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 coloured 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.
For Use with serum, plasma and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.
Interleukin 12 (IL-12), 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.
Biologically active mouse IL-12 is a disulfide-linked, 70 kDa (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 activity, the p40 homodimer has been shown to bind the IL-12 receptor and is an IL-12 antagonist. Mature mouse p35 subunit is composed of 193 amino acid (aa) residues and contains seven cysteines plus one potential N-linked glycosylation site. Mature mouse p40 subunit has 313 aa, with 13 cysteines and five potential N-linked glycosylation sites. Mouse p35 and p40 subunits show 63% and 72% aa identity, respectively, to the human p35 and p40 subunits. Although mouse IL-12 is active on both human and mouse cells, human IL-12 is only active on human cells.
IL-12 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 has also been shown to be a central mediator of the cell-mediated immune response by promoting Th1 development.
Cell surface staining for IL-12 on a human monocytic and mouse macrophage cell line has been reported, suggesting that membrane-associated IL-12 may exist. Cells known to produce IL-12 include macrophages, dendritic cells, monocytes, Langerhans cells, neutrophils, and keratinocytes. Although a human B cell line has been shown to produce IL-12, fresh B cells are apparently not producers of IL-12.
|Kit Components||96 Wells Quantity/Size|
|Aluminium pouches with a Microwell Plate coated with monoclonal antibody to mouse IL-12p70 (8﹡12)||1 plate|
|Mouse IL-12p70 Standard lyophilized, 2000 pg/ml upon reconstitution||2 vials|
|Concentrated Biotin-Conjugate anti-mouse IL-12p70 monoclonal antibody||2 vials|
|Streptavidin-HRP solution||2 vials|
|Standard /sample Diluent||1 bottle|
|Biotin-Conjugate antibody Diluent||1 bottle|
|Streptavidin-HRP Diluent||1 bottle|
|Wash Buffer Concentrate 20x (PBS with 1% Tween-20)||1 bottle|
|Substrate Solution||1 vial|
|Stop Solution||1 vial|
|Adhesive Films||4 pieces|
|Product data sheet||1 copy|
|Storage||Store at 2 - 8°C|
|REPEATABILITY||The coefficient of variation of both intra-assay and inter-assay were less than 10%.|
|SENSITIVITY||The minimum detectable dose was 7pg/mL.|
|ASSAY RANGE||15.6-1000 pg/mL|
|SPECIFICITY||This assay recognizes both natural and recombinant mouse IL-12p70. The factors listed below were prepared at 50ng/ml in Standard /sample Diluent and assayed for cross-reactivity and no significant cross-reactivity or interference was observed.|
Factors assayed for cross-reactivity
|Recombinant mouse||G-CSF, GM-CSF, IL-1α, IL-1β, IL-2 , IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p35, IL-12p40 monomer, IL-12p70, IL-23, TNF-α|
|Recombinant human||G-CSF, IL-6, IL-6sR, IL-12p35, IL-12p40 monomer, IL-12p70, IL-23, GM-CSF|
Data Analysis Assistance
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