Development of a time-resolved fluoroimmunoassay for salmonid insulin-like growth factor-2

In mammals, insulin-like growth factor (IGF)-1 and IGF-2 regulate postnatal growth and embryonic development, respectively. In fish, as in mammals, IGF-1 promotes somatic growth, whereas evidence suggests the possible involvement of IGF-2 in the regulation of somatic growth. However, the measurement of circulating IGF-2 in fish has been hampered, possibly because of interference from IGF-binding proteins (IGFBPs). Extraction of IGFs from IGFBPs is essential before measuring them using immunoassays. We optimized acid-ethanol (AE) extraction method for salmonid IGF-2 and developed a time-resolved fluoroimmunoassay (TR-FIA) using recombinant IGF-2 as the assay component. In TR-FIA, the standard AE-extraction of IGF-1 was not validated for quantifying serum IGF-2 because of the presence of ethanol. Three treatments-vacuum drying, dialysis, and ultrafiltration-were compared after AE extraction, and ultrafiltration by centrifugation was found to be effective. With this extraction method, circulating IGF-2 levels were measured by TR-FIA in fed, fasted, and refed rainbow trout. Serum IGF-2 levels were similar or slightly lower in fed fish than in fasted fish, and refeeding had no positive effect. Serum IGF-2 levels were not correlated with individual growth rates. GH treatment tended to increase circulating IGF-2 levels albeit not statistically significant. Smolting masu salmon had similar but slightly lower serum IGF-2 levels than non-smolting fish, while exhibiting high serum IGF-1 levels. These results suggest that circulating IGF-2 levels were relatively stable. This newly developed assay should facilitate further exploration of the physiological responses and roles of circulating IGF-2 in salmonids.