NL8702235A - IMMUNIC TEST REAGENTS, TOOLS AND METHODS. - Google Patents

Description

-1-

Immunoassay reagents, tools and methods.

The present invention relates to immunoassay reagents, tools and methods thereof.

More particularly, the present invention relates to a novel antibody binding and detection reagent and tools and methods comprising them.

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

The EIA techniques commonly use so-called "heterogeneous" test methods using enzyme-labeled antibodies or antigens attached to ("immobilized on") sensitized surfaces of solid supports, such as test tubes, polystyrene beads or plates with recessed "sources". Such techniques are generally referred to by the abbreviation "ELISA" which stands for "enzyme-linked immunosorbent assay".

In accordance with a 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 nonspecific detector for each "first antibody" either in the free state or bound to its specific antibody in an antigen-antibody complex.

In accordance with one version of the above method, which also responds 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 performed, after which the free specific 870223> -2- 'antibody for said antigen present in the test sample will be attached 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 an enzyme-labeled second antibody is introduced into the tube. This labeled second antibody will also be immobilized by attaching itself to an antibody bound to the surface of the tube 10 via the antigen. The test tube is again emptied, cleaned and filled with a suitable substrate that responds to the enzyme label of the second antibody and measures the enzymatic activity. This activity is directly proportional to the antibody concentration in the test-15 sample.

In recent years, it has been found that the "second antibody" in immunoassay techniques can be replaced by a particular protein, namely the so-called "protein A" which is a fairly large cell wall component of many strains of Staphylococcus aureus. This protein A is covalently bound to the cell wall of the staphylococci but can be solubilized and is capable of binding many classes of immunoglobulin molecules with high affinity. It therefore also binds antibody-antigen complexes.

In addition, the solubilized protein A can be labeled, for example with radioiodine or with enzymes, and enzyme-labeled protein A preparations now become an increasingly common component of immunoassay tools in which they serve as the detector.

30 More recently, Lars Bjorck and others in The

Journal of Immunology Vol. 133 No. 2 Aug. 1984 pp. 969-974 described their findings regarding the purification and properties of Streptococcal protein G as a novel IgG binding receptor.

As described therein, 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 human IgG subclasses and also rabbit, mouse and goat IgG.

40 In another article by Bo Akerstrom and others 8702235-3 * * in The Journal of Immunology Vol. 135 No. Oct 4, 1985 biz. 2589-2592, the greed of protein G for various monoclonal and polyclonal Ig of the G class was investigated and further the use of radiolabeled protein G for binding and detection of antibodies.

The present invention represents yet a further significant improvement on the above ELISA, protein A and protein G techniques and is based on the inventive idea that the enzyme-labeled protein A and radiolabeled protein G can be replaced by intact non-viable stabilized bacterial cells with an active immunoglobulin receptor and an active marker enzyme, which have undergone appropriate pretreatment. For example, staphylococcal cells can effectively and rapidly immunoglobulin (such as a specific "first antibody") under protein A in their cell walls, and G streptococcal cells can similarly bind immunoglobulin under protein G in their cell walls. At the same time, the enzyme label is applied by enzymes naturally present in the cells (ie the endogenous and autotonic enzymes), the activity of which can be easily measured, or is in particularly preferred embodiments of the present invention, as later described, the enzyme label or marker results from a gene for said enzyme placed in a mother cell thereof.

Thus, the present invention provides an immuno-test reagent for binding and detecting antibodies comprising nonviable stabilized bacterial cells with an active immunoglobulin receptor and an active marker enzyme.

In a first embodiment of the present invention, the reagent comprises nonviable stabilized staphylococcal cells.

In another preferred embodiment of the present invention, the reagent comprises nonviable stabilized streptococcal cells.

The reagent of the invention according to the 670223 5 * -4 invention, namely a suspension of suitably treated bacterial cells, has significant advantages as a "detector suspension" over all known detectors of antibodies or complexes thereof, in that it avoids the time and expense involved in: (a) isolating the binding protein or preparing anti-immunoglobulins; (b) preparing the label enzyme to be used; (C) binding the enzyme label to the binding protein - a recognized wasteful step, and / or (b) radiolabelling proteins.

In view of the special properties of the proposed reagent, the invention also provides an enzyme immunoassay test tool comprising one or more containers containing an aqueous suspension of nonviable stabilized bacterial cells with an active immunoglobulin receptor and an active marker enzyme, or a dry stabilized preparation of such cells suitable for in-situ reconstitution.

In accordance with a further aspect of the invention there is provided an immunoassay test tool comprising, in a packaged combination, one or more solid carriers on which at least a portion of the surface is attached to a capture antibody or a bite or antigen to which the specific antibody is to be tested and one or more containers containing 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.

On the basis of the above idea, the invention also provides, in one aspect thereof, a heterogeneous specific binding assay method for determining an unbound antibody in a liquid medium, comprising the steps of: i) incubating a sample of the liquid-40 medium in contact with a solid support with an antigen or bite specific to the antibody affixed to the surface thereof, ii) separating the solid support from the fluid medium sample and cleaning it with aqueous solution to remove from all traces of the sample • iii) incubating the solid support in contact with an aqueous suspension of nonviable stabilized bacterial cells with an active immunoglobulin receptor and an active marker enzyme, iv) separating the solid carrier from said suspension and washing it with aqueous solution, and v) incubating the solid carrier in aa contact with a suitable substrate and testing for enzymatic activity of the marker enzyme on cells held on the solid support 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 an antibody specific for the hapten or antigen to be determined and the reaction mixture to a sample of the liquid medium. incubate in contact with a solid support with the same hapten or antigen mounted on the surface to be examined, in which in step (i) a specific hapten or antigen antibody is added to the test sample, and as the result of binding of the hapten or antigen in the sample, the amount of antibody available for binding to the hapten or antigen attached to the solid surface is reduced proportionally. The above steps ii) to 35 v) above are then performed and the enzymatic activity determined in step v) is inversely proportional to the concentration of the hapten or antigen in the sample. This concentration can be calculated using methods known in immunoassay techniques, for example, by comparison to a series of reference samples 870 2 2.3 5 -6- with known standard hapten or antigen concentrations.

The detector cell suspension of the invention can be similarly used in "sandwich" type immunoassays, namely, in experiments where a "receptor antibody" is immobilized on a solid surface to capture the antigen present in the test sample. , and a "trace" antibody for that antigen is then applied to form a "sandwich". The binding of the "trace" antibody is conventionally exposed by a label contained within that antibody molecule.

In the present method, the detector suspension can be used to eliminate the need to label the second antibody. All that is required is that the detector cells must not bind to the capture antibody. This requirement can be met by using antibodies that are not protein A (eg, chick serum or yolk IgY) or modified antibody lacking the F segment (eg, Fab or F (ab ') ^)

Selective binding to the second antibody can often be observed with conventional antibody preparations, indicating that the capture antibody is confirmed in a configuration that the protein A

binding portion (F segment) of the immunoglobulin unsuitable for binding the detector cell.

A test tool suitable for carrying out the test method of the invention for determining a hapten or antigen in a liquid medium may additionally comprise one or more containers containing standardized solutions of a specific hapten or antigen antibody to be tested. The test tools of the invention optionally include further packaged auxiliary reagents or test method solutions such as buffer solutions, standardized reference solutions of the analyte to be determined, substrate solutions and detector reagents to determine the activity of the endogenous enzyme serving as a label, Etc. Such assay tools which include 8702235 -7- a lyophilized stabilized preparation of Staphylococcus aureus cells may also additionally include packaged aqueous medium to form the required aqueous suspension of those cells in situ.

In preferred embodiments of the invention there is also provided an examination tool further comprising an absorbent reagent carrier thereby incorporating a reagent specific for that marker enzyme.

As noted above, the staphylococcal cells of the invention have been suitably treated before use to ensure that the cell suspension is safe (i.e. non-pathogenic), standardized and stable while the binding capacity and activity of the marker enzyme selected to serve as the 15 label. It has been found that these requirements can be met, for example, by confirming the staphylococcal cells by treatment with 0.5% formaldehyde solution at room temperature for 2 hours followed by heating for about 5-15 min at about 50-70 ° C. The treated cells can be stored, e.g.

as a 10% solution with a preservative such as sodium azide at 4 ° C or as a lyophilized grain of staphylococcal cells stored in airtight sealed ampoules. Another possibility is that a safe and stable reagent can be obtained by freeze-drying a suspension of staphylococcal cells and storing the product in airtight sealed ampoules. The lyophilized cells can be reconstituted in situ to be used by suspending them in a suitable liquid medium.

It should be noted that although stabilized staphylococcal cells are available commercially, the standard treatment for stabilization thereof under normal conditions renders all endogenous enzymes found thereon inactive and therefore are nonviable stabilized staphylococcal cells with an active immunoglobulin receptor and an active marker enzyme heretofore unavailable and have not been suggested to be used as immunoproofing reagents.

In an embodiment of the invention, 870223 5 «η -8- is to investigate marker enzyme endogenous catalase and the catalase activity of the staphylococcus cells.

In particularly preferred embodiments of the invention, the reagents used are nonviable stabilized staphylococcal cells having an active receptor for immunoglobulin and an active marker enzyme, the gene for that enzyme being introduced into a parent cell thereof by genetic engineering procedures per se be famous.

Therefore, for use in the present invention, a genetically engineered strain of the Cowan Staphylococcus was prepared which differs from the original strain in that it has a gene for the formation of lactamase.

The general advantage of applying this approach, that is, the introduction of a new gene, lies in the fact that one cannot rely on enzyme markers that happen to be present in the original strain. Therefore, a new hereditary marker more suitable for immuno-challenge can be added to the existing repertoire of the endogenous enzymes.

The specific advantages of the lactamase gene lie in the fact that it meets all of the following requirements: 1. The enzyme is largely cell-bound.

2. The enzyme is fully accessible to the substrate.

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

4. The enzyme has a very high turnover rate.

5. The substrate is stable, not expensive and its use does not pose any health risks or safety precautions.

6. The test of the lactamase reaction is simple, fast and very sensitive.

7. The trial does not require instrumentation and a permanent record of results can be held without any additional action.

870 2 23 5 -9-

As indicated above, several types of Streptococcus pyogenes are known to generate cell wall proteins that can serve as receptors for the Fc portion of immunoglobulins. The receptor specificity of such proteins may vary with the type of the producing strain. Of particular interest are C and G type streptococci that produce potent receptors of broad specificity. Such bacterial cells can be used as reagents in a manner analogous to that described for the Cowan I cells. However, the following differences should be noted: 1. The broader specificity of streptococcal Fc receptors allow the extension of the present methodology to the detection of immunoglobulins that do not adequately bind protein A.

2. The streptococcal cells do not have a 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 with the genetic modification of. the Cowan strain, as described above, will provide insertion of suitable genes with additional enzyme labels, which may be more advantageous in immunoassay diagnostic tools.

From the foregoing, it will be apparent that streptococcal cells possessing Fc receptors may themselves serve as a reagent source for detecting a variety of immunoglobulins. If preferred, such cells can be used in combination with the Cowan reagent described above. The combined use of both types of cells can take one of several forms: 1. The reagent may consist of a mixture of cells of both types. Each type can carry its own distinctive enzyme label or a single S70 2 23 5 * -10-f test can be used if a similar enzyme activity is chosen as a label for both cell types.

2. The mixed cells are bound together (eg with glutaraldehyde) to form a combined reagent, which is now a single enzyme label (eg lactamase) but two types of Fc receptors.

3. A combined reagent is generated by interaction with immunoglobulins present or contemplated in the assay.

Although the invention will now be described in connection with certain preferred embodiments of the present invention so that aspects thereof can be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. In contrast, it is intended to encompass all alternatives, modifications and equivalents as they 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 particulars shown are given by way of example only and for illustrative discussion of preferred embodiments of the invention and serve to clarify what is believed to be the most useful and clearest description of procedures, principles and inventive aspects.

EXAMPLE I

Preparation of stabilized suspension of Staphyloccus aureus cells.

Staphylococcus aureus (strain Cowan I, ATCC), was grown in TSB (Trypticase Soy Broth, Difco), in 500 ml Erlenmeyer flasks, on a shaker, for 18 hours at 37 ° C. The cells were then spun down (10 min at 10,000 rpm), washed twice with phosphate buffered saline (PBS), pH 7.3 and 40 suspended in 10 volumes of PBS (pH 7.3) containing 670 2 23 5 -11- 0.5% 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 7.3). the suspension was incubated at 60 ° C for 10 min with 5 gentle shaking, as previously spun and resuspended in 10 volumes of PBS (pH 7.3). Samples of that suspension streaked on AB3 (Difco) plates failed to display growth after 24 hours at 37 ° C. After 12 months of 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 II.

Preparation of Viresdried Stabilized Staphylococcus 15 Aureus Reagent.

Staphylococcus aureus (strain Cowan I, ATCC) was grown and harvested as described in Example I above. The cells were washed with PBS, resuspended in 10 volumes of fresh PSB, incubated at 60 ° C for 10 min and placed in 0.5 ml aliquots in lyophilization ampoules. After freeze-drying, the airtight sealed ampoules were stored at room temperature (22-25 ° C).

Before use, the contents of one ampoule were suspended in 5.0 ml PBS containing 0.01% merthiolate.

EXAMPLE III.

Detection of rabbit anti-BSA.

Microtiter plates (U-type, Nunc) were activated by filling the wells with 100 liters of 0.2% glutaraldehyde solution in borate buffer, pH9.0, and incubation at 56 ° C for 3 hours. The plates were then washed 10 times with twice distilled water and coated with antigen by filling the wells with 100 liters of bovine serum albumin (BSA) (50 g / ml) incubating at 37 ° C for 2 hours and at 4 ° for 18 hours C to keep. After three washes with PBS, the unoccupied surfaces were blocked by filling the wells with 100 liters of ovalbumin (1.0 mg / ml) and incubating at 37 ° C for 2 hours, followed by three washes with PBS.

Comparative sources were prepared as above with the BSA omitted.

40 Antibody for BSA (anti-BSA) was prepared in §70 2 23 5 -12-1 * rabbits. Normal rabbit serum (NRS) served as a nonspecific comparison material. Serial dilutions were made in PBS, and 100 liters each were added to the antigen coated sources. After incubation at 37 ° C for 30 minutes, the wells were washed three times with PBS. For detection of the antibody retained by the antigen, a proportional portion (100 liters) of a staphylococcal cell suspension (prepared according to Examples I and II above) was added to each source. After 20 min incubation at 37 ° C, the sources 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, according to the method described in Israeli patent no. 36496.

RESULTS.

The test system described above was used to compare the sensitivity of the detectors, ie the staphylococcal cell suspensions prepared according to Examples I and II above with the sensitivity of a radioiodinated protein A preparation, conventionally used in sensitive solid phase radioimmunoassays. The results are summarized in Table A.

TABLE A

25 Detector Anti-BSA Anti-BSA Anti-BSA NRS

prepared according to 1: 1000 1: 3000 1: 10,000 1: 1000

Example I * positive positive positive negative 30 Example II * positive positive positive negative

Radioiodinated protein A positive positive negative negative 35 (*) Based on catalase activity shown 5 min. After the addition of the substrate, namely 50 liters of a 6% hydrogen peroxide solution, §702 23 5 5 -13-

EXAMPLE IV

A gene for constitutive penicillinase formation (pen I ~) was introduced into the Cowan I strain of Staphylococcus aureus. The gene was generated in a plasmid (pi258 Pen 5 1443) that hosted the Staphylococcus aureus strain (RN 453) (Novick, R.P., and Brodsky, R., 1972). Plasmid Replication Studies: I. Replication of Unconfirmed Plasmids in S Aureus. J. Mol. Biol 68: 285-302), and was transferred with phage o 11. The transfer was as described by Novick (Novick, R.P., 1967). A stabilized suspension of these cells with lactamase as their marker enzyme was then prepared using the procedure of Example I.

EXAMPLE V.

15 Detection of antibodies to brucella in bovine sera.

A polystyrene Petri dish (Miniplate standard size) was activated at 8 equidistant preselected sites by applying 50 liters of a 25% solution of glutaraldehyde (Merck) to each site.

20 After 150 sec. at 24 ° C, the dish was cleaned with water and 10 liters of the antigen suspension was added to each spot. The antigen suspension consisted of heat-killed (90 min. At 72 ° C) cells from Brucella abortion, suspended in PS (0.5% phenol and 0.85% NaCl 25 in distilled water) at an optical density corresponding to 200 Klett units. The spots were thoroughly cleaned and the Petri dish was covered with a 5 ml layer of a blocking solution consisting of BSA (20 mg / ml) in PBS. After 2 hours at 37 ° and 16 hours at 4 ° 30, the blocking solution was decanted and the Petri dish was cleaned twice with PBS, then twice with 0.05% Tween 20 in PBS and again with PBS. The dish was then air dried and 20 liters of bovine sera samples, diluted as indicated in Table B, were added to the 35 labeled spots. After 60 min at 37 °, the dish was cleaned and dried as before, and each spot took up 20 liters of a detector suspension.

The detector suspension consisted of the genetically modified staphylococcal cells of Example IV suspended in 40 PBS at OD corresponding to 200 KU (Klett units).

8702235 # -14-

After cleaning and air drying, the lactamase activity of the detector cells retained on the sites was tested by placing a developer strip on each site. The developer strip was 5 a section (12 x 5 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 10-lactamase, was tested by determining the time required for the appearance of a white circle at the center of the blue-black detector strip. The white circle indicated that iodine had been taken up by penicilloic acid and was therefore removed from the complex giving the dark color to the detector strip. The penicilloic acid was the product of hydrolysis of an-lactam (here penicillin) catalyzed by-lactamase. Therefore, the decolorization rate of the circular region above the marked spot was inversely proportional to the amount of lactamase retained at that spot.

TABLE · B

Serum No. Agglutination Complement Test Titer * Confirmation Titer ** 25 _titer * _ 2 1:10 1: 5 1: 200 6 1:20 1: 5 1: 200 32 Negative Negative 1: 1000 53 1:80 1: 5 1: 1000 30 77 negative negative negative 26 1:40 1:50 1: 2000 111 1: 1250 - 1: 16000

Normal negative negative negative

Bovine Serum 35 * As determined by the Kimron Veterinary Institute, Beth Dagan, Israel.

** The highest dilution (in PBS) showing decolorization in <10 min.

870 2 23 5 * -15-

It will be appreciated by 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 its essential accessories, and it is therefore desirable that the present embodiments and examples be considered illustrative and non-limiting in all respects, reference being made to the appended claims rather than the foregoing description, and all. changes that fall within the meaning and scope of equivalence of the claims are therefore deemed to be included therein.

- conclusions - 870223 5

Claims (17)

1. Immunoassay reagent for binding and detecting antibodies characterized by nonviable (non-viable) stabilized bacterial cells with an active immunoglobulin receptor and an active marker enzyme. The immunoassay reagent for binding and detecting antibodies according to claim 1, characterized by non-viable stabilized staphylococcus cells. The immunoassay reagent for binding and detecting antibodies according to claim 1, characterized by non-viable stabilized streptococcus cells. 4. A test tool for enzyme immunoassay 15 characterized by one or more containers containing an aqueous suspension of nonviable stabilized bacterial cells with an active receptor for immunoglobulin and an active marker enzyme, or a dry stabilized preparation of such cells, suitable for in- situ reconstitution. Test tool according to claim 4, characterized in that said cells are a Cowan I strain of Staphylocuccus aureus cells containing a gene for the formation of β-lactamase, said gene for said enzyme being placed in a mother cell thereof. Test tool according to claim 4, characterized in that said cells are group G streptococcal strain G 148 cells. Test tool according to claim 4, characterized in that it also comprises one or more solid carriers with at least part of the surface thereof secured with a capture antibody or a bite. or antigen against which the specific antibody is to be tested. Test tool according to claim 4, characterized in that the tool also comprises one or more containers containing standardized solutions of a specific hapten or antigen antibody to be examined. Test tool according to claim 4, characterized in that the tool further comprises an absorbent reagent carrier which receives a reagent specific for that marker enzyme thereon. 10. Heterogeneous specific binding assay method for determining an unbound antibody in a liquid medium, characterized by: i) incubating a sample of said liquid medium in contact with a solid support which has an antigen or bites on its surface specific for that antibody has been confirmed; ii) separating the solid support from that liquid medium sample and cleaning it with aqueous solution to remove all traces of that sample; Iii) incubating the solid support in contact with an aqueous suspension of non-viable stabilized bacrerial cells with an active immunoglobulin receptor and an active marker enzyme; Iv) separating the solid support from that suspension and washing it with aqueous solution; and v) incubating that solid support in contact with a suitable substrate and testing for enzymatic activity of the marker enzyme. to cells held on the solid support surface. 870 2 23 5 * -18- A method according to claim 10 suitable for determining a bite or antigen in a liquid medium, characterized in that the solid support has fixed on its surface the same bite or antigen to be tested, and in step i ) a specific antibody for the hapten or antigen is added to the test sample, and as a result of binding the hapten or antigen in said sample, the amount of antibody available for binding to the hapten or antigen attached to the solid surface. , is reduced proportionately. 12. A method according to claim 10, characterized in that the aqueous suspension contains staphylococcal cells stabilized by treatment with heat and formaldehyde in order to maintain a binding capacity and the activity of the marker enzyme. 13. A method according to claim 12, characterized in that the marker enzyme is endogenous catalase and the catalase activity of the staphylococcal cells is tested. Method according to claim 10, characterized in that the solid support is incubated with an aqueous suspension of non-viable stabilized staphylococcal cells with an active receptor for immunoglobulin and an active marker enzyme, wherein the gene for the enzyme has been introduced into a mother cell thereof. 15. A method according to claim 10, characterized in that the aqueous suspension contains stabilized streptococcal cells. 16. A method according to claim 15, characterized in that the marker enzyme is endogenous hemolysin and the hemolysin activity of the streptococcal cells is tested. & 70 2 225 * __ V- * -19- 17. A method according to claim 10, characterized in that the aqueous suspension contains a Cowan I strain of Staphylococcus aureus containing a gene for the production of β-lactamase, the gene for the enzyme 5 being introduced into a mother cell thereof. 6 7 0 2 20 i>