Isolation and characterization of antigen-specific plasmablasts using a novel flow cytometry-based Ig capture assay
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...
| Main Authors: | , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
American Association of Immunologists
2017
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| _version_ | 1826283296501268480 |
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| author | Pinder, C Kratochvil, S Cizmeci, D Muir, L Guo, Y Shattock, R McKay, P |
| author_facet | Pinder, C Kratochvil, S Cizmeci, D Muir, L Guo, Y Shattock, R McKay, P |
| author_sort | Pinder, C |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-07T00:56:47Z |
| format | Journal article |
| id | oxford-uuid:8859a283-7f28-4918-967a-a376fc7b6e1e |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T00:56:47Z |
| publishDate | 2017 |
| publisher | American Association of Immunologists |
| record_format | dspace |
| spelling | oxford-uuid:8859a283-7f28-4918-967a-a376fc7b6e1e2022-03-26T22:16:43ZIsolation and characterization of antigen-specific plasmablasts using a novel flow cytometry-based Ig capture assayJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:8859a283-7f28-4918-967a-a376fc7b6e1eEnglishSymplectic Elements at OxfordAmerican Association of Immunologists2017Pinder, CKratochvil, SCizmeci, DMuir, LGuo, YShattock, RMcKay, PWe 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. |
| spellingShingle | Pinder, C Kratochvil, S Cizmeci, D Muir, L Guo, Y Shattock, R McKay, P Isolation and characterization of antigen-specific plasmablasts using a novel flow cytometry-based Ig capture assay |
| title | Isolation and characterization of antigen-specific plasmablasts using a novel flow cytometry-based Ig capture assay |
| title_full | Isolation and characterization of antigen-specific plasmablasts using a novel flow cytometry-based Ig capture assay |
| title_fullStr | Isolation and characterization of antigen-specific plasmablasts using a novel flow cytometry-based Ig capture assay |
| title_full_unstemmed | Isolation and characterization of antigen-specific plasmablasts using a novel flow cytometry-based Ig capture assay |
| title_short | Isolation and characterization of antigen-specific plasmablasts using a novel flow cytometry-based Ig capture assay |
| title_sort | isolation and characterization of antigen specific plasmablasts using a novel flow cytometry based ig capture assay |
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