Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays

Koppert S, Wolf C, Becza N, Sautto GA, Franke F, Kürten S, Ross TM, Lehmann P, Kirchenbaum GA (2021)

Publication Type: Journal article

Publication year: 2021

Journal

Cells MDPI

Book Volume: 10

Journal Issue: 8

DOI: 10.3390/cells10081843

Abstract

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.

Authors with CRIS profile

Stefanie Kürten Lehrstuhl für Mikroskopische Anatomie und Molekulare Zellbiologie

Involved external institutions

CTL Europe GmbH DE Germany (DE) University of Georgia (UGA) US United States (USA) (US)

How to cite

APA:

Koppert, S., Wolf, C., Becza, N., Sautto, G.A., Franke, F., Kürten, S.,... Kirchenbaum, G.A. (2021). Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays. Cells, 10(8). https://doi.org/10.3390/cells10081843

MLA:

Koppert, Sebastian, et al. "Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays." Cells 10.8 (2021).

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