SARS-CoV-2 δ变异株S抗原含量检测方法的建立及验证

Development and verification of a method for detection of S antigen content of SARS-CoV-2 δ variant

目的 建立严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)δ变异株S抗原含量的检测方法,并进行验证。方法 采用δ变异株重组表达的受体结合域(receptor binding domain,RBD)蛋白分别免疫山羊和家兔,制备抗RBD多克隆抗体。以制备的两种抗RBD多克隆抗体为包被抗体和一抗,HRP标记的羊抗兔IgG抗体为酶标抗体,建立用于检测S抗原含量的双抗体夹心ELISA法。棋盘滴定法确定包被抗体和一抗,同时优化3种抗体稀释度。验证方法的准确性、精密性、线性范围及专属性。采用建立的方法检测SARS-CoV-2 δ株灭活疫苗(Vero细胞)原液及其中间品的S抗原含量。结果 兔抗RBD多克隆抗体及羊抗RBD多克隆抗体的效价分别为1∶32 000和1∶64 000,蛋白浓度分别为2.26和5.41 mg/mL。最佳包被抗体为羊抗RBD多克隆抗体,一抗为兔抗RBD多克隆抗体,包被抗体、一抗及酶标抗体最佳稀释度分别为1∶4 000、1∶8 000和1∶16 000。40、20、10 U/mL3种浓度S抗原内部参考品重复6次检测结果的回收率为83.5%~103.0%;重复6次测定3批SARS-CoV-2 δ株灭活疫苗(Vero细胞)原液抗原含量的CV均<7.5%,2名实验员对3批上述疫苗原液重复检测3次结果的CV均<7.5%;S抗原含量理论值在10~40 U/mL范围内,与测定值呈良好的线性关系,线性回归方程为:y=0.942 3 x+0.049 2,R~2=0.995 4,定量限为10 U/mL;建立的方法与甲型肝炎灭活疫苗(人二倍体细胞)、四价流感病毒裂解疫苗、Vero细胞宿主蛋白、Vero细胞培养上清液等无交叉反应。SARS-CoV-2 δ株灭活疫苗(Vero细胞)原液及中间品3次检测结果的CV均<15.0%。结论 建立的双抗体夹心ELISA法具有良好的准确性、精密性、专属性,可用于SARS-CoV-2 δ株灭活疫苗(Vero细胞)原液及中间品中S抗原的定量检测。

Objective To develop and verify a method for the detection of S antigen content of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)δ variant.Methods Recombinant receptor binding domain(RBD)protein of δmutant was used to immunize goats and rabbits to prepare anti-RBD polyclonal antibodies.A double antibody sandwich ELISA was developed using the goat anti-RBD polyclonal antibody as coating antibody,the rabbit anti-RBD polyclonal antibody as primary antibody,and the goat HRP-labeled anti-rabbit IgG polyclonal antibody as detection antibody to detect S antigen content.The coating antibody and primary antibody were determined by chessboard titration,and the dilution of three kinds of antibodies were optimized.The method was verified for the accuracy,precision,linear range and specificity and used to detect the S antigen content of SARS-CoV-2 δ inactivated vaccine(Vero cell)bulk and its intermediates.Results The titers of rabbit anti-RBD polyclonal antibody and goat anti-RBD polyclonal antibody were 1∶32 000 and 1∶64 000,and the protein concentrations were 2.26 and 5.41 mg/mL,respectively.The optimal coating antibody was goat anti-RBD polyclonal antibody,and the primary antibody was rabbit anti-RBD polyclonal antibody.The optimal dilutions of coating antibody,primary antibody and enzyme-labeled antibody were 1∶4 000,1∶8 000 and 1∶16 000 respectively.The recoveries of the internal reference samples of S antigen at concentrations of 40,20 and 10 U/mL detected for 6 times were within 83.5% ~ 103.0%.The CV values of antigen content in 3 batches of SARS-CoV-2 δ strain inactivated vaccine(Vero cell)in the 6 repeated tests were all less than 7.5%,and the CV values of results of 3 repeated tests for 3 batches of the above-mentioned vaccine bulks by 2 experimenters were less than 7.5%.The theoretical value of S antigen content was in the range of 10 ~ 40 U/mL,which showed a good linear relationship with the measured value.The linear regression equation was y = 0.942 3 x + 0.049 2,R~2= 0.995 4,and the quantitative limit was 10 U/mL.The developed method had no cross reaction with inactivated hepatitis A vaccine(human diploid cells),quadrivalent split influenza vaccine,Vero cell host protein and Vero cell culture supernatant.The CV values of the detection results for 3 times of the bulk and intermediate of SARS-CoV-2 δ strain inactivated vaccine(Vero cell)were all less than 15.0%.Conclusion The double antibody sandwich ELISA method developed in this study had good accuracy,precision and specificity,and might be used for the quantitative detection of S antigen in the bulk and intermediate of inactivated vaccine(Vero cell)against SARS-CoV-2 δ strain.