The Dual-Color Canine IFN-g/IL-17 ELISpot assay is designed for the simultaneous detection of IFN-g and IL-17 secreting cells at the single cell level and can be used to simultaneously quantitate the frequency of canine IFN-g and IL-17 secreting cells. ELISpot assays are well suited for monitoring immune responses to various treatments and therapies and have been used for the quantitation of antigen-specific CD4+ and/or CD8+ T cell responses. Other methods for assessment of antigen-specific T cell responses, such as chromium release assays with quantitation by limiting dilution, are tedious and require previous in vitro expansion of T cells for several days. These assays typically are not suitable for measuring infrequent T cell responses that occur at less than 1 in 1000. ELISpot assays are highly reproducible and sensitive and can be used to measure responses with frequencies well below 1 in 100,000. ELISpot assays do not require prior in vitro expansion of T cells and are suitable for high-throughput analysis using only small volumes of primary cells. As such, ELISpot assays are useful tools for research in vaccine development and for the monitoring of various clinical trials.
Test Principle:The enzyme-linked immunospot (ELISpot) assay was originally developed for the detection of individual B cells secreting antigen-specific antibodies (1, 2). This method has since been adapted for the detection of individual cells secreting specific cytokines or other antigens (3, 4). ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique. A monoclonal antibody specific for canine IFN-g and a monoclonal antibody specific for canine IL-17 have been pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37°C CO2 incubator for a specified period of time. During this incubation period, the immobilized antibodies in the immediate vicinity of the secreting cells bind secreted IFN-g and IL-17. After washing away any cells and unbound substances, a biotinylated polyclonal antibody specific for canine IFN-g and a horseradish peroxidase-conjugated polyclonal antibody specific for canine IL-17 are added to the wells. Following a wash to remove any unbound antibodies, alkaline-phosphatase conjugated to streptavidin is added. Unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added. After washing the BCIP/NBT from the wells with tap water, an AEC chromogen solution is then added to the wells. A blue-black colored precipitate forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual IFN-g secreting cell. A red precipitate also forms and appears as spots, with each red spot representing an individual IL-17 secreting cell. The spots can be counted with an ELISpot reader system or using a stereomicroscope.
Kit Components:Canine IFN-g/IL-17 Microplate: 1x96-well PVDF-backed microplate coated with a monoclonal antibody specific for canine IFN-g and a monoclonal antibody specific for canine IL-17
Detection Antibody Concentrate A: 150ml of a 120X concentrated solution of biotinylated polyclonal antibody specific for canine IFN-g with preservatives
Detection Antibody Concentrate B: 250ml of a 60X concentrated solution of horseradish peroxidase-conjugated polyclonal antibody specific for canine IL-17 with preservatives
Streptavidin-AP Concentrate A: 150ml of a 120X concentrated solution of Streptavidin conjugated to Alkaline Phosphatase with preservatives
Dilution Buffer: 1x12ml of a buffer for diluting Detection Antibody Concentrates with preservatives
Dilution Buffer: 2x12ml of a buffer for diluting Streptavidin-AP Concentrate A with preservatives
Wash Buffer Concentrate: 50ml of a 10X concentrated solution of a buffered surfactant with preservative
BCIP/NBT Chromogen: 1x12ml of a stabilized mixture of 5-Bromo-4-Chloro-3¢ Indolylphosphate p-Toluidine Salt (BCIP) and Nitro Blue Tetrazolium Chloride (NBT)
AEC Chromogen: 300ml of 2% 3-Amino-9-Ethyl-Carbazole (AEC) in stabilizing buffer
AEC Chromogen Buffer: 12ml of 0.1% H2O2 in Acetate Buffer for diluting AEC Chromogen
Dual-Color ELISpot Schematic: A full-color diagram of the assay principle.
Storage and Stability:Store components at 4°C. Stable for at least 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
