AFP

GWB-F49883

Alpha Fetoprotein (AFP) is a 68 kDa glycoprotein which is normally only produced in the fetus during its development. It is a normally produced by the liver and yolk sac of the fetus. AFP levels decrease soon after birth and probably has no function in normal adults. It binds the hormone estradiol to keep it from affecting the fetal brain. Its measurement during pregnancy has been useful to detect certain abnormalities - specifically if high levels of AFP are found in amniotic fluid it can indicate a developmental defect in the baby. In some patients who are not pregnant a tumor can produce AFP thus it can be used as a tumour marker. AFP is the main tumour marker (along with HCG) to diagnose testicular cancer and its values over time can have significant effect on the treatment plan. Like all tumour markers the detection of AFP by itself is not diagnostic of anything although if it is detected it is certainly advisable to rule out the diseases could cause levels to rise. The primary reason tumor markers are used are to measure the success of a treatment (e. g. chemotherapy) if levels of AFP are going down it is an indication that a disease is improving. New research exhibits that an isoform of AFP which binds Lens culinaris agglutinin (AFP-L3) can be particularly useful in early identification of aggressive tumors associated with hepatocellular carcinoma (HCC). Principles of the assay: The AFP assay is based on simultaneous binding of human AFP to two monoclonal antibodies; one is immobilized on the microplate the other is soluble and conjugated with horseradish peroxidase (HRP). Microtiter strip wells are precoated with anti-AFP IgG antibodies. AFP in samples and standards binds to the immobilised antibodies on the surface of the microtiter wells and the second soluble anti-AFP antibody-enzyme conjugate binds to the immobile antibody-AFP-complex during the first incubation. Afterwards a bound/free separation is performed by solid-phase washing. The immune complex is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this product is proportional to the amount of AFP in samples and standards. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorption at 450 nm is read using an ELISA microwell plate reader. Storage and Stability: The reagents are stable up to the expiry date stated on the label when stored at 2. . . 8 & deg; C. After first use the standard solutions are still stabile for another 6 months if stored at 2?8 & deg; C. Limitations of the Test: Sample(s) which are contaminated microbiologically should not be used in the assay. Highly lipemeic or haemolysed specimen(s) should similarly not be used. It is important that the time of reaction in each well is held constant for reproducible results. Pipetting of samples should not extend beyond ten minutes to avoid assay drift. If more than one plate is used it is recommended to repeat the dose response curve. Addition of the substrate solution initiates a kinetic reaction which is terminated by the addition of the stop solution. Therefore the addition of the substrate and the stopping solution should be added in the same sequence to eliminate any time deviation during reaction. Plate readers measure vertically. Do not touch the bottom of the wells. Failure to remove adhering solution adequately in the aspiration or decantation wash step(s) may result in poor replication and spurious results. References Wisdom G. B. (1976) Clin. Chem. 22/8 1243 - 1255Shome B. and Parlow A. F. (1974) J. Clin. Endocr. Metab. 39 199 ? 202Uotila M. Ruoslahti E. Engvall E. (1981) J. Immunol. Methds 42 11-15Acosta A. A. M. D. and Wright G. L. (1983) J. of Clinical Immunoassays 6 41Jacobsen G. K. (1983) Acta Path Microb. Immun. Scand. 91 183 ? 190Kohn J. Oee A. H. McElwain T. J. (1976) Lancet 2 433 ? 436Waaldmann T. A. and McIntire K. R. (1974) Cancer 34 1510 - 1515