The ELISpot (Enzyme Linked Immuno-Spot) assay provides an effective method of measuring the antibody or cytokine production of immune cells on the single cell level. The popularity of this assay has seen resurgence in recent years as researchers attempt to gain a better understanding of immune responses in a variety of applications. The following protocol is an example of a typical ELISpot assay for quantifying the number cells producing interferon-gamma (IFN-γ) in response to antigen or non-specific activation using phytohemagglutinin (PHA). It may be optimized as necessary for other applications.
Analyze plate using imaging system.
Image of a typical ELISpot well, note that the spots are clear, focused and easy to distinguish. The image represents the number of cells secreting IFN-γ in response to CEF peptide pool stimulation of HPBMC. The assay was performed using MultiScreenR ImmobilonR-P plates (MSIPS4W10), antibodies and enzyme conjugate from Mabtech AB (part number 3420-2A) and substrate from Moss, Inc. (part number NBTH-100). Wells were imaged using the Zeiss KS ELISpot imaging system.
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