Comparison of the effectiveness of various procedures for reducing or elimination insulin-like growth factor-binding protein interference with insulin-like growth factor-I radioimmunoassays on porcine sera

R. S. Frey, M. R. Hathaway, W. R. Dayton

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43 Scopus citations

Abstract

We have examined the efficacy of various methods for reducing the interference of insulin-like growth factor-binding proteins (IGFBPs) with insulin-like growth factor-I (IGF-I) radioimmunoassays (RIAs) run on porcine sera. Acid-ethanol (AE) extraction, AE extraction followed by cryoprecipitation, glycyl-glycine (GG) extraction, GG extraction followed by Sephadex G-50 chromatography in 1 mol acetic acid/l (GG/G-50), and Sep-Pak chromatography were analysed. To provide a range of IGF-I and IGFBP levels, sera obtained from control, hypophysectomized, diabetic and somatotrophin-treated pigs were used. Recoveries of IGF-I added to sera prior to treatments other than Sep-Pak chromatography ranged from 85 to 105% and were not significantly different. In contrast, Sep-Pak chromatography gave extremely variable recoveries. 125I-Labelled IGF-I ligand blotting showed that GG extraction followed by acid G-50 chromatography was by far the most effective method of removing or inactivating IGFBPs in porcine sera. Consequently, this procedure was used as a standard against which to compare other extraction procedures. GG extraction alone removed or inactivated low molecular weight binding proteins but appeared to have little effect on IGFBP-3. AE extraction reduced the level of IGFBP-3 but had little effect on lower molecular weight binding proteins. Even though none of the tested procedures completely removed or inactivated the binding proteins, all samples yielded IGF-I displacement curves that were parallel to that obtained for IGF-I standard. Despite yielding parallel displacement curves, sera extracted by various methods gave dramatically different apparent IGF-I levels when subjected to IGF-I RIA. IGF-I RIA of GG extracted sera yielded IGF-I values that were closest to those obtained for identical serum samples subjected to glycyl-glycine extraction followed by G-50 chromatography. For sera from control, hypophysectomized, diabetic and somatotrophin-treated pigs, the relationship of the IGF-I level in GG-extracted sera to that in GG-extracted, acid G-50 chromatographed (GG/G-50) sera was √ GG = 1.13 √ GG/G-50 - 0.23 (r2 = 0.98). Consequently, GG extraction can be used to remove IGFBP interference with IGF-I RIAs of porcine sera from normal, hypophysectomized, diabetic and somatotroph-treated animals.

Original languageEnglish (US)
Pages (from-to)229-237
Number of pages9
JournalJournal of Endocrinology
Volume140
Issue number2
DOIs
StatePublished - 1994

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