IL-12/IL-23 p40, Canine, ELISpot BioAssay(TM) Kit (IL-12, IL-23 p40, Interleukin-12 Subunit beta, IL-12B, Cytotoxic Lymphocyte Maturation Factor 40kD Subunit, CLMF p40, IL-12 Subunit p40, IL12B)

Canine IL-12/IL-23 p40 is a 40kD glycoprotein that shows considerable structural similarity to the extracellular domain of hematopoietin receptors. It is synthesized as a 329aa precursor with a 22 aa signal sequence and a 307 aa mature region. It contains one 90 aa fibronectin type III domain and one 75 aa Ig C2-like region. There are two intrachain disulfide bonds and two potential N-linked glycosylation sites. p40 is an inducible molecule synthesized in response to APC-activating substances such as LPS and CpG. Once made, it can be secreted as a monomer, homodimer, heterodimer linked to p35 (IL-12), and a heterodimer linked to p19 (IL-23). Relative to p35, it exists in a 10-1000 fold excess. Canine p40 is 94%, 85%, 84%, 65%, and 65% aa identical to feline, human, porcine, rat and mouse p40, respectively. Cells known to express p40 include macrophages, dendritic cells, monocytes, Langerhans cells, neutrophils, keratinocytes, plasmacytoid dendritic cells, and microglia. p40 binds to the 100kD subunit (Rb1) of the heterodimeric IL-12 receptor. This subunit is shared by both the receptors for IL-12 and IL-23. As noted, p40 exists as a homodimer, as a subunit of IL-12, and as a subunit of IL-23. As a component of IL-12, p40 contributes to the potentiation of effector Th1 responses but not necessarily to their initiation. As a component of IL-23, p40 has activity on memory T cells, inducing proliferation and IFN-g production. As a homodimer (p40)2, its activities are less certain. It is inhibitory to IL-12 activity in mice, attributable mostly to competitive inhibition. In humans, however, (p40)2 shows little competitive inhibition, principally due to low dimer formation and low affinity binding to Rb1. It does, however, have stimulatory activity on a number of cell types, and may actually signal through a poorly-defined receptor complex composed of Rb1 and an 85kD subunit (1-6). The Canine IL-12/IL-23 p40 ELISpot assay is designed for the detection of IL-12/IL-23 p40 secreting cells at the single cell level, and it can be used to quantitate the frequency of canine IL-12/IL-23 p40 secreting cells. ELISpot assays are well suited for monitoring immune responses to various stimuli, treatments and therapies, and they have been used for the quantification of antigen-specific CD4+ and/or CD8+ T cell responses. Other methods for the assessment of antigen-specific T cell responses, such as the chromium release assay 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 they are suitable for high-throughput analysis using only small volumes of primary cells. As such, ELISpot assays are useful tools for research in areas as diverse as antigen recognition, vaccine development, cytokine secretion and the monitoring of various clinical trials.\n\nIntended Use:\nFor the quantitative determination of the frequency of cells releasing canine IL-12/IL-23 p40.\n\nTest Principle:\nThe enzyme-linked immunospot (ELISpot) assay was originally developed for the detection of individual B cells secreting antigen-specific antibodies (7, 8). This method has since been adapted for the detection of individual cells secreting specific cytokines or other antigens (9, 10). ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique. A polyclonal antibody specific for canine IL-12/IL-23 p40 has 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 degrees C CO2 incubator for a specified period of time. During this incubation period, the immobilized antibody in the immediate vicinity of the secreting cells binds secreted IL-12/IL-23 p40. After washing away any cells and unbound substances, a biotinylated polyclonal antibody specific for canine IL-12/IL-23 p40 is added to the wells. Following a wash to remove any unbound biotinylated 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 and appears as spots at the sites of cytokine localization, with each individual spot representing an individual IL-12/IL-23 p40 secreting cell. The spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope.\n\nKit Components:\nCanine IL-12/IL-23 p40 Microplate 1x 96-well PVDF-backed microplate coated\nwith polyclonal antibody specific for canine IL-12/IL-23 p40.\nDetection Antibody Concentrate 150ml of a 120-fold concentrated solution of biotinylated polyclonal antibody specific for canine IL-12/IL-23 p40 with preservatives.\nStreptavidin-AP Concentrate A 150ml of a 120-fold concentrated solution of Streptavidin conjugated to Alkaline Phosphatase with preservatives.\nDilution Buffer 1 12ml of a buffer for diluting Detection Antibody Concentrate with preservatives.\nDilution Buffer 2 12ml of a buffer for diluting Streptavidin-AP Concentrate A with preservatives.\nWash Buffer Concentrate 50ml of a 10-fold concentrated solution of a buffered surfactant with preservative.\nBCIP/NBT Chromogen 12ml of a stabilized mixture of 5-Bromo-4-Chloro-3

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SPECIFICATIONS

Catalog Number

I8434-30H1

Size

1Kit

References

1. Buttner, M. et al. (1997) Cytokine 10:241. 2. Park, A.Y. and P. Scott (2001) Scand. J. Immunol. 53:529. 3. Trinchieri, G. et al. (2003) Immunity 19:641. 4. Brombacher, F. et al. (2003) Trends Immunol. 24:207. 5. Lankford, C.S. and D.M. Frucht (2003) J. Leukoc. Biol. 73:49. 6. Abdi, K. (2002) Scand. J. Immunol. 5:1.7. Czerkinsky, C.C. et al. (1983) J. Immunol. Methods 65:109. 8. Sedgwick, J.D. and P.G. Holt (1983) J. Immunol. Methods 57:301. 9. Czerkinsky, C.C. et al. (1984) J. Immunol. Methods 72:489.10. Helms, T. et al. (2000) J. Immunol. 164:3723.