US4471058A
Expired / Lapsed
US
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Method for the detection and/or determination of a polyvalent antigen using at least two different monoclonal antibodies
Assignee: Michigan State University
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Filing: 1982-07-26
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Granted: 1984-09-11
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Expires: 2002-07-26
Abstract
Abstract A method for the detection and/or determination of an antigen having at least two separate antibody combining sites or antigenic determinants (i.e. a polyvalent antigen) using at least two different monoclonal antibodies which bind to the separate sites is described. In particular the method utilizes Protein A with a carrier to immobilize a first monoclonal antibody which in turn, can bind to one antigenic determinant on the antigen. The second antibody with a label is provided in a solution and binds to the second antigenic determinant on the antigen. A test kit is described for practicing the method. Novel anti-PGH synthase and anti-PGI2 synthase antibodies and hybridoma cells producing such antibodies are described.
Inventors
- 👤 William L. Smith
- 👤 David L. Dewitt
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 12:45
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/573; C; C07; C07K; C07K16/00; C07K16/40; G01N33/543; G01N33/54306; G01N33/554; G01N33/563; G01N33/569; G01N33/56911; G01N33/56938; G01N33/577; Y; Y10; Y10S; Y10S436/00; Y10S436/807; Y10S436/808; Y10S436/809
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
249 words
In a method for determination of polyvalent antigens by reaction with monoclonal antibodies specific for the antigen, wherein the antigen has at least two separate different combining sites for the antibodies the improvement which comprises: (a) providing a first murine IgG monoclonal antibody which combines with a first antigen combining site and which is bound to an immobilizing carrier by means of Staphylococcus aureus Protein A directly or indirectly by means of a conjugal antibody between the IgG antibody and the protein A and a solution containing a second murine IgG monoclonal antibody which combines with a second combining site on the antigen and not to the carrier and which is labeled so as to be detectable, wherein the antibodies are produced by different hybridoma cells and are specific for the same polyvalent antigen but having different combining sites on the antigen, and wherein the second antibody does not react with Protein A or reacts only slowly with Protein A so as not to interfere with determination of the antigen; (b) reacting the antigen in a solution with the first antibody bound to the carrier to provide a first antibody-antigen complex with the carrier; (c) reacting the first complex with the second antibody in a solution to provide a first and second antibody-antigen complex with the carrier which is separable from the solution; (d) separating the first antibody-second antibody-antigen complex from unbound second antibody in solution; and (e) determining the presence of label in the first antibody-second antibody-antigen complex.
Claim 2
Dependent Claim
249 words
In a method for determination of polyvalent antigens by reaction with monoclonal antibodies specific for the antigen, wherein the antigen has at least two separate different combining sites for the antibodies the improvement which comprises: (a) providing a first murine IgG monoclonal antibody which combines with a first antigen combining site and which is bound to an immobilizing carrier by means of Staphylococcus aureus Protein A directly or indirectly by means of a conjugal antibody between the IgG antibody and the protein A and a solution containing a second murine IgG monoclonal antibody which combines with a second combining site on the antigen and not to the carrier and which is labeled so as to be detectable, wherein the antibodies are produced by different hybridoma cells and are specific for the same polyvalent antigen but having different combining sites on the antigen, and wherein the second antibody does not react with Protein A or reacts only slowly with Protein A so as not to interfere with determination of the antigen; (b) reacting the antigen in a solution with the first antibody bound to the carrier to provide a first antibody-antigen complex with the carrier; (c) reacting the first complex with the second antibody in a solution to provide a first and second antibody-antigen complex with the carrier which is separable from the solution; (d) separating the first antibody-second antibody-antigen complex from unbound second antibody in solution; and (e) determining the presence of label in the first antibody-second antibody-antigen complex.
Claim 3
Dependent Claim
45 words
The method of claim 1 wherein the carrier contains Protein A of killed Staphylococcus aureus cells, wherein the first antibody binds to the Protein A and the antigen and wherein the second antibody does not bind to Protein A in the presence of the antigen.
Claim 4
Dependent Claim
16 words
The method of claim 2 wherein the carrier is attenuated cells of Staphylococcus aureus ATCC 12598.
Claim 5
Dependent Claim
12 words
The method of claim 2 wherein the carrier is Protein A sepharose.
Claim 6
Dependent Claim
11 words
The method of claim 1 wherein the antigen is PGH synthase.
Claim 7
Dependent Claim
12 words
The method of claim 1 wherein the antigen is PGI 2 synthase.
Claim 8
Dependent Claim
48 words
The method of claim 1 wherein the carrier is attenuated cells of Staphylococcus aureus containing protein A, wherein the antigen is PGH synthase and wherein the second antibody does not bind the protein A in the presence of the antigen and the first antibody does bind protein A.
Claim 9
Dependent Claim
38 words
The method of claim 1 wherein the carrier is attenuated cells of Staphylococcus aureus carrier protein A complexed with a conjugal anti-mouse antibody which binds the protein A and which binds the first antibody which binds the antigen.
Claim 10
Dependent Claim
44 words
The method of claim 8 wherein the conjugal antibody is rabbit anti-mouse antibody, wherein the first antibody is secreted by hybridoma cell isn-1 deposited as ATCC No. HB8123 and the second antibody is secreted by hybridoma cell isn-3 deposited as ATCC No. HB 8122.
Claim 11
Dependent Claim
49 words
The method of claim 1 wherein the first antibody is secreted by one hybridoma cell selected from cyo 1, 5 and 7 deposited as ATCC HB 8124, HB 8126 and HB 8127, respectively and the second antibody is secreted by hybridoma cell cyo 3 deposited as ATCC HB 8125.
Claim 12
Dependent Claim
34 words
The method of claim 1 wherein the first antibody is derived from hybridoma cells isn-1 deposited as ATCC HB 8123 and the second antibody is secreted by hybridoma cell isn-3 deposited as ATCC-HB 8122.
Claim 13
Dependent Claim
133 words
As a mercantile kit test kit for a polyvalent antigen which comprises in combination: (a) a first container containing an IgG monoclonal antibody bound to Protein A immobilized on a second container containing a carrier; and (b) a second container containing a labeled second IgG monoclonal antibody in a solution, wherein in use the amount of the second antibody which combines with the antigen is determined by the label in direct correlation with the amount of antigen, wherein the first and second antibodies are produced by different hybridoma cells and are both specific for the antigen but combine different antigenic determinants on the antigen and wherein the second antibody does not react with Protein A or reacts only slowly with Protein A so as not to interfere with a determination of the antigen.
Claim 14
Dependent Claim
133 words
As a mercantile kit test kit for a polyvalent antigen which comprises in combination: (a) a first container containing an IgG monoclonal antibody bound to Protein A immobilized on a second container containing a carrier; and (b) a second container containing a labeled second IgG monoclonal antibody in a solution, wherein in use the amount of the second antibody which combines with the antigen is determined by the label in direct correlation with the amount of antigen, wherein the first and second antibodies are produced by different hybridoma cells and are both specific for the antigen but combine different antigenic determinants on the antigen and wherein the second antibody does not react with Protein A or reacts only slowly with Protein A so as not to interfere with a determination of the antigen.
Claim 15
Dependent Claim
18 words
The test kit of claim 12 wherein the carrier is attenuated cells of Staphylococcus aureus containing Protein A.
Claim 16
Dependent Claim
81 words
The test kit of claim 12 wherein the first antibody is secreted by a hybridoma cell selected from cyo 1, 5 and 7, deposited as ATCC HB 8124, HB 8126, and HB 8127, respectively, wherein the carrier is attenuated cells of Staphylococcus aureus containing Protein A and wherein the second labeled antibody is secreted by hybridoma cell cyo-3 deposited as ATCC HB 8125, and does not bind to the protein A of the Staphylococcus aureus when complexed with the first antibody.
Claim 17
Dependent Claim
60 words
The test kit of claim 12 wherein the carrier is attenuated cells of Staphylococcus aureus, wherein the second labeled antibody is secreted by hybridoma cell isn-3 deposited as ATCC HB 8122 with the label added, wherein the first antibody is unlabeled and is secreted by hybridoma cell isn-1 deposited as ATCC HB 8123 and wherein the assay is rapidly completed.
Claim 18
Dependent Claim
15 words
The test kit of claim 14 or 15 wherein the label is radioisotope 125 I.
Claim 19
Dependent Claim
21 words
Monoclonal antibodies produced by hybridoma cells cyo-1,-3,-5 and -7 and deposited as ATCC HB 8124, HB8125, HB8126 and HB 8127 respectively.
Claim 20
Dependent Claim
21 words
Monoclonal antibodies produced by hybridoma cells cyo-1,-3,-5 and -7 and deposited as ATCC HB 8124, HB8125, HB8126 and HB 8127 respectively.
Claim 21
Dependent Claim
17 words
Monoclonal antibodies produced by hybridoma cells isn-1 and isn-3 and deposited as ATCC HB8123 and HB8122 respectively.
Claim 22
Dependent Claim
17 words
Monoclonal antibodies produced by hybridoma cells isn-1 and isn-3 and deposited as ATCC HB8123 and HB8122 respectively.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1987-09-11 | 1988-03-12 | 1988-09-11 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1991-09-11 | 1992-03-12 | 1992-09-11 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1995-09-11 | 1996-03-12 | 1996-09-11 | $7700.00 | Window Closed / Lapsed |