A Method for Measuring the Binding Affinity and Capacity of Growth Hormone Binding Protein in Human Serum Using FPLC to Separate Bound and Free Ligand

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Abstract
A method was developed for the measurement of growth hormone binding protein (II-GHBP) in human serum, lodinated human growth hormone (hGH), freshly purified on Sephacryl 200-HR, was incubated overnight at room temperature with human serum in the presence of different concentrations of radioinert hGH. Free and bound hGH were separated on a Superose 12 column by a fully automated FPLC system. The column was eluted with 0.05 M phosphate buffer, pH 7.5, containing 0.15 M NaCl. Concentrations of bound and free hormone were calculated from the elution pattern. Binding affinity and capacity were calculated by Scatchard plot analysis of the binding data. The data were corrected for endogenous hGH in the samples. The intra-assay variation in the percentage bound GH was 4% whereas the interassay variation in binding capacity was found to be 12%. The ll-GHBP levels in serum from healthy female and male volunteers were 1350±530 (mean ± S. D.; n = 29) and 900 ± 205 fmole/l (n = 22) respectively (pa.) differed between serum from female (0.31 ±0.07 nM–1) and male subjects (0.43 ± 0.11 nM–1; p < 0.01). The advantage of the present method over published methods for ll-GHBP assay is that binding capacity and affinity are determined. The disadvantage is that, even with the automated FPLC, the method is slow since 6 FPLC runs are required for every sample. Current efforts are directed to improve this disadvantage.