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.
For the quantitative determination of human interleukin 12p70 (IL-12p70) concentrations in cell culture supernates, serum, and plasma.
Principle of the assay: This assay 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.
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