Elsevier

Methods

Volume 29, Issue 3, March 2003, Pages 260-269
Methods

T-cell epitope mapping using the ELISPOT approach

https://doi.org/10.1016/S1046-2023(02)00348-1Get rights and content

Abstract

The ELISPOT assay is particularly well suited to measure both clonal size and effector function of low-frequency antigen-specific T-cell populations directly ex vivo. Typically, an ELISPOT assay is performed with a freshly obtained sample (or cryopreserved sample) using less than 24 h of culture. Additionally, this assay allows for the simultaneous analysis of hundreds of variables in parallel from a single tissue specimen. Because of these capabilities, this assay has found widespread use in the context of direct ex vivo immune diagnostic monitoring in humans. Herein we describe the rationale, methodology, and useful hints for performing T-cell epitope mapping using ELISPOT analysis, and review typical results of such an analysis.

Section snippets

Why ELISPOT: a highly sensitive measure of the clonal size of antigen-specific T-cell populations in vivo

During an immune response low-frequency antigen-specific naive T cells clonally expand. The overall clonal size reached defines the effector cell mass and therefore the magnitude of the acquired immune repertoire. During initial encounter with the antigen, naive T cells differentiate into memory cells with specific cytokine producing or killing effector function that defines the quality of the immune response. The scope of immune diagnostics therefore has to account for both clonal size and

Why use peptide libraries: a comprehensive approach to define the engaged T-cell repertoire

The ELISPOT assay takes advantage of the fact that memory T-cell populations do not secrete effector cytokine until they encounter cognate antigen [9]. To detect such T cells, the appropriate form of cognate antigen needs to be provided. Because T cells recognize processed antigen, the mode of antigen administration and the type of APC defines what determinant is presented to the test cell population in the recall assay. Soluble antigens added to cell culture are preferentially presented on

Validation of peptide library approach

The basic tenet of testing libraries using an IFN-γ ELISPOT approach is that peptide-induced IFN-γ production signifies in vivo-primed memory cells. In the experimental setting, this issue has been addressed by testing naive versus immunized mice. BALB/c mice were immunized with hen egg lysozyme (HEL) using adjuvants that induce either type 1 or type 2 immunity [8]. HEL is one of the best defined prototype antigens with respect to determinant hierarchy/immunodominance. Splenocytes were assayed

Predicted versus actually recognized T-cell determinants

An increasing number of T cell determinants have been established for the different MHC alleles empirically and considerable progress has been made toward an understanding of MHC–peptide binding motifs [28], [29], [30], [31], [32], [33] and making predictions as to candidate T-cell determinants. Such approaches have offered the potential to select candidate peptides for immune monitoring when it is necessary to limit (for feasibility purposes) the repertoire of test peptides (e.g., when using

Cytokine-specific antibodies

Several vendors sell IFN-γ antibodies that perform excellently in ELISPOT assays, including Pharmingen/BD, R&D Systems, Endogen, and Mabtech. Antibodies that have been produced for flow cytometry application have frequently been deaggregated, which results in poor ELISPOT assay performance; one must be careful to use ELISPOT-grade reagents. Some vendors have established that their product permits immune monitoring at single-cell resolution (recommended). Because spot sizes, even for a T-cell

References (33)

  • J.D. Sedgwick et al.

    J. Immunol. Methods

    (1986)
  • J.M. Versteegen et al.

    J. Immunol. Methods

    (1988)
  • C. Ekerfelt et al.

    J. Immunol. Methods

    (2002)
  • T.W. Tobery et al.

    J. Immunol. Methods

    (2001)
  • H.G. Rammensee

    Curr. Opin. Immunol.

    (1995)
  • D.D. Anthony et al.

    Clin. Immunol.

    (2002)
  • J.D. Sedgwick et al.

    J. Exp. Med.

    (1983)
  • T. Helms et al.

    J. Immunol.

    (2000)
  • A. Karulin et al.

    J. Immunol.

    (2000)
  • M.D. Hesse et al.

    J. Immunol.

    (2001)
  • H.C. Yip et al.

    J. Immunol.

    (1999)
  • S. Ehlers et al.

    J. Exp. Med.

    (1991)
  • V. Engelhard

    Annu. Rev. Immunol.

    (1994)
  • E.E. Sercarz et al.

    Annu. Rev. Immunol.

    (1993)
  • G. Gammon et al.

    Immunol. Rev.

    (1987)
  • G. Gammon et al.

    J. Exp. Med.

    (1991)
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