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Analysis of institutional authors

Phelps, AAuthorPazos-Castro, DAuthorTome-Amat, JAuthor

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

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

Authors: 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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Abstract

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.

Keywords

AntibodyAntigensAssayB-lymphocytesBloodElispotFlow cytometryGenerationGerminal centerMemoryResponsesVaccine

Quality index

Bibliometric impact. Analysis of the contribution and dissemination channel

The work has been published in the journal Nature Protocols due to its progression and the good impact it has achieved in recent years, according to the agency WoS (JCR), it has become a reference in its field. In the year of publication of the work, 2024 there are still no calculated indicators, but in 2023, it was in position 2/85, thus managing to position itself as a Q1 (Primer Cuartil), in the category Biochemical Research Methods. Notably, the journal is positioned above the 90th percentile.

Independientemente del impacto esperado determinado por el canal de difusión, es importante destacar el impacto real observado de la propia aportación.

Según las diferentes agencias de indexación, el número de citas acumuladas por esta publicación hasta la fecha 2025-06-08:

  • WoS: 1
  • Scopus: 4
  • OpenCitations: 2

Impact and social visibility

From the perspective of influence or social adoption, and based on metrics associated with mentions and interactions provided by agencies specializing in calculating the so-called "Alternative or Social Metrics," we can highlight as of 2025-06-08:

  • The use, from an academic perspective evidenced by the Altmetric agency indicator referring to aggregations made by the personal bibliographic manager Mendeley, gives us a total of: 57.
  • The use of this contribution in bookmarks, code forks, additions to favorite lists for recurrent reading, as well as general views, indicates that someone is using the publication as a basis for their current work. This may be a notable indicator of future more formal and academic citations. This claim is supported by the result of the "Capture" indicator, which yields a total of: 53 (PlumX).

With a more dissemination-oriented intent and targeting more general audiences, we can observe other more global scores such as:

  • The Total Score from Altmetric: 83.68.
  • The number of mentions on the social network X (formerly Twitter): 18 (Altmetric).

Leadership analysis of institutional authors

This work has been carried out with international collaboration, specifically with researchers from: Canada; United States of America.

There is a significant leadership presence as some of the institution’s authors appear as the first or last signer, detailed as follows: First Author (DIAZ PERALES, ARACELI) .