Human interleukin-5 (IL-5) ELISpot Kit is designed for the quantitative detection and enumeration of cells secreting human IL-5 in a suspension of peripheral blood mononuclear cells (PBMCs). ELISpot is commonly used for evaluating specific immune responses, such as determining the frequency of activated T cell subsets in response to antigenic stimulation. IL-5 is a signature cytokine secreted by activated T helper 2 (Th2) cells. It plays an important role in humoral immunity, mediating immune responses against large extracellular pathogens such as helminths, as well as allergic inflammation. ELISpot is highly sensitive and can detect secreted analytes at a single-cell level. This assay is used in research of infectious diseases, cancers, allergies, and autoimmune diseases.
The assay is based on a modified version of the sandwich ELISA method, in which cells are added to ELISpot plates pre-coated with capture antibodies specific for the cytokine. The cytokine is then captured throughout the culture period and is detected by the addition of a biotinylated detection antibody, followed by streptavidin-alkaline phosphatase (SA-ALP), which binds to the biotinylated antibody. Addition of the chromogenic enzyme substrate 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP)/nitroblue tetrazolium (NBT), results in the formation of colored spots. The number, size, and intensity of these spots are directly proportional to the frequency of cytokine-secreting cells in the sample and the amount of analyte secreted.
Figure 1. Human IL-5 ELISpot Kit Detects IL-5-Secreting T Cells Following Polyclonal Stimulation.
ELISpot analysis of human peripheral blood mononuclear cells (PBMCs) isolated from whole blood using Lymphoprep™ (Catalog #18060) density gradient medium or EasySep™ Direct Human PBMC Isolation Kit (Catalog #19654). Cells were cultured in ImmunoCult™-XF T Cell Expansion Medium (Catalog #10981) in the absence of stimuli (negative control) or presence of Anti-human CD3 mAb (CD3-2), azide free (included in kit) or phytohemagglutinin (PHA). Cells were seeded at 2.5 x 10^5 cells/well on IL-5 pre-coated white plate and incubated at 37°C, 5% CO2 for 18 hours. Following incubation, ELISpot plates were processed as per the instructions on the Product Information Sheet. Wells were imaged and counted using an ELISpot reader.
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