A novel bioassay based on human growth hormone (hGH) receptor mediated cell proliferation: measurement of 20K-hGH and its modified forms.
Previously we introduced the full-length hGH receptor (hGHR) into the mouse pro-B cell line, Ba/F3, and obtained stable transfectant (Ba/F3-hGHR), which could grow in response to 20K- and 22K-hGH in a dose-dependent manner(1). In the present study, we established a new bioassay system based on the proliferation of the Ba/F3-hGHR in combination with the eluted stain assay (ESTA). The Ba/F3-hGHR assay is completed in 18 h and requires only 10(-6)-fold amount of GH sample (1.8 ng) as compared with the rat weight gain assay. The validation study shows that the Ba/F3-hGHR assay is specific for hGH, precise (RSD = 1.1-19.7%) and ultrasensitive (lower limit of working range = 18.7 pg/mL). Four modified forms of recombinant 20K-hGH (oxidized, deamidated, des-Phe(1)and cleaved form) all of which are newly identified were measured by the Ba/F3-hGHR assay and the rat weight gain assay with our in-house recombinant 20K-hGH as standard. The oxidized and deamidated 20K-hGH were fully active, however the des-Phe(1)and cleaved 20K-hGH had significantly reduced activities in both assays. These findings suggest that the Ba/F3-hGHR assay is useful as an alternative to the rat weight gain assay.
Ikeda
M
M
Life Science Laboratories, Mitsui Chemicals Inc., Mobara, Chiba, Japan.
Wada
M
M
Fujita
Y
Y
Takahashi
S
S
Maekawa
K
K
Honjo
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay / Q000379:methods; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli; D019382:Human Growth Hormone / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D011986:Receptors, Somatotropin / Q000187:drug effects / Q000502:physiology*; D014407:Tumor Cells, Cultured
D020033:Protein Isoforms; D011986:Receptors, Somatotropin; D019382:Human Growth Hormone