Chapter 28 : Methods for Detection of Antigen-Specific T Cells by Enzyme-Linked Immunospot Assay (ELISPOT)

Abstract:

The assay most widely utilized is the enzyme-linked immunospot assay (ELISPOT). The ELISPOT was first described in 1983 as an alternative to plaque-forming assays for the detection of antibody-secreting cells. This assay may be adapted for use with synthetic peptides, including overlapping peptide pools. In an effort to increase the sensitivity of ELISPOT, several groups have developed modifications of the standard protocol involving the addition of exogenous cytokines, costimulatory antibodies, or antigen-presenting cells. The ELISPOT is relatively new, and considerable methodological variation exists among laboratories. As a positive control for cytokine release, many laboratories rely on polyclonal stimuli. However, these reagents may give rise to a nearly confluent lawn of spots. Statistical methods for evaluating ELISPOT results and determining the appropriate positive cutoff have varied considerably between laboratories. A summary of statistical methods reported in the recent literature is given. Standardization and validation of any bioassay is a lengthy process and will require many multicenter studies. As ELISPOT readers continue to improve and statistical methods achieve broader acceptance, the trend towards increasing standardization is likely to accelerate rapidly within the next few years.