Measurement of growth hormone-binding protein in the rat by a ligand immunofunctional assay.
We have developed a ligand immunofunctional assay (LIFA) for quantifying the circulating functional GH-binding protein (GHBP) in the rat. This two-site solid-phase assay uses a capture monoclonal antibody (4.3) specific to the hydrophilic C-terminal segment of rat GHBP (rGHBP), saturation of binding with human GH, and a detection system of rabbit antihuman GH polyclonal antibody and peroxidase-conjugated antirabbit immunoglobulin G antibody. Results were compared with Scatchard estimates derived by immuno-precipitation with monoclonal antibody 4.3. This assay was used to determine the GHBP levels in male and female rats and to investigate the diurnal properties and dynamics of GH and GHBP interaction 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; beta = 0.55; r2 = 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 peaks (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 to secretory pulses of GH. However, the GH bursts significantly altered the distribution of the GH-GHBP complex in male rats. By saturation and mass analysis, the greater GH pulsatile secretion in male rats resulted in occupancy of GHBP from less than 5% at nadir to about 80% at secretory peaks, in contrast to the less than 5-15% range of GHBP occupancy 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 rLIFA shows good correlation to Scatchard analysis using an identical antibody. We conclude that this assay provides a rapid, sensitive, and accurate measurement of the circulating functional GHBP in the rat, and that it facilitates the study of GH and GHBP dynamics under a range of physiological conditions.
Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales, Australia.
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KC
Leung
W J
WJ
Millard
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Peters
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I
Markus
W R
WR
Baumbach
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Barnard
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Ho
D000818:Animals; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins / Q000097:blood* / Q000276:immunology; D002940:Circadian Rhythm; D005260:Female; D013006:Growth Hormone / Q000097:blood*; D007124:Immunoenzyme Techniques; D008297:Male; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000276:immunology; D012737:Sex Factors
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D011994:Recombinant Proteins; D013006:Growth Hormone; C051815:somatotropin-binding protein