This mouse MCP-1 enzyme-linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for mouse MCP-1. Standards or samples are then added to the appropriate microtiter plate wells and incubated. Mouse MCP-1, if present, will bind and become immobilized by the antibody pre-coated on the wells. A biotin conjugated antibody specific for mouse MCP-1 is added to each well. The biotin conjugated antibody will bind to the mouse MCP-1 on the plate. The microtiter plate wells are thoroughly washed to remove unbound biotin conjugate and other components of the sample. Avidin has a very high affinity to biotin. In order to quantify the amount of mouse MCP-1 present in the sample, a standardized preparation of avidin conjugated horseradish peroxidase (HRP) is added to each well. Avidin-HRP will bind to the biotin on plate during incubation. The wells are thoroughly washed to remove all unbound Avidin-HRP conjugate and a TMB(3,3'5,5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only wells to which mouse MCP-1, Biotin conjugate and Avidin-HRP are attached will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.

In order to measure the concentration of mouse MCP-1 in the samples, this kit contains two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant/ urine testing). According to the testing system, the provided standard is diluted (2-fold) with the appropriate Calibrator Diluent and assayed at the same time as the samples. This allows the operator to produce a standard curve of Optical Density (O.D.) versus MCP-1 concentration (pg/mL). The concentration of MCP-1 in the samples is then determined by comparing the O.D. of the samples to the standard curve.