MEASUREMENT OF GROWTH HORMONE-BINDING PROTEIN IN THE RAT BY A LIGAND IMMUNOFUNCTIONAL ASSAY

We have developed a ligand immunofunctional assay (LIFA) for quantifyi ng the circulating functional GH-binding protein (GHBP) in the rat. Th is two-site solid-phase assay uses a capture monoclonal antibody (4.3) specific to the hydrophilic C-terminal segment of rat GHBP (rGHBP), s aturation of binding with human GH, and a detection system of rabbit a ntihuman GH polyclonal antibody and peroxidase-conjugated antirabbit i mmunoglobulin G antibody. Results were compared with Scatchard estimat es derived by immuneprecipitation with monoclonal antibody 4.3. This a ssay was used to determine the GHBP levels in male and female rats and to investigate the diurnal properties and dynamics of GH and GHBP int eraction in 15-min blood sampling over a 6-h period. The dynamic range of the rLIFA was 0.15-20.0 nM recombinant rGHBP, with intraassay and interassay coefficients of variation of 10.5% (n = 20) and 12.9% (n = 12), respectively. Serum GHBP levels determined by the rLIFA and those derived from Scatchard estimates were strongly correlated (n = 8; bet a = 0.55; r(2) = 0.89; P = 0.0005). Male rats had lower GHBP levels (6 .5 +/- 0.7 nM; mean +/- SE; n = 14) than female rats (35.4 +/- 2.7 nM; n = 15; P = 0.0001). In the diurnal study, male rats had higher GH pe aks (312.5 +/- 121.6 ng/ml; n = 7) than female rats (96.5 +/- 15.4 ng/ ml; n = 9; P < 0.0001). In contrast to the pulsatile secretion of GH, GHBP levels in both sexes remained stable and showed no relationship t o secretory pulses of GH. However, the GH bursts significantly altered the distribution of the GH-GHBP complex in male rats. By saturation a nd mass analysis, the greater GH pulsatile secretion in male rats resu lted in occupancy of GHBP from less than 5% at nadir to about 80% at s ecretory peaks, in contrast to the less than 5-15% range of GHBP occup ancy in female rats. In male rats, greater than 80% of GH at secretory peaks existed in the free form, whereas in female rats, 16-23% of GH existed in the free form during pulsatile secretion. In summary, the r LIFA shows good correlation to Scatchard analysis using an identical a ntibody. We conclude that this assay provides a rapid, sensitive, and accurate measurement of the circulating functional GHBP in the rat, an d that it facilitates the study of GH and GHBP dynamics under a range of physiological conditions.