用MTT法代替^3H—TdR法对重组人粒细胞集落刺激因子制剂活性测定的研究

Study on the Bioassay of Recombinant Human Granulocyte Colony Stimulating Factor Preparation by Tetrazolium Salt Method Instead of ~3H-TdR Method

本文对基因重组粒细胞集落刺激因子 (rh G- CSF)制剂用四氮唑盐 (MTT)法代替[3H]胸腺嘧啶核苷 (3H- Td R)渗入法进行生物活性测定作了研究。对 MTT法中一些主要条件进行了比较试验 ,结果表明当 NFS- 60细胞株的初始浓度为 6× 10 5个 / ml,MTT浓度为 6mg/ ml,MTT孵育时间为 6h时 ,可提高 MTT测定方法的灵敏度。用 MTT法和 3H- Td R法对 rh G- CSF生物活性测定结果作比较 ,8批 rh G- CSF制剂的两法结果之比平均为 1.0 0 ,变异系数 (RSD)为 5% ,实验证实两法测定的结果具有一致性 ,因此完全可以用 MTT法来代替 3H- Td R法进行 rh G- CSF的活性测定 。

The bioassay of recombinant human granulocyte colony stimulating factor (rhG CSF) preparation is studied by tetrazolium salt (MTT) method instead of 3H TdR incorporation method The main steps of the MTT method are examined in detail The sensitivity of the MTT method is improved, when initial concentration of NFS 60 cells is 6×10 5/ml,the concentration of MTT is 6 mg/ml and the incubation period with MTT is 6h We have compared the values obtained by the MTT method and the 3H TdR method The mean bioactivity ratio of rhG CSF measured for 8 samples by two methods is 1 00 The relative standard deviation (RSD) is 5% The estimates of rhG CSF potency measured by either MTT or 3H TdR method are almost equal We concluded that bioactivity of rhG CSF can be measured by MTT method instead of 3H TdR method, so the cost of test and the definite drawback of requiring radioisotopes will be overcome