This hCG 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 to hCG. Standards or samples are added to the microtiter plate wells and incubated. The plate wells are washed to remove any unbound standard or sample, hCG, if present, will bind to the antibody pre-coated on the wells. A standardized preparation of horseradish peroxidase (HRP)-conjugated monoclonal antibody specific to hCG is added to each well to “sandwich” the hCG 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-benzidene) substrate solution is added to each well. This enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain hCG 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 hCG in the sample, this 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 hCG concentration (mIU/mL). The concentration of hCG in the samples is then determined by comparing the O.D. of the samples to the standard curve.