Isolation and Characterization of Antigen-Specific Plasmablasts Using a Novel Flow Cytometry-Based Ig Capture Assay

• Published in: The Journal of immunology (1950) 2017-12, Vol.199 (12), p.4180-4188
• Main Authors: Pinder, Christopher L, Kratochvil, Sven, Cizmeci, Deniz, Muir, Luke, Guo, Yanping, Shattock, Robin J, McKay, Paul F
• Format: Article
• Language: English
• Subjects: Assaying; Clinical trials; Cloning; Cryopreservation; Enzyme-linked immunosorbent assay; Flow cytometry; Immunoglobulins; Lymphocytes B; Peripheral blood mononuclear cells; Vaccination; Viral envelope proteins
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.1701253

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.