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Tumor Markers: CEA 
 
Carcinoembryonic Antigen
  คาซิโนเอ็มบริโอนิค แอนติเจน

 Tumor Markers 
  ตัวช่วยบ่งชี้ภาวะการเกิด
  มะเร็งของอวัยวะต่างๆ
PSA
   ภาวะมะเร็งต่อมลูกหมาก
CEA
   ภาวะมะเร็งลำไส้
AFP
   ภาวะมะเร็งตับ
b-HCG
   
CA125
  
Ovarian Cancer 
   Antigen
CA 15-3
  
Breast Cancer 
   Antigen
CA19-9
 
Gastrointestinal 
  Cancer Antigen
AP
  Acid Phosphatase


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  Tumor Markers: CEA Carcinoembryonic Antigen      
Tumor Antigens : Carcinoembryonic Antigen
Include 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.

Tumor marker, 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 blood.

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.

Although not 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 laboratory error.

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.

 

Enzyme Immunoassay for the Quantitative Determination of Carcinoembryonic Antigen (CEA) in Human Serum

 For In Vitro Diagnostic Use Only
Store at 2 to 8°C.

CEA Enzyme Immunoassay

Intended Use
For the quantitative determination of the Cancer Antigen CEA concentration in human serum.

Introduction
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.

Principle of the Test
The CEA ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay. 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 450 nm.

Reagents

Materials 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.

Materials required but not provided:
Precision pipettes:  0.05ml, 0.1ml, 0.2ml, and 1ml.
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 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.

Reagent Preparation
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

Assay Procedure

1.        Secure the desired number of coated wells in the holder.

2.        Dispense 50 l of standard, specimens, and controls into appropriate wells.  

3.        Dispense 100 l of Enzyme Conjugate Reagent to each well.


4.        Thoroughly mix for 30 seconds.  It is very important to have a complete mixing in this 
       setup.

5.        Incubate at room temperature (18-25 C) for 60 minutes.

6.        Remove the incubation mixture by emptying plate content into a waste container. 

7.        Rinse and empty 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 100 l of TMB solution into each well.  Gently mix for 5 seconds.

10.     Incubate at room temperature for 20 minutes.

11.     Stop the reaction by adding 100 l of Stop Solution to each well.

12.     Gently mix for 30 seconds. It is important to make sure that all the blue color changes to yellow color completely.

13.     Read the optical density at 450nm with a microtiter plate 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 CEA 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 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.

CEA (ng/ml)

Absorbance (450nm)

0

0.019

3

0.105

12

0.362

30

0.814

60

1.390

120

2.032

         Expected Values and Sensitivity

The 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.

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.

                                    References


[i] Engall, E., Methods in Enzymology, Volume 70, Van Vunakis, H. and Langone, J. J. (eds.), Academic Press, New York, 419-492(1980).

[ii] Uotila, M., Ruoslahti, E. and Engvall, E., J. lmmunol. Methods, 42, 11-15 (1981).

1 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.

2 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.

3 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, 1987;3-16.

4 Zamcheck N. and Martin E.W.  Sequential Carcinoembryonic Antigen Levels in Pancreatic Cancer: Some Clinical Correlations.  Cancer 1981;47:1620-1627.

5 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.


 

 

 
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