The C11.5 antibody reacts with human IL-12 p40 monomer and p70 heterodimer, but not p35 monomer. The immunogen used to generate the C11.5 hybridoma was the CHO-expressed recombinant human IL-12 p70 heterodimer. p40 has also been described as a subunit of IL-23 and thus it is possible that the C11.5 antibody will crossreact with IL-23. This antibody is routinely tested by flow cytometric analysis. Other applications were tested during antibody development only or reported in the literature. Expression of IL-12 p40/p70 by activated CD14+ human PBMCs. Ficoll-separated human PBMCs were primed for 2 hrs with recombinant human IFN-γ (10 ng/mL, Cat. No. 554616), then activated with IFN-γ (10 ng/mL) and LPS (100 ng/mL, Sigma) in the presence of 2 μM GolgiStop™ (Cat. No. 554724) for an additional 22 hr. Cells were harvested, stained with FITC-mouse anti human CD14 antibody (Cat. No. 555397), fixed, permeabilized, and then stained with 0.125 μg of PE-C11.5 antibody (Cat. No. 554575), following Pharmingen's staining protocol (left panel). The data reflect gating on monocytes, based on forward and side scattered light signals. To demonstrate specificity of staining, the binding of the PE-C11.5 antibody was blocked by preincubation of the fixed/permeabilized cells with the unlabeled C11.5 antibody (5 μg, Cat. No. 554573) prior to staining (right panel). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
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