| Since 2019,Coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has brought about serious infections and large numbers of deaths worldwide,causing damage to the global health system and human health.During the widespread transmission of SARS-CoV-2,a variety of lineage mutant strains have been produced,and their infectivity,viral load and pathogenicity have changed.Some mutant strains have shown immune escape to partial neutralizing antibodies induced by natural infection or vaccination,which poses great challenges for disease prevention and clinical treatment.Therefore,rapid screening and identification of fully human broad-spectrum neutralizing antibodies against SARS-CoV-2 could provide an effective strategy for its treatment.In this study,the peripheral blood mononuclear cells(PBMC)of recovered patients were used to obtain antigen-specific memory B cells through magnetic bead sorting combined with flow sorting technology.The recombinant fully human antibodies were cloned and expressed in vitro using single B cell cloning technology.The binding and neutralization abilities of antibodies were identified by enzyme linked immunosorbent assay(ELISA)and pseudovirus neutralization test.Finally,multiple monoclonal antibodies targeting spike(S)protein of SARS-CoV-2 were identified.The study established the magnetic bead sorting combined with flow sorting technology.Sorted by Pan B magnetic beads,the proportion of memory B cells in the sample was increased from 1.36%to 7.36%,improving the efficiency of subsequent flow sorting.By comparing the difference between fresh blood collected from on-site and cryopreserved samples,this plan could effectively remove dead cells,and increased the viability of cell samples from 80%to 95%after cryopreservation and recovery.The proportion of antigen-specific memory B cells was not significantly different between the two sample types.The study optimized the construction process of the antibody gene expression vector,and directly amplified and extended the antibody gene into the expression vector through Overlap PCR,which improved the efficiency of vector construction.Single B cell PCR results showed that the efficiency of amplification of the antibody genes was over 90%.By sequencing and comparing the expression vectors,a total of 174 recombinant antibody sequences were obtained.In the study,174 strains of fully human antibodies were recombined and expressed using HEK293F suspension culture.Through ELISA binding test,33 strains of antibodies targeting Spike protein were identified,of which 15,19,28 and 8 antibody strains bound to NTD,RBD,S1 and S2,respectively were further determined.The results of the polyreactive binding test showed that all S2-binding antibodies could cross-recognize SARS-CoV-2 and SARS-CoV,and some RBD antibodies could cross-recognize SARSCoV-2 and SARS-CoV,and were subsequently identified as antibodies binding to Class 3 regions.The study found that JYTA108 and JYTA172 binding to RBD Class 1 and Class 2 regions,had broad-spectrum neutralizing activity,and could neutralize the prototype,Alpha,Gamma,lota,Epsilon,Delta,Lambda,BA.1,BA.2,BA.2.12.1,BA.2.75 and BA.4/5 pseudoviruses,but their neutralizing activity in Omicron and its sublineage mutants was decreased.JYTA034 bound to the RBD Class 3 region and had a broadspectrum binding activity.It could bind to all mutant RBD proteins detected by prototype,Beta,Delta,C.1,BA.1 and XBB strains.There was no significant difference in the binding activity among the tested mutant strains.To further improve the throughput of antibody detection and screening,this study used Homogeneous Time Resolved Fluorescence(HTRF)to construct a high-throughput RBD-specific antibody detection system:Using 2.5 μL of 40 nM RBD protein,the antibody concentration ranged from 0.003 μg/mL to 40.8 μg/mL could be detected.This study optimized the single B cell PCR cloning antibody process,established the HTRF high-throughput screening RBD antibody technology,and improved the screening platform for fully human antibodies against new pathogens.Multiple antibodies against S protein were successfully isolated and identified using PBMC samples from COVID survivors.Our study preliminarily clarified the action sites and mechanisms of those antibodies,providing data reference and theoretical basis for potential treatment. |
