CA1294871C - Immunoassay reagents, kits and methods - Google Patents
Immunoassay reagents, kits and methodsInfo
- Publication number
- CA1294871C CA1294871C CA000547275A CA547275A CA1294871C CA 1294871 C CA1294871 C CA 1294871C CA 000547275 A CA000547275 A CA 000547275A CA 547275 A CA547275 A CA 547275A CA 1294871 C CA1294871 C CA 1294871C
- Authority
- CA
- Canada
- Prior art keywords
- cells
- enzyme
- antigen
- viable
- hapten
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56938—Staphylococcus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Abstract
IMMUNOASSAY REAGENTS, KITS AND METHODS
ABSTRACT OF THE DISCLOSURE
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 immunoglobuiin and an active marker enzyme, or a dry stabilized preparation of such cells, suitable for in-situ reconstitution and methods for the use thereof.
ABSTRACT OF THE DISCLOSURE
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 immunoglobuiin and an active marker enzyme, or a dry stabilized preparation of such cells, suitable for in-situ reconstitution and methods for the use thereof.
Description
~L~3 ~ ~37 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.
.
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 carriers, such as test tubes, polystyrene beads or plates provided with recessed "wells". Such techniques are generally ~e~erred 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 : ~
~L~ 3t7 ~
serves as a non-specific detector for any "first antibody" either in the Free state or when bound to its specific antigen in an antigen-antibody complex.
In accordance with one version of the above methodi which reactsalso 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 antibody and the enzymatic activity is measurede This activity is directly proportional to the antibody concentration in the test sa~ple.
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 cell wall of the staphylococci but can be solubilized and ~is 394~
capable of binding many classes of immunoglobulin molecules, with high affinity. It thus also binds antibody-antigen complexes. The solubilized protein A can, furthermore, be labeled, e.g. with radio-iodine 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 ~indings with 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 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.
. . .
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 proteln 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
~9~
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 bacter-ial cells having an active receptor for immunoglobulin and an active marker enzyme, which have undergone an appropriate pre-treatment. Thu~, e.g., staphylococcal cells can effectively and rapidly bind immunoglobulin (such as any specific "first antibody") by virtue of the pxotein A in their cell walls and G streptococcal cells can similarly bind immunoglobulin by virtue of the protein G in their cell walls. At the same timP the enzyme-label is provided by enzymes which are naturally present in the cells (i.e. the endogenous or auto-chthonous 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 in one aspect, provide~
a method of immunoassay for binding and detecting anti-bodies which u~es both an active receptor for an immunoglobulin, and an endogenous active marker enzyme as a label, both being present in non~viable stabilized bacterial cells and both having been heritable and inherently formed therein befora stabilization.
In a first preferred embodiment of the present _ . ,`~
1L;~94~
invention said reagent compris~s non-viable stabilized staphylococcus cells.
In another preferred embodiment o~ the present invention said reagent comprise~ non-viable stabilized 5treptococcus cells.
The reagent used in accordance with th~ 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 ~tep, and/or (d) radiolabeling of proteins.
The present invention also provides a test kit for enzyme immunoassay according to the above-described method comprising one or more containers holding an aqueous suspension of non-viable stabilized bacterial c~lls having an active receptor for i~munoglobulin and an endogenous, heritable active marker enzyme formed and produced by bacterial cells, or a dry s~abilized preparation o~ such cells, suitable for in-situ reconstitution.
,~ ' ~9~
The test kit for carrying out immunoassay may further comprise, in a packaged combination, one or more solid carriers having ~ixed 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, one or more containers holding standardized solutions of a speci~ic antibody to the hapten or antigen which i5 to be assayed and/or an adsorbant reagent carrier incorporating therein a reagent specific to the endogenous active marker enzyme.
In accordance with one embodiment of the immunoassay method, there is provided a heterogenous specific binding assay m~thod for determining an unbound antibody in a liquid medium, comprising the steps of:
i) incubating a sample of the liquid medium in contact with a solid carrier having fixed on its surface an antigen or hapten specific to : the antibody;
ii) separating the solid carrier from the liquid medium sample and rinsing it with aqueous solution to remove all traces o~ the sample;
iii~ incubating the solid carrier in contact with an aqueous suspension of non-viable stabilized bacterial cells having an endogenou~ active marker enzyme, the enzyme having been ,~ ' 8~L
`~
heritabl~ and inherently formed in the cells before stabilization;
iv) separating the solid carrier from the suspension and washin~ it with aqueous solution; and v) incubating the solid carrier in contact with a suitable substrate and assaying for enz~matic activity of the endogenous active 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 o~ 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 ~ixed on its sur~ace the same hapten or antigen which is to be assayed 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 o~ antibody available for binding ~o the hapten or antigen fixed on the solid sur~ace 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~ ~his 8a 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 - g -capture antigen present in the test sample, and a "tracer" antibody to that antigen is then applied to form a "sandwich". The binding o~ 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. (e.g. IgY from chicken serum or from yolk) or modified antibody in which the Fc segment is missing (e.g. Fab or F(ab')2).
Selective binding to the second antibody can often be observed with conventional antibody preparatlons, 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 holdiny 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 reagents or solutions for carrylng out the assay method, such as buffer solutions, standardized referer)ce ~ :
' :
.~,. . .
' 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 ~or 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 above9 the staphylococcal cells of the present invention are appropriately treated before use in order to ensure that the cell suspension is safe ti.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 temperature for 2 hours followed by heating for about 5-15 mins.
at about 50-70C~ The treated cells can be preserved, e.g., as a 10%
suspension with a preservative such as sodium azide at ~C 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 .
3L~9~
sealed ampoulesO The freezedried cells can be reconstituted in situ for use by suspending them in a suitable liguid medium.
It is to be noted that while stabilized staphylococcal cells are commercially available the standard treatment for stabilization thereof normally inactivates any endogenous enzymes found thereon and thus non-viable stabilized staphylococcal cells having an active receptor for immunoglobulin and an active marker enzyme heretofore have not been available and also have not been suqgested for use as immunoassay reagents.
In one embodiment of the present invention said marker enzyme is endogenous catalase and tha catalase activity of the staphylococcal cells is assayed.
In especially preferred embodiments o~ the present invention there are used a~ reagents non-viable stabilized staphylococcal cells having an active receptor for immunoglobulin and an act~ve marker enzyma, 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 wa~
prepared a genetically engineered strain o~ the Cowan staphylococcus which differs from the original strain in having a gene for the formation of B-lactamase.
~ "
,~ ..
, The general advantage of using this approach o~
inserting a new gene is in not having to rely on enzyme markers which happen to be present in the original strain. Thus there can be added to the existing repertoire o~ the endogenous enzymes a new heritable marker which is more suitable for immunoassay.
The specific advantage~ of the gene for ~-lactamase are in that it fulfills all of the following requirements:
1. The enzyme is largely cell~ound.
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.
.
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 carriers, such as test tubes, polystyrene beads or plates provided with recessed "wells". Such techniques are generally ~e~erred 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 : ~
~L~ 3t7 ~
serves as a non-specific detector for any "first antibody" either in the Free state or when bound to its specific antigen in an antigen-antibody complex.
In accordance with one version of the above methodi which reactsalso 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 antibody and the enzymatic activity is measurede This activity is directly proportional to the antibody concentration in the test sa~ple.
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 cell wall of the staphylococci but can be solubilized and ~is 394~
capable of binding many classes of immunoglobulin molecules, with high affinity. It thus also binds antibody-antigen complexes. The solubilized protein A can, furthermore, be labeled, e.g. with radio-iodine 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 ~indings with 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 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.
. . .
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 proteln 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
~9~
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 bacter-ial cells having an active receptor for immunoglobulin and an active marker enzyme, which have undergone an appropriate pre-treatment. Thu~, e.g., staphylococcal cells can effectively and rapidly bind immunoglobulin (such as any specific "first antibody") by virtue of the pxotein A in their cell walls and G streptococcal cells can similarly bind immunoglobulin by virtue of the protein G in their cell walls. At the same timP the enzyme-label is provided by enzymes which are naturally present in the cells (i.e. the endogenous or auto-chthonous 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 in one aspect, provide~
a method of immunoassay for binding and detecting anti-bodies which u~es both an active receptor for an immunoglobulin, and an endogenous active marker enzyme as a label, both being present in non~viable stabilized bacterial cells and both having been heritable and inherently formed therein befora stabilization.
In a first preferred embodiment of the present _ . ,`~
1L;~94~
invention said reagent compris~s non-viable stabilized staphylococcus cells.
In another preferred embodiment o~ the present invention said reagent comprise~ non-viable stabilized 5treptococcus cells.
The reagent used in accordance with th~ 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 ~tep, and/or (d) radiolabeling of proteins.
The present invention also provides a test kit for enzyme immunoassay according to the above-described method comprising one or more containers holding an aqueous suspension of non-viable stabilized bacterial c~lls having an active receptor for i~munoglobulin and an endogenous, heritable active marker enzyme formed and produced by bacterial cells, or a dry s~abilized preparation o~ such cells, suitable for in-situ reconstitution.
,~ ' ~9~
The test kit for carrying out immunoassay may further comprise, in a packaged combination, one or more solid carriers having ~ixed 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, one or more containers holding standardized solutions of a speci~ic antibody to the hapten or antigen which i5 to be assayed and/or an adsorbant reagent carrier incorporating therein a reagent specific to the endogenous active marker enzyme.
In accordance with one embodiment of the immunoassay method, there is provided a heterogenous specific binding assay m~thod for determining an unbound antibody in a liquid medium, comprising the steps of:
i) incubating a sample of the liquid medium in contact with a solid carrier having fixed on its surface an antigen or hapten specific to : the antibody;
ii) separating the solid carrier from the liquid medium sample and rinsing it with aqueous solution to remove all traces o~ the sample;
iii~ incubating the solid carrier in contact with an aqueous suspension of non-viable stabilized bacterial cells having an endogenou~ active marker enzyme, the enzyme having been ,~ ' 8~L
`~
heritabl~ and inherently formed in the cells before stabilization;
iv) separating the solid carrier from the suspension and washin~ it with aqueous solution; and v) incubating the solid carrier in contact with a suitable substrate and assaying for enz~matic activity of the endogenous active 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 o~ 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 ~ixed on its sur~ace the same hapten or antigen which is to be assayed 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 o~ antibody available for binding ~o the hapten or antigen fixed on the solid sur~ace 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~ ~his 8a 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 - g -capture antigen present in the test sample, and a "tracer" antibody to that antigen is then applied to form a "sandwich". The binding o~ 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. (e.g. IgY from chicken serum or from yolk) or modified antibody in which the Fc segment is missing (e.g. Fab or F(ab')2).
Selective binding to the second antibody can often be observed with conventional antibody preparatlons, 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 holdiny 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 reagents or solutions for carrylng out the assay method, such as buffer solutions, standardized referer)ce ~ :
' :
.~,. . .
' 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 ~or 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 above9 the staphylococcal cells of the present invention are appropriately treated before use in order to ensure that the cell suspension is safe ti.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 temperature for 2 hours followed by heating for about 5-15 mins.
at about 50-70C~ The treated cells can be preserved, e.g., as a 10%
suspension with a preservative such as sodium azide at ~C 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 .
3L~9~
sealed ampoulesO The freezedried cells can be reconstituted in situ for use by suspending them in a suitable liguid medium.
It is to be noted that while stabilized staphylococcal cells are commercially available the standard treatment for stabilization thereof normally inactivates any endogenous enzymes found thereon and thus non-viable stabilized staphylococcal cells having an active receptor for immunoglobulin and an active marker enzyme heretofore have not been available and also have not been suqgested for use as immunoassay reagents.
In one embodiment of the present invention said marker enzyme is endogenous catalase and tha catalase activity of the staphylococcal cells is assayed.
In especially preferred embodiments o~ the present invention there are used a~ reagents non-viable stabilized staphylococcal cells having an active receptor for immunoglobulin and an act~ve marker enzyma, 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 wa~
prepared a genetically engineered strain o~ the Cowan staphylococcus which differs from the original strain in having a gene for the formation of B-lactamase.
~ "
,~ ..
, The general advantage of using this approach o~
inserting a new gene is in not having to rely on enzyme markers which happen to be present in the original strain. Thus there can be added to the existing repertoire o~ the endogenous enzymes a new heritable marker which is more suitable for immunoassay.
The specific advantage~ of the gene for ~-lactamase are in that it fulfills all of the following requirements:
1. The enzyme is largely cell~ound.
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 i~ 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 require~ no instrumentation and a permanent record of the results can be retained without any additional manipulations.
~s indicated hereinbefore several types of Streptococcus pyoqenes are known to produce cell wall proteins which can serve as receptors for the Fc portion '~?' '' .
J ~,~, :
9gL~
12a o~ immunoglobulins. The receptor specificity of such proteins may vary with the type of the producing strain. Of 12~37:~
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 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 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 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:
' ~2~
1. The reagent may consist of a mixture of cells of both typesO 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.
B-lactamase) but two kinds of Fc receptors.
lo 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 thereo~ may be more fully understood and appreciated, it i5 not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equi~alents as may be included within the scope of the invention as de~ined by the appended claims. Thus, the ~ollowing examples which include preferred embodiments will serve to illustrate tha practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion o~ preferred embodiments of the present invention only and are ' ~
..
~z~
presented in the cause of providing what is believed to be the most useful and readily understood description o~
formulation procedures as well as of the principles and conceptual aspects of the invention.
Pre~aration of stabilized suspension of Staphylococcus aureus cell~
Staphylococcus aureus (strain Cowan I, ATCC), was grown in TSB (Trypticase Soy Broth, Difco), in 500 ml Erlenmeyer flasks, on a reciprocal shaker, for 18 hours at 37C. The cells were 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 suspended in 10 volumes o~ PBS (pH
7.3). The suspension wa~ incubated for lQ minutes at 60C under gentle shaking, spun as before and resuspended in 10 volume~ of P~S (pH 7.3). Samples of that suspension, streaked on AB3(Difco) plates, failed to show growth after 24 hours at 37C. After 12 months storage at 4~C the suspension showed no 3ign5 of change in binding and catalytic properties. Before use the suspension was diluted 1:10 in PBS.
, .. .
:
~LZ9~
Preparation of freeze-dried stab;lized Staph,ylococcus au~ c~a~
Staphylococcus aureus (strain Cowan I, ATCC) was cultivated and harvested as described in ~xmaple 1 above. The cells were washed with PBS, resuspended in 10 volumes of fresh TSB~ inc~bated for 10 minutes at 60C and dispensed in 0.5 ml portions into lyophili~ation-ampoules.
After freeze-drying the sealed ampoules were stored at room temperature t22-25C). For use, the contents of one ampoule were suspended in 5.0 ml PBS containing 0.01% merthiolate.
Detection of Rabbit anti-BSA
Microtitr~e plates (U type, Nunc) wre activated by filling the wells with 100 1 of 0.2% glutaraldehyde solution in bor~te buffer, pH
9.0, and incubation for 3 hours at 56C. The plates were then washed 10 times with twice-distilled water and coated with antigen by filling the wells with 100 1 of bovine serum albumin (BSA) (50 g/ml), incubating for 2 hours at 37C and storing at 4C for 18 hours. After 3 washes with PBS the unoccupied surfaces were blocked by filling the wells with 100 1 of ovalbumin (1.0 mg/ml) and incubating for 2 hours at 37C, 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 made in PBS, and 100 ~1 of each were added to the antigen coated wells. After 30 minutes incubation at 37C the wells were washed thrice with PBS. For detection o~ the antibody retained by the antigen, an ali~uot (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 Israeli Patent Specification No. 36496.
~SS~E
The test system dsscribed above was used to compare the sensitivity o~ the detectors, i.e., the staphylococcal 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.
* - Trademark ~L~9~
~E~
Detector prepared Anti-BSA ~nti-BSA Anti-BSA NRS
according to1:1000 1:3QOO 1:10000 1:1000 Example 1* Pos. Pos. Pos. Neg.
Example 2* Pos. Pos. Pos. Neg.
-10 Radioiodinated Protein A Pos. Pos. NegO Neg.
(*) Based on catalase activity displayed 5 minutes after th~ addition of the substrate, namely 50 1 of a 6%
hydrogen peroxide solution.
.
, ~L2~7~
A gene for constitutive penicillinase Pormation (pen I-) was inserted into the Cowan I strain of Staphylococcus aureus. The gene originated in a plasmid (PI258 pen I443) hosted by strain (RN 453) of Staphylococcus au~eus (Novick. R.P., and Brodsky, R., 1972)~ Studies on plasmid replication: I. Replication of unestablished plasmids in S ~ureu~. J.Mol.Biol 68:
285-302), and was transduced with phage o 11. The transduction was as des~ribed 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 thes~ cells having B-lactamase as their marker enzyme was then prepared by the procedure of Example 1.
EXAMPLE S
Detection of an~ibodies to ~rucella i~ bovine sera A polystyrene Petri dish (standard size, Miniplate) was activated at 8 preselected, equidistant spots by 20- placing 50 ~1 of a 25% solution o~ glutaraldehyde (Merck) at each spot. Aftsr 150 sec at 24C the dish was rinsed with water and 10 ~1 of the antigen suspension wa added to each spot. The antigen suspension consisted of heat-killed (90 min at 72C
cells o~ B~ucella abortus, suspended in PS (0~5% phenol and 0.85% NaCl in distilled water) at optical density corresponding to 200 Klett Units. The spots were 8~
,,, 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 hre at 4 the blocking solution was poured o~f and the Petri dish was rinsed twice with PBS, then twice with O.05% ~ween* 20 in PBS and again with PB~. 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 60 min at 37, the dish was rinsed and dried as before, and each spot received 20 ~1 o~ a detector suspe~sion. The detector suspension consisted of the genetically modified staphylococcal cells of example 4 suspended in PBS at OD corresponding to 200 XU. After rinsing and air drying the B-lactamase activity of the det~ctor cells retained on the spots was tested by placing on each spot a developer ætrip. The dQveloper 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 (1.0%
w/v) and penicillin (50 mM) in P~S. The activity o~ the marker enzyme of the d~tector cells, namely B-lactamase, was assayed by determining the time required for the appearance o~ a white circle in the center of the blue-hlack 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 * - Trademark ~L~9~71 20a to the detector strip. The penicilloic acid is the product of hydrolysis of a ~-lactam (here penicillin) catalysed by B-lactama~e. Hence the rate o~ *he decolorization of the circular area above the marked spot is inversely proportional to the amount of B-lactamase retained on that spot.
.~
~9~18~
Serum Agglutination Complement Present No. Titre* Fixation Test Titre* Titre*~
2 1:10 1:5 1:200 6 1:20 1:5 1 200 32 Neg Neg l:1000 53 1:80 1:5 l:1000 77 Neg Neg Neg 26 1:40 1:50 1:2000 111 1:1250 - 1:16000 Normal Neg Neg Neg Bovine Serum .
* As determined at the Kimron Veterinary Institute, Beth Dagan, - Israel ** The highest dilution (in PBS) which showed decolorization in ~ 10 mi n.
- 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 fore-going description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced ~herein. ~ :
- .: -
~s indicated hereinbefore several types of Streptococcus pyoqenes are known to produce cell wall proteins which can serve as receptors for the Fc portion '~?' '' .
J ~,~, :
9gL~
12a o~ immunoglobulins. The receptor specificity of such proteins may vary with the type of the producing strain. Of 12~37:~
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 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 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 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:
' ~2~
1. The reagent may consist of a mixture of cells of both typesO 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.
B-lactamase) but two kinds of Fc receptors.
lo 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 thereo~ may be more fully understood and appreciated, it i5 not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equi~alents as may be included within the scope of the invention as de~ined by the appended claims. Thus, the ~ollowing examples which include preferred embodiments will serve to illustrate tha practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion o~ preferred embodiments of the present invention only and are ' ~
..
~z~
presented in the cause of providing what is believed to be the most useful and readily understood description o~
formulation procedures as well as of the principles and conceptual aspects of the invention.
Pre~aration of stabilized suspension of Staphylococcus aureus cell~
Staphylococcus aureus (strain Cowan I, ATCC), was grown in TSB (Trypticase Soy Broth, Difco), in 500 ml Erlenmeyer flasks, on a reciprocal shaker, for 18 hours at 37C. The cells were 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 suspended in 10 volumes o~ PBS (pH
7.3). The suspension wa~ incubated for lQ minutes at 60C under gentle shaking, spun as before and resuspended in 10 volume~ of P~S (pH 7.3). Samples of that suspension, streaked on AB3(Difco) plates, failed to show growth after 24 hours at 37C. After 12 months storage at 4~C the suspension showed no 3ign5 of change in binding and catalytic properties. Before use the suspension was diluted 1:10 in PBS.
, .. .
:
~LZ9~
Preparation of freeze-dried stab;lized Staph,ylococcus au~ c~a~
Staphylococcus aureus (strain Cowan I, ATCC) was cultivated and harvested as described in ~xmaple 1 above. The cells were washed with PBS, resuspended in 10 volumes of fresh TSB~ inc~bated for 10 minutes at 60C and dispensed in 0.5 ml portions into lyophili~ation-ampoules.
After freeze-drying the sealed ampoules were stored at room temperature t22-25C). For use, the contents of one ampoule were suspended in 5.0 ml PBS containing 0.01% merthiolate.
Detection of Rabbit anti-BSA
Microtitr~e plates (U type, Nunc) wre activated by filling the wells with 100 1 of 0.2% glutaraldehyde solution in bor~te buffer, pH
9.0, and incubation for 3 hours at 56C. The plates were then washed 10 times with twice-distilled water and coated with antigen by filling the wells with 100 1 of bovine serum albumin (BSA) (50 g/ml), incubating for 2 hours at 37C and storing at 4C for 18 hours. After 3 washes with PBS the unoccupied surfaces were blocked by filling the wells with 100 1 of ovalbumin (1.0 mg/ml) and incubating for 2 hours at 37C, 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 made in PBS, and 100 ~1 of each were added to the antigen coated wells. After 30 minutes incubation at 37C the wells were washed thrice with PBS. For detection o~ the antibody retained by the antigen, an ali~uot (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 Israeli Patent Specification No. 36496.
~SS~E
The test system dsscribed above was used to compare the sensitivity o~ the detectors, i.e., the staphylococcal 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.
* - Trademark ~L~9~
~E~
Detector prepared Anti-BSA ~nti-BSA Anti-BSA NRS
according to1:1000 1:3QOO 1:10000 1:1000 Example 1* Pos. Pos. Pos. Neg.
Example 2* Pos. Pos. Pos. Neg.
-10 Radioiodinated Protein A Pos. Pos. NegO Neg.
(*) Based on catalase activity displayed 5 minutes after th~ addition of the substrate, namely 50 1 of a 6%
hydrogen peroxide solution.
.
, ~L2~7~
A gene for constitutive penicillinase Pormation (pen I-) was inserted into the Cowan I strain of Staphylococcus aureus. The gene originated in a plasmid (PI258 pen I443) hosted by strain (RN 453) of Staphylococcus au~eus (Novick. R.P., and Brodsky, R., 1972)~ Studies on plasmid replication: I. Replication of unestablished plasmids in S ~ureu~. J.Mol.Biol 68:
285-302), and was transduced with phage o 11. The transduction was as des~ribed 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 thes~ cells having B-lactamase as their marker enzyme was then prepared by the procedure of Example 1.
EXAMPLE S
Detection of an~ibodies to ~rucella i~ bovine sera A polystyrene Petri dish (standard size, Miniplate) was activated at 8 preselected, equidistant spots by 20- placing 50 ~1 of a 25% solution o~ glutaraldehyde (Merck) at each spot. Aftsr 150 sec at 24C the dish was rinsed with water and 10 ~1 of the antigen suspension wa added to each spot. The antigen suspension consisted of heat-killed (90 min at 72C
cells o~ B~ucella abortus, suspended in PS (0~5% phenol and 0.85% NaCl in distilled water) at optical density corresponding to 200 Klett Units. The spots were 8~
,,, 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 hre at 4 the blocking solution was poured o~f and the Petri dish was rinsed twice with PBS, then twice with O.05% ~ween* 20 in PBS and again with PB~. 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 60 min at 37, the dish was rinsed and dried as before, and each spot received 20 ~1 o~ a detector suspe~sion. The detector suspension consisted of the genetically modified staphylococcal cells of example 4 suspended in PBS at OD corresponding to 200 XU. After rinsing and air drying the B-lactamase activity of the det~ctor cells retained on the spots was tested by placing on each spot a developer ætrip. The dQveloper 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 (1.0%
w/v) and penicillin (50 mM) in P~S. The activity o~ the marker enzyme of the d~tector cells, namely B-lactamase, was assayed by determining the time required for the appearance o~ a white circle in the center of the blue-hlack 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 * - Trademark ~L~9~71 20a to the detector strip. The penicilloic acid is the product of hydrolysis of a ~-lactam (here penicillin) catalysed by B-lactama~e. Hence the rate o~ *he decolorization of the circular area above the marked spot is inversely proportional to the amount of B-lactamase retained on that spot.
.~
~9~18~
Serum Agglutination Complement Present No. Titre* Fixation Test Titre* Titre*~
2 1:10 1:5 1:200 6 1:20 1:5 1 200 32 Neg Neg l:1000 53 1:80 1:5 l:1000 77 Neg Neg Neg 26 1:40 1:50 1:2000 111 1:1250 - 1:16000 Normal Neg Neg Neg Bovine Serum .
* As determined at the Kimron Veterinary Institute, Beth Dagan, - Israel ** The highest dilution (in PBS) which showed decolorization in ~ 10 mi n.
- 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 fore-going description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced ~herein. ~ :
- .: -
Claims (14)
1. A method of immunoassay for binding and detecting antibodies which uses both an active receptor for an immunoglobulin, and an endogenous active marker enzyme as a label, both being present in non-viable stabilised bacterial cells and both having been heritable and inherently formed therein before stabilisation.
2. A heterogeneous specific binding assay method for determining an unbound antibody in a liquid medium, as claimed in claim 1, 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 stabilised bacterial cells having an endogenous active marker enzyme, the enzyme having been heritable and inherently formed in the cells before stabilisation:
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 endogenous active marker enzyme on cells retained on the solid carrier surface.
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 stabilised bacterial cells having an endogenous active marker enzyme, the enzyme having been heritable and inherently formed in the cells before stabilisation:
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 endogenous active marker enzyme on cells retained on the solid carrier surface.
3. A method according to claim 2 adapted for determining a hapten or antigen in a liquid medium wherein said solid carrier has fixed 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 antigen available for binding to the hapten or antigen fixed to the solid surface is proportionately reduced.
4. A method according to claim 1 or 2, wherein the bacterial cells are stabilized by treatment with heat and formaldehyde to preserve their immunoglobulin binding capacity and the activity of the endogenous marker enzyme.
5. A method as claimed in claim 1 or 2, wherein the non-viable, stabilized bacterial cells comprise staphylococcal cells.
6. A method according to claim 1 or 2, wherein the non-viable stabilized bacterial cells comprise staphylococcal cells, the endogenous active marker enzyme is catalase and the catalase activity of the staphylococcal cells is assayed.
7. A method according to claim 2 comprising incubating said solid carrier with an aqueous suspension of non-viable stabilized staphylococcal cells having an active receptor for immunoglobulin and an endogenous active marker enzyme, the gene for said enzyme having been inserted into a parental cell thereof.
8. A method according to claim 1 or 2, wherein the non-viable stabilised bacterial cells comprise a Cowan I strain of Staphylococcus aureus containing a gene for the formation of .beta.-lactamase, the gene for the enzyme having been inserted into a parent cell thereof.
9. A method as claimed in claim 1 or 2, wherein the non-viable stabilised bacterial cells comprise streptococcal cells.
10. A method according to claim 1 or 2, wherein the non-viable stabilized- bacterial cells comprise streptococcal cells, the endogenous active marker enzyme is hemolysin and the hemolysin activity of the streptococcal cells is assayed.
11. A test kit for an enzyme immunoassay according to the method of claim 1, comprising one or more containers holding an aqueous suspension of non-viable stabilized bacterial cells having an active receptor for immunoglobulin and an endogenous active marker enzyme, or a dry stabilised preparation of such cells, suitable for in-situ reconstitution.
12. A test kit according to claim 11 further comprising one or more solid carriers having fixed on at least portion of the surface thereof a capture antibody or a hapten or antigen the specific antibody to which is to be assayed.
13. A test kit according to claim 12, which kit additionally comprises one or more containers holding standardised solutions of a specific antibody to the hapten or antigen which is to be assayed.
14. A test kit according to claim 13, which kit additionally comprises an absorbent reagent carrier incorporating thereon a reagent specific to the endogenous active marker enzyme.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IL80083 | 1986-09-19 | ||
| IL80083A IL80083A0 (en) | 1986-09-19 | 1986-09-19 | Immunoassay reagents,kits and methods |
| IL80313A IL80313A0 (en) | 1986-09-19 | 1986-10-15 | Immunoassay reagents,kits and methods |
| IL80313 | 1986-10-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1294871C true CA1294871C (en) | 1992-01-28 |
Family
ID=26321590
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000547275A Expired - Lifetime CA1294871C (en) | 1986-09-19 | 1987-09-18 | Immunoassay reagents, kits and methods |
Country Status (9)
| Country | Link |
|---|---|
| AU (1) | AU594503B2 (en) |
| CA (1) | CA1294871C (en) |
| CH (1) | CH675309A5 (en) |
| DE (1) | DE3731227A1 (en) |
| FR (1) | FR2604259B1 (en) |
| GB (1) | GB2197468B (en) |
| IL (1) | IL80313A0 (en) |
| IT (1) | IT1223303B (en) |
| NL (1) | NL8702235A (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1989010974A1 (en) * | 1988-05-11 | 1989-11-16 | Trustees Of The Sisters Of Charity Of Australia | Enzyme immunoassay system |
| US5759774A (en) * | 1988-05-18 | 1998-06-02 | Cobe Laboratories, Inc. | Method of detecting circulating antibody types using dried or lyophilized cells |
| DE3936256A1 (en) * | 1989-10-31 | 1991-05-02 | Max Planck Gesellschaft | TEST FOR THE DIFFERENTIAL QUANTIFICATION OF THE FREE BZW. THE PEPTIDASE COMPLEXED PROTEINASE INHIBITOR (ALPHA) (ARROW ABBEERTS) 2 (ARROW ABUTE) MACROGLOBULIN |
| WO2008136861A2 (en) * | 2006-11-22 | 2008-11-13 | 3M Innovative Properties Company | Inactivated and dried biological preparations |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3990947A (en) * | 1975-03-07 | 1976-11-09 | Warner-Lambert Company | Composition for detecting fibrinogen, fibrinogen split products and fibrin split products |
| FR2405301A1 (en) * | 1977-10-04 | 1979-05-04 | Api Labor | SUBSTRATES AND METHOD FOR THE RAPID IDENTIFICATION OF BACTERIA OF THE GENUS STREPTOCOCCUS |
| SE7712244L (en) * | 1977-10-31 | 1979-05-01 | Jonsson U R S | PROCEDURE FOR PREPARATION OF KILLED BACTERIA |
| US4471058A (en) * | 1982-07-26 | 1984-09-11 | Board Of Trustees Operating Michigan State University | Method for the detection and/or determination of a polyvalent antigen using at least two different monoclonal antibodies |
-
1986
- 1986-10-15 IL IL80313A patent/IL80313A0/en unknown
-
1987
- 1987-09-10 GB GB8721276A patent/GB2197468B/en not_active Expired - Lifetime
- 1987-09-16 CH CH3575/87A patent/CH675309A5/fr not_active IP Right Cessation
- 1987-09-17 FR FR878712904A patent/FR2604259B1/en not_active Expired - Lifetime
- 1987-09-17 IT IT21950/87A patent/IT1223303B/en active
- 1987-09-17 AU AU78623/87A patent/AU594503B2/en not_active Ceased
- 1987-09-17 DE DE19873731227 patent/DE3731227A1/en not_active Withdrawn
- 1987-09-18 NL NL8702235A patent/NL8702235A/en not_active Application Discontinuation
- 1987-09-18 CA CA000547275A patent/CA1294871C/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| CH675309A5 (en) | 1990-09-14 |
| AU594503B2 (en) | 1990-03-08 |
| GB2197468B (en) | 1991-03-13 |
| IT1223303B (en) | 1990-09-19 |
| AU7862387A (en) | 1988-03-24 |
| GB8721276D0 (en) | 1987-10-14 |
| IT8721950A0 (en) | 1987-09-17 |
| GB2197468A (en) | 1988-05-18 |
| FR2604259A1 (en) | 1988-03-25 |
| DE3731227A1 (en) | 1988-06-30 |
| IL80313A0 (en) | 1987-01-30 |
| FR2604259B1 (en) | 1991-05-10 |
| NL8702235A (en) | 1988-04-18 |
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