Abstract:

In order to solve the problems of high cost， narrow detection linear range and poor source controllability of commercial kits for determining the content of p24 protein in lentivirus vector， a pair of mouse monoclonal antibodies against p24 protein were used as capture antibodies and detection antibodies， and the detection antibodies were coupled with biotin to establish a double antibody sandwich ELISA method for quantitative detection of p24 protein. The best working concentration of coated antibody and detection antibody in double antibody sandwich ELISA were determined by square matrix titration. The linearity range， lower limit of quantification， accuracy， precision and specificity of the standard curve were investigated. Six batches of lentivirus vector samples produced by our company were used to detect p24 content and sample stability were investigated for method fitness. The experimental results showed that the optimal working concentrations of coated antibody and biotin labeled antibody in double antibody sandwich ELISA were 0.8 μg·mL^-1 and 0.005 μg·mL^-1， the method had the best linearity in the concentration range of 1.25~80.00 ng·mL^-1， and the correlation coefficient r²>0.95. The recovery of intra and inter assay for high， medium and low quality control sample were 80%~120%， the coefficient of variation was less than 10.0%， and the lower limit of quantitative detection was 1.25 ng·mL^-1. The detection results of p24 protein content of 6 batches of the same lentivirus vector were within 30% deviation， and the detection results of storage stability of 4 batches of samples within 8 hours were within 10% deviation. The double antibody sandwich ELISA method has been optimized， developed and fully validated， which could be used for quantitative detection of lentivirus vector p24 protein content， and provide important data support and theoretical basis for lentivirus vector process development， quality control and quality consistency between batches.

Key words: lentivirus vector, ELISA, p24 protein

摘要：

测定慢病毒载体p24蛋白含量的商业化试剂盒存在成本高、检测线性范围窄和货源可控性差等不足，为解决上述问题，采用由一对鼠抗p24蛋白的单克隆抗体作为捕获抗体与检测抗体，其中检测抗体进行生物素偶联来建立p24蛋白定量的双抗体夹心ELISA检测方法。通过方阵滴定法确定双抗夹心ELISA实验中的包被抗体与检测抗体的最佳工作浓度。对该方法进行了标准曲线线性范围、定量下限、准确度、精密度、特异性等的考察。取本公司自主研发生产的6个批次的慢病毒载体样品进行p24蛋白定量检测和放置稳定性来考察方法的适用性。实验结果显示，双抗夹心ELISA方法中包被抗体和生物素标记检测抗体的最优工作浓度分别为0.8 μg·mL^-1和0.005 μg·mL^-1，方法在1.25~80.00 ng·mL^-1浓度区间有最佳线性，相关系数r²>0.95。批内和批间检测高、中、低质控品的回收率在80%~120%之间，变异系数均小于10.0%，定量检测下限为1.25 ng·mL^-1。同一种慢病毒载体6批次的p24蛋白含量检测结果均在30%偏差范围内，4批次样本放置8 h的稳定性检测结果偏差均在10%范围内。对双抗体夹心ELISA法进行了优化开发和全面验证，旨在提升慢病毒载体p24蛋白含量的定量检测水平，以及为慢病毒载体工艺开发、质量控制和批次之间质量的一致性研究提供重要的数据支撑和理论依据。

关键词: 慢病毒载体, ELISA, p24蛋白