Measurement of Intact Insulin-Like Growth Factor-Binding Protein-3 in Human Plasma Using a Ligand Immunofunctional Assay 1

Abstract

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is a fundamental mechanism in the regulation of IGF-I bioavailability in the bloodstream. Its measurement by Western immunoblotting provides only semiquantitative estimation. We have developed a ligand immunofunctional assay (LIFA) for quantifying human (h) intact IGFBP-3 in biological fluids. IGFBP-bound IGFs are dissociated and separated by acid pH ultrafiltration, and a monoclonal antibody specific to the first 160 amino acids of IGFBP-3 is used to capture hIGFBP-3 in a solid-phase assay. The complex is then incubated with (125)I-IGF-I, which binds to intact IGFBP-3 but not to its proteolytic fragments. Binding specificity was demonstrated in competition experiments with unlabeled IGF. Nonspecific binding was 1.4%. The fragments comprising residues 1-160 and 1-95 of recombinant hIGFBP-3 [corresponding to the major proteolytic fragments of approximately 30 kDa and (glycosylated) 20 or (nonglycosylated) 16 kDa detected in serum by Western immunoblotting, respectively] fail to bind (125)I-IGF-I when complexed with the monoclonal antibody. Similarly, no binding of (125)I-IGF-I was obtained in the LIFA when applied to plasmas from pregnant women during the final 3 months of pregnancy, where the characteristic 42- to 39-kDa doublet of intact IGFBP-3 is undetectable. The standard curve was established using a pool of plasmas (EDTA) from healthy adults, for which standardization with glycosylated recombinant hIGFBP-3 yielded an intact IGFBP-3 content of 2 microg/mL. The dynamic range of the LIFA was 0.50-3.75 microL equivalent of the plasma pool in a total volume of 300 microL per assay tube, with a sensitivity threshold of approximately 1 ng intact IGFBP-3. Unknown plasma samples were studied at three concentrations. Intra- and interassay variations were 3.6% and 4%, respectively. In 31 healthy adults, the mean plasma concentration of intact IGFBP-3 was 2.24 +/- 0.08 (SEM) mg/L, and that of total IGFBP-3 measured by immunoradiometric assay was 3.27 +/- 0.14 mg/L. The calculated mean proportion of proteolysed IGFBP-3 was 29.4 +/- 1.9%. In these subjects, a close correlation was found between intact and total IGFBP-3 (r = 0.71, P = 0.0001). The LIFA for IGFBP-3, therefore, provides accurate and sensitive measurement of intact IGFBP-3, the form with the functional capacity to sequester IGF-I in the bloodstream by association with the acid-labile subunit in 140-kDa complexes. In combination with total IGFBP-3 and IGF-I assays, the LIFA opens new perspectives in investigating the regulation of IGFBP-3 proteolysis and IGF-I bioavailability in man.