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Competitive Binding Assay for Determination of Rat Insulin-Like Growth Factor Binding Protein-3

Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, Sydney, NSW 2065, Australia

Address all correspondence and requests for reprints to: Dr. Jan Frystyk, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia.

We describe a novel competitive assay for rat insulin-like growth factor (IGF)-binding protein-3 (rIGFBP-3) based on the ability of IGFBP-3 to form a ternary complex with the acid labile subunit (ALS) in the presence of IGF. Human (h)ALS was bound to test tubes pre-coated with anti-human ALS antibody. The assay depends on competition between a covalent complex of ¹²⁵I-hIGF-I and hIGFBP-3, added as tracer, and hIGFBP-3 or rIGFBP-3 in standards and test samples, for binding to the immobilized hALS. Purified natural hIGFBP-3 served as standard. Human IGFBP-3 and rIGFBP-3 were able to compete for tracer binding in the presence, but not in the absence, of IGF-I. Before assay, rat serum samples were acidified to denature endogenous ALS. Standards ranged from 0.10 (lower detection limit) to 20 ng/tube. Rat serum, semipurified rIGFBP-3, human serum and purified hIGFBP-3 diluted in parallel. The level of rIGFBP-3 was 1.63 ± 0.28 mg/l (mean ± SEM) in young rats and increased to 3.41 ± 0.26 mg/l (p < 0.05) in old rats (n = 5–6). Fasting for 3 days reduced rIGFBP-3 from 2.41 ± 0.27 to 1.33 ± 0.14 mg/l (p < 0.05). Levels of rIGFBP-3 were reduced in hypophysectomized (0.16 ± 0.04 mg/l; p < 0.05) and diabetic rats (1.04 ± 0.30 mg/l; p < 0.05), and normal in insulin-treated diabetic rats (2.49 ± 0.04 mg/l; ns), when compared to controls (2.79 ± 0.22 mg/l). Changes in levels of IGFBP-3 paralleled those of immunoreactive rALS. We conclude that this assay provides a novel method of quantitating functional IGFBP-3 in rat serum.

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