Differences in defining residues relevant to antibody binding by ELISA and proteolysis protection at the level of peptide antigenic determinants.
The interaction of monoclonal antibody (mAb) 1C3 with bovine beta-casein was analyzed by two methods. The competitive enzyme-linked immunosorbent assay in which mAb 1C3 bound competitively to a peptide in solution or beta-casein adsorbed to wells in microtitration plates showed that beta-casein (f194-200) (peptide of residues 194 to 200 of beta-casein) was the shortest peptide among the tested peptides that had binding ability for mAb 1C3. Protection against proteinase digestion was analyzed by incubating proline-specific endopeptidase, carboxypeptidase Y or aminopeptidase T with beta-casein (f193-202) in the presence of mAb 1C3 or nonspecific mAb 31A4. Proteolysis of peptide bonds between residues 200 and 201, and 201 and 202 was depressed in the presence of mAb 1C3. However, peptide bonds between 193 and 194, and 194 and 195 were cleaved in the presence of mAb 1C3 as easily as in the presence of mAb 31A4, suggesting that the region of residues 200 to 202 was obscured by, or within the antibody binding site, but that the region of residues 193 to 195 was not. The apparent antibody binding site shown from the protection against proteolysis by mAb was clearly not identical to the shortest antigen peptide, beta-casein (f194-200), indicated from the competitive binding assay.
Kobayashi
T
T
Department of Agricultural Chemistry, University of Tokyo, Japan.
Ametani
A
A
Yamauchi
K
K
Kaminogawa
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000032:analysis; D001667:Binding, Competitive; D002364:Caseins / Q000276:immunology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002364:Caseins; D010446:Peptide Fragments; D010455:Peptides; D010450:Endopeptidases