Abstract
An improved in vivo body weight gain bioassay for the potency determination of human growth hormone (hGH) has been set up in “little” mice (lit/lit), a mutant derived from the C57BL/6J strain. This improved assay now has a detection limit of the order of 0.05 µg/mouse/day, which corresponds to a sensitivity about 20-fold higher than that of the most sensitive in vivo assay reported up to now: the tibia test in hypophysectomized rats or mice. This sensitivity was achieved mainly by introduction of a careful pre-assay selection and of a three injections per day schedule. The utilization of these conditions in a 2×2 factorial assay design allowed the potency determination of recombinant DNA-derived hGH (rec-hGH) in bacterial extracts with acceptable accuracy and precision, together with the greatest economy of material, only 0.24 mg of unknown and standard hormone preparation being sufficient for an entire 10-animal assay. This contrasts to a minimum of 2.7 mg that are necessary for an economical assay in hypophysectomized rats. The same assay procedure was also used to demonstrate the in vivo bioactivity of hGH secreted into a culture medium from transduced human primary keratinocytes. The growth curve constructed with n=8 little mice presented a highly significant correlation (r=0.939, p<0.001) and a slope=0.016 g/mouse/day. It was thus possible to prove, for the first time, the in vivo bioactivity of rec-hGH secreted by transplantable human epidermal cells, utilized as an experimental model for somatic gene therapy.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bellini M.H., Bartolini P. In vivo bioassay for the potency determination of human growth hormone in dwarf “little” mice. Endocrinology 132: 2051, 1993.
Bristow A.F., Gaines-Das R., Jeffcoate S.L., Schulster D. The first international standard for somatotropin: report of an international collaborative study. Growth Reg. 5: 133, 1995.
Greenspan F.S., Li C.H., Simpson M.E., Evans H.M. Bioassay of hypophyseal growth hormone: the tibia test. Endocrinology 45: 455, 1949.
Papkoff H., Li C.H. Hypophyseal growth hormone. In: Dorfman R.I. (Ed.), Methods in Hormone Research. Academic Press, New York, 1962, vol. 2, p. 671.
Morgan J.R., Barrandon Y., Green H., Mulligan R.C. Expression of an exogenous growth hormone gene by transplantable human epidermal cells. Science 237: 1476, 1987.
Teumer J., Lindahl A., Green H. Human growth hormone in the blood of athymic mice grafted with cultures of hormone-secreting human keratinocytes. FASEB J. 4: 3245, 1990.
Vogt P.M., Thomson S., Andree C., Liu P., Breuing K., Hatzis D., Brown H., Mulligan R.C., Eriksson E. Genetically modified keratinocytes transplanted to wounds reconstitute the epidermis. Proc. Natl. Acad. Sci. USA. 91: 9307, 1994.
Jensen V.B., Jensen T.G., Jensen P.K.A., Rygaard J., Hansen B.S., Fogh J., Kolvraa S., Bolund L. Gene transfer into cultured human epidermis and its transplantation onto immunodeficient mice: an experimental model for somatic gene therapy. J. Invest. Dermatol. 103: 391, 1994.
Lesniak M.A., Gorden P., Roth J., Gavin III J.R. Binding of 125I-human growth hormone to specific receptors in human cultured lymphocytes. J. Biol. Chem. 249: 1661, 1974.
Chen B.F., Chang W.C., Chen S.T., Chen D.S., Hwang L.H. Long-term expression of the biologically active growth hormone in genetically modified fibroblasts after implantation into a hypophysectomized rat. Hum. Gene Ther. 6: 917, 1995.
Mathor M.B. Gene expression studies with human growth hormone gene transfected cells for a possible utilization in gene therapy. Ph D thesis work, IPEN-CNEN/University of São Paulo — Brazil, 1994.
Eicher E.M., Beamer W.G. Inherited ateliotic dwarfism in mice. J. Hered. 67: 87, 1976.
Jansson J.O., Downs T.R., Beamer W.G., Frohman L.A. Receptor associated resistance to growth hormone-releasing factor in dwarf “little” mice. Science 232: 511, 1986.
Wilson D.B., Wyatt D.P., Gadler R.M., Baker C.A. Quantitative aspects of growth hormone cell maturation in the normal and little mutant mouse. Acta. Anat. 131: 150, 1988.
Zarrow M.X., Yochim J.M., McCarthy J.L. Experimental Endocrinology. A sourcebook of Basic Techniques. Academic Press, New York, 1964, p. 308.
Bartolini P., Marques de Assis L., Schwarz I., Pieroni R.R. An accurate determination of human growth hormone content in different pituitary extracts, using a radioimmunoassay with polyacrylamide gel electrophoresis as a bound-free separation system. Clin. Chim. Acta 79: 223, 1977.
Lowry D.B., Rosebrough N.J., Farr A.L., Randall R.J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265, 1951.
Ribela M.T.C.P., Bartolini P. The use of recombinant human growth hormone for radioiodination and standard preparation in radioimmunoassay. J. Immunol. Methods 159: 269, 1993.
Zambruno G., Marchisio P.C., Marconi A., Vaschieri C., Melchiori A., Giannetti A., De Luca M. Transforming growth factor-β1 modulates β1 and β5 integrin receptors and induces the de novo expression of the α v β heterodimer in normal human keratinocytes: implications for wound healing. J. Cell Biol. 129: 853, 1995.
Bartolini P., Muramoto E. Bioassay of hGH: standardization of more economical assay designs. Horm. Metab. Res. 16: 614, 1984.
Rodbard D. Statistical estimation of the minimal detectable concentration (sensitivity) for radioligand assays. Anal. Biochem. 90: 1, 1978.
Wilhelmi A.E. Growth hormone bioassay. In: Berson S.A., Yalow R.S. (Eds.), Methods in Investigative and Diagnostic Endocrinology. North Holland, Amsterdam, 1973, vol. 2A, p. 296.
Groesback M.D., Parlow A.F. Highly improved precision of the hypophysectomized female rat body weight gain bioassay for growth hormone by increased frequency of injections, avoidance of antibody formation, and other simple modifications. Endocrinology 120: 2582, 1987.
European Pharmacopoeia Convention. Human growth hormone for injection. In: Maisonneuve S.A. (Ed.), European Pharmacopoeia, ed 2, part II, 11th fascicule. Sainte Ruffine, France, 1987, p. 556.1.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bellini, M.H., Mathor, M.B., De Luca, M. et al. Ultrasensitive in vivo bioassay detects bioactive human growth hormone in transduced primary human keratinocytes. J Endocrinol Invest 21, 1–6 (1998). https://doi.org/10.1007/BF03347278
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF03347278