S/D残留量对SARS-CoV-2假病毒中和抗体检测方法的影响

目的： 评价有机溶剂/表面活性剂（solvent and detergent，S/D）法灭活后S/D残留量对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的假病毒中和抗体检测方法（pseudotyped virus based neutralization assay，PBNA）的影响。方法： 在严重急性呼吸综合征（severe acute respiratory syndrome，SARS）和2019冠状病毒病（coronavirus disease，COVID-19）患者恢复期血浆中加入0.1~10倍《中华人民共和国药典》（简称《中国药典》）规定残留标准[100 μg·mL^-1聚山梨酯80（吐温80）+10 μg·mL^-1磷酸三丁酯（TNBP）]浓度的S/D，模拟灭活后S/D残留量。用PBNA检测加入S/D前后的中和抗体水平，比较S/D对中和方法的影响。将不同浓度的S/D加入到SARS-CoV和SARS-CoV-2假病毒中进行灭活处理1、2和6 h，检测假病毒的滴度，评价S/D对冠状病毒假病毒的灭活效果。结果： 当S/D残留低于《中国药典》残留上限（100 μg·mL^-1吐温80+10 μg·mL^-1 TNBP）时，SARS-CoV-2中和抗体EC₅₀平均值与无S/D组的PBNA检测结果无统计学差异；当S/D残留大于2.5倍《中国药典》残留标准时，S/D对PBNA有显著影响。含有10 mg·mL^-1吐温80+1 mg·mL^-1 TNBP的S/D试剂，室温静置1 h即可以灭活SARS-CoV和SARS-CoV-2假病毒，假病毒滴度下降分别达到5.11和4.80 log滴度，其灭活效果随着S/D的浓度下降而下降，S/D浓度为1%时无灭活效果。结论： S/D残留量对SARS-CoV-2的PBNA有显著影响，为保障PBNA的检测准确性，血浆中S/D残留量应不超过《中国药典》规定的标准，S/D试剂具有灭活2种冠状病毒假病毒的作用。

Objective: To evaluate the influence of residual solvent and detergent(S/D),which is used to inactivate SARS-CoV-2 in convalescent plasma(CP),on pseudotyped virus based neutralization assay(PBNA). Methods: To simulate S/D residue after inactivation,S/D from 0.1 to 10-fold amounts of what is listed in Pharmacopoeia of the People's Republic of China (Chinese Pharmacopoeia)[100 μg·mL^-1 polysorbate 80(Tween 80) + 10 μg·mL^-1 tributyl phosphate(TNBP)] were added into CPs from SARS(severe acute respiratory syndrome,SARS) or COVID-19 (coronavirus disease,COVID-19) infected individuals. The titers of neutralizing antibody before and after S/D addition were detected by PBNA. The influence of S/D on the detection was investigated. After 1,2 and 6 hours of treatment,the titers of pseudotyped virus were detected to evaluate the inactivation effect of S/D on coronavirus pseudotyped viruses. Results: When S/D residue did not exceed the residue standard (100 μg·mL^-1 Tween 80+10 μg·mL^-1 TNBP) stipulated in Chinese Pharmacopoeia,no significant difference was observed between the S/D and control groups for EC₅₀ values of SARS-CoV-2 neutralizing antibody. When S/D residue exceeded the 2.5 folds of residue standard,a significant inhibitory effect was presented on neutralizing antibody detection. S/D containing 10 mg·mL^-1 Tween 80+1 mg·mL^-1 TNBP,could inactivate both pseudotyped viruses after 1-hour incubation at room temperature. The titers of pseudotyped virus decreased 5.11 and 4.80 log for SARS-CoV and SARS-CoV-2 respectively. When the S/D concentration was reduced to 1%,the inactivation effect disappeared. Conclusion: Residual S/D has significant inhibitory effect on evaluating neutralizing antibody when its concentration is higher than its standard in Chinese Pharmacopoeia. To ensure the accuracy of PBNA detection,the S/D residue in plasma should not exceed the standard in Chinese Pharmacopoeia. S/D reagent has the function of inactivating SARS-CoV and SARS-CoV-2 pseudotyped viruses.

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