Technical Notes: Human IL-12 Immunoassay

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
Cells (1) Activation Conditions (2) IL-12 (3) p40 (4) p40/IL-12
pg/mL ratio
NC37 None <5 36 >7
PMA + A23187 9 5203 578
Monocytes None <5 5 >1
IFN-gamma <5 131 >26
LPS 5 3241 >648
IFN-gamma + LPS 18,565 68,200 3.7
SAC 97 3981 41
IFN-gamma + SAC 2,535 20,640 8
  1. 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.
  2. 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).
  3. The immunoactivity of IL-12 was measured with a Quantikine IL-12 immunoassay kit (Catalog # D1200) specific for only the heterodimeric IL-12.
  4. 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.

References

  1. Wolf, S.F. et al. (1994) Stem Cells 12:154.
  2. Zeh, H.J. et al. (1994) in The Cytokine Handbook, 2nd edition, Thomson, A. editor, Academic Press, New York, p 239.
  3. Gillessen, S. et al. (1995) Eur. J. Immunol. 25:200.
  4. Ling, P. et al. (1995) J. Immunol. 154:116.
  5. D’Andrea, A. et al. (1992) J. Exp. Med. 176:1387.
  6. Wahl, L.M. and P. D. Smith (1995) in Current Protocols in Immunology, Coligans, J. et al. editors, Wiley Interscience, p. 7.6.2.
  7. Gately, M.K. et al. (1995) in Current Protocols in Immunology, Coligans, J. et al. editors, Wiley Interscience, p. 6.16.3.
  8. Hayes, M.P. et al. (1995) Blood, 86:646.
  9. Cassatella, M.A. et al. (1995) Eur. J. Immunol. 25:1.