ELISA Capture: The OX-81 antibody is useful as a capture antibody in a sandwich ELISA for measuring rat IL-4 protein levels in conjunction with biotinylated mouse anti-rat IL-4 monoclonal antibody (Cat. No. 555090) as detection antibody and recombinant rat IL-4 protein (Cat. No. 555107) as the standard. The OX-81 antibody should be pretitrated in the range of 1.0 to 4.0 µg/ml to determine its optimal concentration for ELISA. To obtain linear standard curves, doubling dilutions of recombinant rat IL-4 protein (e.g., ranging from ~5,120 to 5 pg/ml) are recommended for inclusion in each ELISA plate. For specific methodology, please visit our web site, www.bdbiosciences.com, and go to the protocols section or the chapter on ELISA in the Immune Function Handbook.
Note: This ELISA pair shows no cross-reactivity with any of the cytokines or chemokines tested (e.g., rat IL-2, IL-6, IL-10, GM-CSF, IFN-γ, TNF; human IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, 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-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).
Note: This ELISA pair is recommended primarily for measuring cytokine from experimental cell culture systems. These ELISA reagents are not recommended for assaying serum or plasma samples. For measuring rat IL-4 in serum or plasma our rat IL-4 BD OptEIA™ Set (Cat. No. 555198) is specially formulated and recommended.
Immunofluorescent Staining and Flow Cytometric Analysis: The PE conjugated OX-81 antibody (Cat. No. 555082) is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate IL-4 producing cells.
A useful control for demonstrating specificity of staining is either of the following: 1) pre-block the fluorochrome-conjugated OX-81 antibody with ligand (e.g., recombinant rat IL-4, Cat No. 555107) prior to staining, or 2) pre-block the fixed/permeabilized cells with unlabelled OX-81 antibody (Cat. No. 555080) prior to staining. The intracellular staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe.
Neutralization: This antibody can be used for in vitro or in vivo neutralization of rat IL-4 bioactivity. The no azide/low endotoxin (NA/LE) format of the OX-81 clone is recommended for in vitro or in vivo assays (Cat. No. 555078).