Comparative Use of Immunological Methods & Ligand Blotting of Insulin-like Growth Factor Binding Proteins in Serum and Other Biological Fluids

Abstract

There is controversy as to which methodology most accurately detects the Insulin-like growth factor binding proteins (IGFBPs). We have compared immunological and ligand binding techniques to assess the IGFBPs in serum from non-pregnant women and women in late pregnancy, fetal cord serum and amniotic fluid. A specific radioimmunoassay for IGFBP-3 measured mean levels of 3.5 and 3.7μg/ml for non-pregnancy and pregnancy serum, respectively, 0.7μg/ml for fetal cord serum and 5.1μg/ml for amniotic fluid. In contrast, Western-ligand blot of fluid showed IGFBP-3 as a 40 kDa doublet in non-pregnancy serum and fetal cord serum, unlike pregnancy serum or amniotic fluid, where IGFBP-3 was undetectable by standard blot analysis. However, analysis of fluids at 25-50 times more volume showed that IGFBP-3 was detectable as a 40 kDa doublet in pregnancy serum and amniotic fluid. The molecular mass of immunoreactive IGFBP-3 was examined by Western-immunoblot, probed with antibody α IGFBP-31l. As in Westem-ligand blot, intact IGFBP-3 (40 kDa) was easily detected in non-pregnancy serum and fetal cord serum, but required greater volumes of sample for detection in pregnancy serum and amniotic fluid. In addition, smaller immunoreactive fragments were found in all fluids: a faint 27 and 22 kDa band was found in non-pregnancy serum and fetal cord serum, with more dominant bands of 31-27, 26, 25, 24 and 22 kDa in pregnancy serum and 31-26, 25, 22, 19-17 kDa in amniotic fluid. To address the obvious discrepancies between ligand and immunological analysis, samples were assessed for IGFBP-3 protease activity. Non-pregnancy serum proteolyzed 14% of the substrate, pregnancy serum 61%, fetal cord serum 33%, and amniotic fluid 50-61%. Thus, fluids with apparently low IGFBP-3 by Western-ligand blot, but high IGFBP-3 by RIA, showed high levels of protease activity. Previously, IGFBP-proteases have been shown to degrade IGFBP to smaller fragments with a reduced affinity for IGF. We conclude that the discrepancy between ligand binding techniques and immunological techniques can be explained, at least in part, by the presence of IGFBP-protease activity.