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 unique 35 kDa (p35) subunit and a common 40 kDa (p40) subunit that is also present in IL-23. Monomers of 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). 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-12 p70 (5). Most of the free p40 subunits secreted by the various human cell lines examined have been found to exist as monomers (1). 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, 6). In the mouse system, p40 homodimers are produced in vivo and function as IL-12 antagonists (7). Polymorphisms exist in the mouse IL-12/IL-23 p40 sequence.
For the quantitative determination of mouse interleukin 12/23p40 (IL-12/IL-23p40) 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-12/IL-23p40 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-12/IL-23p40 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/IL-23p40 is added to the wells and binds to the combination of capture antibody- IL-12/IL-23p40 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/IL-23p40 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/IL-23p40 standard dilutions and IL-12/IL-23p40 sample concentration determined.
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