IL-12, a cytokine produced by macrophages and B lymphocytes, has multiple effects on T-cells and NK cells, including stimulation of cytotoxic activity, proliferation, and promotion of Th1 development and IFN-gamma and TNF production.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 have no IL-12 activity, the p40 dimer has been shown to bind the IL-12 receptor and to be an IL-12 antagonist.3,4
Free p35 has not been detected in supernatant solutions of cultured cells expressing only p35 or both p35 and p40 mRNAs.1 In contrast, p40 is secreted in excess of IL-12 in cells expressing both p35 and p40 mRNAs.5 This presents an analytical problem for either an immunoassay, which may cross react with monomeric or dimeric p40, or a bioassay, for which dimeric p40 can act as an antagonist.
We show here that heterodimeric IL-12 (p70) can be accurately measured in the presence of excess p40 using R&D Systems’ IL-12 ELISA kit. The IL-12 level measured with the Quantikine kit was also shown to correlate well with the IL-12 level measured using bioassays.
The concentration of secreted p40 can be several hundred-fold higher than that of IL-12 (Table 1). In agreement with previous reports8,9, production of p40 and IL-12 by monocytes was optimally induced by a combination of IFN-gamma and LPS, and levels of IL-12 agree with those reported by Cassatella et al.9 In contrast, levels of IL-12 from NC37 and SAC-stimulated monocytes are considerably lower than those reported by D’Andrea et al.5, who used a double-determinant IL-12 RIA that exhibited significant cross-reactivity with p40, while levels of p40 are consistent with theirs.
|Table 1. IL-12 and p40 Production by NC37 cells and Human Monocytes|
|Cells (1)||Activation Conditions (2)||IL-12 (3)||p40 (4)||p40/IL-12|
|PMA + A23187||9||5203||578|
|IFN-gamma + LPS||18,565||68,200||3.7|
|IFN-gamma + SAC||2,535||20,640||8|
We measured IL-12 bioactivity with PHA-activated T-lymphoblasts as the responding cells. Bioactive IL-12 in media conditioned by IFN-gamma + LPS or IFN-gamma + SAC. 18 - 23 ng/mL or 3 - 5 ng/mL IL-12 could be neutralized by anti-IL-12 neutralizing antibody (Catalog # AB-219-NA), in agreement with the levels measured by immunoassay.
In R&D Systems’ Quantikine IL-12 and Quantikine HS IL-12 Immunoassay kits, the monoclonal antibody (capture antibody) is specific for heterodimeric IL-12, with no reactivity with free p40. The high levels of IL-12 reported in some cases may, therefore, represent an over estimation from use of antibodies that cross-react with p40. Because of the excess p40 in many situations, IL-12 levels can be measured accurately only if the ELISA is highly specific.