Interleukin-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 activated human B lymphoblastoid cell lines. IL-12 is produced by macrophages and B lymphocytes and has multiple effects on T-cells and NK cells, including stimulation of cytotoxic activity, proliferation, and promotion of Th1 development as well as IFNγ and TNF production. IL-12 is a disulfidelinked, 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 have no IL-12 activity, the p40 dimer has been shown to bind the IL-12 receptor and to be an IL-12 antagonist. Free p35 has not been detected in supernatant solutions of cultured cells expressing only p35 or both p35 and p40 mRNAs. In contrast, p40 is secreted in excess of IL-12 in cells expressing both p35 and p40 mRNAs. The p40 subunit of IL-12 has been shown to have extensive amino acid sequence homology to the extracellular domain of the human IL-6 receptor while the p35 subunit shows distant but significant sequence similarity to IL-6, G-CSF, and chicken MGF. These observations have led to the suggestion that IL-12 might have evolved from a cytokine/soluble receptor complex. Human and mouse IL-12 share 70% and 60% amino acid sequence homology in their p40 and p35 subunits, respectively. IL-12 apparently shows species specificity with human IL-12 reportedly showing minimal activity in the murine system.

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-12p40 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-12p40 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-12p40 is added to the wells and binds to the combination of capture antibody-IL-12p40 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-12p40 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-12p40 standard dilutions and IL-12p40 sample concentration determined.

Figure 1:Schematic diagram of the assay

1. Aluminium pouches with a Microwell Plate coated with monoclonal antibody to human IL-12p40 (812)

2. 2 vials human IL-12p40 Standard lyophilized, 4000 pg/ml upon reconstitution

3. 2 vials concentrated Biotin-Conjugate anti-human IL-12p40 monoclonal antibody

4. 2 vials Streptavidin-HRP solution,

5. 1 bottle Standard /sample Diluent

6. 1 bottle Biotin-Conjugate antibody Diluent

7. 1 bottle Streptavidin-HRP Diluent

8. 1 bottle Wash Buffer Concentrate 20x (PBS with 1% Tween-20)

9. 1 vial Substrate Solution

10. 1 vial Stop Solution

11. 4 pieces Adhesive Films

12. package insert

NOTE: [96 Tests]

Unopened Kit：Store at 2 -8° C. Do not use past kit expiration date. opened/ReconstitutedReagents：Please refer to the datasheets for detail information.

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