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Enzyme-linked immunospot assays for direct ex vivo measurement of vaccine-induced human humoral immune responses in blood

Nature Protocols volume 8, pages 1073–1087 (2013)Cite this article

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Abstract

The enzyme-linked immunospot (ELISPOT) assay was originally developed to enumerate antigen-specific antibody-secreting cells (ASCs), and has subsequently been adapted for various applications, including the detection cytokine-secreting cells. Owing to its exceptionally high sensitivity, the ELISPOT has proven to be especially useful for detecting discrete populations of active cells (e.g., antigen-specific cells). Because of its versatility, the ELISPOT assay is used for a wide range of applications, including clonal analyses of immune responses after vaccination or after immunotherapy. Here we describe standard protocols for the detection of human ASCs specific to virtually any vaccine antigen after enrichment of circulating plasmablasts. In addition, a protocol is described for the measurement of mucosal ASC responses after prior immunomagnetic enrichment of mucosally derived blood lymphocytes. The protocols described allow rapid (∼6–8 h) detection of specific ASCs in small (1–2 ml) samples of blood and can be performed in resource-poor settings.

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Figure 1: Comparative kinetics of serological and blood ASC responses after immunization.
Figure 2: Schematic diagram of one-step and two-step ASC ELISPOT.
Figure 3: Influence of antigen coating concentration and blocking.
Figure 4: Comparison of Ficoll gradient and whole-blood lysis techniques for the detection of vaccine-specific IgG- and IgA-ASCs.
Figure 5: Detection of magnetically enriched blood ASCs.
Figure 6: Example of ELISPOT plate layout.
Figure 7: Anticipated results of the ELISPOT assay.

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Acknowledgements

We thank M. Ali for advice on data analyses and P. Sharma for technical and logistical support. This article describes protocols that have matured from studies supported in part by the Bill & Melinda Gates Foundation (grants OPP1017157 and PROVIDE), the European Union Seventh Framework Program (grant 280873 ADITEC), the World Health Organization (WHO; Global Polio Eradication Program), PATH Vaccine Solutions (EVI Program), Institut National de la Santé et de la Recherche Médicale (INSERM, France), the Swedish Agency for International Cooperation (SIDA) and the Swedish National Research Council. Merck Vaccines (USA), Green Cross Vaccines (South Korea), Sanofi-Pasteur (France), Crucell (the Netherlands) and the National Institute for Public Health and the Environment (RIVM, the Netherlands) are kindly acknowledged for providing reagents and vaccines. The International Vaccine Institute is supported by the governments of the Republic of Korea, Sweden and the Netherlands.

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Authors and Affiliations

  1. Laboratory Sciences Division,

    Giulietta Saletti, Nicolas Çuburu, Jae Seung Yang, Ayan Dey & Cecil Czerkinsky

  2. International Vaccine Institute, Seoul, South Korea

    Giulietta Saletti, Nicolas Çuburu, Jae Seung Yang, Ayan Dey & Cecil Czerkinsky

  3. Göteborg University Vaccine Research Center,

    Cecil Czerkinsky

  4. Göteborg, Sweden

    Cecil Czerkinsky

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  1. Giulietta Saletti
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Contributions

G.S., N.Ç. and C.C. designed the experiments. G.S., N.Ç., J.S.Y. and A.D. performed the experiments and analyzed data. G.S., A.D. and C.C. wrote the paper.

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Correspondence to Cecil Czerkinsky.

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Saletti, G., Çuburu, N., Yang, J. et al. Enzyme-linked immunospot assays for direct ex vivo measurement of vaccine-induced human humoral immune responses in blood. Nat Protoc 8, 1073–1087 (2013). https://doi.org/10.1038/nprot.2013.058

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