重组人粒细胞巨噬细胞集落刺激因子生物学活性测定TF-1细胞/MTT比色法的优化及其验证

Optimization and verification of TF-1 cells / MTT colorimetry for determination of biological activity of recombinant human granulocyte macrophage colony stimulating factor

目的对检测重组人粒细胞巨噬细胞集落刺激因子（recombinant human granulocyte macrophage colony stimulating factor,rh GM-CSF）生物学活性的TF-1细胞/MTT比色法进行优化及验证。方法以rh GM-CSF依赖细胞株TF-1为靶细胞,采用MTT比色法测定rh GM-CSF生物学活性,对比色法中裂解液种类、裂解时间、细胞浓度、MTT作用时间等进行优化,并对优化后的方法进行准确度和精密度验证。结果优化后的TF-1细胞/MTT比色法：将供试品及标准品稀释至18 IU/ml,做2倍系列稀释,共8个稀释度,50μl/孔;加入6×10^5个/ml TF-1细胞悬液,50μl/孔,37℃培养48-52 h;加入MTT溶液,20μl/孔,37℃培养5 h;加入15%SDS裂解液,150μl/孔,裂解过夜,检测A570值。3种不同浓度的rh GM-CSF标准品活性测定结果回收率为104%;rh GM-CSF标准品6次检测活性平均值为18.6 IU/ml,变异系数为5.37%;rh GM-CSF标准品3 d活性检测结果的平均变异系数为5.18%。结论采用优化后MTT比色法检测rh GM-CSF生物学活性,准确度和精密度均较高,可应用于rh GM-CSF活性的检测。

Objective To optimize the verify the TF-1 cells / MTT colorimetry for determination of biological activity of recombinant human granulocyte macrophage colony stimulating factor（rh GM-CSF）. Methods The biological activity of rh GM-CSF was determined by MTT colorimetry using rh GM-CSF-dependent cell strain TF-1 as target. The lysis agent,lysis time, cell concentration and treatment time with MTT were optimized, and the method after optimization was verified for accuracy and precision. Results The optimized TF-1 cells / MTT colorimetry was as follows： either test sample or standard was diluted to a concentration of 18 IU / ml, the diluted 2-fold serially to eight dilutions, 50 μl for each well.TF-1 cell suspension at a concentration of 6 × 10~5 cells / ml was inoculated, 50 μl for each well, incubated at 37 ℃ for48 ~ 52 h, added with MTT solution, 20 μl for each well, and further incubated at 37 ℃ for 5 h, then added with 15%SDS, 150 μl for each well, incubated overnight and determined for A570 value. The recovery rates of activities of three batches of rh GM-CSF standard were 104%, while the mean activity in six tests was 18. 6 IU / ml, with a coefficient of variation（CV） of 5. 37%. However, the mean CV of determination results of activity on 3 working days was 5. 18%.Conclusion The optimized TF-1 cells / MTT colorimetry showed high accuracy and precision, which was suitable for the determination of biological activity of rh GM-CSF.