Principle of Assay
This ultra-sensitive TSH 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 TSH. Standards or samples are then added to the microtiter plate wells and TSH, if present, will bind to the antibody pre-coated on the wells. In order to quantitate the amount of TSH present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated monoclonal antibody, specific for TSH are added to each well to “sandwich” the TSH immobilized on the plate. The microtiter plate undergoes incubation, and then the wells are thoroughly washed to remove all unbound components. Next, 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 TSH 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.
In order to measure the concentration of TSH in the sample this Ultra-sensitive Human TSH ELISA Kit includes a set of calibration standards (6 standards). The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density (O.D.) versus TSH concentration (mIU/mL). The concentration of TSH in the samples is then determined by comparing the O.D. of the samples to the standard curve.
