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Quantification of Insulin-Like Growth Factor I (IGF-I) Without Interference by IGF Binding Proteins

Department of Physiology, Loma Linda University School of Medicine, Loma Linda, California 92350

Address correspondence and requests for reprints to: Daisy D. De León, Ph.D., Loma Linda School of Medicine, 101 Alumni Hall, Loma Linda, California 92350.

	Abstract

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A chemiluminescent dot blot assay has been developed by our laboratory for rapid determinations of IGF-I in serum-free conditioned media (CM) collected from cultured cells. In contrast to IGF-I radioimmunoassays (RIAs), the IGF binding proteins (IGFBPs) did not interfere with the dot blot assay and did not require the laborious (and sometimes ineffective) removal of IGFBPs. Although all six IGFBPs were shown to bind to ¹²⁵I IGF-I, none interfered with IGF-I detection on nitrocellulose dot blots. In contrast, an RIA using the same Oncogene monoclonal antibody (clone 82-9A) showed interference by IGFBP-1, IGFBP-2, and IGFBP-4. The IGF-I dot blot assay was sensitive (0.125 - 8.0 ng IGF-I), specific (assay crossreactivity with IGF-II is less than 1%), and reproducible (intra-assay variance 6%; inter-assay variance <12%) when chemiluminescence was quantified by phosphorimager and Molecular Analyst software (BioRad). The apparent sensitivity of the enhanced chemiluminescence (ECL) reagent to serum, precludes the use of this assay for IGF-I determination in serum or in serum-containing media.

Received December 26, 1996.

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