Principle of Assay
The Yes Biotech Neonatal TSH quantitative enzyme immunoassay described as a solid phase enzyme linked immunosorbent assay (ELISA). Monoclonal antibodies, specific to TSH, have been bound to the surface of each microplate well. During the course of the assay, a blood sample (collected on filter paper) is added to the microplate wells with Sample Buffer and incubated overnight. After washing the microplate to remove the filter paper and unbound component of the sample, a standardized preparation of horseradish peroxidase-conjugated monoclonal antibody specific for TSH b unit is added to each well and incubated. The TSH, if present in the sample, will bind to the antibody on the coated well and will form an Antibody-TSH-Antibody-HRP “sandwich”. The microplate wells are thoroughly washed to remove unbound conjugate.
Next, a TMB (3,3', 5,5' tetramthyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a 15-minute 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 ± 2 nm.
In order to measure the concentration of TSH in the test sample, this neonatal TSH ELISA Kit includes calibration standards and controls. The calibration standards and controls are assayed at the same time as the test samples and allow for the operator to produce a standard curve of optical density versus TSH mIU/mL, serum. Therefore, by comparing the optical density of the samples to this standard curve, the concentration of the TSH in the test samples is then determined.
