Additional Information

Assay Principle

The Eagle Biosciences Mouse Interleukin 12 p70 (IL-12 p70) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-12 p70 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-12 p70 present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for IL-12 p70 is added to the wells and binds to the combination of capture antibody- IL-12 p70 in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of IL-12 p70 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven IL-12 p70 standard dilutions and IL-12 p70 sample concentration determined.

1. Prepare all reagents and working standards as directed in the previous sections.
2. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
3. Add 100 µL of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µL) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5. Add 100 µL of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C.
6. Repeat the aspiration/wash.
7. Add 100 µL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37°C. Avoid placing the plate in direct light.
8. Repeat the aspiration/wash.
9. Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
10. Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
11. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length;610-650nm is acceptable)

Assay Background

Interleukin 12 (IL-12 p70), also known as natural killer cell stimulatory factor (NKSF) or cytotoxic lymphocyte maturation factor (CLMF) is a pleiotropic cytokine originally identified in the medium of a human B-lymphoblastoid cell line (1-3).  Biologically active mouse IL-12 p70 is a disulfide-linked, 70kDa (p70) heterodimeric glycoprotein composed of a 40 kDa (p40) subunit and a 35 kDa (p35) subunit. While the p40 and p35 subunits by themselves do not have IL-12 p70   activity, the p40 homodimer has been shown to bind the IL-12 p70receptor and is an IL-12 p70    antagonist (4, 5). Mature mouse p35 subunit is composed of 193 amino acid (aa) residues and contains seven cysteines plus one potential N-linked glycosylation site (6). Mature mouse p40 subunit has 313 aa, with 13 cysteines and five potential N-linked glycosylation sites (6). Mouse p35 and p40 subunits show 63% and 72% aa identity, respectively, to the human p35 and p40 subunits (3, 6). Although mouse IL-12 p70 is active on both human and mouse cells, human IL-12 p70 is only active on human cells.

IL-12 p70 has been shown to have multiple effects on T lymphocytes and natural killer (NK) cells. Some of these effects include the induction of IFN-γ and TNF production by T and NK cells, the enhancement of cytotoxic activity of T and NK cells and the stimulation of T and NK cell proliferation. IL-12 p70 has also been shown to be a central mediator of the cell-mediated immune response by promoting Th1 development (7-11).  Cell surface staining for IL-12 p70 on a human monocytic and a mouse macrophage cell line has been reported, suggesting that membrane-associated IL-12 p70 may exist (12). Cells known to produce IL-12 p70    include macrophages, dendritic cells, monocytes, Langerhans cells, neutrophils, and keratinocytes. Although a human B cell line has been shown to produce IL-12 p70(2), fresh B cells are apparently not producers of IL-12 p70.