Epitope mapping of a series of human thymidylate synthase monoclonal antibodies.
We have reported previously the development and application of several monoclonal antibodies to thymidylate synthase (TS). In this study, we used a series of overlapping 17-mer peptides that spanned the entire TS protein to map the epitope recognized by three TS monoclonal antibodies (TS 106, TS 109, and TS 110). Using an ELISA, we identified two peptides (R126-F142 and L131-R147) that bound all three antibodies, which suggests that each antibody recognized a similar epitope on TS. A second set of peptides, representing sequential single-residue truncations from either the amino terminus or the carboxyl terminus starting with a G129-E145 17-mer, was synthesized. A 10-amino acid sequence P133-F142 (PVYGFQWRHF) was identified as the binding epitope for all three antibodies. Further investigation via substitution mutational analysis of each residue within this epitope revealed that residues F137, W139, R140, H141, and F142 were critical for maximal binding of TS 106 and TS 110. TS 109 showed a similar pattern except in regard to R140, with which there was no apparent loss of binding. In addition to the utility of the three antibodies in detecting and measuring TS levels, identification of the binding locus permits the potential application of these antibodies in the investigation of TS enzymatic and regulatory function.
Behan
K A
KA
National Cancer Institute, Medicine Branch at the National Naval Medical Center, Bethesda, Maryland 20889-5105, USA.
Johnston
P G
PG
Allegra
C J
CJ
D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology*; D002277:Carcinoma / Q000276:immunology; D003110:Colonic Neoplasms / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006801:Humans; D007150:Immunohistochemistry; D008958:Models, Molecular; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology*; D013940:Thymidylate Synthase / Q000032:analysis / Q000276:immunology*
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D013940:Thymidylate Synthase