Chapter 30
The Quantification of Antigen-Specific T Cells by IFN-γ
ELISpot
Bassel Akache and Michael J. McCluskie
Abstract
The enzyme-linked immune absorbent spot (ELISpot) assay allows for the quantification of the number of
cells producing a particular secreted analyte. As T lymphocytes secrete cytokines such as interferon (IFN)-γ
upon binding of the T cell receptor with its cognate antigen epitope, IFN-γ ELISpot allows for the
measurement of antigen-specific T cells in an immune sample. Immune cells are isolated from the vaccinated
subject and incubated with the epitope/antigen of interest on polyvinylidene difluoride (PVDF)-lined
microplates precoated with a capture antibody to IFN-γ. Cytokine spots are then detected utilizing an
IFN-γ-specific detection antibody and an enzyme-linked conjugate. Here, we describe the quantification of
OVA-specific CD8 and CD4 T cells from mouse splenocytes to measure vaccine-induced cellular responses.
Key words Vaccines, ELISpot, T lymphocyte, Interferon-γ, Ovalbumin
1 Introduction
The immune system employs a cellular immune response to fight
various disease indications, such as cancer or viral infections. Malignant or infected cells are killed through direct interaction with
lymphocytes [1]. As such, vaccine platforms and adjuvants capable
of inducing strong antigen-specific CD8 T cell responses have been
developed and evaluated for use in novel vaccines targeting these
types of indications. To measure the activity of such vaccines/
adjuvants, a reliable method to quantitate the number of antigenspecific T cells is required. Enzyme-linked immunosorbent spot
(ELISpot) is a modification of a regular sandwich ELISA, where
the assay input is live cells instead of protein-containing extract
[2]. It is routinely used to measure the immunogenicity of vaccines
in preclinical and clinical vaccine studies where cellular immune
responses are thought to be an important correlate/contributor
to vaccine efficacy [3–6]. In addition, these assays have been used to
measure the number of T cells generated spontaneously by the
body in response to a viral infection (RSV, influenza, etc.) or
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_30, © Springer Science+Business Media, LLC, part of Springer Nature 2021
525
The Quantification of Antigen-Specific T Cells by IFN-γ
ELISpot
Bassel Akache and Michael J. McCluskie
Abstract
The enzyme-linked immune absorbent spot (ELISpot) assay allows for the quantification of the number of
cells producing a particular secreted analyte. As T lymphocytes secrete cytokines such as interferon (IFN)-γ
upon binding of the T cell receptor with its cognate antigen epitope, IFN-γ ELISpot allows for the
measurement of antigen-specific T cells in an immune sample. Immune cells are isolated from the vaccinated
subject and incubated with the epitope/antigen of interest on polyvinylidene difluoride (PVDF)-lined
microplates precoated with a capture antibody to IFN-γ. Cytokine spots are then detected utilizing an
IFN-γ-specific detection antibody and an enzyme-linked conjugate. Here, we describe the quantification of
OVA-specific CD8 and CD4 T cells from mouse splenocytes to measure vaccine-induced cellular responses.
Key words Vaccines, ELISpot, T lymphocyte, Interferon-γ, Ovalbumin
1 Introduction
The immune system employs a cellular immune response to fight
various disease indications, such as cancer or viral infections. Malignant or infected cells are killed through direct interaction with
lymphocytes [1]. As such, vaccine platforms and adjuvants capable
of inducing strong antigen-specific CD8 T cell responses have been
developed and evaluated for use in novel vaccines targeting these
types of indications. To measure the activity of such vaccines/
adjuvants, a reliable method to quantitate the number of antigenspecific T cells is required. Enzyme-linked immunosorbent spot
(ELISpot) is a modification of a regular sandwich ELISA, where
the assay input is live cells instead of protein-containing extract
[2]. It is routinely used to measure the immunogenicity of vaccines
in preclinical and clinical vaccine studies where cellular immune
responses are thought to be an important correlate/contributor
to vaccine efficacy [3–6]. In addition, these assays have been used to
measure the number of T cells generated spontaneously by the
body in response to a viral infection (RSV, influenza, etc.) or
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_30, © Springer Science+Business Media, LLC, part of Springer Nature 2021
525