Development of a novel flow cytometry method for detecting pneumococcal‐specific B cells

Antigen‐specific B cell identification by flow cytometry is crucial for investigating their immunophenotype, subset distribution, and kinetics post‐infection or immunization. Methods using biotinylated polysaccharide antigens have been described, but there is still room for improvement regarding sensitivity and applicability. The aim of this study was the development and validation of a multimer bead‐based method for detecting pneumococcal polysaccharide serotypes (PS)‐specific B cells following pneumococcal immunization. PS was chemically biotinylated and mounted on anti‐biotin beads, and labeled with phycoerythrin (PE)‐conjugated anti‐biotin antibody to form a PS‐multimer used for cell staining. Labeled beads were washed to remove excess fluorochrome and diminish non‐specific labeling and background noise. Optimal ratios of PS‐bead conjugate to PE and PS‐multimer to cells were determined with titration assays. Comparison between the PS‐multimer and a PS‐PE monomer revealed enhanced detection of PS‐specific cells and considerable signal amplification, attributed to the multimeric form of the detection probe and increased availability of antigen epitopes. To validate the specificity of the method, a competition assay using unbound PS was performed. Following pre‐incubation with increasing PS concentrations, detection of PS‐specific B cells with the PS‐multimer was inhibited in a stepwise manner. Pre‐incubation with excess PS completely blocked the fluorescent signal. This novel bead‐based flow cytometry approach is a sensitive method demonstrating high specificity. It generated enhanced signals, provided clear‐cut results, and was easily applicable, not requiring B cell pre‐enrichment. It could be modified to adapt other antigens of interest, especially polysaccharides and proteins that could be used to probe antigen‐specific B cell responses. The study of such responses may elucidate the underlying mechanisms involved in the establishment of long‐term protection, provide evidence‐based rationale for improving currently available vaccines and vaccination strategies, and pave the way for future vaccine development.