ELISA: The purified 2H5 antibody (Cat. No. 551217) is useful as a capture antibody for a sandwich ELISA for measuring mouse MCP-1 protein levels. Purified 2H5 antibody can be paired with the biotinylated 4E2/MCP antibody (Cat. No. 554444) as the detecting antibody, with recombinant mouse MCP-1 (Cat. No. 554590) as the standard. Purified 2H5 antibody should be titrated 6 - 10 µg/ml to determine optimal concentration for ELISA capture. To obtain linear standard curves, doubling dilutions of mouse MCP-1 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. For specific methodology, please visit the protocols section of the Immune Function Handbook, which is also posted on our web site, www.bdbiosciences.com.
Note 1: This reagent is recommended primarily for assay of cytokine from experimental cell culture systems. For detection of MCP-1 in mouse serum or plasma samples, the BD OptEIA™ Mouse MCP-1 ELISA Set is recommended.
Note 2: 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, TCA-3, 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, TNF-β, VEGF; rat IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF).
Neutralization: The NA/LE™ 2H5 antibody (Cat. No. 554440) is useful for neutralization of mouse MCP-1 bioactivity. A suitable NA/LE™ hamster IgG isotype-matched control is the G235-2356 antibody (Cat. No. 554709).
Immunofluorescent Staining and Flow Cytometric Analysis: The 2H5 antibody is useful for immunofluorescent staining and flow cytometric analysis to identify and enumerate MCP-1 producing cells within mixed cell populations. PE conjugated 2H5 antibody (Cat. No. 554443) is especially suitable for these experiments. The use of a specificity control, such as recombinant mouse MCP-1 (Cat No. 554590) or unlabeled 2H5 antibody (Cat. No. 554441) is recommended. For specific methodology, please visit the protocols section or the Immune Function Handbook, which is also posted on our web site, www.bdbiosciences.com.