Alpha Fetoprotein ELISA - (AFP)

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

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES

SUMMARY
Alpha-fetoprotein (AFP, also sometimes called alpha-1-fetoprotein, alpha-fetoglobulin, or alpha fetal protein) is a protein that in humans is encoded by the AFP gene. The AFP gene is located on the q arm of chromosome 4 (4q25). AFP is a major plasma protein produced by the yolk sac and the liver during fetal development. It is thought to be the fetal form of serum albumin. AFP binds to copper, nickel, fatty acids and bilirubin and is found in monomeric, dimeric and trimeric forms.

PRINCIPLE OF THE TEST
The AFP ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay system utilizes a rabbit 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 antibody-enzyme 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 450 nm.

EXAMPLE OF STANDARD CURVE
Results of a typical standard run with optical density readings at 450 nm shown in the Y axis against Alpha Fetoprotein concentrations shown in the X axis. This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and standard curve.