SOLID-PHASE ASSAY FOR INSULIN-LIKE GROWTH-FACTOR (IGF) BINDING TO IGF-BINDING PROTEIN-3 - APPLICATION TO THE STUDY OF THE EFFECTS OF ANTIBODIES AND HEPARIN

The availability and activity of insulin-like growth factors (IGF-I an d IGF-II) are largely determined by a group of IGF-binding proteins (I GFBPs). We have developed a new assay to characterize the interaction between the IGFs and IGFBP-3. In this assay, recombinant IGFBP-3 (5 ng ) was immobilized on plastic microtitre wells, after which radiolabell ed IGF-I or -II was allowed to bind. The assay is highly specific, sin ce neither IGF bound to control wells blocked with albumin. By constru cting both saturation and competition binding curves, equivalence of b inding between the radiolabelled and native IGF ligands could be demon strated. From these curves, reliable specific activities of the tracer s were calculated. Scatchard plots of both types of data produced iden tical results for dissociation constants and number of binding sites. The affinity of IGF-II was twice as high as the affinity of IGF-I (dis sociation constants of 44 and 102 pM respectively). The assay was used to show that polyclonal anti-IGFBP-3 antibodies could block binding o f IGF. Alkylating agents and NaCl were without effect, but chaotropic salts such as CaCl2 and NaSCN decreased IGF binding to IGFBP-3. IGFBP- 1 and IGFBP-3, but not an N-terminal fragment of IGFBP-3, could effect ively block binding of both IGF-I and IGF-II to the solid-phase IGFBP- 3. Increasing concentrations of heparin had little or no effect on IGF -I binding, but strongly inhibited IGF-II binding. This was shown to b e a consequence of a decrease in both the affinity and the number of b inding sites. Possibly, the interaction of IGFBP-3 with heparin or hep arin-like structures in vivo can lead to the selective release of IGF- II from this binding protein. Our results with heparin also suggest th at the binding sites on IGFBP-3 for IGF-I and IGF-II are not completel y identical. This assay can be applied to the study of various aspects of the interaction between the IGFs and IGFBP-3, such as the effects of interfering substances and structure-function relationships of both moieties of the complex.