Description

Nori Porcine IL-12 ELISA Kit Summary

Alternative names for IL-12: Interleukin 12 P35, IL12 P35

Alternative names for porcine: pig, swine

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Interleukin 12 (IL12) is the founding member of the IL12 family of heterodimeric cytokines, which have important immunological functions. IL12 is composed of two disulfide-linked subunits of 35 kDa and 40 kDa, respectively. The 35 kDa subunit (p35) is an α helical protein homologous to IL6 and GCSF.The 40 kDa subunit (p40) contains one fibronectin type III and one Ig C2-like domain, and has a high degree of structural homology to type I cytokine receptors. Whereas p35 subunit is unique to IL-12, the p40 subunit is also utilized in IL23. Mature rat p40 contains 313 aa and can exist in multiple forms, including monomer, homodimer, heterodimer linked to p19 (forming IL 23), and heterodimer linked to p35 (forming IL12). The expression of p40 is upregulated by substances such as LPS and CpG that activate antigen presenting cells.

Cells known to produce IL12 include macrophages, dendritic cells, monocytes, Langerhans cells, neutrophils, and keratinocytes. The activities of IL12 are mediated by the receptor complex composed of two type I transmembrane proteins: a 100 kDa ligand binding subunit (IL12 Rβ1) and a 130 kDa signal transducing subunit (IL12 Rβ2). IL-12 mediates enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes and potentiates the expansion and late activation of Th1 CD4+ T cells. IL-12 also has anti-angiogenic activity (1-4). IL-12 is linked with autoimmunity, and normally stops allergies to food developing (5).

References

1. Park, A.Y. and P. Scott (2001) Scand. J. Immunol. 53:529.
2. Trinchieri, G. et al. (2003) Immunity 19:641.
3. Brombacher, F. et al. (2003) Trends Immunol. 24:207.
4. Lankford, C.S. and D.M. Frucht (2003) J. Leukoc. Biol. 73:49
5. Temblay JN, et al. J Allergy Clin Immunol. 2007 Sep;120 (3):

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