重组人干扰素β1a生物学活性MTS/PMS检测方法的建立

Development of a MTS/PMS colorimetric assay for activity of recombinant human interferon β1a

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 develop a MTS / PMS colorimetric assay for activity of recombinant human interferon β la （IFNI3 la）. Methods A method for determination of biological activity of IFNβ1a was developed using MTS coupled with PMS as staining solu- tion. The concentration and CPE time of cells, working concentration of MTS as well as time for staining were optimized, based on which dose-effect curves were plotted. The developed method was verified for reproducibility and accuracy, and the results were compared with those of crystal violet staining. Results The optimal concentration and CPE time of cells were 1- 105 cells/ml and 24 - 36 h, while the optimal working concentration of MTS and time for staining were 2 mg/ml and 40 min, respectively. The rela- tionship coefficients （R^2） of S-shaped effect curves of IFN^la for protection of Wish cells were more than 0. 99. The coefficient of variation （CV） of activities of samples added to the wells in the same positions of different plates was 6%-20%, while that to the different wells on the same plate was 13% - 17%. The recovery rate of cells for determination of IFNβ1a activity was 86% -121%. The effect curve of the developed method for determination of recombinant human IFNβ1a activity was in a reverse S shape, with a mean R2 value of more than 0. 99 which was higher than that by crystal violet staining. In addition, the developed method was more stable than crystal violet staining. Conclusion A MTS/PMS colorimetric assay was successfully developed, which was suitable for routine determination of activity of recombinant human IFNβ1a.