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Measurement of Insulin-Like Growth Factor-II in Physiological Fluids and Tissues. I. An Improved Extraction Procedure and Radioimmunoassay for Human and Rat Fluids^*

Lilly Laboratory for Clinical Research, Eli Lilly Co., and the Departments of Biochemistry and Molecular Biology, Pharmacology, and Medicine, Indiana University School of Medicine Indianapolis, Indiana 46202

Abstract

The measurement of serum insulin-like growth factors (IGFs) in serum is complicated by the presence of high affinity IGF-binding proteins. The accurate measurement of IGFs by radioligand binding assays requires that the interference from binding proteins be eliminated. Acid-gel chromatography, the standard method for removing binding proteins, is laborious and time consuming. Alternative methods for extracting serum IGFs include the use of HCl-ethanol treatment and reverse phase minicolumns. However, these methods are unsuitable for use with serum for some species, such as rat and sheep, due to incomplete removal of binding proteins. We developed a fast protein liquid chromatography size-exclusion chromatographic method for characterizing the presence of IGF-binding proteins in physiological fluids and used this method to systematically investigate different combinations of acids and organic solvents as potential extraction methods for IGFs. We developed and validated an improved extraction procedure that uses formic acid, Tween-20, and acetone. The new extraction method was used in conjunction with purified biosynthetic human IGF-II and a commercially available anti-IGF-II monoclonal antibody in the development of an improved RIA for IGF-II. The new RIA is sensitive (5.0 pg/tube), specific (IGF-I cross-reactivity, <1%), and reproducible [interassay precision (coefficient of variation), <9.2%). We measured the serum concentrations of IGFII in adults and found a significant difference between normal subjects and individuals with insulin-dependent diabetes mellitus. (Endocrinology 128: 805–814,1991)

Footnotes

^* Presented in part at the 71st Annual Meeting of The Endocrine Society, Seattle, WA, 1989.

Received July 30, 1990.

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