LIGAND BLOT ANALYSIS OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS USING BIOTINYLATED INSULIN-LIKE GROWTH-FACTOR-I

A nonradioactive method for the detection of insulin-like growth facto r-binding proteins (IGFBPs) was developed utilizing human recombinant insulinlike growth factor-I (IGF-I) biotinylated with N-hydroxysuccini mido-biotin. Human plasma samples were separated by 15% SDS polyacryla mide gel electrophoresis, and proteins were transferred to nitrocellul ose by Western blotting. The nitrocellulose sheets were incubated over night with IGF-I-biotin at 4 degrees C. The next day streptavidin-pero xidase was added for 1 h, and IGFBPs were visualized by an enhanced ch emiluminescence system. Five characteristic bands with molecular weigh ts of 41 and 39 (IGFBP-3), 34 (IGFBP-2), 30 (IGFBP-1) and 24 kD (IGFBP -4) were detected. Binding was specific for IGFs, since unlabeled IGF- I inhibited IGF-I-biotin binding. IGFBP ligand blots with biotinylated IGF-I and I-125-IGF-I yielded comparable results. The suitability of the new assay for clinical purposes was demonstrated by several clinic al examples. In summary, a rapid, reliable, nonradioactive assay for q ualitative analysis of IGFBPs has been developed.