Detection of autoantigens by immunoblotting using a peroxidase-anti-peroxidase complex.
An immunoblotting procedure using a peroxidase-anti-peroxidase (PAP) complex was developed for the detection of autoantigens in crude mixtures by human autoimmune sera. Thymus proteins were transferred to a nitrocellulose sheet after electrophoresis in polyacrylamide gels and probed with a 1:100 dilution of serum. The location and extent of immunoglobulin G (IgG) binding was determined by sequential reaction with: rabbit anti-human IgG, goat anti-rabbit IgG and rabbit peroxidase-anti-peroxidase complex. The peroxidase was allowed to react with chloronaphthol and low levels of autoantigen/autoantibody complex were detectable with virtual absence of background colour. The inclusion of human IgG and its pepsin-generated fragment provided a means of controlling and calibrating the blotting procedure.
D000818:Animals; D000941:Antigens / Q000032:analysis*; D001324:Autoantigens / Q000032:analysis*; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007124:Immunoenzyme Techniques; D007223:Infant; D008970:Molecular Weight; D010209:Paper; D011817:Rabbits; D012261:Ribonucleoproteins; D012967:Sodium Dodecyl Sulfate; D055517:snRNP Core Proteins; D000097585:SS-B Antigen
D000941:Antigens; D001324:Autoantigens; D003101:Collodion; D012261:Ribonucleoproteins; D012967:Sodium Dodecyl Sulfate; D055517:snRNP Core Proteins; D000097585:SS-B Antigen