Human Immunoglobulin A (IgA) 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 IgA in a suspension of peripheral blood mononuclear cells (PBMCs). ELISpots are commonly used to evaluate immune responses, such as the frequency and isotype of circulating antigen-specific memory B cells or antibody-secreting cells (ASCs). Soluble IgA plays an important role in neutralizing invading pathogens. 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 IgA-secreting cells. This assay is based on a modified version of the sandwich ELISA method, where cells are added to ELISpot plates pre-coated with either capture antibodies specific for human IgA (protocol 1) or an antigen of choice (protocol 2). Secreted IgA is then captured throughout the culture period, before the cells are washed away. IgA 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 IgA-secreting cells in the sample and amount of analyte 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 #200-1008/200-1009).
Figure 1. Human IgA ELISpot Antibody Pair Kit Detects Total IgA+ 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 48 hours. Cells were then seeded at 5 x 10^4 cells/well on ELISpot plates pre-coated with Anti-human IgA mAb (MT57), 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 IgA. Wells were imaged and counted using an ELISpot reader.
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