Radioimmunoassays for fish tail neuropeptides: I. Development of assay and measurement of immunoreactive urotensin I in Catostomus commersoni brain, pituitary, and plasma.

Antisera were raised in rabbits using highly purified urotensin I (UI) or UI (4-41) conjugated with high-molecular-weight proteins. Antiserum 2U4, at a final dilution of 1:300,000, gave 50% binding to iodinated UI (specific activity 40-120 microCi/micrograms). Carp (Cyprinus carpio) and sucker (Catostomus commersoni) UI peptides cross-reacted completely with the antiserum. Results from cross-reactivity tests using various tryptic and Staphylococcus aureus protease fragments of UI indicated that the antigenic sites of the main antibody(ies) are directed against a part sequence in the C-terminal region of the peptide although antibodies with lower concentration or affinity, recognizing N-terminal region(s) of the peptide, are also present. No cross-reactivity was seen with insulin, secretin, vasoactive intestinal peptide or a structural UI-homolog, the ovine hypothalamic corticotropin-releasing factor, up to molar concentrations. Sauvagine, another structurally homologous peptide from frog skin, showed only 0.1% cross-reactivity. The urophysis-specific proteins, urophysins B and D, showed a low degree of cross-reactivity. Assays of serial dilutions of urophysis extracts from teleostean species yielded parallel displacement curves, except in the case of the pacific goby, Gillichthys mirabilis. Varying concentrations of immunoreactive UI were present in different regions of C. commersoni brain, spinal cord, and pituitary, and also in plasma. The measurement of 17.5 +/- 2.1 fmol UI/ml of plasma indicates that this assay may be used for the study of circulating UI peptide(s).