Chapter title |
Detection of Cross-Reactive B Cells Using the FluoroSpot Assay
|
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Chapter number | 6 |
Book title |
Handbook of ELISPOT
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-8567-8_6 |
Pubmed ID | |
Book ISBNs |
978-1-4939-8566-1, 978-1-4939-8567-8
|
Authors |
Peter Jahnmatz, Niklas Ahlborg, Jahnmatz, Peter, Ahlborg, Niklas |
Abstract |
B cell ELISpot enables a sensitive analysis of antigen-specific B cells at the single cell level but is limited to the analysis of reactivity with a single antigen. By reversing the B cell ELISpot and using anti-IgG capture antibodies instead of coated antigen, the specificity of antibodies secreted by B cells can be defined using soluble tagged antigen for detection. When combining this approach with fluorescent detection of the antigen in a B cell FluoroSpot assay, reactivity with multiple antigens can be defined. In the protocol described herein, splenocytes from a mouse immunized with an antigen were analyzed for their reactivity with the antigen used for immunization and for cross-reactivity with a different but structurally related antigen. Using this assay, we found that at least 15% of the B cells displayed detectable cross-reactivity. B cell FluoroSpot utilizing multiple antigens provides a tool for a single-cell analysis of B cell cross-reactivity, for example, with variable and polymorphic antigens found in various pathogens; or analysis of other types of immune responses where analysis of cross-reactivity is of interest. It is also possible to simultaneously analyze B cell reactivity to completely different antigens. |
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Unknown | 3 | 100% |
Demographic breakdown
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