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Phelps, AAutor o coautorPazos-Castro, DAutor o coautorTome-Amat, JAutor o coautor

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Production and use of antigen tetramers to study antigen-specific B cells

Publicat a:Nature Protocols. 19 (3): 727-751 - 2024-03-01 19(3), DOI: 10.1038/s41596-023-00930-8

Autors: Phelps, A; Pazos-Castro, D; Urselli, F; Grydziuszko, E; Mann-Delany, O; Fang, A; Walker, TD; Guruge, RT; Tome-Amat, J; Diaz-Perales, A; Waserman, S; Boonyaratanakornkit, J; Jordana, M; Taylor, JJ; Koenig, JFE

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B cells generate antibodies that provide protection from infection, but also cause pathology in autoimmune and allergic conditions. Antigen-specific B cells can be detected by binding their surface antibody receptors with native antigens conjugated to fluorescent probes, a technique that has revealed substantial insight into B cell activation and function. This protocol describes the process of generating fluorescent antigen tetramer probes and delineates a process of enriching large samples based on antigen-specificity for high-resolution analyses of the antigen-specific B cell repertoire. Enrichment of tetramer-binding cells allows for detection of antigen-specific B cells as rare as 1 in 100 million cells, providing sufficient resolution to study naive B cells and IgE-expressing cells by flow cytometry. The generation of antigen tetramers involves antigen biotinylation, assessment of biotin:antigen ratio for optimal tetramer loading and polymerization around a streptavidin-fluorophore backbone. We also describe the construction of a control tetramer to exclude B cells binding to the tetramer backbone. We provide a framework to validate whether tetramer probes are detecting true antigen-specific B cells and discuss considerations for experimental design. This protocol can be performed by researchers trained in basic biomedical/immunological research techniques, using instrumentation commonly found in most laboratories. Constructing the antigen and control tetramers takes 9 h, though their specificity should be assessed before experimentation and may take weeks to months depending on the method of validation. Sample enrichment requires similar to 2 h but is generally time and cost neutral as fewer cells are run through the flow cytometer.

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