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目的建立重组人干扰素β1a(Recombimant human interferon beta1a,rhIFNβ1a)生物学活性MTS/PMS检测方法。方法将MTS和PMS偶联作为染色液,建立IFNβ1a生物学活性检测方法,对细胞浓度、细胞病变时间、MTS工作浓度和染色时间进行优化,并绘制效应曲线。对建立的方法进行重复性、准确性验证,并与结晶紫染色法进行比较。结果优化后的MTS/PMS法的最佳反应条件为:细胞浓度1×105个/ml,细胞病变时间24～36 h,MTS工作浓度2 mg/ml,染色时间40 min;A570/630值与IFNβ1a保护Wish细胞S型效应曲线的相关系数(R2)均达0.99以上。不同检测板、相同加样位置的变异系数在6%～20%之间;相同检测板、不同加样位置的变异系数在13%～17%之间;检测IFNβ1a细胞收集液的回收率在86%～121%之间。该法检测rhIFNβ1a生物学活性效应曲线呈反“S”型,线型较好,R2值均在0.99以上,均比结晶紫染色法的R2值高,且比结晶紫染色法更稳定。结论已建立了rhIFNβ1a生物学活性MTS/PMS检测方法,适用于常规定量测定rhIFNβ1a的生物学活性。
OBJECTIVE: To establish a method for the bioassay of recombinant human interferon β1a (rhIFNβ1a) by MTS / PMS assay. Methods The biological activity of IFNβ1a was detected by coupling MTS and PMS as staining solution. The cell concentration, cytopathic effect time, MTS working concentration and staining time were optimized and the effect curve was drawn. The repeatability and accuracy of the established method were verified and compared with the crystal violet staining method. Results The optimized reaction conditions of MTS / PMS were as follows: cell concentration 1 × 105 cells / ml, cytopathic effect time 24 ~ 36 h, MTS working concentration 2 mg / ml, staining time 40 min; A570 / 630 value and The correlation coefficient (R2) of IFNβ1a for protecting S-shape curve of Wish cells reached above 0.99. The coefficient of variation (CV) between different test plates and the same sample loading position was between 6% and 20%. The coefficient of variation of the same test plate and different sample loading positions was between 13% and 17% % ~ 121% between. The method of detecting the biological activity of rhIFNβ1a showed an inverted “S” shape, with good linearity and R2 values above 0.99, both higher than the R2 value of the crystal violet staining method and more stable than the crystal violet staining method. Conclusion The biological activity of rhIFNβ1a MTS / PMS assay has been established and is suitable for the routine quantitative determination of rhIFNβ1a biological activity.