Description
An enzyme immunoassay for the quantitative determination of free beta subunit of human chorionic gonadotropin (free b-hCG) concentration in serum.
The free b-hCG ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay.[i] The assay system utilizes a unique monoclonal antibody directed against a distinct antigenic determinant on the free b-hCG. Mouse monoclonal anti-free-b-hCG antibody is used for solid phase immobilization (on the microtiter wells). A goat anti whole hCG antibody is in the antibody-enzyme (horseradish peroxidase) conjugate solution. The test sample is allowed to react sequentially with the two antibodies, resulting in
the free b-hCG molecules being sandwiched between the solid phase and enzyme-linked antibodies. After two separate 30 minute incubations at 37 °C, the wells are washed with water to remove unbound labeled antibodies. A solution of TMB Reagent is added and incubated for 20 minutes, resulting in the development of a blue color. The color development is stopped with the addition of Stop Solution changing the color to yellow. The concentration of b-hCG is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.
