Preparation And Storage
Recommended Assay Procedures
ELISA Capture: IL-12 p40 ELISA: The purified C15.6 antibody is useful as a capture antibody for a sandwich ELISA measuring the total amount of p40 in solution (complexed as homodimer or heterodimer, as well as free monomer). Purified C15.6 antibody can be paired with the biotinylated C17.8 antibody (Cat. No. 554476) as the detecting antibody, with recombinant mouse IL-12 p70 protein (Cat. No. 554592; see figure) or IL-12 p40 protein (Cat. No. 554594; data not shown) as the standard. The purified C15.6 antibody should be titrated 6-10 µg/ml to determine optimal concentration for ELISA capture. To obtain linear standard curves, doubling dilutions of mouse IL-12 protein ranging from ~4,000 to 30 pg/ml are recommended for inclusion in each ELISA plate. For maximal sensitivity, an overnight incubation (4°C) of samples/standards with the coated capture antibody is recommended.
Note: For testing mouse IL-12 p40 in complex biological fluids such as serum or plasma, the BD OptEIA™ mouse IL-12 p40 ELISA Set is recommended (Cat. No. 555165). For testing mouse IL-12 p70 in serum or plasma BD OptEIA™ mouse IL-12 p70 ELISA Set is recommended (Cat. No. 555256).
Blocking Control for Intracellular Staining: The purified C15.6 antibody can be used as a blocking control to demonstrate specificity of IL-12 staining by directly-conjugated C15.6. To perform this control, the fixed/permeabilized cells (~1 million) can be incubated with 1-10 µg of purified C15.6 antibody for 20 minutes at 4°C, prior to staining with directly-conjugated C15.6 (e.g., 0.1 - 0.5 µg mAb/1 million cells). The intracellular cytokine staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or the chapter on intracellular staining and flow cytometry in the Immune Function Handbook.
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.
The C15.6 monoclonal antibody specifically binds to both free and complexed (homodimer p80 and heterodimer p70) forms of the p40 subunit of mouse interleukin-12 (IL-12). The immunogen used to generate the C15.6 hybridoma was recombinant mouse IL-12 p70 protein. p40 has also been described as a subunit of IL-23 and thus it is possible that the C15.6 antibody will crossreact with IL-23.
This antibody is routinely tested by ELISA capture. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.
Development References (4)
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Gately MK, Chizzonite R, Presky DH. Measurement of Human and Mouse Interleukin-12. In: Cooligan J, Kruisbeek A, Margulies D, Shevach E, Storber W, ed. Current Protocols in Immunology. New York: John Wiley and Sons; 1995:6-16.
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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. (Biology). View Reference
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Prussin C, Metcalfe DD. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods. 1995; 188(1):117-128. (Methodology: IC/FCM Block). View Reference
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Wysocka M, Kubin M, Vieira LQ, et al. Interleukin-12 is required for interferon-gamma production and lethality in lipopolysaccharide-induced shock in mice. Eur J Immunol. 1995; 25(3):672-676. (Clone-specific: ELISA). View Reference
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