ELISA Capture: The purified MQ2-13A5 antibody (Cat. No. 554543) is useful as a capture antibody for a sandwich ELISA for measuring human IL-6 protein levels. Purified MQ2-13A5 antibody can be paired with biotinylated MQ2-Human IL-6 39C3 antibody (Cat. No. 554546) as the detecting antibody, with recombinant human IL-6 (Cat. No. 550071) as the standard. Purified MQ2-13A5 antibody should be titrated 1-4 µg/ml to determine optimal concentration for ELISA capture. To obtain linear standard curves, doubling dilutions of human IL-6 ranging from ~2,000 to 15 pg/ml are recommended for inclusion in each ELISA plate. For specific methodology, please visit the protocols section or chapter on ELISA in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com.
Note 1: This ELISA pair shows no cross-reactivity with any of the cytokines tested (e.g., 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; human IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 p70, IL-12 p40, 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-β, VEGF; rat IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF).
Note 2: This ELISA pair is recommended primarily for measuring cytokine from experimental cell culture systems. These ELISA reagents are not recommended for assay of serum samples. The BD OptEIA™ ELISA Set (Cat. No. 555220) and Kit (Cat. No. 550799) are specially-formulated for serum cytokine measurement.
1. Blocking Control for Intracellular Staining: The purified MQ2-13A5 antibody can be used as a blocking control to demonstrate specificity of
IL-6 staining by the FITC-MQ2-13A5 antibody (Cat. No. 554544) or the PE-MQ2-13A5 antibody (Cat. No. 554545). To perform this control, the fixed/permeabilized cells (~ 1 million) can be incubated with 1-10 µg of purified MQ2-13A5 antibody (Cat. No. 554543) for 20 minutes at 4°C, prior to staining with the conjugated antibody (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.
2. IF/Flow: The MQ2-13A5 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-6 producing cells within mixed cell populations. The directly- conjugated MQ2-13A5 antibodies are especially suitable for these studies. For specific methodology, please visit the protocols section or chapter on intracellular staining in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com.
3. Western Blot: The MQ2-13A5 antibody (Cat. No. 554543) has been found useful for Western blot. Please note that this application is not routinely tested at BD Biosciences.
3. Neutralization: The NA/LE formulation of the MQ2-13A5 antibody (Cat. No. 554541) is useful for neutralization of human IL-6 bioactivity.