Enzyme Immunoassay for the determination of Free Beta-Subunit of Human Chorionic Gonadotropin (Free β-hCG) in Human Serum

Product Description
Human Chorionic Gonadotropin (hCG) is a glycoprotein hormone normally produced by placenta during pregnancy. The hormone is present in blood and urine around seven to thirteen days following implantation of the fertilized ovum.
Structurally intact hCG molecules consist of two non-covalently linked polypeptide subunits, the alpha and beta chain subunits. Measurement of intact hCG and of the alpha subunit of hCG appears to give similar results in blood and urine but not the levels of beta subunit. In the normal second-trimester maternal sera, the level of intact hCG range from 20,000 mIU/ml to 50,000 mIU/ml (1ng = 15mIU).
In contrast, the levels of either free b or free β-hCG are on average one half of 1% of hCG levels. hCG and the free subunits appear not to be useful as serological markers for nontrophoblastic tumors; however, the absolute increase of β-hCG level in choriocarcinoma patients clearly differentiates it from normal pregnancy.
Recent studies showed a significant increase in the level of free β-hCG subunit in trisomy 21 cases as compared with controls. Hence, it has been suggested that free β-hCG subunit assay in a combination of maternal serum AFP could be effective in a screening protocol for trisomy 21.

Principle
The free β-hCG ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay system utilizes a unique monoclonal antibody directed against a distinct antigenic determinant on the b-subunit of the hCG molecule.
Mouse monoclonal anti-β-hCG antibody is used for solid phase immobilization (on the microtiter wells). A goat anti-β-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 β-hCG molecules being sandwiched between the solid phase and enzyme-linked antibodies. After incubation 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 β-hCG is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450nm.

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