Establishment Of ELISA Detection Method For SARS-CoV-2 Neutralizing Antibody

COVID-19 is acute infectious respiratory infectious disease caused by SARS-Co V-2infection.Since December 2019,the disease has spread rapidly around the world.SARS-Co V-2 is recognized by RBD on the surface spike protein S1 and bind to the host cell receptor ACE2,which mediates viral infection of target cells.Therefore,the RBD region of SARS-Co V-2 S protein is deemed to be the main target for the study of antiviral drugs and vaccines.At present,specific domestic drugs for the treatment of SARS-Co V-2have not yet been listed,so far,vaccination of COVID-19 vaccine is by far the most effective means of prevention.The Nab produced by the body after vaccination can help the body resist virus infection.The detection of protective Nab level induced by the vaccine is an indirect method to evaluate the protection of the vaccine against the vaccinated population,and the titer of Nab is usually used.Although the traditional neutralization test based on live virus is the gold standard for Nab determination,this method takes a long time,low flux,high requirements for laboratory safety and operator proficiency,and is not suitable for large-scale population Nab detection;while the appropriate PBNA method can be comparable to the detection effect of real virus,while reducing the biosafety level requirements of the experimental environment.Nevertheless,PBNA still has to be operated and detected at the cellular level,which is time-consuming and labor-consuming,which hinders its widespread application in clinical laboratories.The ELISA method is easy,fast,high-throughput,and can meet the detection needs of large-scale samples.At present,it has been reported that a variety of SARS-Co V-2Nab ELISA detection methods,whether competitive method or the indirect method,are based on the principle of receptor-ligand binding or inhibitory binding,and the antigens used are recombinant RBD proteins.In this study,two eukaryotic recombinant expression plasmids pc DNA3.1（-）-RBD₂₁₉and pc DNA3.1（-）-RBD₂₂₃ with altered amino acid length SARS-Co V-2 RBD were successfully constructed.Two kinds of recombinant RBD proteins with high purity were obtained after transfection by HEK293F cells,and the purity was 91.2%（219-RBD）and93.7%（223-RBD）,respectively.Western blot results showed that they could be recognised by specific RBD monoclonal antibodies.Two recombinant proteins,219-RBD and223-RBD,were used as detection antigens to establish an indirect ELISA detection method,and two established indirect Elisa methods were used to detect 60 serum of volunteers immunized with COVID-19 whole virus inactivated vaccine.The consequences of PBNA neutralization antibody as the gold standard,219-RBD and 223-RBD as the detection antigen,the qualitative detection sensitivity of antibody was 96.43%and 94.64%,respectively.The consistency analysis showed that the values of kappa and kappa were0.783 and 0.702,respectively.Semi-quantitative results of RBD and 223-RBDELISA antibodies were compared with those of PBNA,and the Pearson correlation coefficients were 0.81 and 0.73 respectively.In the consistency analysis of the Bland-Altman method,98.3%of the two kinds of indirect ELISA fell within the 95%consistency limit,so the indirect ELISA results of the two kinds of RBD proteins had statistically acceptable correlation and consistency with the PBNA results.However,consistency between219-RBD and gold standard is significantly higher than that of 223-RBD,so it is more suitable to be used as a candidate antigen for neutralizing antibody detection in SARS-Co V-2 vaccine.To sum up,using these two recombinant RBD proteins as coating antigens,two indirect ELISA detection methods were established,and the two indirect ELISA were used to detect the level of neutralizing antibodies in the serum of volunteers immunized with new crown inactivated vaccine.The results showed that 219-RBD as a coated antigen had high correlation and consistency with PBNA,which could be utilized to detect neutralizing antibodies in COVID-19 vaccine.It not only provides a new idea for the evaluation and screening of neutralizing antibody against a novel coronavirus vaccine and the development of the kit,but also provides a methodological reference for the detection and screening of neutralizing antibody by COVID-19 RBD polymer.