PROTOCOL FOR THE ALPHA-FETOPROTEIN EIA KIT
By IMMUNOS - Catalogue #KH2005
Note: 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.
SPECIMENT COLLECTION & 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 & 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 percent of the healthy subjects have AFP levels less than 8.5 ng/ml.
AFP may be elevated in pregnancies with neural tube defects and low in pregnancies with
Down syndrome. 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.
For pricing or other inquiries send an E-mail to: sales@immunos.com.
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Materal Serum Screening: Double, Triple, or Quad Screening Information By ARUP Lab
Analytical sensitivity of 0.5 ng/mL.
