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

Abstract

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.