牛痘病毒GM-CSF注射液中GM-CSF活性及表达量的检测

Determination of activity and expression level of GM-CSF in vaccinia virus/GM-CSF injection

目的建立牛痘病毒粒细胞巨噬细胞集落刺激因子(granulocyte macrophage colony stimulating factor,GM-CSF)注射液中GM-CSF活性及表达量的梯度分析法。方法采用TF-1细胞增殖法,四参数方程拟合检测不同浓度梯度样品的GM-CSF生物学活性;采用ELISA法,二次方程拟合检测不同浓度梯度样品的GM-CSF表达量。结果牛痘病毒GM-CSF注射液及标准品的浓度与GM-CSF的生物学活性呈量效依赖关系,随着GM-CSF浓度的增加,其GM-CSF的生物学活性增强,数据经四参数方程拟合后呈S型曲线,相关系数分别为0.953和0.997,将剂量反应曲线中直线部分的浓度对数和相应的吸光度值作直线回归,两回归线的斜率b之间差异无统计学意义(P>0.05);用该方法检测1批牛痘病毒GM-CSF注射液感染上清中GM-CSF活性,平均值为(181±16)GCU,变异系数为8.84%。牛痘病毒GM-CSF注射液及标准品的浓度与GM-CSF表达量呈量效依赖关系,随着稀释倍数的增加,GM-CSF的表达量也降低,采用二次方程进行拟合,r2均﹥0.99;用该方法检测1批牛痘病毒GM-CSF注射液感染上清中GM-CSF表达量,均符合表达量与感染浓度成正比升高的规律和趋势,且均>15 ng/m L。结论建立的方法稳定、可靠、适用性强,能表明病毒感染量与GM-CSF表达活性及表达量间的量效关系,对同类产品的质量控制具有借鉴意义。

Objective To develop a gradient assay of activity and expression level of granulocyte macrophage colony stimulating factor（GM-CSF） in vaccinia virus/GM-CSF injection. Methods The biological activities of GM-CSF in samples at various concentration gradients were tested by using TF-1 cell proliferation and four-parameter equation fitted method. The GM-CSF expression levels of samples at various concentration gradients were determined by ELISA and quadratic equation fitted method. Results The biological activities of GM-CSF of vaccinia virus/GM-CSF injection and its standard increased with the increasing GM-CSF concentration. S-shaped curves were obtained after the data on vaccinia virus/GM-CSF injection and its standard were fitted by four-parameter equation,with correlation coefficients of 0. 953 and 0. 997 respectively. The logs of concentration in linear part of dose-response curve were subjected to linear regression against the corresponding absorbance,and the result showed no significant difference between the slopes b of the regression lines of vaccinia virus/GM-CSF injection and its standard（P〉 0. 05）. The GM-CSF activities in one batch of supernatant infected with vaccinia virus/GM-CSF injection were determined by the method,and the result showed a mean activity of（181 ± 16） GCU,with a variation coefficient of 8. 84%. The GM-CSF expression level decreased with the increasing dilution of vaccinia virus/GM-CSF injection and its standard,and the r^2 values in quadratic equation fitting were more than 0. 99. The GM-CSF expression levels in one batch of supernatant infected with vaccinia virus/GMCSF injection were determined by the method,and the results were consistent with the regularity and trend that the expression increases with the increasing infectious concentration,all of which were more than 15 ng/m L. Conclusion The developed method is stable,reliable and adaptable,which reflects the relationship between the quantity of virus infection and the activity as well as expression level of GM-CSF,and provides a reference for quality control of products of the same kind.