title: 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.
authors.affiliation: Graduate School of Science and Engineering, Science section, and Venture Business Laboratory, Ehime University, Matsuyama, Japan. shimazaki.yoji.my@ehime-u.ac.jp
authors.forename: Youji
authors.initials: Y
authors.lastname: Shimazaki
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kohno
authors.forename: Izumi
authors.initials: I
authors.lastname: Fukui
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Koyashiki
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001360:Avidin / Q000737:chemistry / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011145:Polyvinyls; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism*; D013552:Swine; D014168:Transferrin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D005465:Fluorocarbon Polymers
chemical_list: D000324:Adrenocorticotropic Hormone; D000906:Antibodies; D001360:Avidin; D000939:Epitopes; D055767:Immobilized Proteins; D008567:Membranes, Artificial; D010446:Peptide Fragments; D011145:Polyvinyls; D013205:Staphylococcal Protein A; D014168:Transferrin; D014357:Trypsin; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers