Use of Intramolecularly, Covalently Cross-Linked Proteins As Binding Partners In Immunoassays

Abstract

The invention concerns the use of intramolecularly, covalently cross-linked proteins and covalently cross-linked reverse transcriptase from HIV as immunological binding partners in immunoassays. It also concerns immunological test procedures for detecting an analyte in a sample in which intramolecularly, covalently cross-linked proteins are used as binding partners, and it further concerns intramolecularly, covalently cross-linked reverse transcriptase from HIV and a method for producing this reverse transcriptase.

1 . A method for detecting an antibody in a sample comprising the steps of:
 (a) combining the sample with an intramolecularly, covalently cross-linked protein antigen in the absence of detectable intermolecular multimers of the antigen, wherein the antigen specifically binds with the antibody or with a substance bound to the analyte to form a complex,   (b) adding to the combination formed in step (a) a binding partner provided with a label that combines with the complex formed in step (a) to produce a detectable signal, and   (c) determining the signal produced in step (b) as a measure of the analyte in the sample.   
   
   
       2 . The method of  claim 1  wherein the protein antigen is selected from the group consisting of DNA and RNA polymerases. 
   
   
       3 . The method of  claim 1  wherein the protein antigen is prostate specific antigen. 
   
   
       4 . A method for detecting an antibody in a sample comprising the steps of:
 (a) contacting the sample with a composition comprising an intramolecularly, covalently cross-linked protein antigen, wherein said composition comprises less than about 5% of total intermolecular multimers of the protein antigen relative to the eluted peak intramolecularly, covalently cross-linked protein antigen as determined in a gel permeation chromatograph, wherein the intramolecularly, covalently cross-linked protein antigen specifically binds with the antibody or with a substance bound to the antibody to form a complex,   (b) adding to the combination formed in step (a) a binding partner provided with a label that combines with the complex formed in step (a) to produce a detectable signal, and   (c) determining the signal produced in step (b) as a measure of the analyte in the sample.   
   
   
       5 . The method of  claim 4  wherein intermolecular multimers of the protein antigen are undetectable using SDS-PAGE analysis when 500 ng of said composition is loaded per lane. 
   
   
       6 . The method of  claim 4  wherein intermolecular multimers of the protein antigen are undetectable using gel permeation chromatography analysis. 
   
   
       7 . The method of  claim 4  wherein the protein is selected from the group consisting of DNA and RNA polymerases. 
   
   
       8 . The method of  claim 4  wherein the protein antigen is cross-linked using a cross-linking agent selected form the group consisting of MHS (3-maleimidobenzoyl-N-hydroxysuccinimide ester), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), DSS (disuccinimidylsuberate), HSAB (N-hydroxysuccinimidyl-4-azidobenzoate), and sulfo-SANPAH (sulfosuccinimidyl-6(4′-amido-2′-nitrophenylamido)hexanoate). 
   
   
       9 . The method of  claim 8  wherein the intramolecularly, covalently cross-linked protein antigen is further modified to comprise a signal generating group or a ligand for binding to a solid support. 
   
   
       10 . The method of  claim 9  wherein the signal generating group is selected from the group consisting of enzymes, fluorescent groups and chemiluminescent groups. 
   
   
       11 . The method of  claim 9  wherein the ligand is biotin or streptavidin. 
   
   
       12 . The method of  claim 1  wherein the intramolecularly, covalently cross-linked protein antigen is further modified to comprise a signal generating group or a ligand for binding to a solid support. 
   
   
       13 . The method of  claim 1  wherein the intramolecularly, covalently cross-linked protein antigen is covalently linked to biotin or streptavidin.