Mouse anti human mcp 1 antibody

A 96-well plate (Nunc ‘Maxisorp’, Thermo Fisher Scientific) was coated with 100 μl of an affinity-purified mouse anti-human MCP-1 antibody (R&D Systems, Abingdon, UK) at a concentration of 2 μg/ml diluted in PBS. The plate was covered with an adhesive strip (Appleton Woods, Birmingham, UK) and placed at 4^oC overnight to allow adsorption of antibody to the walls of the microplate. Recombinant human MCP-1 (R&D Systems) was used to create a reference concentration series to form a 9-point standard series (9.76–2500 pg/ml). A ‘blank’ sample comprising diluent alone was also included. Following washing with buffer, an affinity-purified biotinylated polyclonal goat anti-human MCP-1 antibody (100 ng/ml; R&D systems, Minneapolis, MN, USA) was added and signal amplification performed with streptavidin-HRP conjugate. MCP-1 intensity was measured by absorbance (450 nm) in the presence of 3,3′,5,5′-tetramethylbenzidine (TMB; Cambridge Bioscience, Cambridge, UK) in an ELx800 spectrophotometric microplate reader (BioTek, Winooski, VT, USA).