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INTRODUCTIONHuman growth hormone (hGH) is mainly produced inthe somatotrophic cells of the pituitary and is theproduct of the GH-N gene. The GH-N gene encodes aGH protein of 22 kDa (22K-hGH) and its splice variantprotein of 20 kDa (20K-hGH)2. The biological activity of20K-hGH was originally reported to be similar to thatfound for 22K-hGH by the body weight gain assay inhypophysectomized rats3, which was supported bysubsequent studies4,5. In contrast, in rat Nb2 cell growthassay the biological activity of 20K-hGH was much lessthan that of 22K-hGH6. In addition, in immunofunctionalassay (IFA), which is supposed to measure the bindingaffinity to the GH receptor, the activity of 20K-hGH waslower than that of 22K-hGH7. These discrepancies sug-gest that the biological activity obtained with 20K-hGHcan vary considerably depending on the assay employed.From the clinical point of view, the biological activity of20K-hGH should depend on its ability to activate humanGH receptor (hGHR), since the activation of hGHR is aprerequisite for inducing the somatogenic signal8.In an earlier paper, Roswall et al established a novelbioassay based on hGHR activation using a mouseA novel bioassay based on humangrowth hormone (hGH) receptormediated cell proliferation:measurement of 20K-hGH and itsmodified formsM. Ikeda1, M. Wada1, Y. Fujita1, S. Takahashi2, K. Maekawa1and M. Honjo11Life Science Laboratories, Mitsui Chemicals Inc., Chiba, Japan and 2Institute of Biological Science, Mitsui Pharmaceuticals Inc., Chiba, JapanSummaryPreviously we introduced the full-length hGH receptor (hGHR) into the mouse pro-B cell line, Ba/F3, andobtained stable transfectant (Ba/F3-hGHR), which could grow in response to 20K- and 22K-hGH in a dose-dependentmanner1. In the present study, we established a new bioassay system based on the proliferation of the Ba/F3-hGHRin combination with the eluted stain assay (ESTA). The Ba/F3-hGHR assay is completed in 18 h and requires only10–6-fold amount of GH sample (1.8 ng) as compared with the rat weight gain assay. The validation study shows thatthe Ba/F3-hGHR assay is specific for hGH, precise (RSD = 1.1–19.7%) and ultrasensitive (lower limit of workingrange = 18.7 pg/mL). Four modified forms of recombinant 20K-hGH (oxidized, deamidated, des-Phe1and cleavedform) all of which are newly identified were measured by the Ba/F3-hGHR assay and the rat weight gain assay with ourin-house recombinant 20K-hGH as standard. The oxidized and deamidated 20K-hGH were fully active, however thedes-Phe1and cleaved 20K-hGH had significantly reduced activities in both assays. These findings suggest that theBa/F3-hGHR assay is useful as an alternative to the rat weight gain assay. © 2000 Harcourt Publishers LtdKey words: Ba/F3-hGHR, cell proliferation, ESTA, bioassay, 20K-hGH, oxidized 20K-hGH, deamidated 20K-hGH,des-Phe120K-hGH, cleaved form 20K-hGHReceived 7 February 2000Revised 5 June 2000Accepted 15 August 2000Correspondence to: Mitsufumi Wada PhD, Life Science Laboratories, MitsuiChemicals Inc., 1144 Togo, Mobara, Chiba 297–0017, Japan. Tel: +81 47525 6728; Fax: +81 475 25 6553; E-mail: mitsufumi.wada@mitsui-chem.co.jp1096–6374/00/050248+08 $35.00/0© 2000 Harcourt Publishers LtdGrowth Hormone & IGF Research 2000, 10, 248–255doi:10.1054/ghir.2000.0163, available online at http://www.idealibrary.com on