Direct quantitation of growth hormone binding protein in human serum by a ligand immunofunctional assay: comparison with immunoprecipitation and chromatographic methods.

Abstract

Current methods for measuring GH-binding protein (GHBP) are laborious, require separation of GHBP complex, and may be affected by endogenous GH content of the sample. Such methods estimate binding activity and GHBP concentration can only be obtained by Scatchard analysis. We have developed and validated a 2-site immunofunctional assay for the direct quantitation of GHBP in human serum employing a capture monoclonal antibody against GHBP and a polyclonal antibody against hGH. Results were compared with binding activity data and Scatchard-derived capacity estimates obtained by immunoprecipitation and gel chromatography procedures. Serum samples were obtained from 21 subjects with GH deficiency, 24 patients with acromegaly, and 56 normal subjects; 12 of whom were postmenopausal women who were studied before and during oral estrogen treatment. Using the immunofunctional assay, serum GHBP concentrations in normal subjects ranged from 0.14-3.28 nmol/L, was positively related to body mass index (BMI, P = 0.0004) and negatively related to age (P = 0.015). Women had significantly higher levels (0.99 +/- 0.12 vs. 0.63 +/- 0.09 nmol/L; P = 0.0191) than age and BMI-matched men. GHBP levels were not different between normal, acromegalic, or GH-deficient subjects. Oral estrogen therapy in postmenopausal women increased serum GHBP concentrations up to 5-fold. There was a significant nonlinear relationship between the immunofunctional assay measurements and binding activity by either immunoprecipitation (r = 0.84) or chromatographic (r = 0.73) methods; increase in GHBP concentrations was not reflected in proportionate increase in both activity assays. Estrogen-induced changes in circulating GHBP levels were greatest with the immunofunctional assay followed by Scatchard-derived values from immunoprecipitation and chromatographic methods. We conclude that ligand immunofunctional assay measurements of GHBP are higher but show good agreement with binding activity or Scatchard derived estimates from immunoprecipitation and chromatographic methods. This assay provides a direct and practical tool for rapid, accurate and sensitive estimation of GHBP concentration in serum.