A Homologous Radioimmunoassay for the Measurement of Urotensin II in the Euryhaline Flounder,Platichthys flesus

Abstract

A sensitive and specific homologous radioimmunoassay (RIA) has been developed to measure tissue and circulating levels of the fish caudal neurosecretory system neuropeptide, urotensin II (UII), in the euryhaline flounderPlatichthys flesus.A polyclonal antiserum was raised against flounder UII in rabbit; UII-125I was produced by the iodogen method and purified by HPLC. Antiserum specificity to flounder UII was demonstrated through lack of cross-reactivity with several small peptides and parallelism with standard curves for serial dilutions of UII in plasma and urophysial extracts. Biological activity of the peptide measured by UII RIA was confirmed by bioassay. Plasma intra- and interassay coefficients of variation were 9 and 18% (n=5 andn=3), respectively, nonspecific binding constituted 4.6% (±1.42%,n=8) of total counts, and the limit of RIA detectability was estimated as 1.5 × 10−16M UII/assay tube. Plasma samples were subject to a reversed-phase liquid chromatography purification protocol which had an extraction efficiency of 63% (±10%,n=6) and showed consistent recovery of UII over a range of plasma volumes and peptide concentrations. Plasma UII concentrations in seawater (SW)-adapted flounder (3.80±0.77×10−11M,n=7) were significantly higher than those in freshwater (FW)-adapted fish (1.10±0.15×10−11M,n=7). This variation coincided with differences in plasma osmolality and Na+levels. No differences were found, however, between urophysial UII concentrations in SW-adapted (3.71±1.78×10−10M UII/gland,n=7) and FW-adapted (2.53±1.33×10−10M UII/gland,n=7) flounder.