Human IL-12 p70 ELISA Assay Kit

$405.00

The Eagle Biosciences Human Interleukin 12p70 (IL-12p70) ELISA Assay Kit (enzyme-linked immunoassay kit) is intended for the quantitative determination of human Interleukin 12p70 (IL-12p70) concentrations in cell culture supernates, serum, and plasma. The Eagle Biosciences Human Interleukin 12p70 (IL-12p70) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: L7031-K01 Categories: , ,

Human IL-12 p70 ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 7 pg/mL
Dynamic Range: 31.25 – 2000 pg/mL
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl

Additional Information

Assay Background


IL-12, also known as natural killer cell stimulatory factor (NKSF) or cytotoxic lymphocyte maturation factor (CLMF), is a pleiotropic cytokine produced primarily by antigen-presenting cells (monocytes/macrophages, dendritic cells and B lymphocytes). IL-12 has multiple effects on T lymphocytes and natural killer (NK) cells, including the ability to stimulate cytotoxicity, proliferation, cytokine production and Th1 subset development (1, 2).  IL-12 is a disulfide-linked, 70 kDa (p70) heterodimeric glycoprotein composed of a 40 kDa (p40) subunit and a 35 kDa (p35) subunit. The p40 and p35 subunits by themselves do not have IL-12 activity, but the homodimer of p40 has been shown to bind the IL-12 receptor and is an IL-12 antagonist (3, 4).

The genes for human p40 and p35, found on chromosomes 5 and 3, respectively, are independently regulated (1, 5). The expression of p35 mRNA has been found to be nearly ubiquitous. However, p35 subunits have not been detected in culture supernates of cells expressing only p35 or both p35 and p40 mRNAs (1). In cells expressing both p35 and p40 mRNAs, p40 mRNA is expressed to a higher level and free p40 subunits not associated with p35 subunits are secreted together with heterodimeric IL-12p70 (6).

In the culture supernates of various activated human monocytes where free p40 is present in vast excess over p70, the levels of p70 measured by bioassays are consistent with those measured using a p70-specific immunoassay, suggesting that p40 monomers are not efficient IL-12 antagonists (1, 7). Currently, the physiological role of free p40 subunits is not clear.

Assay Principle


The Eagle Biosciences Human Interleukin 12p70 (IL-12p70) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-12p70 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-12p70 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-12p70 is added to the wells and binds to the combination of capture antibody-IL-12p70 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-12p70 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-12p70 standard dilutions and IL-12p70 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 30nm as the reference wave length;610-650nm is acceptable)

Manual

Product Manual


Publications

References


1. Wolf, S.F. et al. (1994) Stem Cells 12:154.
2. Hendrazak, J.A. and M.J. Brunda (1995) Laboratory Investigation 72:619.
3. Gillessen, S. et al. (1995) Eur. J. Immunol. 25:200.
4. Ling, P. et al. (1995) J. Immunol. 154:116.
5. Sieburth, D. et al. (1992) Genomics 14:59.
6. D’Andrea, A. et al. (1992) J. Exp. Med. 176:1387.
7. Tsang, M.L-S. and J.A. Weatherbee (1996) J. of NIH Research 8:68.