Alpha-fetoprotein (AFP) is a glycoprotein of 590aa containing 3.4% carbohydrate content with a molecular weight of 61-75kD. AFP is normally produced in the developing embryo and fetus by the fetal yolk sac, the fetal gastrointestinal tract, and eventually by the fetal liver. In humans, AFP levels decrease gradually after birth, reaching adult levels by 8 to 12 months. Normal adult AFP levels are low and AFP has no known function in normal adults. The biologic role of AFP has not been defined yet. Because of its biochemical similarity to albumin, it has been postulated that AFP could be a carrier protein. It may have an immunoregulatory function during pregnancy. Increased serum levels are found in some tumors, such as hepatocellular carcinoma (HCC), hepatoblastoma, and germ cell tumors. Although total AFP is a useful serological marker for diagnosis of HCC, the false-negative or positive rate with AFP level is very high. AFP-L3, an isoform of AFP which binds Lens culinaris agglutinin, can be particularly useful in early identification of aggressive tumors associated with HCC. AFP mRNA, the circulating genetic markers, also has been used in monitoring distal metastasis or postoperative recurrence of HCC.
Intended Use
Human Alpha Fetoprotein (human AFP) ELISA kit is to be used for the in vitro quantitative determination of human AFP in human serum, human plasma, cell lysate and buffered solution. The assay will recognize both native and recombinant human AFP. This kit has been configured for research use only and is not to be used in diagnostic procedures.
Sensitivity: |The minimal detectable dose of human AFP was calculated to be 1.82ng/ml, by subtracting two standard deviations from the mean of 12 zero standard replicates (ELISA buffer, S0) and intersecting this value with the standard curve obtained in the same calculation.
Specificity
The following substances were tested and found to have no cross-reactivity: human serum albumin, human prostate specific antigen(PSA), human carcinoembryonic antigen(CEA), human neuron specific enolase(NSE), human Vitamin D binding protein(VDBP), Hemoglobin
Test Principle
The design of this assay is based on a sandwich Enzyme-Linked Immunosorbent Assay (ELISA). The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific to human AFP. Samples are pippetted into these wells. Nonbound AFP and other components of the sample should be removed by washing, then biotin-conjugated monoclonal antibody specific to AFP added. In order to quantitatively determine the amount of AFP present in the sample, Avidin conjugated to Horseradish Peroxidase (HRP) should be added to each microplate well. The final step, a TMB-substrate solution added to each well. Finally, a sulfuric acid solution is added and the resulting yellow colored product is measured at 450nm. Since the increases in absorbency is directly proportional to the amount of captured AFP.
Kit Components
Microplate: 1x96 wellsIncubation buffer: 1x30mlWashing buffer, (20x): 2x25mlStandard protein: 1x1vialStandard/sample dilution buffer: 1x25mlSecondary antibody: 1x1vialAV-HRP, (100X): 1x150ulSecondary antibody/AV-HRP dilution buffer: 1x25mlSubstrate (TMB): 1x15mlStop solution: 1x15ml
Storage and Stability
Store all components at 4°C. Stable for at least 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
References
1. Yao DF et al. 2007, Hepatobiliary Pancreat Dis Int. 6(3):241-247. 2. Ding X et al. 2005, World J Gastroenterol. 11(17):2656-2661. 3. Bader D et al. 2004, Clin Chim Acta. 349(1-2):15-23 4. Ball D et al. 1992, Am J Med Sci. 303(3):157-159.
