Preparation And Storage
Recommended Assay Procedures
ELISA Detection (IL-12 p70 protein ELISA): The biotinylated C8.6 antibody (Cat. No. 554660) is useful as a detection antibody for a sandwich ELISA for specifically measuring human IL-12 p70 protein levels. Biotinylated C8.6 antibody can be paired with the purified rat anti-human IL-12 p70 specific 20C2 (Cat. No. 555065) as the capture antibody and recombinant human IL-12 p70 protein (Cat. No. 554613) as the standard. Biotinylated C8.6 antibody should be titrated (0.5 - 2.0 µg/ml) to determine optimal concentration for ELISA detection. To obtain linear standard curves, doubling dilutions of human IL-12 p70 ranging from ~2000 to 15 pg/ml are recommended for inclusion in each ELISA plate. For testing IL-12 p70 in serum or plasma BD OptEIA™ Set (Cat. No. 555183) or BD OptEIA™ Kit (Cat. No. 559258) is recommended.
Note 1: This ELISA for measuring human IL-12 p70 protein levels is not interfered with by a 50-fold excess (by mass) of the human p40 monomer. This assay is specific for measuring human IL-12 p70 protein. It has been reported that activated PBMC produce an excess of free p40 protein over the p70 heterodimer.
ELISA Detection (IL-12 p40 protein ELISA): The biotinylated C8.6 antibody (Cat. No. 554660) is useful as a detection antibody for specifically measuring human IL-12 p40 protein levels, with no reactivity to human IL-12 p70 protein. Biotinylated C8.6 antibody can be paired with the purified (exclusively p40 specific) C8.3 antibody (Cat. No. 551227) as the capture antibody with recombinant human IL-12 p40 (Cat. No. 554633) as the standard. For testing IL-12 (p40) in complex biological fluids like serum or plasma BD OptEIA™ Set (Cat. No. 555171) or BD OptEIA™ Kit (Cat. No. 551116) is recommended.
Note 2: These ELISA pairs show no cross-reactivity with any of the cytokines or chemokines tested (e.g., human IL-1α, IL-1β, IL-2, IL-3, IL-4. IL-5, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-13, IL-15, G-CSF, GM-CSF, IFN-γ, lymphotactin, MCP-1, MCP-2, MIP-1α, MIP-1β, NT-3, PDGF-AA, sCD23, SCF, TNF, Lt-α, VEGF; mouse IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 p70, IL-15, GM-CSF, IFN-γ, MCP-1, TCA-3, TNF; rat IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF).
Neutralization: The C8.6 antibody has been reported to be useful for neutralization of human IL-12 p70 bioactivity. A formulation that does not contain sodium azide formulation (NA/LE™) is available (Cat. No. 554659) and is recommended for functional assay. This application is not routinely tested at BD Biosciences Pharmingen.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
Companion Products
The C8.6 antibody reacts with human IL-12 p40 monomers, homodimers, and p70 heterodimers, but not p35 subunits. The immunogen used to generate the C8.6 hybridoma was the purified p40 subunit of human IL-12. The C8.6 antibody is a neutralizing antibody and has been reported to strongly inhibit three different biological activities of IL-12 (i.e., IFN-γ induction), mitogenic effects on PHA blasts, and enhancement of NK cell-mediated cytotoxicity. C8.6 has been shown to immunoprecipitate IL-23.
This antibody is routinely tested by ELISA as a detection antibody. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.
Development References (3)
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D'Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M, Trinchieri G. Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med. 1993; 178(3):1041-1048. (Clone-specific: Neutralization). View Reference
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D'Andrea A, Rengaraju M, Valiante NM, et al. Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells. J Exp Med. 1992; 176(5):1387-1398. (Clone-specific). View Reference
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Oppmann B, Lesley R, Blom B, et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.. Immunity. 2000; 13(5):715-25. (Clone-specific: Immunoprecipitation). View Reference
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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.