suspension of these cells having - lactamase as their marker enzyme was then prepared by the procedure of Example 1.

EXAMPLE 5

Detection of antibodies to brucella in bovine sera A polystyrene Petri dish (standard size, Miniplate) was activated at 8 preselected, equidistant spots by placing 50 1 of a 25% solution of glutaraldehyde (Merck) at each spot. After 150 sec 40 at 24'C the dish was rinsed with water and 10 1 of the antigen suspension was added to each spot. The antigen suspension consisted of heat-killed (90 min at 72'C cells of Brucella abortus, suspended in PS (0.5% phenol and 0.85% NaCl in distilled water) at optical density correspond ing to 200 Klett Units. The spots were extensively rinsed and the Petri dish overlayed with 5 ml of a blocking solution consisting of BSA (20 mg/ml) in PBS. After 2 hrs at 37' and 16 hrs at 4' 45 the blocking solution was poured off and the Petri dish was rinsed twice with PBS, then. twice with 0.05% Tween 20 in PBS and again with PBS. The dish was then air- dried and 20 1 samples Of bovine sera, diluted as indicated in Table 2, were added to the marked spots. After min at 370, the dish was rinsed and dried as before, and each spot received 20 1 of a detector suspension. The detector suspension consisted of the genetically modified staphylococ- 50 cal cells of example 4 suspended in PBS at OD corresponding to 200 KU. After rinsing and air drying the -lactamase activity of the detector cells retained on the spots was tested by placing on each spot a developer strip. The developer strip was a section (12 x 15 mm) of Whatman No. 3 filter paper impregnated with a reagent solution containing iodine (12 mM), potassium iodide (63 mM), soluble starch (1.0% w/v) and penicillin (50 mM) in PBS. The activity of the marker enzyme of the detector cells, namely - lactamase, was assayed by determining the time required for the appearance of a white circle in the centre of the blue-black detector strip. The white circle indicated that iodine has been taken up by penicilloic acid and thus removed from the complex which gave the dark color to the detector strip. The penicilloic acid is the product of hydrolysis of a -lactam (here penicillin) catalysed by -lactamase. Hence the rate of the decolorization of the circular area above the marked spot is inversely proportional to the amount of -lactamase retained on that spot.

j i 60 1 1 i 1 6 GB2197468A 6 TABLE 2

Serum Agglutination Complement Present 5 NO. Titre Fixation Test Titre Titre 10 2 1.10 1.5 1.200 6 1: 20 1.5 1.200 32 Neg Neg 1:1000 15 53 1:80 1:5 1:1000 77 Neg Neg Neg 20 26 1:40 1:50 1.2000 ill 1:1250 - 1:16000 Normal Neg Neg Neg 25 Bovine Serum As determined at the Kimron Veterinary Institute, Beth Dagan, Israel -^' The highest dilution (in PBS) which showed decolorization in < 10 min.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not 40 restrictive, reference being made to the appended claims, rather than to the foregoing descrip tion, and all changes which come within the meaning and range of eclivalency of the claims are therefore intended to be embraced therein.

Claims

CLAIMS 1. An immunoassay reagent for binding and detection of antibodies

comprising non-viable stabilized bacterial cells having an active receptor for immunoglobulin and an active marker enzyme.
2. An immunoassay reagent for binding and detection of antibodies according to claim 1 50 comprising non-viable stabilized staphylococcus cells.
3. An immunoassay reagent for binding and detection of antibodies according to claim 1 comprising non viable stabilized Streptococcus cells.
4. A test kit for enzyme immunoassay comprising one or more containers holding an aqueous suspension of non-viable stabilized bacterial cells having an active receptor for immunoglobu55 lin and an active marker enzyme, or a dry stabilized preparation of such cells, suitable for in-situ 55 reconstitution.
5. A test kit according to claim 4 wherein said cells are a Cowan I strain of Staphylococcus aureus cells containing a gene for the formation of B-lactamase, the gene for said enzyme having been inserted into a parent cell thereof.
6. A test kit according to claim 4 wherein said cells are group G streptococcal strain G148 60 cells.
7. A test kit according to claim 4 further comprising one or more solid carriers having fixed on at least a portion of the surface thereof a capture antibody or a hapten or antigen the specific antibody to which is to be assayed.
8. A test kit according to claim 4, which kit additionally comprises one or more containers 65 7 1-, 15 k GB2197468A 7 holding standardized solution of a specific antibody to the hapten or antigen which is to be be assayed.
9. A test kit according to claim 4 which kit additionally comprises an absorbent reagent carrier incorporating thereon a reagent specific to said marker enzyme. 5
10. A heterogeneous specific binding assay method for determining an unbound antibody in a 5 liquid medium, comprising the steps of: i) incubating a sample of said liquid medium in contact with a solid carrier having fixed on its surface an antigen or hapten specific to said antibody; ii) separating the solid carrier from said liquid medium sample and rinsing it with aqueous solution to remove all traces of said sample; iii) incubating the solid carrier in contact with an aqueous suspension of non-viable stabilized bacterial cells having an active receptor for immunoglobulin and an active marker enzyme; iv) separating the solid carrier from said suspension and washing it with aquous solution; and v) incubating said solid carrier in contact with a suitable substrate and assaying for enzymatic activity of the marker enzyme on cells retained on the solid carrier surface.
11. A method according to claim 10 adapted for determining a hapten or antigen in a liquid medium wherein said solid carrier has fixed on its surface the same hapten or antigen which is to be assayed, and wherein in step (i), a specific antibody to said hapten or antigen is added to the test sample, and as the result of binding of said hapten or antigen in said sample, the amount of antibody available for binding to the hapten or antigen fixed on the solid surface is 20 proportionately reduced.
12. A method acccording to claim 10 wherein said aqueous suspension contains Staphylo coccus cells, stabilized by treatment with heat and formaldehyde to preserve their binding capacity and the activity of the marker enzyme.
13. A method according to claim 12, wherein said marker enzyme is endogenous catalase 25 and the catalase activity of the Staphylococcus cells is assayed.
14. A method according to claim 10 comprising incubating said solid carrier with an aqueous suspension of non-viable stabilized Staphylococcal cells having an active receptor for immunoglo bulin and an active marker enzyme, the gene for said enzyme having been inserted into a parental cell thereof.
15. A method according to claim 10 wherein said aqueous suspension contains stabilized Streptococcus cells.
16. A method according to claim 15 wherein said marker enzyme is endogenous hemolysin and the hemolysin activity of the Streptococcus cells is assayed.
17. A method according to claim 10 wherein said aqueous suspension contains a Cowan 1 35 strain of Staphylococcus aureus containing a gene for the formation of B- lactamase, the gene for said enzyme having been inserted into a parent cell thereof.

Published 1988 at The Patent Office, State House, 66/71 High Holborn, London WC 1 R 4TP. Further copies may be obtained from The Patent Office, Sales Branch, St Mary Cray, Orpington, Kent BR5 3RD. Printed by Burgess & Son (Abingdon) Ltd. Con. 1/87.