Preparation, isolation, and immunochemical studies of the cyanogen bromide peptides from a retinal photoreceptor cell autoantigen, S-antigen.
Purified bovine retinal S-antigen (50,000 m.w.) was treated with cyanogen bromide, producing seven major and several minor fragments. Six of the major and one of the minor components were isolated by reverse phase high performance liquid chromatography. The peptides were characterized with respect to size by urea-SDS-gel electrophoresis, by amino acid composition, and by their ability to bind antibodies, raised in rabbits immunized with purified bovine S-antigen, in both competition and direct enzyme-linked immunosorbent assays. Four of the purified peptides were found, by the direct assay, to bind antibodies in immune sera raised to the intact antigen. Peptides that were negative, or only weakly bound, in the direct enzyme immunoassay were subsequently conjugated to a carrier, poly-L-Glu-Ala-Tyr, and were retested in the enzyme immunoassay in which a peptide of about 25 residues was also found to contain an antigenic determinant. The same five peptides were positive in the competition assays. Isolation of the peptides and gel electrophoresis under reducing and nonreducing conditions revealed that two of the peptides in the reaction mixture were joined by a disulfide linkage.
D000818:Animals; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000276:immunology / Q000302:isolation & purification*; D019393:Arrestin; D001324:Autoantigens / Q000276:immunology / Q000302:isolation & purification*; D002417:Cattle; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification*; D010786:Photoreceptor Cells / Q000032:analysis*; D011817:Rabbits; D012374:Rod Cell Outer Segment / Q000032:analysis* / Q000276:immunology
D000941:Antigens; D019393:Arrestin; D001324:Autoantigens; D010446:Peptide Fragments; D003488:Cyanogen Bromide