一种不依赖于活病毒的中东呼吸综合征冠状病毒受体结合区特异性中和抗体检测方法的建立

Establishment of a novel assay detecting receptor binding domain-specific neutralizing antibody of the Middle East respiratory syndrome coronavirus without live virus

目的拟建立一种不依赖于活病毒和高等级生物安全条件,快速简便检测中东呼吸综合征冠状病毒(MERSCoV)受体结合区(RBD)特异性的中和抗体。方法利用哺乳动物表达系统表达重组rRBD-Fc蛋白;应用流式细胞术检测rRBD-Fc蛋白与Huh-7细胞结合的最佳浓度,建立通过流式细胞术检测rRBD-Fc与Huh-7结合活性的方法,并利用无关蛋白验证结合的特异性;在此基础上,利用RBD特异性中和抗体、RBD特异性非中和抗体、MERS-CoV RBD免疫小鼠血清和无关抗体建立能够检测中和抗体阻断RBD与Huh-7结合作用的RBD特异性中和抗体检测技术方法,并与实毒中和试验进行比较,评价方法的一致性;应用建立的中和抗体检测方法进行血清学检测。结果成功表达了rRBD-Fc蛋白,rRBD-Fc蛋白能够与Huh-7细胞特异性结合,并确定了rRBD-Fc与Huh-7细胞结合的最佳剂量;在此基础上建立了通过流式细胞术检测抗体阻断RBD与Huh-7结合作用的RBD特异性中和抗体检测技术方法,结果表明,检测方法能够区分具有中和活性的RBD特异性中和抗体、rRBD-Fc免疫小鼠血清和其他抗体,具有特异性,且能够反映中和活性的强弱。该方法在检测12株中和抗体时,与传统的实毒中和试验检测结果具有很好的一致性,能够有效应用于血清学检测。结论成功建立了一种不依赖活病毒和高等级生物安全条件,即可快速检测MERS-CoV RBD特异性中和抗体的方法,为针对MERS-CoV疫苗设计和抗体效果快速评价、血清学调查和免疫保护机制研究提供了一种更加简便、安全的技术手段。

This study intends to establish a new assay to detect Middle East Respiratory Syndrome coronavirus（MERSCoV）receptor-binding domain（RBD）-specific neutralizing antibodies,which is safe without using live virus.Recombined rRBD-Fc protein was expressed in mammalian cell expression system.The optimal concentrations of rRBD-Fc,as well as the binding specificity between rRBD-Fc and Huh-7 cells,were confirmed using flow cytometric analysis.Based on the theory that the binding between rRBD-Fc and Huh-7 can be blocked by anti-MERS-CoV neutralizing antibodies,RBD-specific neutralizing antibodies,RBD-specific non-neutralizing antibodies,RBD immune mouse sera,and irrelevant antibodies were used to establish this new detection assay.The developed assay was used to test serum samples,and compared its consistence with a traditional MERS-CoV neutralization test.The results demonstrated that the binding between rRBD-Fc and Huh-7 had specificity,and the established method not only had specificity in differentiating RBD-specific neutralizing antibodies,RBD immune sera,and unrelated antibodies,but also can evaluate neutralizing activity of RBD-specific neutralizing antibodies.The comparison analysis suggests that the new assay was consistent with the live MERS-CoV-based neutralization assay,and thus can be applied to detect anti-MERS-CoV neutralizing antibodies.In summary,we have successfully established a novel assay to detect MERS-CoV RBD-specific neutralizing antibodies,which does not require live virus and high-level biosafety facilities.This assay can be applied as a safe and convenient tool to evaluate efficacy,investigate serology,as well as study protective mechanisms of MERS-CoV vaccines and antibodies.