Description
Human Immunoglobulin M (IgM) ELISpot Antibody Pair Kit is intended for those who want the flexibility of setting up their own ELISpot assay. This kit is designed for the detection and enumeration of cells secreting human IgM in a suspension of peripheral blood mononuclear cells (PBMCs). ELISpot is commonly used to evaluate immune responses, such as the frequency and isotype of circulating antigen-specific memory B cells or antibody-secreting cells (ASCs). ELISpot is highly sensitive and can detect secreted analytes at a single-cell level. This assay is used in research of infectious diseases and vaccines.
This assay can quantify antigen-specific and/or total IgM-secreting cells, depending on how the assay is performed. This assay is based on a modified version of the sandwich ELISA method, in which cells are added to ELISpot plates pre-coated with either capture antibodies specific for human IgM (protocol 1) or an antigen of choice (protocol 2). Secreted IgM is then captured throughout the culture period, before the cells are washed away. Captured IgM is detected by the addition of a biotinylated antigen or detection antibody, followed by SA-ALP, which binds to the biotinylated antigen or 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 antigen-specific or total IgM-secreting cells in the sample and amount of immunoglobulin secreted.
NOTE: This kit includes sufficient reagents for 4 x 96-well ELISpot plates. ELISpot plates (e.g. Millipore Sigma Catalog #MSIPS4W10 / MAIPSWU10) and BCIP/NBT Substrate (Catalog #200-1016) are required for use with this kit and are available for purchase separately. For detecting antigen-specific immune responses, an antigen of choice is also required.
This kit is supplied in two boxes (Catalog #200-1014/200-1015).
