Enzyme Immunoassay for the Quantitative Determination of Alpha-Fetoprotein (AFP) in Human Serum

Product Description
Alpha-fetoprotein (AFP) is a glycoprotein with a molecular weigh of approximately 70,000 daltons. AFP is normally produced during fetal and neonatal development by the liver, yolksac, and in small concentrations by the gastrointestinal tract. After birth, serum AFP concentrations decrease rapidly, and by the second year of life and thereafter only trace amounts are normally detected in serum. Elevation of serum AFP to abnormally high values occurs in several malignant diseases, most notably nonseminomatous testicular cancer and primary hepatocellular carcinoma. In the case of nonseminomatous testicular cancer, a direct relationship has been observed between the incidence of elevated AFP levels and the stage of disease. Elevated AFP levels have also been observed in patients diagnosed with seminoma with nonseminomatous elements, but not in patients with pure seminoma.

In addition, elevated serum AFP concentrations have been measured in patients with other noncancerous diseases, including ataxia telangiectasia, hereditary tyrosinemia, neonatal hyperbilirubinemia, acute viral hepatitis, chronic active hepatitis, and cirrhosis. Elevated serum AFP concentrations are also observed in pregnant women. Therefore, AFP measurements are not recommended for use as a screening procedure to detect the presence of cancer in the general population.

Principle
The AFP ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay system utilizes a goat anti-AFP antibody directed against intact AFP for solid phase immobilization (on the microtiter wells).
A monoclonal anti-AFP antibody conjugated to horseradish peroxidase (HRP) is in the antibodyenzyme conjugate solution. The test sample is allowed to react first with the immobilized rabbit antibody for 30 minutes.
The wells are washed to remove any unbound antigen. The monoclonal-HRP conjugate is then reacted with the immobilized antigen for 30 minutes at room temperature resulting in the AFP molecules being sandwiched between the solid phase and enzyme-linked antibodies.
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 AFP is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450nm.

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