Method for detecting an antigen-specific antibody and reagent for use therein

Abstract

A method for detecting an antigen-specific antibody detection reagent, which includes a cationized, denatured antigenic protein immobilized on a solid-phase surface of a carrier material, is provided. The cationized, denatured antigenic protein has cationized SH groups formed from reaction with a cationizing agent. The detection reagent is capable of specifically binding to an antigen-specific antibody which binds to an epitope of the antigenic protein. The method includes contacting a sample containing the antigen-specific antibody with the detection reagent to bind the antigen-specific antibody with the detection reagent. A labeled secondary antibody is then added to allow the labeled secondary antibody to bind to the antigen-specific antibody; and the detection reagent bound with the antigen-specific antibody is detected.

What is claimed is: 
     
         1 . A method for detecting an antigen-specific antibody contained in a sample, the method comprising the steps of:
 contacting the sample with a reagent for detection of antibody binding to an epitope of an antigenic protein to bind the antigen-specific antibody with the reagent for detection;   wherein the reagent for detection comprises a carrier material and a cationized, denatured antigenic protein immobilized on a solid-phase surface of the carrier material; wherein the cationized, denatured antigenic protein has cationized SH groups formed from reaction with a cationizing agent;   adding thereto a labeled secondary antibody to allow the labeled secondary antibody to bind to the antigen-specific antibody; and   detecting the reagent for detection bound with the antigen-specific antibody.   
     
     
         2 . The method of  claim 1 , wherein the cationized, denatured antigenic protein is a cationized, denatured full-length protein. 
     
     
         3 . The method of  claim 1 , wherein the cationized, denatured antigenic protein is a cationized, denatured membrane protein. 
     
     
         4 . The method of  claim 1 , wherein the cationized, denatured antigenic protein is a cationized, denatured cancer antigenic protein. 
     
     
         5 . The method of  claim 1 , wherein the carrier material is a membrane, wafer, microplate or bead. 
     
     
         6 . The method of  claim 1 , wherein the carrier material is a magnetic microbead. 
     
     
         7 . The method of  claim 1 , wherein the solid-phase surface is a glass, nylon or semiconductor surface. 
     
     
         8 . The method of  claim 1 , wherein the carrier material is a carboxylated polystyrene bead. 
     
     
         9 . The method of  claim 1 , wherein the cationized, denatured antigenic protein is immobilized through a reaction of an antigenic protein amino group with an activated carboxylic acid group on the solid-phase surface. 
     
     
         10 . The method of  claim 1 , wherein the cationized, denatured antigenic protein is indirectly immobilized on the solid-phase surface. 
     
     
         11 . The method of  claim 10 , wherein the cationized, denatured antigenic protein is immobilized indirectly on the solid-phase surface via a biotin-avidin bond. 
     
     
         12 . The method of  claim 1 , wherein the cationized, denatured antigenic protein has cationized SH groups formed from reaction with an alkyl halide cationizing agent. 
     
     
         13 . The method of  claim 12 , wherein the alkyl halide cationizing agent is (3-bromopropyl)-trimethylammonium (TAP-Br). 
     
     
         14 . The method of  claim 1 , wherein the cationized, denatured antigenic protein has cationized SH groups formed from reaction with a cationizing agent comprising a thiosulfonate compound, a mixed disulfide compound, a pyridyl sulfide cationizing agent, or a mixture of two of more thereof. 
     
     
         15 . The method of  claim 1 , wherein the cationizing agent is trimethylammoniopropyl methanethiosulfonate (TAPS-sulfonate). 
     
     
         16 . A reagent for detection of antibody binding to an epitope of an antigenic protein comprising a carrier material and a cationized, denatured antigenic protein immobilized on a solid-phase surface of the carrier material;
 wherein the cationized, denatured antigenic protein has cationized SH groups formed from reaction with a cationizing agent.   
     
     
         17 . The reagent of  claim 16 , wherein the carrier material is a membrane, wafer, microplate or bead. 
     
     
         18 . The reagent of  claim 16 , wherein the cationizing agent comprises a thiosulfonate compound, a mixed disulfide compound, a pyridyl sulfide cationizing agent, alkyl halide cationizing agent or a mixture of two of more thereof. 
     
     
         19 . The reagent of  claim 16 , wherein the cationized, denatured antigenic protein is immobilized indirectly on the solid-phase surface via a biotin-avidin bond. 
     
     
         20 . The reagent of  claim 16 , wherein the cationized, denatured antigenic protein is irreversibly immobilized and indirectly bound to the solid-phase surface via a linker molecule.