Establishment and Application of A Labeling Method for S1-,S2-,and NTD Antigen-specific B Cells and Their Subtypes of SARS-CoV-2
To establish a labeling method for spike(S)1-,S2-,and N-terminal domain(NTD)-specific B cells(and their subtypes)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).This method could be used with flow cytometry to test,or in 10 × immune repertoire sequence to obtain the sequence of antigen-specific B cells and their subtypes.First,S1,S2,and NTD proteins were coupled with fluorescein-streptavidin(600,1200,2400,3600 and 4800 nmol/L),respectively.Antigen-specific B cells were detected by flow cytometry to determine the optimal concentration.Second,the molar ratio of biotin-protein and fluorescein-streptavidin was tested at ratios of 4∶1,5∶1,and 6∶1,respectively.According to the ratio of S1+cluster of differentiation(CD)19+B cells,S2+CD19+B cells,NTD+CD19+B cells,as well as S1+CD19+CD27+B cells,S2+CD19+CD27+B cells,and NTD+CD19+CD27+B cells,the optimal concentration was obtained.Third,the non-specific effects of different types of fluorescein(BV421,Super Bright 600,PE,PE/Cy7 and APC)were tested to evaluate their non-specific binding.Fourth,the established method was repeated thrice to test its stability in flow cytometry.We also tested it with fluorescein-avidin-biotin-containing nucleotide sequences which could be used in a 10 × immune repertoire sequence.First,the optimal concentrations(in nmol/L)of S1,S2,and NTD proteins were 4800,3600,and 3600 nmol/L,respectively.Second,the optimal molar ratio of biotin-S1 protein to fluorescein-avidin was 6∶1,whereas the most suitable molar ratio of biotin-S2 protein and biotin-NTD protein to fluorescein-avidin was 4∶1,respectively.At this concentration,BV421,APC,Super Bright 600,and PE showed weak non-specific binding.Third,repeating this method thrice showed it to be highly stable,with a coefficient of variation of≤20％,and it was suitable for fluorescein-avidin-biotin-containing nucleotide sequences.The established labeling technology for S1-,S2-,and NTD protein-specific B cells(and their subtypes)of SARS-CoV-2 was relatively stable,and could be used in conjunction with flow cytometry and 10 × immune repertoire sequencing.
Flow cytometryBiotin-antigenFluorescein-avidinSARS-CoV-2Antigen-specific B cells
万方数据
维普
