Human IL-5 ELISpot Kit

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ELISpot kits are highly sensitive, microplate-based assays for the detection of cytokine secreting cells. This kit is designed for the detection and enumeration of human IL-5. Complete ELISpot kits are ready-to-run and require no assay development or refinement.

This ELISpot assay employs a capture antibody specific for human IL-5, pre-coated onto a PVDF-backed microplate. Appropriately stimulated cells are pipetted directly into the wells and the immobilized antibody in the immediate vicinity of the secreting cells binds secreted human IL-5. Following wash steps and incubation with a biotinylated detection antibody, alkaline-phosphatase conjugated to streptavidin is added. Unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added. A blue-black colored precipitate forms at the sites of cytokine localization and appears as spots, with each individual spot representing an individual human IL-5 secreting cell. The spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope.

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• Detect and quantitate individual cells secreting human IL-5
• 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

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• Human IL-5 Microplate
• Biotinylated Detection Antibody
• Streptavidin conjugated to Alkaline Phosphatase
• Dilution Buffers
• Wash Buffer Concentrate
• BCIP/NBT Chromogen
• Human IL-5 Positive Control

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• 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