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Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function
Published on: February 16, 2017
Development of a novel flow cytometry method for detecting pneumococcal-specific B cells
Irene Tzovara1, Ioanna Papadatou1, Marianna Tzanoudaki2
1Department of Infectious Diseases - Immunobiology and Vaccinology Research Lab, "Aghia Sophia" Children's Hospital, 1st Department of Pediatrics - National and Kapodistrian University of Athens, Athens, Greece.
Insights
A new multimer bead method enhances detection of pneumococcal polysaccharide-specific B cells using flow cytometry. This sensitive and specific approach improves antigen-specific B cell analysis for vaccine development.
Area of Science:
- Immunology
- Cell Biology
- Vaccinology
Background:
- Flow cytometry is vital for analyzing B cell responses to infection and vaccination.
- Existing methods for detecting polysaccharide-specific B cells lack optimal sensitivity and applicability.
- There is a need for improved techniques to study B cell immunophenotypes and kinetics.
Purpose of the Study:
- To develop and validate a novel multimer bead-based flow cytometry method for detecting pneumococcal polysaccharide (PS)-specific B cells.
- To enhance the sensitivity and specificity of antigen-specific B cell identification.
- To provide a versatile tool for studying B cell responses to various antigens.
Main Methods:
- Chemically biotinylated pneumococcal polysaccharide (PS) was conjugated to anti-biotin beads.
- The PS-conjugated beads were further labeled with phycoerythrin (PE)-conjugated anti-biotin antibody to create a PS-multimer probe.
- Titration assays determined optimal ratios for PS-bead conjugate and PS-multimer to cell staining.
- Specificity was validated using a competition assay with unbound PS.
Main Results:
- The PS-multimer method demonstrated significantly enhanced detection of PS-specific B cells compared to a PS-PE monomer.
- Signal amplification was observed due to the multimeric nature of the probe and increased antigen epitope availability.
- A competition assay confirmed the method's specificity, with signal decreasing in a dose-dependent manner with increasing PS concentrations.
- The method proved sensitive, specific, and easily applicable without requiring B cell pre-enrichment.
Conclusions:
- The developed bead-based flow cytometry approach is a sensitive and specific method for identifying antigen-specific B cells.
- This technique offers enhanced signal detection and clear results, applicable to polysaccharides and potentially other antigens.
- The method can aid in understanding long-term protection mechanisms, improving vaccine strategies, and guiding future vaccine development.
Abstract:
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.
