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Article

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

1
Research & Development Department, Cellular Technology Limited, Shaker Heights, OH 44122, USA
2
Institute of Anatomy and Cell Biology, Friedrich-Alexander University Erlangen-Nürnberg, 91054 Erlangen, Germany
3
Center for Vaccines and Immunology, University of Georgia, Athens, GA 30602, USA
4
Institute of Neuroanatomy, Medical Faculty, University of Bonn, 53115 Bonn, Germany
5
Department of Infectious Diseases, University of Georgia, Athens, GA 30602, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Mats W. Johansson
Cells 2021, 10(8), 1843; https://doi.org/10.3390/cells10081843
Received: 1 June 2021 / Revised: 9 July 2021 / Accepted: 16 July 2021 / Published: 21 July 2021
(This article belongs to the Special Issue Advances in Immune Monitoring)
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. View Full-Text
Keywords: antibody-secreting cell; fluorospot; memory B cell; plasmablast; antigen coating; influenza; EBV; HCMV; SARS-CoV-2; immune monitoring antibody-secreting cell; fluorospot; memory B cell; plasmablast; antigen coating; influenza; EBV; HCMV; SARS-CoV-2; immune monitoring
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MDPI and ACS Style

Köppert, S.; Wolf, C.; Becza, N.; Sautto, G.A.; Franke, F.; Kuerten, S.; Ross, T.M.; Lehmann, P.V.; Kirchenbaum, G.A. Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays. Cells 2021, 10, 1843. https://doi.org/10.3390/cells10081843

AMA Style

Köppert S, Wolf C, Becza N, Sautto GA, Franke F, Kuerten S, Ross TM, Lehmann PV, Kirchenbaum GA. Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays. Cells. 2021; 10(8):1843. https://doi.org/10.3390/cells10081843

Chicago/Turabian Style

Köppert, Sebastian, Carla Wolf, Noémi Becza, Giuseppe A. Sautto, Fridolin Franke, Stefanie Kuerten, Ted M. Ross, Paul V. Lehmann, and Greg A. Kirchenbaum 2021. "Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays" Cells 10, no. 8: 1843. https://doi.org/10.3390/cells10081843

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