Preparation Of SARS-CoV-2 RBD Protein Specific Humanized Neutralizing Monoclonal Antibodies

Coronavirus Disease 2019(COVID-19)has emerged as an infectious disease,caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection.In the course of SARS-CoV-2 infection,the majority of infected patients are unable to effectively eliminate the virus in a short time,and people with underlying diseases or immunodeficiency have a high mortality rate even with the support of medical equipment.Neutralizing antibodies have a high antigen specificity,and methods involving the application thereof can effectively eliminate SARS-CoV-2 in a short time.In our research,a positive correlation was found between the magnitude of antibody responses and disease severity in patients recovered from COVID-19.In addition,the blocking ability of sera was further verified from recovered patients with severe symptoms,and peripheral blood with the blocking ability was selected as the sample resources to sort RBD-specific memory B cells.Specifically,RBD protein specific memory B cells were sorted by flow cytometry,then reverse transcription PCR and nested PCR were used to amplify gene fragment which encoding the heavy chain and light chain variable region of immunoglobulin G(Ig G).Subsequently,molecular cloning technology was used to prepare the Ig G heavy chain and light chain plasmids respectively,which were then successfully expressed in the 293 T eukaryotic expression system.Finally,two RBD protein specific humanized monoclonal antibodies with neutralizing ability were successfully screened by RBD protein binding assay,RBD-h ACE2 protein interaction inhibition assay,SARS-CoV-2 S protein pseudotyped lentiviral-based neutralization assay and SARS-CoV-2authentic virus inhibition assay.The IC50 of these antibodies reached 20ng/ml and 127ng/ml in the SARS-CoV-2 authentic virus assay separately,which may have significant potential in clinical application.