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.
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.
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