Antigens : Carcinoembryonic
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.
CEA: Carcinoembryonic antigen (CEA) is a protein found in many types
of cells but associated with tumors and the developing fetus. CEA is
tested in blood. The normal range is <2.5
ng/ml in an adult non-smoker and <5.0
ng/ml in a smoker. The CEA was one of the first oncofetal
antigens to be described and exploited clinically. It is a complex
glycoprotein of molecular weight 20,000, that is associated with the
plasma membrane of tumor cells, from wich it may be released into the
Although CEA was
first indentified in colon cancer, an abnormal CEA blood level is
specific neither for colon cancer nor for malignancy in general.
Elevated CEA levels are found in a variety of cancers other than
colonic, including pancreatic, gastric, lung, and breast. It is also
detected in benign conditions including cirrhosis, inflamatory bowel
disease, chronic lung disease, and pancreatitis. The CEA was found to
be elevated in up to 19 percent of smokers and in 3 percent of a
healthy control population. Thus, the test for CEA cannot substitute
for a pathological diagnosis.
As a screening
test, the CEA is also inadequate. Since cancer prevalence in a healthy
population is low, an elevated CEA has an unacceptably low positive
predictive value, with excess false positives. Also, since elevated
CEA occurs in the advanced stage of incurable cancer but is low in the
early, curable disease, the likelihood of a positive result affecting
a patient's survival is diminished.
The CEA has been
sugested as having prognostic value for patients with colon cancer.
Preoperative CEA values have been positively correlated with stage and
negatively correlated with disease free survival.
satisfactory for screening a healthy population, CEA has been used to
monitor recurrence. Early data suggested that CEA prededed clinical
relapse by several months. Subsequently, several investigators have
examined intensive, serial CEA monitoring as an indicator for second
look surgery in the hope that relapse could be detected at a time when
surgical ressection for cure was still possible. Criteria for
reoperation included a significant rise of CEA above a base line level
on serial determinations and absence of obvious unresectable disease
on staging workup. Determinations of CEA should be done frequently: at
a minimum of every 3 months and if possible every 1 month to 2 months.
Elevations above baseline should be verified rapidly to exclude
The CEA is of
some use as a monitor in treatment. Usually the CEA returns to normal
within 1 to 2 months of surgery, but if it returns elevated persistent
disease may be indicated. The test is not infallible in patients
treated with radiotherapy and chemotherapy but can be useful in those
whose tumor is not measurable.
The CEA is often
positive in malignancies other than colonic. In cancer of the breast,
lung, pancreas, stomach, and ovary the CEA may be elevated and can be
used to monitor the progress of disease or response to treatement.
Immunoassay for the Quantitative Determination of Carcinoembryonic
Antigen (CEA) in Human Serum
In Vitro Diagnostic Use Only
Store at 2 to 8°C.
the quantitative determination of the Cancer Antigen CEA concentration
in human serum.
Carcinoembroyonic antigen (CEA) is a cell-surface 200-kd glycoprotein.
In 1969, it was reported that plasma CEA was elevated in 35 of
36 patients with adenocarcinoma of the colon and that CEA titers
decreased after successful surgery.
Normal levels were observed in all patients with other forms of
cancer or benign diseases. Subsequent
studies have not confirmed these initial findings, and it is now
understood that elevated levels of CEA are found in many cancers.
Increased levels of CEA are observed in more than 30% of
patients with cancer of the lung, liver, pancreas, breast, colon, head
or neck, bladder, cervix, and prostate. Elevated plasma levels are
related to the stage and extent of the disease, the degree of
differentiation of the tumor, and the site of metastasis. CEA is also
found in normal tissue.
of the Test
The CEA ELISA test is based on the principle of a solid phase
enzyme-linked immunosorbent assay.[i][ii]
The assay system utilizes a monoclonal antibody directed against a
distinct antigenic determinant on the intact CEA molecule is used for
solid phase immobilization (on the microtiter wells). A goat anti-CEA
antibody conjugated to horseradish peroxidase (HRPO) is in the
antibody-enzyme conjugate solution. The test sample is allowed to
react simultaneously with the two antibodies, resulting in the CEA
molecules being sandwiched between the solid phase and enzyme-linked
antibodies. After a 1 hour 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 with the
addition of Stop solution changing the color to yellow. The
concentration of CEA is directly proportional to the color intensity
of the test sample. Absorbance is measured spectrophotometrically at
provided with the kit:
Antibody-coated microtiter plate
with 96 wells.
Lyophilized CEA standards containing; 0, 3, 12, 30, 60, and 120ng/ml
CEA. I set.
Enzyme Conjugate Reagent, 13 ml.
TMB Reagent (One-Step), 11ml.
Stop Solution (1N HCl), 11 ml.
required but not provided:
0.05ml, 0.1ml, 0.2ml, and 1ml.
Disposable pipette tips.
Vortex mixer or equivalent.
Absorbent paper or paper towel.
Microtiter plate reader.
Specimen Collection and Preparation
should be prepared from a whole blood specimen obtained by acceptable
medical techniques. This kit is for use with serum samples without
of Test Kit and Instrumentation
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.
All reagents should be brought to room temperature (18-25 C)
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
Secure the desired number of coated wells in the holder.
l of standard, specimens, and controls into appropriate wells.
l of Enzyme Conjugate Reagent to each well.
Thoroughly mix for 30 seconds.
It is very important to have a complete mixing in this
Incubate at room temperature (18-25
C) for 60 minutes.
Remove the incubation mixture by emptying plate content into a
Rinse and empty the microtiter wells 5 times with distilled
or deionized water.
do not use tap water)
Strike the wells sharply onto absorbent paper or paper towels
to remove all residual
l of TMB solution into
each well. Gently mix for
Incubate at room temperature for 20 minutes.
Stop the reaction by adding 100
l of Stop Solution to each well.
Gently mix for 30 seconds. It is important to make sure that all the blue color changes to yellow
the optical density at 450nm with a microtiter plate reader within
Calculate the average absorbance values (A450) for each set of
reference standards, control, and samples.
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.
the mean absorbance value for each sample, determine the corresponding
concentration of CEA in ng/ml from the standard curve.
of Standard Curve
Results of a typical standard run with optical density readings at
450nm shown in the Y axis against CEA 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
most complete study of CEA is a compilation of collaborative studies
in which CEA values in 35,000 samples from more than 10,000 patients
and controls were analyzed. Of 1425 normal persons who did not smoke, 98.7% had values
less than 5.0 ng/ml. It
is recommended that each laboratory establish its own normal range. The minimum detectable concentration of CEA by this
assay is estimated to
be 1.0 ng/ml.
of the Procedure
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
The wash procedure is critical. Insufficient washing will
result in poor precision and falsely elevated absorbance readings.
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.
Engall, E., Methods in Enzymology, Volume 70, Van Vunakis, H. and
Langone, J. J. (eds.), Academic Press, New York, 419-492(1980).
Uotila, M., Ruoslahti, E. and Engvall, E., J. lmmunol. Methods, 42,
Gold P, Freedman S O.
Demonstration of tumor specific antigen in human colonic
carcinoma by immunologic tolerance and absorption techniques. J
Exp Med 1965;127:439-462.
Thompson D P M, Krupey J, Freedman S O, et al.
The radioimmunoassay of circulating carcinoembryonic
antigen of the human digestive system.
Proc Natl Acad Sci USA 1969;64:161-167.
Schwartz M K.
Tumor markers in diagnosis and screening.
In: Ting S W, Chen J S, Schwartz M K, eds.
Human tumor markers, Amsterdam: Elsevier Science,
Zamcheck N. and Martin E.W.
Sequential Carcinoembryonic Antigen Levels in Pancreatic
Cancer: Some Clinical Correlations.
Mughal A.W., Hortobagyi G. N., Fritsche H.A., Buzdar A.U. Yap
H-Y., and Blumenschein G.R.
Serial Plasma Carcinoembryonic Antigen Measurements During
Treatment of Metastatic Breast Cancer.
JAMA 1983; 259:1881-1886.