hCG is a one-step immunoassay, based on the principle of the sandwich method. The assay system utilizes high affinity and specificity monoclonal antibodies (enzyme conjugated and immobilized) directed against a distinct antigenic determinant on the beta sub unit of hCG molecule. The test sample is allowed to react simultaneously with the two antibodies, resulting in the hCG molecules being sandwiched between the solid phase and enzyme-linked antibodies. The unbound components are removed by washing. After addition of the solution containing TMB and hydrogen peroxide, the wells with bound conjugate develop a blue color which is converted to yellow after the reaction has been stopped with sulphuric acid. The color intensity is directly proportional to the concentration of hCG in the specimen and can be read at 450 nm.