J. Grulichhenn et al., LIGAND BLOT ANALYSIS OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS USING BIOTINYLATED INSULIN-LIKE GROWTH-FACTOR-I, Hormone research, 49(1), 1998, pp. 1-7
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.