Production and Characterization of Monoclonal Antibodies against the Vasoconstrictive Peptide Human Urotensin-II

Abstract

We report the production and characterization of four monoclonal antibodies (MAbs) against human urotensin-II (hU-II). The antibodies were raised against human hU-II, which contains the C-terminus cyclic ring (CFWKYC) that is conserved across species. Multiple selection assays were applied to ensure antibody potency and reactivity against the ring structure. The MAbs reacted via ELISA with hU-II bound to plastic, immunoprecipitated [¹²⁵I-Y⁹] hU-II, bound to biotinylated hU-II in BIAcore analysis and, by Western analysis, recognized the full-length human preprourotensin-II expressed in transfected HEK293 cells. All four MAbs cross-reacted with porcine A, porcine B, rat, mouse, and goby U-II in ELISA. By competitive RIA, hU-II(5-11) (identical to the C-terminus of goby U-II) reacted equivalently to hU-II and goby U-II. The IC₅₀s were 0.8 nM for one MAb and 1.6 nM for the others. All four MAbs reacted 15-fold less potently with hU-II(5-10) and 50-fold less potently with hU-II(5-10) amide. Thus, the ring structure and terminal Val/Ile comprise the binding site for this group of MAbs. This panel of antibodies could be useful tools to help delineate the biology and pharmacology of U-II. They may also be of diagnostic value in monitoring hU-II in body fluids.