DETECTION AND EPITOPE MAPPING OF IMMUNOREACTIVE HUMAN ENDOTHELIN-1 USING ELISA AND A SURFACE-PLASMON RESONANCE-BASED BIOSENSOR

A surface plasmon resonance-based biosensor (BIA technology) and enzym e-linked immunosorbent assays (ELISA) have been used for detecting and characterizing human endothelin (ET), a potent vasoactive 21 amino ac id polypeptide. Antibodies produced against the isoform ET-1 and its C -terminal eptapeptide ET-1(15-21) have been characterized with respect to their binding capacity to the two isoforms ET-1 and ET-3, the non- secreted portion of the precursor molecule Big.ET-1(22-38), the C-term inal of ET-1, six analogues of ET 1(16-21) each containing a substitut ion with Ala of a single amino acid in positions 16-21, respectively, and three synthetic cyclic peptides mimicking the N-terminal portion o f ET-1. Antibodies reacting with ET-1 also bound to ET-1(16-21) and, w ith less affinity, to ET-3 but did not cross-react with Big.ET-1(22-38 ). Ala substitution in positions 16, 17 and 19 of ET-(16-21) hardly af fected the antibody binding capacity of ET-1(16-21), whereas Ala subst itution of Asp(18), Ile(20) and, in particular, Trp(21), inhibited its immunoreactivity. The C-terminus thus represents an immunodominant ep itope in ET-1 and is important for antibody binding. Epitope mapping u sing as antibody pairs polyclonal anti-ET-1 and monoclonal anti-ET-1(1 5-21) antibodies indicated the presence of another immunogenic domain in the N-terminal portion of the molecule. There was excellent agreeme nt between the epitopes determined using ELISA and BIA analyses. (C) 1 997 Elsevier Science Limited.