A homologous radioimmunoassay for the measurement of urotensin II in the euryhaline flounder, Platichthys flesus

A sensitive and specific homologous radioimmunoassay (RIA) has been develop ed to measure tissue and circulating levels of the fish caudal neurosecreto ry system neuropeptide, urotensin II (UII), in the euryhaline flounder Plat ichthys flesus. A polyclonal antiserum Mi as raised against flounder UII in rabbit; UII-I-125 was produced by the iodogen method and purified by HPLC. Antiserum specificity to flounder UII was demonstrated through lack of cro ss-reactivity with several small peptides and parallelism with standard cur ves for serial dilutions of UII in plasma and urophysial extracts. Biologic al activity of the peptide measured by UII RIA was confirmed by bioassay. P lasma intra- and interassay coefficients of variation were 9 and 18% (n = 5 and n = 3), respectively, nonspecific binding constituted 4.6% (+/-1.42%, 8) of total counts, and the limit of RIA detectability was estimated as 1.5 x 10(-16) M UII/assay tube. Plasma samples were subject to a reversed-phas e liquid chromatography purification protocol which had an extraction effic iency of 63% (+/-10%, n = 6) and showed consistent recovery of UII over a r ange of plasma volumes and peptide concentrations. Plasma UII concentration s in seawater (SW)-adapted flounder (3.80 +/- 0.77 x 10(-11) M, n = 7) were significantly higher than those in freshwater (FW)-adapted fish (1.10 +/- 0.15 x 10(-11) M, n = 7). This variation coincided with differences in plas ma osmolality and Na+ levels. No differences were found, however, between u rophysial UII concentrations in SW-adapted (3.71 +/- 1.78 x 10(-10) M UII/g land, n = 7) and FW-adapted (2.53 +/- 1.33 x 10(-10) M UII/gland, n = 7) fl ounder (C) 1999 Academic Press.