Radioimmunoassay for insulin-like growth factor-I: solutions to some potential problems and pitfalls.
This report describes essential requirements for the validation of a radioimmunoassay (RIA) for insulin-like growth factor-I (IGF-I) and presents solutions to some problems and pitfalls commonly observed. The preparation of IGF-I to be used as radioligand or standard has to be selected carefully since some IGF-I preparations are contaminated with variants which demonstrate different potencies for different antisera used in the RIA. Accurate assessment of IGF-I levels in blood plasma requires an efficient extraction method for the IGF-binding proteins (IGFBPs). Extraction methods to remove the influence of IGFBPs in the RIA were compared using blood plasma of considerable differences in IGF-I/IGFBP ratios. Acidification of plasma before column chromatography on Sephadex G-75 (G75) is generally considered to be the most reliable extraction method, but it is very time-consuming. The acid-ethanol extraction (AE) of plasma is not valid in many situations. Non-parallel displacement to the IGF-I standard was observed with AE-extracted plasma samples in the RIA. In addition, a comparison of IGF-I values obtained in the RIA after AE or G75 extraction of fetal ovine plasma has shown no significant correlation. We report an extraction technique based on a modified AE extraction followed by cryo-precipitation (AEC). AEC extraction on blood plasma reduced residual IGFBPs to a level that did not interfere in the assay. Furthermore, AEC-extracted plasma samples showed parallel displacement in the RIA to highly purified preparations of authentic IGF-I. We observed high correlations, with a slope close to unity, of IGF-I values obtained in the RIA using the AEC or G75 extraction for plasma from different species including adult and fetal sheep, rat, mouse and man. The AEC extraction provides a rapid and simple alternative to G75 extraction for blood plasma from a variety of species provided that high-affinity antisera are used for the RIA.
Breier
B H
BH
Department of Paediatrics, University of Auckland, New Zealand.
Gallaher
B W
BW
Gluckman
P D
PD
D000818:Animals; D002352:Carrier Proteins / Q000097:blood; D002850:Chromatography, Gel / Q000379:methods; D003080:Cold Temperature; D006801:Humans; D007106:Immune Sera; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000032:analysis*; D051379:Mice; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D015203:Reproducibility of Results; D012756:Sheep
D002352:Carrier Proteins; D007106:Immune Sera; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I