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During more than 25 years of application in immunological sciences, ELISPOT has been established as a routine, robust, versatile, and reliable assay. From basic research to clinical immune monitoring, ELISPOT is being used to address the quantification and (to a lesser extent) functional characterization of immune cells secreting different molecules in the context of health and disease, immune intervention, and therapy in humans and other species [Kalyuzhny (Ed.) (2005) Handbook of Elispot: methods and protocols, Vol. 302, Humana Press Inc., Totowa, NJ]. Over the last decade, ELISPOT assays have been increasingly implemented as an immune-monitoring tool in clinical trials [Schmittel et al. J Immunother 23:289-295, 2000; Whiteside Immunol Invest 29:149-162, 2000; Nagata et al. Ann N Y Acad Sci 1037:10-15, 2004; Cox et al. (2005) Cellular immune assays for evaluation of vaccine efficacy in developing countries., In Manual of Clinical Immunology Laboratory (Rose, N. R., Hamilton, R. G., and Detrick, B., Eds.), p 301, ASM Press, Washington, DC; Cox et al. Methods 38:274-282, 2006]. While the principles of the original protocol have changed little since its first introduction [Czerkinsky J Immunol Methods 110:29-36, 1988], individual laboratories have adapted assay procedures based on experimental needs, availability of reagents and equipment, obtained recommendations, and gained experience, leading to a wide disparity of applied ELISPOT protocols with inevitable consequences. This chapter addresses the resulting challenges for ELISPOT use in clinical trial settings, and discusses the influence of harmonization strategies as a tool for overcoming these challenges. Furthermore, harmonization is discussed in the context of assay standardization and validation strategies.

Over the past decade, ELISPOT has become well-established as a mainstream technology for the study of immune responses in vivo mainly due to its unique ability to detect rare antigen-specific lymphocytes ex vivo. The primary readout for ELISPOT assays has traditionally been the measurement of the frequency of analyte-secreting cells within a test population. While it has been generally appreciated that ELISPOT is a high-information-content assay system in which spot morphologies provide additional valuable information on the amount of analyte secreted by individual cells as well as the kinetics of the secretory process, the precise relationships involved have not been fully characterized and the specific relevant information -conveyed by spot morphologies has remained largely unexplored. In an attempt to bridge this gap, we formulated an in silico kinetic model for spot formation and derived a solution for the model in both a general and a numerical form. Both solutions suggested a logarithmic relationship between spot size and cell productivity. This chapter involves an in-depth analysis of the relationship between observed spot morphologies and cells' secretory functions (as well as an examination of additional assay parameters), and predictions based on the mathematical model are verified under experimental assay conditions where possible.

Intracellular responses to external pathogens/stimuli are crucial to the host's response to infection. The methods used to analyse these responses fall into many categories. Activation of proteins as part of a signal cascade can be screened for using conventional immunoblotting techniques or immunoprecipitation to identify the presence of modified proteins or protein complexes. Transcription factor activity can be screened for by EMSA or ELISAs to identify DNA binding of these factors. Finally, expression of activated genes can be quantified using real-time PCR methods. Here, we will show how to perform these assays and discuss the relative merits of each.