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Quantitation of human IL-12 heterodimer and p40
R&D
L-12, a cytokine produced by macrophages
and B lympho-
F&e 1. IL-12 and pd@ Production by NC37 cells and Human Monocytes
cytes, has multiple effects on T-cells and NK cells, including stimulating cytotoxic activity, proliferation, promotion of Thl development
SYSTEMS
Cell
and IFN-y production? * IL-12 is a disul-
Activation Conditions
fide-linked, 70 kDa (~70) heterodimeric glycoprotein composed of a 40-kDa (~40) subunit and a 35-kDa (~35) subunit. The p40 and ~35
IL-12 p40/1L-I2 ratio P4B (PgimL) (PglmL)
None
<5 9
36
>7
5203
577
None
es
F
>I
IFN-y
131
>26
LPS
<5 <5
324
expressing only p35 or both p35 and p40 mRNAs.’ 1.1contrast, ~40
IFN-y + LPS SAC
18,565 97
68,200 3981
3.7 41
is secreted in excess of IL-12 in cells expressing both p35 and p40
IFN-y + SAC
2,535
20,640
8
NC 37
subunits by themselves have no IL-12 activity, but the p40 dimer
PMA+A23
binds to the IL-12 receptor and is an IL-12 antagonist”,4 in z~itro. However, most of the p40 secreted by various cell lines has been
Monocytes
reported to exist as monomers.’ Free ~35 has not been detected in supernatants
of cultured cells
I87
I
>648
mRNAs.5 This presents an analytical problem for either an IL-12 immunoassay, which may cross react with dimeric or monomeric ~40, or a bioassay, which may be antagonized by dimeric ~40. We show here that the heterodimeric IL-12 (~70) can be accurately quantitated
in the presence of excess p40 using R&D Systems’
Quantikine
IL-12 ELISA kit. The IL-12 level measured with the
~40 but not heterodimeric
esults and Discussion
Quantikine kit was also shown to correlate well with the IL-12 level measured using bioassays.
IL-12. An insect cell expressed recombi-
nant preparation of monomeric p40 was used as the calibrator. Results in Table 1 are a mean of two determinations.
The concentration
of secreted p40 can be several hundred-fold
higher than that of IL-12 (Table 1). In agreement with previous reports,8,9 production of p40 and IL12 by monocytes was optimally induced by a combination of IFN-y and LPS, and levels of IL-12 Cells Human EBV-transformed in RPM1 supplemented
lymphoblastoid
cells (NC37) were grown
with 10% FCS. Human monocytes were pre-
pared from buffy coat cell= by size sedimentation.”
The resulting
cells were 65% monocytes, 21% lymphocytes and 12 % neutrophils. NC37 cells (1~10~ cells/mC) were stimulated for 24 hrs at 37’C with 10 ng/mL
PMA and 25 ng/mL
Monocytes (1~10~ cells/ml
calcium ionophore A23187.
in RPM1 1640 supplemented
with 5%
FCS) were cultured overnight with or without recombinant human IFN-y (lOOng/mL) and then 24 hr with or without addition of Ll’S (lmg/mL)
lower than those reported by D’Andrea et ~1.‘~who used a doubledeterminant IL-12 RIA with significant cross-reactivity while levels of p40 are consistent with theirs. We
measured
IL-12
bioactivity
with
of IL-12 was measured with a Quantikine IL-12
with ~40,
PHA-activated
T-lymphoblasts as the responding cells.’ In media conditioned by IFN-y + LPS and IFN-y + SAC, we measured lB-23 ng/mL and 3-S ng/mL IL-12, respectively, in agreement with the levels measured by immunoassay. The IL-12 bioactivity could be neutralized by anti-IL-12 neutralizing antibody. In R&D Systems’ Quantikine IL-12 and Quantikine
or SAC (0.0074% wt/vol, Pansorbin).
II_-I2 Assays The immunoactivity
agree with those reported by Cassatella et nl.’ In contrast, levels of IL-12 from NC-37 and SAC-stimulated monocytes are considerably
HS IL-12
Immunoassay kits, the monoclonal capture antibody is completely specific for heterodimeric IL-12, with no reactivity with free ~40.
IL-12. The p40 subunit was measured using the Quantikine IL-12
The high levels of IL-12 reported in some cases may, therefore, IFpresent an overestimation from use of antibodies that cross-react
detection antibody and a microtiter plate coated with a monoclonal
with ~40. Because of the excess p40 in many situations, lL-12 levels
antibody (clone U31052.1, at 4 trg/mL) that specifically captures free
can be measured accurately only if the ELISA is highly specific.
immunoassay
kit (Cat. # D1200) specific for only the heterodimeric
References 1. Wolf, 5. F. c, n/. (1994) Smn C,G
5. b.
3. 4.
12,15¶-168 Zeh, H. 1. rt sf. (1994) in The Cytokine Handbook, 2nd edition, Academic Press, New York, pp 234-256 Gillessen, S. etnl. (1995) Esr. /, bw~~rnol.25, 2Oa-206 Ling. P. et al. (1995) /. btma~~of. 1% 116-127
For
more
2.
BeI@
information
please
0800 IO 468 Damnartc8001
caH:
U&O 123555
I IO3
85 92. Deutxhla,,nd:0,30
Thomson. A. editor.
7. 8. 9.
D’Andra, A. ptui. (lY92) j. Exp. Mrd. 176.1387-33Y8 Wahl, I. M. and l? LX Smith (1995) in Current Protocols in Immunology, Ct%Jn+. 1. c’t0’. editors, Wiley Interscience, p 7.6.2 Gately, M. K. et nf. (1995) in Current t’rotocok in bnmunotogy, Co&an\. I. ct ~11. cdltw. Wiley Interscience, p 6.16.3 Hays M. I’. cf nl. (1995) Blood86.646650 Cassatella, M.A. ct RI. (1995) Eur. /. Inwwnol. 25.1-5
USA: I-800-343-7475
I IOlbY France:05 9072 49 Nederlamk060
225607. Noge:8CO / 1033 Sver@:O2079
Enter number E9 on RES card or use fax form
3! 49 Switzerland: I58 2492
L-12, a cytokine produced by macrophages
and B lympho-
F&e 1. IL-12 and pd@ Production by NC37 cells and Human Monocytes
cytes, has multiple effects on T-cells and NK cells, including stimulating cytotoxic activity, proliferation, promotion of Thl development
SYSTEMS
Cell
and IFN-y production? * IL-12 is a disul-
Activation Conditions
fide-linked, 70 kDa (~70) heterodimeric glycoprotein composed of a 40-kDa (~40) subunit and a 35-kDa (~35) subunit. The p40 and ~35
IL-12 p40/1L-I2 ratio P4B (PgimL) (PglmL)
None
<5 9
36
>7
5203
577
None
es
F
>I
IFN-y
131
>26
LPS
<5 <5
324
expressing only p35 or both p35 and p40 mRNAs.’ 1.1contrast, ~40
IFN-y + LPS SAC
18,565 97
68,200 3981
3.7 41
is secreted in excess of IL-12 in cells expressing both p35 and p40
IFN-y + SAC
2,535
20,640
8
NC 37
subunits by themselves have no IL-12 activity, but the p40 dimer
PMA+A23
binds to the IL-12 receptor and is an IL-12 antagonist”,4 in z~itro. However, most of the p40 secreted by various cell lines has been
Monocytes
reported to exist as monomers.’ Free ~35 has not been detected in supernatants
of cultured cells
I87
I
>648
mRNAs.5 This presents an analytical problem for either an IL-12 immunoassay, which may cross react with dimeric or monomeric ~40, or a bioassay, which may be antagonized by dimeric ~40. We show here that the heterodimeric IL-12 (~70) can be accurately quantitated
in the presence of excess p40 using R&D Systems’
Quantikine
IL-12 ELISA kit. The IL-12 level measured with the
~40 but not heterodimeric
esults and Discussion
Quantikine kit was also shown to correlate well with the IL-12 level measured using bioassays.
IL-12. An insect cell expressed recombi-
nant preparation of monomeric p40 was used as the calibrator. Results in Table 1 are a mean of two determinations.
The concentration
of secreted p40 can be several hundred-fold
higher than that of IL-12 (Table 1). In agreement with previous reports,8,9 production of p40 and IL12 by monocytes was optimally induced by a combination of IFN-y and LPS, and levels of IL-12 Cells Human EBV-transformed in RPM1 supplemented
lymphoblastoid
cells (NC37) were grown
with 10% FCS. Human monocytes were pre-
pared from buffy coat cell= by size sedimentation.”
The resulting
cells were 65% monocytes, 21% lymphocytes and 12 % neutrophils. NC37 cells (1~10~ cells/mC) were stimulated for 24 hrs at 37’C with 10 ng/mL
PMA and 25 ng/mL
Monocytes (1~10~ cells/ml
calcium ionophore A23187.
in RPM1 1640 supplemented
with 5%
FCS) were cultured overnight with or without recombinant human IFN-y (lOOng/mL) and then 24 hr with or without addition of Ll’S (lmg/mL)
lower than those reported by D’Andrea et ~1.‘~who used a doubledeterminant IL-12 RIA with significant cross-reactivity while levels of p40 are consistent with theirs. We
measured
IL-12
bioactivity
with
of IL-12 was measured with a Quantikine IL-12
with ~40,
PHA-activated
T-lymphoblasts as the responding cells.’ In media conditioned by IFN-y + LPS and IFN-y + SAC, we measured lB-23 ng/mL and 3-S ng/mL IL-12, respectively, in agreement with the levels measured by immunoassay. The IL-12 bioactivity could be neutralized by anti-IL-12 neutralizing antibody. In R&D Systems’ Quantikine IL-12 and Quantikine
or SAC (0.0074% wt/vol, Pansorbin).
II_-I2 Assays The immunoactivity
agree with those reported by Cassatella et nl.’ In contrast, levels of IL-12 from NC-37 and SAC-stimulated monocytes are considerably
HS IL-12
Immunoassay kits, the monoclonal capture antibody is completely specific for heterodimeric IL-12, with no reactivity with free ~40.
IL-12. The p40 subunit was measured using the Quantikine IL-12
The high levels of IL-12 reported in some cases may, therefore, IFpresent an overestimation from use of antibodies that cross-react
detection antibody and a microtiter plate coated with a monoclonal
with ~40. Because of the excess p40 in many situations, lL-12 levels
antibody (clone U31052.1, at 4 trg/mL) that specifically captures free
can be measured accurately only if the ELISA is highly specific.
immunoassay
kit (Cat. # D1200) specific for only the heterodimeric
References 1. Wolf, 5. F. c, n/. (1994) Smn C,G
5. b.
3. 4.
12,15¶-168 Zeh, H. 1. rt sf. (1994) in The Cytokine Handbook, 2nd edition, Academic Press, New York, pp 234-256 Gillessen, S. etnl. (1995) Esr. /, bw~~rnol.25, 2Oa-206 Ling. P. et al. (1995) /. btma~~of. 1% 116-127
For
more
2.
BeI@
information
please
0800 IO 468 Damnartc8001
caH:
U&O 123555
I IO3
85 92. Deutxhla,,nd:0,30
Thomson. A. editor.
7. 8. 9.
D’Andra, A. ptui. (lY92) j. Exp. Mrd. 176.1387-33Y8 Wahl, I. M. and l? LX Smith (1995) in Current Protocols in Immunology, Ct%Jn+. 1. c’t0’. editors, Wiley Interscience, p 7.6.2 Gately, M. K. et nf. (1995) in Current t’rotocok in bnmunotogy, Co&an\. I. ct ~11. cdltw. Wiley Interscience, p 6.16.3 Hays M. I’. cf nl. (1995) Blood86.646650 Cassatella, M.A. ct RI. (1995) Eur. /. Inwwnol. 25.1-5
USA: I-800-343-7475
I IOlbY France:05 9072 49 Nederlamk060
225607. Noge:8CO / 1033 Sver@:O2079
Enter number E9 on RES card or use fax form
3! 49 Switzerland: I58 2492