新型冠状病毒S蛋白抗体的制备及初步应用

Preparation and preliminary application of antiserum against S protein of SARS-CoV-2

目的制备新型冠状病毒S蛋白抗体,初步建立检测新型冠状病毒灭活疫苗抗原含量的方法。方法使用新型冠状病毒重组S蛋白分别免疫羊和兔,获得抗血清。采用间接ELISA法和微量中和试验检测抗血清效价,免疫印迹试验检测抗体特异性。采用蛋白G纯化树脂分别对羊和兔抗血清进行亲和层析纯化,获得抗体。以纯化后的羊抗体作为包被抗体,兔抗体作为显示抗体,建立新型冠状病毒灭活疫苗抗原含量检测方法。结果对羊进行4次免疫后,抗血清ELISA效价达220000,中和抗体效价达1536。对兔进行3次免疫后,抗血清ELISA效价达220000,中和抗体效价达4096。羊和兔抗体均可与新型冠状病毒S蛋白特异性结合。应用纯化后的抗体,成功建立了检测抗原含量的双抗体夹心ELISA方法,该方法线性良好,R^(2)>0.99,可用于新型冠状病毒灭活疫苗原液抗原含量的检测。结论成功制备了羊和兔抗新型冠状病毒S蛋白高效价抗体,并初步建立了新型冠状病毒灭活疫苗抗原含量检测的双抗体夹心ELISA方法。

Objective To prepare antiserum against S protein of SARS-CoV-2 and preliminarily develop a method for determination of antigen content in inactivated SARS-CoV-2 vaccine.Methods Goats and rabbits were immunized with recombinant S protein of SARS-CoV-2,and the obtained antisera were determined for titer by indirect ELISA and neutralization assay and for specificity by Western blot,then purified by protein G resin affinity chro-matography.A sandwich ELISA for determination of antigen content of inactivated SARS-CoV-2 vaccine was developed by using purified goat antibody as capture antibody and purified rabbit antibody as detection antibody.Results The titer of antiserum of goats after immunization for 4 times reached 220000 by ELISA and 1536 by neutralization assay,while that of rabbits after immunization for 3 times reached 220000 by ELISA and 4096 by neutralization assay.Both goat and rabbit antibodies showed specific binding to the S protein of SARS-CoV-2.A double antibody sandwich ELISA method for determination of antigen content was successfully developed by using the purified antibodies,which showed a good linearity with a R^(2) value of more than 0.99.Conclusion High titer goat and rabbit antisera against S protein of SARSCoV-2 were prepared successfully,and double antibody sandwich ELISA method for determination of antigen content of inactivated SARS-CoV-2 vaccine was preliminarily developed.