Principle of Assay
This MCP-3 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 MCP-3. Standards or samples are then added to the appropriate microtiter plate wells and incubated. MCP-3, if present, will bind and become immobilised by the antibody pre-coated on the wells. The microtiter plate wells are thoroughly washed to remove unbound MCP-3 and other components of sample. In order to quantitate the amount of MCP-3 present in the sample, a standardised preparation of horseradish peroxidase (HRP)-conjugated monoclonal antibody specific for MCP-3 is added to each well to "sandwich" the MCP-3 immobilized during the first incubation. The microtiter plate then undergoes a second incubation. The wells are thoroughly washed to remove all unbound HRP-conjugated antibodies 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 those wells that contain MCP-3 and enzyme-conjugated antibody 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 MCP-3 in the samples, this kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing.) According to the testing system, the provided standard is diluted (2-fold) with 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-3 concentration (pg/ml). The concentration of MCP-3 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
