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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 and validate the TF-1 cell / MTT assay for detecting the biological activity of recombinant human granulocyte macrophage colony stimulating factor (rh GM-CSF). Methods rh GM-CSF-dependent cell line TF-1 was used as target cells. The biological activity of rh GM-CSF was assayed by MTT colorimetric method. The lysate type, lysis time, cell concentration and MTT action time were optimized. And the optimized method is verified by accuracy and precision. Results Optimized TF-1 cell / MTT colorimetric method: dilute the test sample and standard to 18 IU / ml, make 2 times of serial dilution, a total of 8 dilutions, 50μl / hole; add 6 × 10 ~ 5 Ml / ml TF-1 cell suspension, 50 μl / well and incubate at 37 ° C for 48-52 h; add MTT solution, 20 μl / well and incubate at 37 ° C. for 5 h; lysed overnight by adding 15% SDS lysate, A570 value detection. The recovery of rh GM-CSF standard assay was 104%. The average assay activity of rh GM-CSF standard was 18.6 IU / ml and the coefficient of variation was 5.37%. The rh GM-CSF standard The average coefficient of variation of 3 days activity test results was 5.18%. Conclusion The optimized rhTGF-MTT method was used to detect the biological activity of rh GM-CSF. The accuracy and precision of rh GM-CSF were high and could be applied to the detection of rh GM-CSF activity.