CCL13, also known as Monocyte Chemoattractant Protein-4 (MCP-4), is a CC chemokine that acts as a chemoattractant for monocytes, eosinophils and T cells and as an activator of basophils. It signals through the CCR2 and CCR3 receptors. Hybridoma: Produced from a hybridoma resulting from the fsion of a mouse myeloma with B cells obtained from a mouse immunized with purified, E. coli-derived, recombinant human CCL13 (rhCCL13). Endotoxin Level: < 0.1 EU per 1ug of the antibody as determined by the LAL method. Applications: Neutralization of Bioactivity, Flow Cytometry, ELISA capture Recommended Dilution: Neutralization of Human CCL13 Bioactivity: The exact concentration of antibody required to neutralize human CCL13 activity is dependent on the cytokine concentration, cell type, growth conditions and the type of activity studied. The Neutralization Dose50 (ND50) for this antibody is defined as that concentration of antibody required to yield one-half maximal inhibition of the cytokine activity on a responsive cell line, when that cytokine is present at a concentration just high enough to elicit a maximum response. Flow Cytometry: This antibody was validated for flow cytometry using IL-1beta treated A549 cells. For intracellular staining to detect human CCL13, cells must first be fixed and permeabilized using 4% paraformaldehyde and 0.1% saponin in PBS. Dilute this antibody to 25ug/mL and add 10ul of the diluted solution to 1-2.5 x 10e5 cells in a total reaction volume not exceeding 200ul. The binding of unlabeled monoclonal antibodies may be visualized by adding 10ul of a 25ug/mL stock solution of a secondary developing reagent such as goat anti-mouse IgG conjugated to a fluorochrome. ELISA Capture: This antibody can be used as a capture antibody in a human CCL13 ELISA in combination with the biotinylated polyclonal detection antibody. A general protocol is provided on the next page. Using plates coated with 100ul/well of the capture antibody at 2ug/mL, in combination with 100ul/well of the detection antibody, an ELISA for sample volumes of 100ul can be obtained. To arrive at the optimal dose range for this ELISA, set up a two-fold dilution series of the protein standard starting with 0.5ng/ml. Optimal dilutions to be determined by the researcher. Storage and Stability: Lyophilized powder may be stored at 4C for short-term only. Reconstitute to nominal volume by adding sterile 40-50% glycerol and store at -20C. Reconstituted product is stable for 12 months at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
