Interleukin 6, Canine, BioAssay(TM) ELISpot Kit (IL-6, IL6)

Interleukin 6 (IL-6) is a pleiotropic a-helical cytokine that plays important roles in acute phase reactions, inflammation, hematopoiesis, bone metabolism, and cancer progression. IL-6 activity is central to the transition from acute inflammation to either acquired immunity or chronic inflammatory disease. It is secreted by multiple cell types as a 22-28kD phosphorylated and variably glycosylated molecule (1-4). Mature canine IL-6 is 187aa in length and shares 76%, 59%, 38%, and 40% aa sequence identity with feline, human, mouse, and rat IL-6, respectively (5). IL-6 induces signaling through a cell surface heterodimeric receptor complex composed of a ligand binding subunit (IL-6 R) and a signal transducing subunit (gp130). IL-6 binds to IL-6 R, triggering IL-6 R association with gp130 and gp130 dimerization (6). gp130 is also a component of the receptors for CLC, CNTF, CT-1, IL-11, IL-27, LIF, and OSM (7). Soluble forms of IL-6 R are generated by both alternate splicing and proteolytic cleavage (3). In a mechanism known as trans-signaling, complexes of soluble IL-6 and IL-6 R elicit responses from gp130-expressing cells that lack cell surface IL-6 R (3). Trans-signaling enables a wider range of cell types to respond to IL-6, as the expression of gp130 is ubiquitous while that of IL-6 R is predominantly restricted to hepatocytes, leukocytes, and lymphocytes (3). Soluble splice forms of gp130 block trans-signaling from IL-6/IL-6 R but not from other cytokines that utilize gp130 as a coreceptor (4, 8). \n\nThe Canine IL-6 ELISpot assay is designed for the detection of IL-6 secreting cells at the single cell level, and it can be used to quantify the frequency of canine IL-6 secreting cells. ELISpot assays are well suited for monitoring cellular responses to various stimuli, treatments and therapies, and they have been used specifically for the quantification of a multitude of physiologic responses. Other methods for the assessment of cytokine-secreting cells are tedious and require previous in vitro expansion of cytokine secreting cells for several days. These assays typically are not suitable for measuring infrequent 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 IL-6 secreting cells, and thus 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, and the monitoring of various clinical trials.\n\nIntended Use:\nFor the quantitative determination of the frequency of cells releasing canine IL-6.\n\nTest Principle:\nThe enzyme-linked immunospot (ELISpot) assay was originally developed for the detection of individual B cells secreting antigen-specific antibodies (9, 10). This method has since been adapted for the detection of individual cells secreting specific cytokines or other antigens (11, 12). ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique. A polyclonal antibody specific for canine IL-6 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-6. After washing away any cells and unbound substances, a biotinylated polyclonal antibody specific for canine IL-6 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-6 secreting cell. The spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope.\n\nKit Components:\nIL-6 Microplate 1x96 wells PVDF-backed microplate coated with polyclonal\nantibody specific for canine IL-6.\nDetection Antibody Concentrate 150ml of a 120X concentrated solution of biotinylated polyclonal antibody specific for canine IL-6 with preservatives.\nStreptavidin-AP Concentrate A 150ml of a 120X concentrated solution of Streptavidin conjugated to Alkaline Phosphatase with preservatives.\nDilution Buffer 1x12ml of a buffer for diluting Detection Antibody Concentrate with preservatives.\nDilution Buffer 2x12ml of a buffer for diluting Streptavidin-AP Concentrate A with preservatives.\n10X Wash Buffer Concentrate 50ml of a 10X concentrated solution of a buffered surfactant with preservative.\nBCIP/NBT Chromogen 12ml of a stabilized mixture of 5-Bromo-4-Chloro-3

Prices direct from United States Biological

Quick response times

Exclusive Biosave savings/discounts

SPECIFICATIONS

Catalog Number

I8428-04N

Size

1Kit

References

1. Van Snick, J. (1990) Annu. Rev. Immunol. 8:253. 2. Hodge, D.R. et al. (2005) Eur. J. Cancer 41:2502. 3. Jones, S.A. (2005) J. Immunol. 175:3468. 4. Rose-John, S. et al. (2006) J. Leukoc. Biol. 80:227. 5. Kukielka, G.L. et al. (1995) Circulation 92:1866. 6. Murakami, M. et al. (1993) Science 260:1808. 7. Muller-Newen, G. (2003) Sci. STKE 2003:PE40. 8. Mitsuyama, K. et al. (2006) Clin. Exp. Immunol. 143:125. 9. Czerkinsky, C.C. et al. (1983) J. Immunol. Methods 65:109. 10. Sedgwick, J.D. and P.G. Holt (1983) J. Immunol. Methods 57:301. 11. Czerkinsky, C.C. et al. (1984) J. Immunol. Methods 72:489. 12. Helms, T. et al. (2000) J. Immunol. 164:3723.