Epitope mapping of bovine serum albumin using monoclonal antibodies coupled with a photoreactive crosslinker.
Antigenic determinants (epitopes) on bovine serum albumin (BSA) to anti-BSA monoclonal antibodies (mAbs) were assessed by means of photoaffinity labeling. Anti-BSA mAbs, AB3 and AB6, were modified with an 125I-labeled photoreactive crosslinker, sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) and then bound with BSA. The complex yielded was irradiated with UV light to facilitate covalent binding of the modified mAb with BSA. Reduction of the product with 2-mercaptoethanol revealed that radioactivity of 125I-SASD was specifically introduced into BSA, probably near the mAb combining site(s). The radiolabeled or native BSA was digested with V8 protease and the peptides produced were assessed for their amino acid sequence, the radiolabeled amino acids contained, and their reactivity to mAb. The results indicated that AB3 mAb recognized an epitope in the region of Ala-537 to Asp-554 on a linear part of the BSA molecule, whereas AB6 mAb probably recognized an epitope of the assembled form present in a peptide of Asp-299 to Glu-338.
Ueno
H
H
Department of Hygenic Chemistry, Faculty of Pharmaceutical Science, Tohoku University, Sendai.
Masuko
T
T
Wang
J
J
Hashimoto
Y
Y
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018604:Epitope Mapping / Q000379:methods*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010449:Peptide Mapping; D010777:Photochemistry; D012710:Serum Albumin, Bovine / Q000276:immunology*; D012711:Serum Albumin, Radio-Iodinated; D013388:Succinimides / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D001386:Azides; D003432:Cross-Linking Reagents; D012711:Serum Albumin, Radio-Iodinated; D013388:Succinimides; D012710:Serum Albumin, Bovine; C047257:sulfosuccinimidyl 2-(4-azidosalicylamido)ethyl-1,3'-dithiopropionate