Paper
XU Jinhua, ZHAO Wenpeng, ZHAO Lili, CAO Yu, PENG Wei, LIU Zhong
2023, 54(04): 545-553.
The establishment of the fibroblast growth factor 21(FGF21)-Fc fusion protein in vitro activity
test can be divided into binding assay at the protein level and biological assay at the cell level. This study established a
homogeneous time-resolved fluorescence(HTRF) method to detect the binding activity of FGF21-Fc fusion protein and
β-Klotho ligand protein. And the reporter gene cells were constructed independently to test biological activity. When
the FGF21-Fc fusion protein binded with β-Klotho, the FGF receptor and the intracellular signal transduction were
activated to further affect luciferase activity, which could be used to monitor and quantitatively measure biological activity
indirectly. During the establishment process of the binding assay, the sample and ligand concentrations, dilution ratios of
donor and acceptor, and incubation time were optimized comprehensively. And methodological verification was carried
out to prove that the HTRF technology had good repeatability and specificity and this method could be used to detect the
binding activity of FGF21-Fc fusion protein and β-Klotho. Later, the fetal bovine serum concentration, density of report
gene cells, plate type, and cells in the plate form in bioactivity assay were optimized. And methodological verification was
carried out to prove that the reporter gene method had good repeatability and specificity and this method could be used to
test the biological activity of FGF21-Fc fusion protein. In this study, a quality control method for FGF21-Fc fusion protein
was established, which provided a reference for analysis technology of drugs with the same activity detection difficulties,
filling the gap at home and abroad.
