Epitope mapping of monoclonal antibodies to tumor necrosis factor-alpha by synthetic peptide approach.
A monoclonal antibody (mAb) against human tumor necrosis factor-alpha (TNF-alpha), designated 3B10, neutralizes biological activity of TNF-alpha, while another anti-TNF-alpha mAb 10F10 does not. In Western blot analysis, both mAbs bound to SDS-denatured TNF-alpha, indicating that the epitopes recognized by the mAbs are sequential but not conformational. To map precisely the epitopes of the mAbs, 76 overlapping octapeptides corresponding to an entire sequence of TNF-alpha were synthesized and their abilities to react with the mAbs were examined by enzyme-linked immunosorbent assay (ELISA). 3B10 bound to only one peptide at position 81-88 of TNF-alpha, SRIAVSYQ, whereas 10F10 was reactive with three overlapping peptides, ANALLANG (33-40), ALLANGVE (35-42), and LANGVELR (37-44). These results demonstrate that the 81-88 and 37-40 regions are important for the recognition of TNF-alpha by 3B10 and by 10F10, respectively. In solid-phase ELISA, 3B10 inhibited the binding of TNF-alpha to soluble TNF receptors, sTNF-RI and sTNF-RII. In contrast, 10F10 exerted little effect on the binding. TNF-alpha was detected by sandwich-type ELISA where 3B10 alone was used for both capture and detection, suggesting that 3B10 did not interfere with the trimer formation of TNF-alpha. The results obtained in this study suggest that the 81-88 region of TNF-alpha may participate in the receptor binding and that 3B10 neutralizes the activities of TNF-alpha by blocking the region.
Nagahira
K
K
Suntory Institute for Biomedical Research, Osaka, Japan.
Fukuda
Y
Y
Terakawa
M
M
Hashino
J
J
Nasu
T
T
Nakazato
H
H
Nakanishi
T
T
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000494:pharmacology; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000276:immunology; D017433:Protein Structure, Secondary / Q000187:drug effects; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000276:immunology*
D000911:Antibodies, Monoclonal; D010455:Peptides; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha