Single-Cell Analysis of B Cell/Antibody Cross-Reactivity Using a Novel Multicolor FluoroSpot Assay

The Journal of Immunology : Official Journal of the American Association of Immunologists
Alexandros HadjilaouEva Harris

Abstract

Dengue is a major public health problem globally. It is caused by four antigenically distinct serotypes of dengue virus (DENV1-4), and although serotype-specific and strongly neutralizing cross-reactive immune responses against the four DENV serotypes are thought to be protective, subneutralizing Abs can contribute to increased disease severity upon secondary infection with a different DENV serotype. Understanding the breadth of the immune response in natural DENV infections and in vaccinees is crucial for determining the correlates of protection or disease severity. Transformation of B cell populations to generate mAbs and ELISPOT assays have been used to determine B cell and Ab specificity to DENV; however, both methods have technical limitations. We therefore modified the conventional ELISPOT to develop a Quad-Color FluoroSpot to provide a means of examining B cell/Ab serotype specificity and cross-reactivity on a single-cell basis. Abs secreted by B cells are captured by an Fc-specific Ab on a filter plate. Subsequently, standardized concentrations of all four DENV serotypes are added to allow equal stoichiometry for Ag binding. After washing, the spots, representing individual B cells, are visualized using four fluorescent...Continue Reading

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Citations

Sep 30, 2017·Cells·Josué da Costa Lima-JuniorFátima Conceição-Silva
Nov 11, 2018·The Journal of Immunology : Official Journal of the American Association of Immunologists·Awadalkareem AdamAnuja Mathew
Feb 28, 2019·Nature Communications·Paulina AndradeEva Harris
Apr 19, 2019·JCI Insight·Matthew H CollinsAravinda M de Silva
Aug 5, 2017·Frontiers in Microbiology·Wen-Yang TsaiWei-Kung Wang
Dec 7, 2019·Journal of Immunological Methods·Peter JahnmatzNiklas Ahlborg
Mar 30, 2018·Current Opinion in Virology·Leah C Katzelnick, Eva Harris
Mar 19, 2021·European Journal of Immunology·Olivia T M BucheliKlaus Eyer

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