AU594503B2

AU594503B2 - Immunoassay reagents, kits and methods

Info

Publication number: AU594503B2
Application number: AU78623/87A
Authority: AU
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: 1987-09-17
Publication date: 1990-03-08
Anticipated expiration: 2007-09-17
Also published as: FR2604259B1; GB2197468A; NL8702235A; IT1223303B; GB8721276D0; CA1294871C; AU7862387A; CH675309A5; IL80313A0; IT8721950A0; FR2604259A1; GB2197468B; DE3731227A1

Description

Ij 594503 S F Ref: 37039 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: This document contains the amnendments made under Section 49 and is correct for printing.

tr f t Name and Address of Applicant: Yissum Research Development Company of the Hebrew University of Jerusalem 46 Jabotinsky Jerusalem

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SAddress for Service: I S

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Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia

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t r rf C Complete Specification for the invention entitled: Immunoassay Reagents, Kits and Methods The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/3 i i

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i: i IMMUNOASSAY REAGENTS, KITS AND METHODS IMMUNOASSAY REAGENTS, KITS AND METHODS i; i ABSTRACT OF THE DISCLOSURE t c Ct C Cr C C re

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The invention provides 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, as well as providing 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 immunoglobulin and an active marker enzyme, or a dry stabilized preparation of such cells, suitable for in-situ reconstitution and methods for the use thereof.

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-2- The present invention relates to immunoassay reagents, kits and methods.

More particularly the present invention relates to a novel 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 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.

04 4 0 a o *o o 0 o o a o e 00 0 0* 0 A 06 oo °j .4 4 t0 13 I 0 0 II Among the EIA techniques there are commonly used the so-called "heterogenous" assay methods employing enzyme-labeled antibodies or antigens attached to ("immobilized on") sensitized surfaces of solid t C 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 t 1 i: Ci; Fi i -CI~--3~CI~ I r -3- I t C

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C) Cf* serves as a non-specific detector for any "first antibody" either in the free state or when bound to its specific antigen in an antigenantibody ccmplex.

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 mixtur 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 enzymelabel of the second antibody and the enzymatic activity is measured.

This activity is directly proportional to the antibody concentration in the test sample.

q1 In recent years it has been found that the "second antibody" in d 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 cell wall of the staphylococci but can be solubilized and is 1.a -Cr: i solubilized protein A can, furthermore, be iabeled, 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

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t regard to the purification and properties of Streptococcal Protein G, as a novel IgG-binding receptor.

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

StIn 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 j i .4 4 o o o 4 4 4 4 re 9r 1 I I

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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, 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 the endogeneous or autochtonous enzymes), the activity of which can be readily measured, or in especially preferred embodiments of the present invention, as described hereinafter, the enzyme label or marker is the result of a gene for said enzyme having been inserted in 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 immunoglobulin and an active marker enzyme.

In a first preferred embodiment of the present invention said reagent comprises non-viable stabilized staphylococcus cells, i pl_

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4 tIC Cr~r *449 4 1 r i 0I 2 i r 4 I n -s~y I, tP te 8* 8 i:i o 8 8 8I 8 I ICr I I -6- 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: isolating the binding protein or preparing antiimmunoglobulins; preparing the enzyme to be used as label; linking the enzyme-label to the binding protein a notoriously wasteful step, and/or 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 suspension of non-viable stabilized bacterial cells having an active receptor for immunoglobulin and an active marker enzyme, or a dry stabilized preparation of such cells, suitable for in-situ reconstitution.

(1 I 'P i ii In accordance with a further aspect of the invention there is provided a test kit for carrying out imnmunoassay, comprising in a 14 C 0 i.

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mi 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 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 if

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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 determination 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 o°o 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 Th wherein in step 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 S* binding to the hapter 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 S- 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 I hapten or antigen.

IThe 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

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t l ~d' ;i capture antigen present in the test sample, and a "tracer" antibody to that antigen is then applied to form a "sandwich". The binding of the "tracer" antibody is conventionally revealed by a label incorporated in that antibody molecule.

In the present method the detector suspension can be used to eliminate the need for. labeling 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 Protein A. IgY from chicken serum or from yolk) or modified antibody in which the Fc segment is missing Fab or F(ab') 2 I I t4Cr

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If~ 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 determining 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 0 be assayed. The test kits according to the invention optionally further comprise packaged auxiliary reagents or solutions for carrying out the assay method, such as buffer solutions, standardized reference

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solutions of the analyte to be determined, substrate solutions and detector reagents for measuring the activity of the endogeneous enzyme serving as label, etc. Such test kits which comprise a lyophilized 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 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 temperature for 2 hours followed by heating for about 5-15 mins.

at about 50 0 -70 0 C. The treated cells can be preserved, as a suspension with a preservative such as sodium azide at 4°C or as a freeze-dried pellet of staphylococcal cells stored in sealed ampoules.

Alternatively, a safe and stable reagent can be obtained by freezedrying a suspension of staphylococcal cells and storing the product in

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ir 11 sealed ampoules. The freeze-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 inactivates any endogenous enzymes found thereon and thus non-viable stabilized Staphylococcus cells having an active receptor for immunoglobulin 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 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 B-lactamase.

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itt6 Iir C 4 i -I A q"" 1' CUII~- 12 The gene is present existing which is general advantage of using this approach of inserting a new in not having to rely on enzyme markers which happen to be in the original strain. Thus there can be added to the repertoire of the endogenous enzymes a new heritable marker more suitable for immunoassay.

9 r 'C n rl I r'yudc4jll: l ti_ o The specific advantages of the gene for B-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 B-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 ii i:, 1 ~:ln~n~n rr--: kr-cn it 1 r ii ii 13 4

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t oL r 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 I cells. The following differences must be noted, however: 1. The broader specificity of streptococcal F receptors allows c 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 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 F receptors 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: i1 1

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14 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 with glutaraldehyde) to form a combined r6sn. which now carries a single enzyme label .P-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 "0 that purpose.

II '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 S 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 include preferred embodiments will serve to illustrate .the practice of this invention, it being understood that the partib culars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the pres-nt invention only and are presented in the cause of providing what is believed to be the L/ 1 t r p pr

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~1~ i 11 9 C cC C 15 most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of thb invention.

EXAMPLE 1 Preparation of stabilized suspension of Staphylococcus aureus cells Staphylococcus aureus (strain Cowan I, ATGC), was grown in TSB (Trypticase Soy Broth, Difco), in 500 ml Erlenmeyer flasks, on a reciprocal shaker, for 18 hours at 37 0 C. The cells were then spun down min at 10,000 RPM), washed twice with pho-sphate buffered saline (PBS), pH 7.3 and suspended in 10 volumes of PBS (PH 7.3) containing formaldehyde. After 2 hours at room temperature the cells were spun down and washed as before, and then suspended in 10 volumes of PBS (pH 7. The suspension was incubated for 10 minutes at 60 0

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under gentle shaking, spun as before and resuspended in 10 volumes of PBS (PH 7. Samples of that suspens-Ion, streaked on AB3(Difco) plates, failed to show growth after 24 hours at 37 0 GC. After 12 months storage at 4 0 GC the suspension showed no signs of change in binding and catalytic properties. Before use the suspension was diluted 1:10 in

PBS.

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I0 t t. 4 EXAMPLE 2 Preparation of freeze-dried stabilized Staphylococcus aureus reagent Staphylococcus aureus (strain Cowan I, ATCC) was cultivated and harvested as described in Exmaple 1 above. The cells were washed with PBS, resuspended in 10 volumes of fresh TSB, incubated for 10 minutes at 600C and dispensed in 0.5 ml portions into lyophilization-ampoules.

After freeze-drying the sealed ampoules were stored at room temperature (22-25 0 For use, the contents of one ampoule were suspended in 5.0 ml PBS containing 0.01% merthiolate.

EXAMPLE 3 Detection of Rabbit anti-BSA Microtitre plates (U type, Nunc) wre activated by filling the wells with 100 1p of 0.2% glutaraldehyde solution in borate buffer, pH and incubation for 3 hours at 560C. The plates were then washed times with twice-distilled water and coated with antigen by filling the wells with 100 pl of bovine serum albumin (BSA) (50 g/ml), incubating for 2 hours at 370C and storing at 40C for 18 hours. After 3 washes with PBS the unoccupied surfaces were blocked by filling the wells with 100 pl of ovalbumin (1.0 mg/ml) and incubating for 2 hours at 370C, followed by 3 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

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*8~31~ 1 W U-D- 17 made in PBS, and 100 p~ of each were added to the antigen coated wells. After 30 minutes incubation at 37 0 C the wells were washed thrice with PBS. For detection of the antibody retained by the antigen, an aliquot (100 pl) of a staphylococcal cell suspension (prepared in accordance with Examples 1 and 2 above) was added to each well. After 20 minutes incubation at 370C 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. 36496.

RESULTS

The test system described above was used to compare the sensitivity of the detectors, i.e. the slephylococcal cell suspensions prepared in accordance with Examples 1 and 2 above with the sensitivity of a radioiodinated protein A preparation, conventionally used in sensitive solid-phase radioimmunoassays. The results are summarized in Table I.

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c CC c r r EC CC :1: TABLE I Detector prepared accord-na to Anti-BSA 1:1000 Anti-BSA 1 :3000 Anti-BSA 1 10000nnnn

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1 innn Example 1* Pos. Pos. Pos. Neg.

t p.a 18 Example 2* Pos. Pos. Pos. Neg.

Radioiodinated Protein A Pos. Pos. Neg. Neg.

Based on catalase activity displayed 5 minutes after the additon of the substrate, namely 50 1 of a 6% hydrogen peroxide solution.

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I I I r It~C 1~ C iii 'K 111 1 19 Sqc a o o *a 0 04 ''ft r t tt Cr EXAMPLE 4 A gene for constitutive penicillinase formation (pen was inserted into the Cowan I strain of Staphylococcus aureus. The gene originated in a plasmid (PI 258 pen 1443) hosted by strain (RN 453) of Staphylococcus aureus (Novick, and Brodsky, 1972). Studies on plasmid replication: I. Replication of unestablished plasmids in S Aureus. J.Mol.Biol 68: 285-302), and was transduced with phage o 11.

The transduction was as described by Novick (Novick, 1967), Properties of a cryptic high frequency transducing phage in Staphylococcus aureus; Virology 33:155-166. A stabilized suspension of these cells having B-lactamase as their marker enzyme was then prepared by the procedure of Example 1.

EXAMPLE 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 ~I of a 25% solution of glutaraldehyde (Merck) at each spot. After 150 sec at 24 0 C the dish was rinsed with water and 10 pil of the antigen suspension was added to each spot. The antigen suspension consisted of heat-killed (90 min at 720C cells of Brucella abortus, suspended in PS phenol and 0.85% NaC1 in distilled water) at optical density corresponding 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 370 and 16 hrs at 40 the blocking solution was poured off and the Petri dish was rinsed twice with PBS, then twice with i

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~rlc -,i c i tr i i jl rT 21 20 ii ii p: 4,: 0.05% Tween 20 in PBS and again with PBS. The dish was then air-dried and 20 pj samples of bovine sera, diluted as indicated in Table 2, were added to the marked spots. After 60 min at 370, the dish was rinsed and dried as before, and each spot received 20 p1 of a detector suspension. The detector suspension consisted of the genetically modified staphylococcal cells of example 4 suspended in PBS at OD corresponding to 200 KU. After rinsing and air drying the B-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 (12x15 mm) of Whatman No. 3 filter paper impregnated with a reagent solution containing iodine (12 mM), potassium iodide (63 mM), soluble starch w/v) and penicillin (50 mM) in PBS. The activity of the marker enzyme of the detector cells, namely f-iactamase, was assayed by determining the time required for the appearance of a white circle in the center 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 B-lactam (here penicillin) catalysed by 3-lactamase. Hence the rate of the decolorization of the circular area above the marked spot is inversely proportional to the amount of 3-lactamase retained on that spot.

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Agglutination Titre* Complement Fixation Titre* .a #9 C t I I C C S

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2 1:10 6 1:20 32 Neg 53 1:80 77 Neg 26 1:40 111 1:1250 Normal Neg Bovine Serum 1:5 1:5 Neg 1:5 Neg 1:50 Neg Present Test Titre** 1:200 1:200 1:1000 1:1000 Neg 1:2000 1:16000 Neg

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As Israel The determined at the Kimron Veterinary Institute, Beth Dagan, highest dilution (in PBS) which showed decolorization in 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 restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

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Claims

1. An immunoassay reagent for binding and detection of comprising non-viable stabilized bacterial cells having receptor for immunoglobulin and an active marker enzyme. antibodies an active \I .1 *r @9 e 9 *9 6 9 94 I

2. An immunoassay reagent for binding and detection of antibodies according to claim 1 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 immunoglobulin and an active marker enzyme, or a dry stabilized preparation of such cells, suitable for in-situ reconstitution.

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 8-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 cells, o- ve ecrees

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. i. I i if i, 1 i A C-r~ -*rrr~~ V I 23 r S I o a P It t r:

8. A test kit according to claim 4, which kit additionally comprises one or more containers holding standardized solutions of a specific antibody to the hapten or antigen which is to 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.

A heterogeneous specific binding assay method for determining an unbound antibody in a 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.

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 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 proportionately reduced. y_ i I 4) i c 1~i i :1 24

12. A method according to claim 10 or 11 wherein said aqueous suspension contains Staphylococcus 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 and the catalase activity of the Staphylococcus cells is assayed.

14. A method according to claim 10 or 11 comprising incubating said solid carrier with an aqueous suspension of non-viable stabilized Staphylococcal 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.

A method according to claim 10 or 11 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 or 11 wherein said aqueous suspension contains a Cowan I strain of Staphylococcus aureus containing a gene for the formation of 3-lactamase, the gene for said enzyme having been inserted into a parent cell thereof.

18. An immunoassay reagent for binding and detection of antibodies substantially as herein described with reference to Example 1, 2 or 3.

19. A test kit for enzyme immunoassay comprising one or more containers holding an immunoassay reagent as defined in claim 18. A heterogeneous specific binding assay method for determining an unbound antibody in a liquid mediu, substantially as herein described with reference to Example 3 or Pt s Ia C C Cb 6t I DATED this EIGHTEENTH day of DECEMBER 1989 Yissum Research Development Company of the Hebrew University of Jerusalem Patent Attorneys for the Applicant SPRUSON FERGUSON Vi