Isolation and Characterisation of Antigen-Specific Plasmablasts Using a Novel Flow Cytometry-Based Immunoglobulin Capture Assay (ICA)
| Autor: | Pinder, Christopher L, Kratochvil, Sven, Cizmeci, Deniz, Muir, Luke, Guo, Yanping, Shattock, Robin J, McKay, Paul F |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
AIDS Vaccines
Cryopreservation Antigens Bacterial Enzyme-Linked Immunospot Assay Hepatitis B Surface Antigens Plasma Cells Vaccination env Gene Products Human Immunodeficiency Virus Cell Separation HIV Antibodies Flow Cytometry Antibodies Bacterial Article Peptide Fragments Antigen-Antibody Reactions HEK293 Cells Tetanus Toxin Blood Preservation Tetanus Toxoid Humans Hepatitis B Vaccines Follow-Up Studies |
| Popis: | We report the development of a novel flow cytometry-based immunoglobulin capture assay (ICA) for the identification and sorting of individual antibody secreting cells based on their antigen reactivity. The ICA represents a fast and versatile tool for single-cell sorting of peripheral plasmablasts, streamlining subsequent antibody analysis and cloning. We demonstrate the utility of the assay by isolating antigen-reactive plasmablasts from cryopreserved PBMC obtained from volunteers vaccinated with a recombinant HIV envelope protein. To show the specificity of the ICA, we produced antigen-specific antibodies from these cells and subsequently verified their antigen reactivity via ELISA. Furthermore, we used the ICA to track antigen-specific plasmablast responses in HIV-vaccine recipients over a period of 42 days 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 antigen-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 antigen-specific cells that can then be captured for downstream applications such as B cell sequencing and/or antibody cloning. We envisage the ICA as being a useful tool in antibody repertoire analysis for future clinical trials. |
| Databáze: | OpenAIRE |
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