Enzyme Immunoassay for the Quantitative Determination of Alpha-Fetoprotein (AFP) in Human Serum
Catalogue #KH2005
Contents
Intended Use
The Alpha-fetoprotein (AFP) Enzyme Immunoassay test kit is intended for the quantitative determination of AFP concentration in human serum. This kit is not licensed for use as a diagnostic test in the United States. It is therefore only sold outside the United States and is marked "for export only."
Outside the United States, the appropriateness of this test kit for research or diagnostic purposes depends on local regulations.
Introduction
AFP is a glycoprotein with a molecular weight 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 of the test
The AFP Quantitative Test Kit is based on a solid phase enzyme-linked immunosorbent assay. The assay system utilizes rabbit anti-AFP antibody for solid phase (microtiter wells) immobilization and a mouse monoclonal anti-AFP antibody in the antibody-enzyme (horseradish peroxidase) conjugate solution. The test specimen (serum) is added to the AFP antibody coated micro titer wells and incubated with the Zero Buffer. If human AFP is present in the specimen, it will bind with the antibody on the well. The well is then washed to remove any residual test specimen, and AFP antibody labeled with horseradish peroxidase (conjugate) is added. The conjugate will bind immunologically to the AFP on the well, resulting in the AFP molecules being sandwiched between the solid phase and enzyme-linked antibodies. After an incubation at room temperature, the wells are washed with water to remove unbound labeled antibodies. A solution of TMB is added and incubated for 20 minutes, resulting in the development of a blue color. The color development is stopped and the color is changed to yellow with the addition of 2N HCl. The extent of color development is measured spectrophotometrically at 450 nm. The concentration of analyte is directly proportional to the color intensity of the test sample.
Materials
Materials provided with the test kits:
- Antibody coated microtiter plate with 96 wells.
- Zero buffer, 13 ml.
- Lyophilized AFP reference standard set, containing ; 0, 5, 20, 50, 150, and 300 ng/ml (WHO, 72/225).
- Enzyme Conjugate Reagent.
- Color Reagent A.
- Color Reagent B.
- 2N HCl.
Materials required but not provided:
- Precision pipettes: 20µl, 50µl, 100µl, 150µl, 200µl, 1ml, and 5ml.
- Disposable pipette tips.
- Distilled water.
- Glass tubes or flasks to mix Color Reagent A and Color Reagent B.
- Absorbent paper or paper towel.
- Graph paper.
Instrumentation
The following equipment items are required to perform this assay:
- A vortex mixer or equivalent to mix reagents.
- A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450nm wavelength is required for use in the absorbance measurement.
Storage of test kits
Unopened test kits should be stored at 2-8°C upon receipt. The microtiter plate should be stored at 2-8°C in a sealed bag with desiccants. This will minimize its exposure to damp air. Opened test kits will remain stable until the expiration date, provided they are stored as described above.
Specimen collection and preparation
- This kit is for use with serum samples prepared from whole blood. Blood should be drawn using standard venipuncture techniques and the serum should be separated from the red blood cells as soon as practical. Avoid grossly hemolytic, lipemic or turbid samples.
- Plasma samples collected in tubes containing EDTA, heparin, or oxalate may interfere with the test procedures and should be avoided.
- Specimens should be capped and may be stored up to 48 hours at 2-8°C, prior to assaying. Specimens held for a longer time can be frozen at -20°C. Thawed samples must be mixed prior to testing.
Reagent preparation
- All reagents should be brought to room temperature (18-25°C ) and mixed by gently inverting or swirling prior to use. Do not induce foaming.
- Add 1 ml of distilled water to reconstitute the lyophilized standards. Allow the reconstituted materials to stand for at least 20 minutes. Mix gently . The reconstituted standards should be stored sealed at 2-8 °C.
- To prepare TMB substrate reagent, , make a 1:1 dilution of Color Reagent A and Color Reagent B at least 15 minutes before use. Mix gently to ensure complete mixing. The prepared TMB substrate reagent is stable at room temperature, in the dark, for up to 3 hours. Discard excess after use.
Assay procedures
- Secure the desired number of coated wells in the holder.
- Dispense 20µl of standard, specimens, and controls into appropriate wells.
- Dispense 100µl of zero buffer into each well.
- Thoroughly mix for 10 seconds. It is very important to mix it completely.
- Incubate at room temperature (18-25°C) for 30 minutes.
- Remove the incubation mixture by emptying plate content into a waste container.
- Rinse and empty the microtiter wells 5 times with running tap or distilled water.
- Strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets.
- Dispense 150µl of Enzyme Conjugate Reagent into each well. Gently mix for 5 seconds.
- Incubate at room temperature for 30 minutes. *Prepare TMB solution 15 minutes before use.
- Remove the incubation mixture by flicking plate content into a waste container.
- Rinse and flick the microtiter wells 5 times with running tap or distilled water.
- Strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets.
- Dispense 200µl of TMB solution into each well. Gently mix for 5 seconds.
- Incubate at room temperature for 20 minutes without shacking.
- Stop the reaction by adding 50µl of 2N HCl to each well.
- Gently mix for 30 seconds to make sure that the blue color changes to yellow color completely.
- Within 30 minutes, read the optical density at 450nm with a microtiter plate reader.
Important Notes
- The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
- It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be competed within 3 minutes. A full plate of 96 well may be used if automated pipetting is available.
- Duplication of all standards and specimens, although not required is recommended.
Calculation of results
Calculate the mean absorbance value (A450) for each set of reference standards, specimens, controls and patient samples. Construct a standard curve by plotting the mean absorbance obtained from each reference standard (Y-axis) against its concentration (X-axis) on graph paper. Use the mean absorbance values for each specimen to determine the corresponding concentration of AFP from the standard curve.
Example of Standard Curve
Results of a typical standard run with the optical density reading at 450nm shown in the Y-axis against the AFP concentrations shown in the X-axis.
Note: 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.
| AFP (ng/ml) | Absorbance (450nm) |
| 0 | 0.012 |
| 5 | 0.127 |
| 20 | 0.455 |
| 50 | 0.952 |
| 150 | 2.150 |
| 300 | 2.932 |
Expected values and sensitivity
In high-risk patients, AFP values between 100 and 350 ng/ml suggest a diagnosis of hepatocellular carcinoma, and levels over 350 ng/ml usually indicate the disease. Approximately 97% of the healthy subjects have AFP levels less than 8.5 ng/ml. It is recommended that each laboratory establish its own normal range. The minimum detectable concentration of AFP by this assay is estimated to be 2.0 ng/ml.
References
- Abelev G I. Alpha-fetoprotein as a marker of embryo-specific differentiation in normal and human tissues. Transplant Rev 1974;20:3-37.
- Hirai H. Alpha fetoprotein. In: Chu T M, ed. Biochemical markers for cancer. New York: Marcel Dekker, 1982:23-59.
- Chan D W, Miao Y C. Affinity chromatographic separation of alpha-fetoprotein variants: Development of a mini-column procedure and application to cancer patients. Clin Chem 1986;32:2143-2146.
- Sell L S. Cancer markers of the 1990s. Clin Lab Med 1990;10:1-37.
- Hirai H, Nishi S, Watabe H et al. Some chemical, experimental and clinical investigations on alpha fetoprotein. In: Hirai H, Miyaji T, eds. Alpha-fetoprotein and hepatoma. Gann Monogr 1973:14:19-34.
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