A simple, rapid immunometric assay for determination of functional and growth hormone‐occupied growth hormone‐binding protein in human serum

We present a sensitive time‐resolved fluorometric immunofunctional assay (TR‐FIA) for direct quantitation of functional growth hormone‐binding protein (GHBP), using an immunoassay kit for growth hormone (GH‐DELFIA). In addition to the immobilized GH antibody, one monoclonal antibody against GHBP was used. This anti‐GHBP was labelled with the chelate of europium. The assay was performed in one step. The detection limit for GHBP was 0.044 nmol L–1 (NBS + 3 SD). The calibration curve was linear in the interval 0.11–8.03 nmol L−1. Average intra‐assay coefficient of variation (CV) was 3.44%. Average interassay CV at GHBP concentrations 0.563 nmol L−1 and 1.40 nmol L−1 were 12% and 6.3% respectively. Analytical recovery in serum ranged from 76% to 127% with a mean of 101 ± 3.6%. Serum GHBP in 102 normal subjects ranged from 0.513 to 3.772 nmol L 1 and was positively related to body mass index (P < 0.001). In growth hormone‐deficient sera GHBP was higher than in control subjects (1.751 ± 0.179 nmol L−1 and 1.257 ± 0.140 nmol L−1 respectively, P < 0.001). Acromegalic patients had lower levels of GHBP than controls (0.946 ± 0.251 and 1.234 ± 0.144 nmol L−1 respectively, P = 0.005). This assay also allowed detection of GH‐complexed GHBP in serum. These results were in agreement with theoretical values calculated from the measured GH and the functional GHBP concentrations. Results were compared with data obtained by a recently reported, validated ligand immunofunctional assay (LIFA), which is fundamentally different. There was a significant linear relationship between the results from the two assays (r = 0.89, P = 0.001). The slope of the regression line was 0.65. In conclusion, this new convenient GHBP TR‐FIA provides a sensitive and precise method for detecting total GHBP as well as complexed GHBP in human serum, and allows easy processing of large numbers of samples.

[1]  M. Blankenstein,et al.  High affinity growth hormone binding protein in plasma of patients with acromegaly and the effect of octreotide treatment , 1992, Clinical endocrinology.

[2]  J. Hocquette,et al.  Evaluation of the growth hormone-binding proteins in human plasma using high pressure liquid chromatography gel filtration. , 1990, The Journal of clinical endocrinology and metabolism.

[3]  L. Carlsson,et al.  Reduced concentration of serum growth hormone-binding protein in children with idiopathic short stature. National Cooperative Growth Study. , 1994, The Journal of clinical endocrinology and metabolism.

[4]  J. Kraner,et al.  Supplemental growth hormone increases the tumor cytotoxic activity of natural killer cells in healthy adults with normal growth hormone secretion. , 1990, Metabolism: clinical and experimental.

[5]  M. Shaw,et al.  Diurnal pattern of plasma growth hormone-binding protein in man. , 1990, The Journal of clinical endocrinology and metabolism.

[6]  Z. Hochberg,et al.  The distal axis of growth hormone (GH) in nutritional disorders: GH-binding protein, insulin-like growth factor-I (IGF-I), and IGF-I receptors in obesity and anorexia nervosa. , 1992, Metabolism: clinical and experimental.

[7]  M. Waters,et al.  Rabbit liver growth hormone receptor and serum binding protein. Purification, characterization, and sequence. , 1988, The Journal of biological chemistry.

[8]  Z. Hochberg,et al.  Stoichiometry of the Pulsating Growth Hormone (GH) Binding to the GH-Binding Protein and the Turnover GH-Receptor , 1994, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[9]  L. Carlsson,et al.  Growth hormone-binding protein determination in plasma: a comparison of immunofunctional and growth hormone-binding assays. , 1993, The Journal of clinical endocrinology and metabolism.

[10]  M. Shaw,et al.  The circulating growth hormone (GH)-binding protein complex: a major constituent of plasma GH in man. , 1988, Endocrinology.

[11]  A C Herington,et al.  Identification and characterization of specific binding proteins for growth hormone in normal human sera. , 1986, The Journal of clinical investigation.

[12]  H. Sadeghi,et al.  Inhibitory effect of growth-enhancing antibody on the interaction between growth hormone and growth hormone binding protein , 1993, Molecular and Cellular Endocrinology.

[13]  William I. Wood,et al.  Growth hormone receptor and serum binding protein: purification, cloning and expression , 1987, Nature.

[14]  M. Waters,et al.  Different modes of growth hormone (GH) administration do not change GH binding protein activity in man , 1993, Clinical endocrinology.

[15]  A. Barkan,et al.  Dynamic changes of growth hormone-binding protein concentrations in normal men and patients with acromegaly: effects of short-term fasting. , 1995, Metabolism: clinical and experimental.

[16]  M. Shaw,et al.  Regulation of plasma growth hormone-binding proteins in health and disease. , 1989, Metabolism: clinical and experimental.

[17]  L. Carlsson,et al.  Growth hormone insensitivity associated with elevated circulating growth hormone-binding protein in children with Alagille syndrome and short stature. , 1993, The Journal of clinical endocrinology and metabolism.

[18]  M. Waters,et al.  Regulation of growth hormone binding protein in man: comparison of gel chromatography and immunoprecipitation methods. , 1993, The Journal of clinical endocrinology and metabolism.

[19]  G. Baumann,et al.  Simple algebraic and graphic methods for the apportionment of hormone (and receptor) into bound and free fractions in binding equilibria; or how to calculate bound and free hormone? , 1989, Endocrinology.

[20]  R. Holl,et al.  Binding protein for human growth hormone: effects of age and weight. , 1991, Hormone research.

[21]  H. Orskov,et al.  Free insulin-like growth factors in human obesity. , 1995, Metabolism: clinical and experimental.

[22]  T. Merimee,et al.  Hormone and receptor studies: relationship to linear growth in childhood and puberty. , 1991, The Journal of clinical endocrinology and metabolism.

[23]  K. Ho,et al.  Direct quantitation of growth hormone binding protein in human serum by a ligand immunofunctional assay: comparison with immunoprecipitation and chromatographic methods. , 1994, The Journal of clinical endocrinology and metabolism.

[24]  B. Sallerin-Caute,et al.  Differential regulation of serum growth hormone (GH)-binding protein during continuous infusion versus daily injection of recombinant human GH in GH-deficient children. , 1993, The Journal of clinical endocrinology and metabolism.

[25]  M. Dattani,et al.  Inhibition of growth hormone bioactivity by recombinant human growth hormone-binding protein in the eluted stain assay system. , 1994, The Journal of endocrinology.

[26]  M. Stolar,et al.  A specific growth hormone-binding protein in human plasma: initial characterization. , 1986, The Journal of clinical endocrinology and metabolism.

[27]  M. Fontoura,et al.  Regulation of Growth Hormone Binding Proteins in Human Plasma , 1991, Acta paediatrica Scandinavica. Supplement.

[28]  Z. Laron,et al.  SERUM GROWTH HORMONE BINDING PROTEIN ACTIVITY IN HEALTHY NEONATES, CHILDREN AND YOUNG ADULTS: CORRELATION WITH AGE, HEIGHT AND WEIGHT , 1989, Clinical endocrinology.

[29]  G. Baumann,et al.  Serum growth hormone-binding protein is unchanged in adult panhypopituitarism. , 1994, The Journal of clinical endocrinology and metabolism.

[30]  A. Rowland,et al.  Ligand-mediated immunofunctional assay for quantitation of growth hormone-binding protein in human blood. , 1991, The Journal of clinical endocrinology and metabolism.