Production and Characterization of Monoclonal Antibodies to the Carboxyl-Terminal Heptapeptide of Endothelin-1

Abstract

Two cell lines, RR5.ET-1 and RR1.ET-1, that produce monoclonal antibodies specific for the carboxyl-terminal heptapeptide of endothelin-1 (ET-1) have been cloned and stabilized. An RIA was developed to facilitate the evaluation and characterization of these monoclonal antibodies. The affinity constant of each MAb for ET-1, as determined by Scatchard analysis, was 5.74 × 10⁸ M^-1 for RR5.ET-1 and 4.15 × 10⁷ M^-1 for RR1.ET-1. The antibodies reacted specifically with the carboxyl-terminus (ET_15-21) and did not cross-react with the amino-terminal amino acids (ET_1-16). As expected, the antibodies cross-reacted with endothelin-2 (ET-2) and endothelin-3 (ET-3), and to a lesser extent, with the closely related sarafotoxins. Both MAbs retained about 55% reactivity with the ET-1 terminal sequence of Asp-Ile-Ile-Trp (ET_18-21) but had no reactivity with the ET sequence His-Leu-Asp-Ile-Ile-Trp-Val-Asn (ET_16-23) nor with Big ET-1 (ET_1-39). These data strongly suggest that the terminal four amino acids of ET-1 are included in the MAb binding site. More importantly, the terminal Trp₂₁ must be free, not linked to Val₂₂ to retain reactivity with either of the MAbs.