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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References
Czerkinsky CC, Nilsson LA, Nygren H et al (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65(1–2):109–121. https://doi.org/10.1016/0022-1759(83)90308-3
Ellebedy AH, Krammer F, Li GM et al (2014) Induction of broadly cross-reactive antibody responses to the influenza HA stem region following H5N1 vaccination in humans. Proc Natl Acad Sci U S A 111(36):13133–13138. https://doi.org/10.1073/pnas.1414070111
Amanna IJ, Carlson NE, Slifka MK (2007) Duration of humoral immunity to common viral and vaccine antigens. N Engl J Med 357(19):1903–1915. https://doi.org/10.1056/NEJMoa066092
Ndungu FM, Lundblom K, Rono J et al (2013) Long-lived Plasmodium falciparum specific memory B cells in naturally exposed Swedish travelers. Eur J Immunol 43(11):2919–2929. https://doi.org/10.1002/eji.201343630
Walsh PN, Friedrich DP, Williams JA et al (2013) Optimization and qualification of a memory B-cell ELISpot for the detection of vaccine-induced memory responses in HIV vaccine trials. J Immunol Methods 394(1–2):84–93. https://doi.org/10.1016/j.jim.2013.05.007
Okamoto Y, Abe T, Niwa T et al (1998) Development of a dual color enzyme-linked immunospot assay for simultaneous detection of murine T helper type 1- and T helper type 2-cells. Immunopharmacology 39(2):107–116. https://doi.org/10.1016/s0162-3109(98)00007-1
Janetzki S, Rueger M, Dillenbeck T (2014) Stepping up ELISpot: multi-level analysis in FluoroSpot assays. Cell 3(4):1102–1115. https://doi.org/10.3390/cells3041102
Gazagne A, Claret E, Wijdenes J et al (2003) A Fluorospot assay to detect single T lymphocytes simultaneously producing multiple cytokines. J Immunol Methods 283(1–2):91–98. https://doi.org/10.1016/j.jim.2003.08.013
Kesa G, Larsson PH, Ahlborg N et al (2012) Comparison of ELISpot and FluoroSpot in the analysis of swine flu-specific IgG and IgA secretion by in vivo activated human B cells. Cell 1(2):27–34. https://doi.org/10.3390/cells1020027
Weinberg A, Muresan P, Richardson KM et al (2015) Determinants of vaccine immunogenicity in HIV-infected pregnant women: analysis of B and T cell responses to pandemic H1N1 monovalent vaccine. PLoS One 10(4):e0122431. https://doi.org/10.1371/journal.pone.0122431
Dosenovic P, Chakrabarti B, Soldemo M et al (2009) Selective expansion of HIV-1 envelope glycoprotein-specific B cell subsets recognizing distinct structural elements following immunization. J Immunol 183(5):3373–3382. https://doi.org/10.4049/jimmunol.0900407
Jahnmatz P, Bengtsson T, Zuber B et al (2016) An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection. J Immunol Methods 433:23–30. https://doi.org/10.1016/j.jim.2016.02.020
Hadjilaou A, Green AM, Coloma J et al (2015) Single-cell analysis of B cell/antibody cross-reactivity using a novel multicolor FluoroSpot assay. J Immunol 195(7):3490–3496. https://doi.org/10.4049/jimmunol.1500918
Areström I, Zuber B, Bengtsson T et al (2012) Measurement of human latent transforming growth factor-beta1 using a latency associated protein-reactive ELISA. J Immunol Methods 379(1–2):23–29. https://doi.org/10.1016/j.jim.2012.02.016
Acknowledgment
The authors would like to thank Kajsa Prokopec, Bernt Axelsson, and Gun Kesa for critical reading of the manuscript. Peter Jahnmatz is a graduate student financed by The Swedish Foundation for Strategic Research (ID14-0070) and Mabtech.
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Jahnmatz, P., Ahlborg, N. (2018). Detection of Cross-Reactive B Cells Using the FluoroSpot Assay. In: Kalyuzhny, A. (eds) Handbook of ELISPOT . Methods in Molecular Biology, vol 1808. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8567-8_6
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DOI: https://doi.org/10.1007/978-1-4939-8567-8_6
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