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 factor-binding proteins (IGFBPs) was developed utilizing human recombinant insulin-like growth factor-I (IGF-I) biotinylated with N-hydroxysuccinimido-biotin. Human plasma samples were separated by 15% SDS polyacrylamide gel electrophoresis, and proteins were transferred to nitrocellulose by Western blotting. The nitrocellulose sheets were incubated overnight with IGF-I-biotin at 4 degrees C. The next day streptavidin-peroxidase was added for 1 h, and IGFBPs were visualized by an enhanced chemiluminescence system. Five characteristic bands with molecular weights 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 (125)I-IGF-I yielded comparable results. The suitability of the new assay for clinical purposes was demonstrated by several clinical examples. In summary, a rapid, reliable, nonradioactive assay for qualitative analysis of IGFBPs has been developed.
Grulich-Henn
J
J
Department of Pediatrics, University of Heidelberg, Germany.
Spiess
S
S
Heinrich
U
U
Schönberg
D
D
Bettendorf
M
M
D000328:Adult; D019921:Biotinylation / Q000379:methods; D015153:Blotting, Western; D002648:Child; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D018969:Insulin-Like Growth Factor Binding Proteins / Q000032:analysis* / Q000737:chemistry; D008297:Male; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D013002:Somatomedins / Q000737:chemistry
D018969:Insulin-Like Growth Factor Binding Proteins; D013002:Somatomedins; D012967:Sodium Dodecyl Sulfate