title: 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.
abstract: The availability and activity of insulin-like growth factors (IGF-I and IGF-II) are largely determined by a group of IGF-binding proteins (IGFBPs). 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 radiolabelled IGF-I or -II was allowed to bind. The assay is highly specific, since neither IGF bound to control wells blocked with albumin. By constructing both saturation and competition binding curves, equivalence of binding between the radiolabelled and native IGF ligands could be demonstrated. From these curves, reliable specific activities of the tracers were calculated. Scatchard plots of both types of data produced identical results for dissociation constants and number of binding sites. The affinity of IGF-II was twice as high as the affinity of IGF-I (dissociation constants of 44 and 102 pM respectively). The assay was used to show that polyclonal anti-IGFBP-3 antibodies could block binding of 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 effectively 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 be a consequence of a decrease in both the affinity and the number of binding sites. Possibly, the interaction of IGFBP-3 with heparin or heparin-like structures in vivo can lead to the selective release of IGF-II from this binding protein. Our results with heparin also suggest that the binding sites on IGFBP-3 for IGF-I and IGF-II are not completely 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.
authors.affiliation: Department of Paediatrics, Wilhelmina Children's Hospital, Utrecht University, The Netherlands.
authors.forename: J A
authors.initials: JA
authors.lastname: Koedam
authors.forename: C M
authors.initials: CM
authors.lastname: Hoogerbrugge
authors.forename: S C
authors.initials: SC
authors.lastname: van Buul-Offers
authors.forename: J L
authors.initials: JL
authors.lastname: Van den Brande
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D000906:Antibodies / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects; D002122:Calcium Chloride / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000378:metabolism; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000276:immunology / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D011869:Radioligand Assay / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013002:Somatomedins / Q000378:metabolism*
chemical_list: D000477:Alkylating Agents; D000906:Antibodies; D018970:Insulin-Like Growth Factor Binding Protein 1; D018972:Insulin-Like Growth Factor Binding Protein 3; D011994:Recombinant Proteins; D013002:Somatomedins; D012965:Sodium Chloride; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D006493:Heparin; D002122:Calcium Chloride