Isolation and Characterization of Antigen-Specific Plasmablasts Using a Novel Flow Cytometry-Based Ig Capture Assay
Christopher L Pinder1, Sven Kratochvil1, Deniz Cizmeci1
1Division of Medicine, Department of Infectious Diseases, Imperial College London, London W2 1PG, United Kingdom; and.
Insights
A new Ig capture assay (ICA) identifies and sorts antigen-reactive antibody-secreting cells. This flow cytometry method aids in antibody analysis and cloning, offering a viable alternative to ELISpot for vaccine response monitoring.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Monitoring antigen-specific antibody responses is crucial for vaccine development and infectious disease research.
- Existing methods for isolating antibody-secreting cells can be time-consuming and may not provide comprehensive phenotypic data.
Purpose of the Study:
- To develop and validate a novel flow cytometry-based Ig capture assay (ICA) for identifying and sorting antigen-reactive antibody-secreting cells.
- To assess the utility of ICA for analyzing plasmablast responses in individuals vaccinated with an HIV envelope protein.
- To compare the performance of ICA with a conventional B cell ELISpot assay.
Main Methods:
- Development of a flow cytometry-based Ig capture assay (ICA) utilizing antigen-specific binding.
- Isolation and sorting of antigen-reactive plasmablasts from peripheral blood mononuclear cells (PBMCs) of HIV-vaccine recipients.
- Production and validation of antigen-specific antibodies via ELISA.
- Head-to-head comparison of ICA with B cell ELISpot assay for monitoring plasmablast responses over 42 days.
Main Results:
- The ICA successfully identified and sorted antigen-reactive plasmablasts from PBMCs of vaccinated volunteers.
- Antigen-specific antibodies produced from ICA-sorted cells demonstrated verified antigen reactivity via ELISA.
- ICA performance was comparable to the conventional B cell ELISpot assay in tracking antigen-specific plasmablast responses.
- ICA facilitated the acquisition of phenotypic information from identified antigen-specific cells.
Conclusions:
- The Ig capture assay (ICA) is a fast, versatile, and effective tool for single-cell sorting of antigen-reactive antibody-secreting cells.
- ICA serves as a viable alternative to ELISpot for monitoring early antigen-specific plasmablast responses post-vaccination or infection.
- The assay's ability to capture phenotypic data and enable downstream applications like B cell sequencing and antibody cloning enhances its utility in antibody repertoire analysis for clinical trials.
Abstract:
We report the development of a novel flow cytometry-based Ig capture assay (ICA) for the identification and sorting of individual Ab-secreting cells based on their Ag reactivity. The ICA represents a fast and versatile tool for single-cell sorting of peripheral plasmablasts, streamlining subsequent Ab analysis, and cloning. We demonstrate the utility of the assay by isolating Ag-reactive plasmablasts from cryopreserved PBMC obtained from volunteers vaccinated with a recombinant HIV envelope protein. To show the specificity of the ICA, we produced Ag-specific Abs from these cells and subsequently verified their Ag reactivity via ELISA. Furthermore, we used the ICA to track Ag-specific plasmablast responses in HIV-vaccine recipients over a period of 42 d and performed a head-to-head comparison with a conventional B cell ELISpot. Results were highly comparable, highlighting that this assay is a viable alternative for monitoring Ag-specific plasmablast responses at early time points after infection or vaccination. The ICA provides important added benefits in that phenotypic information can be obtained from the identified Ag-specific cells that can then be captured for downstream applications such as B cell sequencing and/or Ab cloning. We envisage the ICA as being a useful tool in Ab repertoire analysis for future clinical trials.
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