Human IgG/IgM Dual-Color B Cell ELISpot Kit

Appropriately stimulated cells are pipetted directly into the wells of a PVDF-backed microplate. The PVDF membrane has been pre-coated with capture antibodies specific for IgG/IgM. The microplate is then placed in a humidified 37 °C CO₂ incubator for the desired period of time. During this incubation period, the immobilized capture antibodies in the immediate vicinity of the secreting cells bind secreted IgG/IgM.

At the desired time point, one analyte is detected using a biotinylated detection antibody and alkaline-phosphatase (AP)-conjugated to streptavidin. The second analyte is detected using a horseradish peroxidase (HRP)-conjugated detection antibody. Following incubation with the chromogens BCIP/NBT (substrate for AP, blue-black precipitate) and AEC (substrate for HRP, red precipitate), spots form to indicate the presence of IgG/IgM secreting cells. Each spot represents an individual secreting cell. The spots can be counted with an ELISpot reader system or using a stereomicroscope.

Product Features for Human IgG/IgM Dual-Color B Cell ELISpot Kit

• Detect and quantitate individual cells secreting IgG/IgM
• High sensitivity - ELISpot assays can measure responses with frequencies well below 1 in 100,000 cells
• No in vitro expansion of cells required
• High-throughput - ELISpot assays use only a small number of primary cells

Kit Contents for Human IgG/IgM Dual-Color B Cell ELISpot Kit

• Human IgG/IgM Microplate
• Biotin-conjugated Detection Antibody
• HRP-conjugated Detection Antibody
• Streptavidin-conjugated to Alkaline Phosphatase
• Dilution Buffers
• Wash Buffer Concentrate
• BCIP/NBT Chromogen
• AEC Chromogen
• Human IgG/IgM Positive Controls

Other Reagents Required

• Pipettes and pipette tips
• Deionized or distilled water
• Squirt bottle, manifold dispenser, or automated microplate washer
• 500 mL graduated cylinder
• 37 °C CO₂ incubator
• Sterile culture media
• Dissection microscope or an automated ELISpot reader