Comparison of extraction methods and assay validation for salmon insulin-like growth factor-I using commercially available components.
Insulin-like growth factor-binding proteins (IGFBPs) may interfere with accurate measurement of plasma IGFs in radioimmunoassay (RIA). Although several simplified extraction methods for IGFs have been developed, these methods are not always validated for differing physiological states, developmental stages, and animal species. For teleost fish, neither the necessity of plasma extraction nor the validity of extraction methods for IGF RIA is widely established. We systematically examined the validity of acid-ethanol (AE) extraction, AE extraction followed by cryoprecipitation (AEC extraction), and SP-Sephadex extraction in RIA for salmon IGF-I using commercially available components (GroPep Pty Ltd). Displacement curves of plasma extracted by AE, AEC, and SP-Sephadex were parallel to those of the standard. Measured IGF-I levels in plasma from several developmental stages and under different physiological and experimental conditions were significantly increased by the extractions and comparable to those after acid-size exclusion chromatography (SEC). On Western ligand blotting using digoxigenin-labeled human IGF-I, the intensity of IGFBP bands remaining in plasma were reduced after extraction, although some IGFBPs remained. However, these residual IGFBPs did not interfere measurably with the RIA based on quantitative comparison of IGF-I levels with acid-SEC. We conclude that with this RIA extraction is necessary for measurement of salmon IGF-I in plasma since measured values were routinely lower in unextracted samples, and AE, AEC, and SP-Sephadex extractions are applicable to the IGF-I RIA using the commercially available components. Using the validated RIA for IGF-I, plasma IGF-I levels in nonmaturing and precociously maturing chinook salmon in spring were measured after AE extraction. During spring, nonmaturing and maturing fish fed and grew well, and plasma IGF-I level was significantly correlated with body weight in both fish. This result indicates that circulating IGF-I plays a key role in controlling growth in precociously maturing chinook salmon in spring as in nonmaturing fish.
Shimizu
M
M
Northwest Fisheries Science Center, National Marine Fisheries Service, 2725 Montlake Boulevard East, Seattle, Washington, 98112, USA.
Swanson
P
P
Fukada
H
H
Hara
A
A
Dickhoff
W W
WW
D000818:Animals; D015153:Blotting, Western; D001835:Body Weight; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D000431:Ethanol; D005615:Freezing; D018969:Insulin-Like Growth Factor Binding Proteins / Q000097:blood; D007334:Insulin-Like Growth Factor I / Q000032:analysis*; D008970:Molecular Weight; D017684:Oncorhynchus kisutch / Q000097:blood*
D018969:Insulin-Like Growth Factor Binding Proteins; D000431:Ethanol; D007334:Insulin-Like Growth Factor I