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).
Figure 1. Human IgM ELISpot Antibody Pair Kit Detects Total IgM+ Antibody-Secreting Cells.
ELISpot analysis of peripheral blood mononuclear cells (PBMCs) isolated from whole blood using EasySep™ Direct Human PBMC Isolation Kit (Catalog #19654) and cultured in RPMI 1640 Medium (Catalog #36750) supplemented with 2mM L-glutamine (Catalog #07100) and 10% Fetal Bovine Serum (Catalog #100-0180). Cells were pre-activated with R848, Activator + Recombinant human IL-2, lyophilized (included in kit) or in the absence of stimuli (negative control) at 37°C, 5% CO2 for 72 hours. Cells were then seeded at 1.25 x 10^4 cells/well on ELISpot plates pre-coated with Anti-human ΙgM mAbs (MT11/12), unconjugated and incubated for a further 20 hours. Following incubation, ELISpot plates were processed as per the instructions on the Product Information Sheet for detecting total IgM. Wells were imaged and counted using an ELISpot reader.
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