Antigens : Alpha-Fetoprotein
markers defined by both monoclonal antibodies and polyclonal antisera,
often the so called oncofetal antigens. The oncofetal substances,
present in embryo or fetus, diminish to low levels in the adult but
reappear in the tumor.
is a normal fetal serum protein synthesized by the liver, yolk sac,
and gastrointestinal tract that shares sequence homology with albumin.
It is a major component of fetal plasma, reaching a peak concentration
of 3 mg/ml at 12 weeks of gestation. Following birth, it clears
rapidily from the circulation, having a half life of 3.5 days, and its
concentration in adult serum is less than 20 ng/ml.
AFP is of
importance in diagnosing hepatocellular carcinoma and may be useful in
screening procedures. AFP elevation is more common in areas where
hepatocellular carcinoma is endemic, such as Africa and in patients
who are HBsAg positive.
AFP is a marker
for hepatocellular and germ cell (nonseminoma) carcinoma.1 It is a
glycoprotein produced in large amounts during fetal life and is
homologous to albumin. In healthy adults,
less than 10 µg/L of AFP is found in the circulation.
AFP is elevated in normal pregnancy, benign liver disease (hepatitis,
cirrhosis), as well as in cancer.
An elevated AFP
has been termed by Sell "the single most discriminating
laboratory test indicative of malignant disease now available."
As such, it could be valuable in screening for hepatocellular
carcinoma in high risk populations.
AFP is elevated
in testicular germ cell tumors containing embryonal or endodermal
sinus elements. A defenitive positive marker value is highly sensitive
in indicating relapse or response to treatment.
The AFP is less
frequently elevated in other malignancies such as pancreatic cancers,
gastric cancers, colonic cancers, and bronchogenic cancers. This
elevation was not necessarily associated with liver metastases.
The AFP is
rarely elevated in healthy persons, and a rise is seen in only a few
disease states. Elevation occurs in certain liver diseases, especially
acute viral or drug induced hepatitis and conditions associated with
hepatic regeneration. In general, the elevations are under 500 ng/ml
and do not denote hepatocellular carcinoma. Is also elevated in
ataxia-telangiectasia and in hereditary tyrosinosis.
Thus, AFP is a
useful marker in hepatocellular carcinoma and germ cell tumors, the
only conditions associated with extreme elevations greater than 500 ng/ml.
In both tumors it has value in diagnosis and monitoring of therapy. In
the former, wich is one of the most common tumors worldwide, AFP may
be of use in screening.
Enzyme Immunoassay for the Quantitative Determination of Alpha-Fetoprotein (AFP) in Human Serum
FOR IN VITRO DIAGNOSTIC USE ONLY
Store at 2 to 8ฐC.
AFP Enzyme Immunoassay
For the quantitative determination of the Cancer Antigen AFP concentration in human serum.
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 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 (HRPO) 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-HRPO 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 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.
Materials provided with the kits:
* Rabbit anti-AFP coated microtiter plate with 96 wells.
* Zero Buffer, 13 ml.
* Reference standard set, contains 0, 5, 20, 50, 150, and 300 ng/ml (WHO, 72/225) AFP, lyophilized.
* Enzyme Conjugate Reagent, 18 ml.
* TMB Reagent (One-step), 11 ml.
* Stop Solution (1N HCl), 11 ml.
Materials required but not provided:
* Precision pipettes: 0.02, 0.05, 0.10, 0.15, 0.20, and 1.0 ml.
* Disposable pipette tips.
* Distilled water.
* Vortex mixer or equivalent.
* Absorbent paper or paper towel.
* Graph paper.
* Microtiter plate reader.
SPECIMEN COLLECTION AND PREPARATION
Serum should be prepared from a whole blood specimen obtained by acceptable medical techniques. This kit is for use with serum samples without additives only.
STORAGE OF TEST KIT
Unopened test kits should be stored at 2-8*C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. Opened test kits will remain stable until the expiration date shown, provided it is stored as described above. 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 acceptable for use in absorbance measurement.
1. All reagents should be brought to room temperature (18-25*C) before use.
2. Reconstitute each lyophilized standard with 1.0 ml distilled water. Allow the reconstituted material to stand for at least 20 minutes and mix gently. Reconstituted standards should be stored sealed at 2-8*C
1. Secure the desired number of coated wells in the holder.
2. Dispense 20*l of standard, specimens, and controls into appropriate wells.
3. Dispense 100*l of Zero Buffer into each well.
4. Thoroughly mix for 30 seconds. It is very important to have complete mixing in this setup.
5. Incubate at room temperature (18-25*C) for 30 minutes.
6. Remove the incubation mixture by flicking plate content into a waste container.
7. Rinse and flick the microtiter wells 5 times with distilled or deionized water. (Please do not use tap water)
8. Strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets.
9. Dispense 150*l of Enzyme Conjugate Reagent into each well. Gently mix for 5 seconds.
10. Incubate at room temperature for 30 minutes.
11. Remove the incubation mixture by flicking plate contents into a waste container.
12. Rinse and flick the microtiter wells 5 times with distilled or deionized water.
13. Strike the wells sharply onto absorbent paper to remove residual water droplets.
14. Dispense 100*l TMB solution into each well. Gentle mix for 5 seconds.
15. Incubate at room temperature for 20 minutes.
16. Stop the reaction by adding 100*l of Stop Solution to each well.
17. Gently mix for 30 seconds. It is important to make sure that all the blue color changes to yellow color completely.
18. Read optical density at 450nm with a microtiter reader within 15 minutes.
CALCULATION OF RESULTS
1. Calculate the average absorbance values (A450) for each set of reference standards, control, and samples.
2. Construct a standard curve by plotting the mean absorbance obtained for each reference standard against its concentration in ng/ml on linear graph paper, with absorbance on the vertical (y) axis and concentration on the horizontal (x) axis.
3. Using the mean absorbance value for each sample, determine the corresponding concentration of AFP in ng/ml from the standard curve.
EXAMPLE OF STANDARD CURVE
Results of a typical standard run with optical density readings at 450nm shown in the Y axis against AFP 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.
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
LIMITATIONS OF THE PROCEDURE
1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instructions and with adherence to good laboratory practice.
2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
3. The results obtained from the use of this kit should be used only as an adjunct to other diagnostic procedures and information available to the physician.
1.Engall, E., Methods in Enzymology, Volume 70, Van Vunakis, H. and Langone, J. J. (eds.), Academic Press, New York, 419-492(1980).
2.Uotila, M., Ruoslahti, E. and Engvall, E., J. lmmunol. Methods, 42, 11-15 (1981).
3 Abelev G I. Alpha-fetoprotein as a marker of embryo-specific differentiation in normal and human tissues. Transplant Rev 1974;20:3-37.
4 Hirai H. Alpha fetoprotein. In: Chu T M, ed. Biochemical markers for cancer. New York: Marcel Dekker, 1982:23-59.
5 Chan D W, Miao Y C. Affinity chromatographic separatoin of alpha-fetoprotein variants: Development of a mini-column procedure and application to cancer patients. Clin Chem 1986;32:2143-2146.
6 Sell L S. Cancer markers of the 1990s. Clin Lab Med 1990;10:1-37.
7 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.