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 (1-3).

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 (4, 5). Mature mouse p35 subunit is composed of 193 amino acid (aa) residues and contains seven cysteines plus one potential N-linked glycosylation site (6). Mature mouse p40 subunit has 313 aa, with 13 cysteines and five potential N-linked glycosylation sites (6). Mouse p35 and p40 subunits show 63% and 72% aa identity, respectively, to the human p35 and p40 subunits (3, 6). 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 (7-11).

Cell surface staining for IL-12 on a human monocytic and a mouse macrophage cell line has been reported, suggesting that membrane-associated IL-12 may exist (12). 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(2), fresh B cells are apparently not producers of IL-12.

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.

Figure 1:Schematic diagram of the assay

1. Aluminium pouches with a Microwell Plate coated with monoclonal antibody to mouse IL-12p70 (812)

2. 2 vials mouse IL-12p70 Standard lyophilized, 2000 pg/ml upon reconstitution

3. 2 vials concentrated Biotin-Conjugate anti-mouse IL-12p70 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.

1. Kato, T. et al. (1997) Cell. Immunol. 181:59.

2. Blotta, M.H. et al. (1997) J. Immunol. 158:5589.

3. Gillessen, S. et al. (1995) Eur. J. Immunol. 25:200.

4. Ling, P. et al. (1995) J. Immunol. 154:116

5. Wolf, S.F. et al. (1994) Stem Cells 12:154.

6. Hendrazak, J.A. and M.J. Brunda (1995) Laboratory Investigation 72:619.

7. Scharton-Kersten, T. et al. (1995) J.Immunol.5320:5330

8. Wolf, SF. et al. (1994) stem cells 154:156