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การตรวจวินิจฉัยโรคทางแล็ป

Tumor Markers: CA125  
   Nonmucinous Ovarian 
   Carcinomas
   ตัวบ่งชี้มะเร็งรังไข่

 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: CA125 Nonmucinous Ovarian Carcinomas      
Tumor Antigens : Ovarian Carcinomas
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.

CA 125

CA125 is an antigen present on 80 percent of nonmucinous ovarian carcinomas. It is defined by a monoclonal antibody ( OC125 ) that was generated by immunizing laboratory mice with a cell line established from human ovarian carcinoma. It circulates in the serum of patients with ovarian carcinoma and was therefore investigated for possible use as a marker.

CA125 is often elevated in patients with ovarian cancer, its level following the patient's clinical course. With surgical resection or chemotherapy, the level correlates with patient response. Thus, it is superior to other markers such as CEA.

The CA125 is elevated in other cancers including endometrial, pancreatic, lung, breast, and colon cancer, and in menstruation, pregnancy, endometriosis, and other gynecologic and non gynecologic conditions.

Because of the low prevalence of ovarian cancer, the test is not itself useful in screening.

 


Enzyme Immunoassay for the Quantitative Determination of Ovarian Cancer Antigen CA125 in Human Serum

FOR IN VITRO DIAGNOSTIC USE ONLY
Store at 2 to 8ฐC.

PRODUCT NAMES
CA125 Enzyme Immunoassay

INTENDED USE
For the quantitative determination of the Cancer Antigen CA125 concentration in human serum.

INTRODUCTION
Cancer Antigen 125 (CA125) is a surface antigen associated with epithelial ovarian cancer. In serum, CA125 is associated with a high molecular weight glycoprotein. Published studies have indicated that elevated serum CA125 levels can be found in individuals with serious endometroid, clear-cell and undifferentiated ovarian carcinoma. 
The serum CA125 concentration is greater than 35 units per ml in 60% of women with ovarian cancer and >80% of patients with disseminated ovarian cancer. The serum CA125 is elevated in 1% of normal healthy women, 3% of normal healthy women with benign ovarian diseases, 6% of patients with non-neoplastic conditions (including but not limited to first trimester pregnancy, menstruation, endometriosis, uterine fibrosis, acute salphingitis, hepatic diseases and inflammation of peritoneum, pericardium or pleura). Serial determinations of serum CA125 as well as pelvic examination increase the test specificity. Serum CA125 concentration may be useful in monitoring treatment and distinguishing between good response to treatment and progressive malignant disease with poor therapeutic response. To date, CA125 is the most sensitive marker for residual epithelial ovarian cancer. CA125 may also be elevated in patients with lung, cervical, fallopian tube, and uterine cancer and endometriosis.

PRINCIPLE OF THE TEST
The CA125 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 CA125 molecule is used for solid phase immobilization (on the microtiter wells). A rabbit anti-CA125 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 CA125 molecules being sandwiched between the solid phase and enzyme-linked antibodies. After a 3-hour incubation at 37ฐC, 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 3N HCl changing the color to yellow. The concentration of CA125 is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm. 

REAGENTS
Materials provided with the kit:
ท Murine Monoclonal anti-CA125 coated microtiter plate with 96 wells.
ท Enzyme Conjugate Reagent, 13 ml.
ท CA125 reference standards containing; 0,15,50, 100, 200, and 400Unit/ml of CA125. 1 ml each, ready to use.
ท TMB Reagent (One-Step), 11ml.
ท Stop Solution (1N HCl), 11ml.
Materials required but not provided:
ท Precision pipettes and tips, 0.1ml, 0.2ml, 1ml, and 5ml.
ท Disposable pipette tips.
ท Distilled water.
ท Vortex mixer.
ท Absorbent paper or paper towel.
ท Microtiter plate reader.
ท Graph paper.

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. 

ASSAY PROCEDURE

1. Secure the desired number of coated wells in the holder. Dispense 100*l of CA125 standards, specimens, and controls into the appropriate wells.
2. Dispense 100*l Enzyme Conjugate Reagent into each well.
3. Mix gently for 30 seconds. It is very important to have complete mixing in this setup. 
4. Incubate at 37*C for 90 minutes.
5. Remove the incubation mixture by emptying the plate content into a waste container. 
6. Rinse and empty the microtiter plate 5 times with distilled water or deionized water. (Please do not use tap water)
7. Strike the microtiter plate sharply onto absorbent paper or paper towels to remove all residual water droplets.
8. Dispense 100*l of TMB solution into each well. Gently mix for 10 seconds. Incubate at room temperature, in the dark, for 20 minutes.
9. Stop the reaction by adding 100*l of Stop Solution to each well.
10. Gently mix for 30 seconds. It is important to make sure that all the blue color changes to yellow color completely.
11. 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 U/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 CA125 in U/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 CA125 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 in each experiment. 

CA125 Values (U/ml)

Absorbance (450nm)

0

0.051

15

0.178

50

0.488

100

0.929

200

1.620

400

2.865





EXPECTED VALUES AND SENSITIVITY
Healthy women are expected to have CA125 assay values below 35 U/ml. The minimum detectable concentration of CA125 in this assay is estimated to be 5 U/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

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 Kenemans P, Yedema CA, Bon GG, von Mensdorff-Pouilly S. Ca125 in gynecological pathology a review. Eur J Obstet Gynecol 1993;49:115-124.
4 Saksela F. Prognostic markers in epithelial ovarian cancer. Intl J Gynecol Pathol 1993;12:156-161.
5 Farghaly SA. Tumor markers in gynecologic cancer. Gynecol & Obstet Invest 1992;34:65-72.
6 Welander CE. What do CA 125 and other antigens tell us about ovarian cancer biology. Acta Obstet Gynecol Scand Sup 1992;155:85-93.
7 McGowan L. Pathology of the ovary. Curr Opin on Obstet Gynecol 1991;3:580-586.
8 Niloff JM. Ovarian malignancy. Curr Opin on Obstet Gynecol 1991;3:66-72.
9 Olt G, Berchuck A, Bast RC. The role of tumor markers in gynecologic oncology. Obstet Gynecol Survey 1990;45-:570-577.
10 Diez M, Cerdan FJ, Ortega MD, Torres A, Picardo A, Balibrea JL. Evaluation of serum CA125 as a tumor marker in non-small cell lung cancer. Cancer 1991;67:150-154.
11 Niloff JM, Klug TL, Schaetzl E. Elevation of serum CA125 in carcinomas of the fallopian tube, endometrium, and endocervix. AM J. Obstet Gynecol 1984; 148:1057


 

 

 
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