Isolation and identification of MPER specific antibodies in an HIV-1 infected patient
Objective To isolate membrane-proximal external region(MPER)-specific memory B cells from the peripheral blood mononuclear cells(PBMCs)of an HIV-1 infected patient using flow cytometry,to produce MPER-specific monoclonal antibodies,and to evaluate their binding and neutralization activities.Methods MPER-specific memory B cells were isolated using a biotinylated MPER peptide and flow cytometry single-cell sorting technique.The heavy and light chain variable region genes of the antibody were amplified by reverse transcription polymerase chain reaction(RT-PCR)and cloned into corresponding expression vectors.Following co-transfection into 293F cells,the antibodies were expressed and subsequently assessed for binding ability and neutralizing activity using enzyme-linked immunosorbent assay(ELISA),Biolayer interferometry(BLI),and pseudovirus neutralization assays.Results Twelve monoclonal antibodies were successfully isolated.Among these,504A2 and 504C1 effectively bound to MPER peptides and the transmembrane protein gp41,and both exhibited neutralization capabilities.Specifically,monoclonal antibody 504A2 neutralized pseudoviruses SF162 and MW965.26,while 504C1 specifically neutralized the MPER chimeric pseudovirus HIV2C1.Conclusions This study established a method for sorting MPER-specific monoclonal antibodies and successfully identified two such antibodies with significant neutralizing potential.These findings provide technical support for the further screening of HIV-1 broad-spectrum neutralizing antibodies and the development of diagnostic reagents and vaccine design.
HIV-1membrane-proximal external region(MPER)single B cell sortingmonoclonal antibody
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