Abstract
The enzyme-linked immuno-spot (ELISPOT) technique is a sensitive method used for measurement of elusive immune markers in limited-volume samples. By virtue of the exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific responses), which was not possible before, is now relatively easy. However, development of a method sensitive enough to pinpoint elusive immune markers at the single-cell level is a challenge since there are a number of demands that have to be fulfilled and traps to avoid, achieving a valuable outcome.
To optimize the environment for in vitro culture and analysis of immune spots by ELISPOT, a number of criteria have to be fulfilled: processing of sample and perhaps also cryopreservation of cells before analysis and, for the ELISPOT assay, optimal cell culture, positive and negative controls, antigen concentration, and, finally, development and readout of spots.
If these criteria are fulfilled for your ELISPOT assay, you will likely have the opportunity to pinpoint elusive immune markers at the single-cell level. This chapter describes the ELISPOT assay for detection of cytokines (e.g., IFN-γ and IL-4), with focus on the main criteria that affect the assay. However, this method could be easily adapted to measure other immune markers in small volumes of biological samples.
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References
Czerkinsky CC, Tarkowski A, Nilsson LÅ et al (1984) Reverse enzyme-linked immunospot assay (RELISPOT) for the detection of cells secreting immunoreactive substances. J Immunol Methods 72:489–496
Czerkinsky C, Andersson G, Ekre HP et al (1988) Reverse ELISPOT assay for clonal analysis of cytokine production. J Immunol Methods 110:29–36
Faresjö M (2012) Enzyme linked Immuno-spot; a useful tool in the search for elusive immune markers in common pediatric immunological diseases. Cells 1:141–152
Bull M, Lee D, Stucky J et al (2007) Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials. J Immunol Methods 322:57–69
Axelsson S, Faresjö M, Hedman M et al (2008) Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children. Cryobiol 57:201–208
Kivling A, Nilsson L, Faresjö M (2009) How and when to pick up the best signal from markers associated with T-regulatory cells? J Immunol Methods 345:29–39
Rydén A, Faresjö M (2011) Efficient expansion of cryopreserved CD4 + CD25 + CD127lo/−cells in type 1 diabetes. Res Immunol 1:36–44
Nowell PC (1960) Phytohemagglutinin: an inhibitor of mitosis in cultures of normal human leukocytes. Cancer Res 20:462–466
Gowans JD (1961) The management of rheumatoid arthritis. GP 24:106–116
Kruisbeek AM, Shevach EM (1999) Proliferative assay for T cell function. In: Coligan JE, Kruisbeek AM, Margulis DH et al (ed) Curr Protoc Immunol (IN), Wiley, pp 1–14
Karlsson MGE, Sederholm Lawesson S, Ludvigsson J (2000) Th1-like dominance in high-risk first-degree relatives of type 1 diabetic patients. Diabetologia 43:742–749
Schloot NC, Meierhoff G, Karlsson Faresjö M et al (2003) Comparison of cytokine ELISpot assay formats for the detection of islet antigen autoreactive T cells. Report of the third immunology of diabetes society T-cell workshop. J Autoimmun 21:365–376
Karlsson Faresjö M, Ernerudh E, Ludvigsson J (2004) Cytokine profile in children during the first 3 months after the diagnosis of type 1 diabetes. Scand J Immunol 59:517–526
Husby S, Mestecky J, Moldoveanu Z et al (1994) Oral tolerance in humans. T cell but not B cell tolerance after antigen feeding. J Immunol 152:4663–4670
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Faresjö, M. (2014). The Challenge of Measuring Elusive Immune Markers by Enzyme-Linked Immuno-Spot (ELISPOT) Technique. In: Vancurova, I. (eds) Cytokine Bioassays. Methods in Molecular Biology, vol 1172. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0928-5_1
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DOI: https://doi.org/10.1007/978-1-4939-0928-5_1
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