新型冠状病毒灭活疫苗N蛋白含量双抗体夹心ELISA检测方法的建立与应用

Development and application of a sandwich ELISA method for detection of N protein in SARS-CoV-2 inactivated vaccine

目的:建立新型冠状病毒(SARS coronavirus 2,SARS-CoV-2)灭活疫苗N蛋白含量双抗体夹心ELISA检测方法,并对其进行验证及初步应用。方法:首先包被重组N蛋白,采用间接ELISA方法测定不同抗体对N蛋白的亲和能力。再选取高亲和力的抗体rN003作为包被抗体,rN002作为检测抗体,建立双抗体夹心ELISA检测方法并确定病毒裂解方法。测定该方法的线性范围、准确度、精确度、中间精密度、特异性及灵敏度,并用该方法测定6批SARS-CoV-2灭活疫苗中N蛋白的含量。结果:N蛋白含量在0.018~0.75μg·mL^(-1)范围内该方法呈现良好的线性关系(R^(2)>0.98),回收率在80%~120%之间,CV<10%。6批样品N蛋白含量均占总蛋白的10%~20%,样品均一性较好。结论:本研究成功建立了SARS-CoV-2灭活疫苗N蛋白含量双抗体夹心ELISA检测方法,该方法具有良好的线性、准确度、精确度、中间精密度、特异性及灵敏度,能满足SARS-CoV-2灭活疫苗N蛋白含量的检测。

Objective:To develop a sandwich ELISA method for detection of N protein in SARS-CoV-2 inactivated vaccine,and to further validate and primarily apply it for N protein qutification.Methods:The indirect ELISA method was used to assess the affinity of different anti-N protein antibodies by coating the recombinant N protein.Then the sandwich ELISA system was set up by using antibodies rN003 and rN002 as the capture and detection antibody,respectively.Also,the method for fully lysing virus to release all the N protein was confirmed.Validation of linear range,accuracy,precision,intermediate precision,specificity and LLD were obtained,and N protein content in 6 batches of SARS-CoV-2 inactivated vaccine was determined using this method.Results:N protein content has good linearty within the concentration range of 0.018~0.75μg·mL^(-1)(R^(2)>0.98),with the recovery of 80%~120%and CV<10%.N protein concentrations for 6 batches of samples accounted for 10%~20% of the total protein.All samples showed a good uniformity.Conclusions:The sandwich ELISA method for detection of N protein in SARS-CoV-2 inactivated vaccine was successfully developed with good linearty,accuracy,precision,intermediate precision,selectivity,and sensitivity,which can satisfy the N protein detection of SARS-CoV-2 inactivated vaccine.