Fig. 2: Sample collection and strategies for B cell sequencing and antibody response analysis. | npj Vaccines

Fig. 2: Sample collection and strategies for B cell sequencing and antibody response analysis.

From: Optimizing human B cell repertoire analyses to interpret clinical data and design sequential HIV vaccines

Fig. 2

Samples from peripheral blood, leukapheresis, or fine needle aspirates (FNA) containing B cells can be sorted for isolation of single antigen-specific B cells by flow cytometry using antibodies against specific cell surface markers and fluorochrome- or oligo-conjugated Env proteins recognized by antigen-specific B cell receptors (BCRs). Bone marrow samples, containing plasma cells, can be sorted by using the TRAP assay. Nojima cultures from single B cells generate supernatants that can be screened for antibody binding and neutralization prior to sequencing. Antigen-specific B cells can be sequenced using different methodologies (10x Genomics, SMART-seq, etc), after which paired heavy and light chain HC/LC) V(D)J sequences are assigned to a relevant germline gene database and processed with different bioinformatics tools and pipelines. Serum samples can be analyzed for neutralizing activity, epitope-specific binding assays, and electron microscopy polyclonal antibody epitope mapping (EMPEM). Created with Biorender.com.

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