Quantitation of human IL-12 (p70) and p40 using R&D Systems’ Quantikine ELISA
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
||Activation Conditions (2)
|PMA + A23187
|IFN-gamma + LPS
|IFN-gamma + SAC
- Human EBV-transformed lymphoblastoid cells (NC37 from ATCC# CCL-214) were grown in RPMI supplemented with 10% FCS. Human monocytes were prepared from the buffy coat cells by size sedimentation.6 The resulting cells were 65% monocytes, 21% lymphocytes, and 12% neutrophils.
- NC37 cells (1x106 cells/mL) were stimulated by incubation for 24 hour at 37° C in culture medium containing 10 ng/mL PMA and 25 ng/mL calcium ionophore A23187. IL-12 and p40 were measured in the supernatant solution. Monocytes (1x106 cells/mL in RPMI 1640 supplemented with 5% FCS) were cultured overnight with with or without recombinant human IFN-gamma (100 ng/mL) (Catalog # 285-IF) and then 24 hour with or without addition of LPS (1 mg/mL) or SAC (0.0074% wt/vol, Pansorbin, Calbiochem).
- The immunoactivity of IL-12 was measured with a Quantikine IL-12 immunoassay kit (Catalog # D1200) specific for only the heterodimeric IL-12.
- The p40 subunit was measured using the Quantikine IL-12 detection antibody and a microplate coated with a monoclonal antibody (Catalog # MAB609, 4 mg/mL coating concentration) that specifically captures free p40 but not heterodimeric IL-12. Insect cell-expressed recombinant monomeric p40 was used as the calibrator.
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.
- Wolf, S.F. et al. (1994) Stem Cells 12:154.
- Zeh, H.J. et al. (1994) in The Cytokine Handbook, 2nd edition, Thomson, A. editor, Academic Press, New York, p 239.
- Gillessen, S. et al. (1995) Eur. J. Immunol. 25:200.
- Ling, P. et al. (1995) J. Immunol. 154:116.
- D’Andrea, A. et al. (1992) J. Exp. Med. 176:1387.
- Wahl, L.M. and P. D. Smith (1995) in Current Protocols in Immunology, Coligans, J. et al. editors, Wiley Interscience, p. 7.6.2.
- Gately, M.K. et al. (1995) in Current Protocols in Immunology, Coligans, J. et al. editors, Wiley Interscience, p. 6.16.3.
- Hayes, M.P. et al. (1995) Blood, 86:646.
- Cassatella, M.A. et al. (1995) Eur. J. Immunol. 25:1.