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目的对CHO-K1细胞簇集法检测百日咳毒素(pertussis toxin,PT)毒性进行优化及验证。方法以国家PT标准品为参考品,将PT标准品倍比稀释后与CHO-K1细胞混合培养,显微镜观察PT能引起CHO-K1细胞簇集的程度。对细胞浓度、孵育时间进行优化,并对方法的特异性、敏感度、重复性进行验证。结果方法的最佳细胞浓度为2.5×105个/ml,最佳孵育时间为24 h。只有PT活性成分能使CHO-K1细胞发生簇集,最低检测限为10 pg/ml;2名试验员6次检测结果表明,该方法重复性良好。结论 CHO-K1细胞簇集法可及时、准确地检测出生产过程中PT含量,适用于百日咳发酵过程监测。
Objective To optimize and validate the toxicity of pertussis toxin (PT) by cluster analysis of CHO-K1 cells. Methods The PT standard was used as the reference material, and the PT standard was diluted by times and mixed with CHO-K1 cells. The extent of PT-induced aggregation of CHO-K1 cells was observed under microscope. The cell concentration and incubation time were optimized, and the specificity, sensitivity and reproducibility of the method were verified. Results The best cell concentration was 2.5 × 105 cells / ml and the optimal incubation time was 24 h. Only the PT active ingredient could induce the clustering of CHO-K1 cells with the minimum detection limit of 10 pg / ml. The results of six tests by two testers showed that the method was reproducible. Conclusion CHO-K1 cell clustering method can detect PT content in the production process timely and accurately, and is suitable for the monitoring of the pertussis fermentation process.