Epitope analysis using membrane-immobilized avidin and protein A

Abstract

Adrenocorticotropic hormone (ACTH) and transferrin were trapped by biotinylated anti-ACTH antibody and anti-transferrin antibody, respectively, bound to membrane-immobilized avidin. Polypeptides with the sequences SYSMEHFR, SYSMEHFRWGKPVGK and SYSMEHFRWGKPVGKK were bound to the biotinylated anti-ACTH antibody on the membrane-immobilized avidin after the trapped ACTH was digested with trypsin on the membrane and non-binding polypeptides were washed from the membrane. Further, the polypeptides with the sequence SYSMEHFRWGKPVGK and SYSMEHFRWGKPVGKK were trapped by anti-ACTH antibody bound to membrane-immobilized protein A. The antibody recognized the WGKPVGK region of the antigen, ACTH. Polypeptide with the sequence SMGGKEDLIWELLNQAQEHFGKDK was bound to the biotinylated anti-transferrin antibody on the membrane-immobilized avidin after the trapped transferrin was digested with trypsin on the membrane and non-binding polypeptides were washed from the membrane. Further, the polypeptide with the sequence SMGGKEDLIWELLNQAQEHFGKDK was trapped by anti-transferrin antibody bound to membrane-immobilized protein A. The antibody recognized the SMGGKEDLIWELLNQAQEHFGKDK region of the antigen, transferrin. These results thus indicate that the combined methods of membrane-immobilized avidin and protein A can be applied to examine the epitopes of antigens.