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Development and Clinical Evaluation of a Novel Immunoassay for the Binary Complex of IGF-I and IGF-Binding Protein-1 in Human Serum

Medical Research Laboratories (J.F., K.N.R., S.P.J., H.Ø.), Aarhus University Hospital, DK-8000 Aarhus C, Denmark; and Department of Endocrinology (K.H., M.W.-C.), Odense University Hospital, DK-5000 Odense C, Denmark

Address all correspondence and requests for reprints to: Dr. Jan Frystyk, Institute of Experimental Clinical Research, Aarhus Kommune Hospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark. E-mail: jan{at}frystyk.dk

Correlation studies have suggested that IGF-binding protein (IGFBP)-1 is a dynamic regulator of free IGF-I. To further study this, we developed a monoclonal immunofluorometric assay specific for the binary complex of IGF-I and IGFBP-1 in human serum. An IGFBP-1 antibody, which recognizes all phospho-forms of IGFBP-1, was used for coating. An europium-labeled IGF-I antibody served as tracer. Assay incubation was performed at conditions approaching those in vivo (i.e. pH 7.4, 37 C). The assay was highly specific: no signal was obtained unless both IGF-I and IGFBP-1 were present and neither IGFBP-2, -3, -4, nor IGF-II caused any cross-reaction. The linear standard curve covered 3 orders of magnitude, and within and in-between assay coefficients of variation were less than 5 and 15%, respectively. To study the dynamic relationship between free IGF-I and binary complex formation, seven healthy subjects were fasted for 72 h. Samples were collected every 3 h. During fasting, free IGF-I was reduced by two thirds (P < 0.0001). IGFBP-1 and the binary complex increased in parallel (P < 0.0001), and levels correlated positively in all subjects (0.89 r 0.98; P < 0.0001). Free IGF-I correlated inversely with IGFBP-1 (-0.81 r -0.48; 0.0001 P 0.05) and the binary complex (-0.79 r -0.41; 0.0001 P 0.05). To study overnight fasting levels, we compared healthy controls and patients with type 1 diabetes and chronic renal failure (n = 10), because these patients show profound alterations in their IGF-system. In both groups, the binary complex was increased about 2.5-fold (P < 0.0001), whereas IGFBP-1 was increased by 5- to 6-fold (P < 0.0001). Accordingly, free IGF-I was severely reduced (P < 0.0001). In conclusion, the assay enables us to study the role of IGFBP-1 as a dynamic regulator of free IGF-I. Our results clearly show that IGFBP-1 and free IGF-I are tightly associated peptides. Furthermore, it has now become possible to compare levels of IGF-I carried within the binary complex IGFBP-1:IGF-I in different (patho-) physiological conditions.

This work was supported by Aarhus University-Novo Nordisk Center for Research in Growth and Regeneration (Danish Health Research Council Grant 9700592); the Institute of Experimental Clinical Research, University of Aarhus, Denmark; and the Hørslev Foundation.

Abbreviations: AUC, Area under curve; BMI, body mass index; CRF, chronic renal failure; CV, coefficient of variation; hIGF, human IGF; HSA, human serum albumin; IGFBP, IGF-binding protein; NSB, nonspecific binding; rhIGF, recombinant human IGF.

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