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Tumor Markers: CA15-3 
   (BREAST TUMOUR 
   MARKER
   ตัวบ่งชี้มะเร็งทรวงอก


 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: CA15-3 (BREAST TUMOUR MARKER      
Tumor Antigens : CA15-3 (BREAST TUMOUR MARKER
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.

CA15-3 Test is an immunometric assay for the determination of  CA15-3 antigen in serum. CA15-3 is a high molecular weight (300 - 450 kDa) polymorphic epithelial mucin. The heterogeneous breast cancer- associated mucins consist of a repeated polypeptide core sequence and an outer shell of carbohydrate. Serum CA15-3 values increase with clinical stage of breast cancer, the highest values occurring in metastatic disease. Serial determinations of CA15-3 are most useful as an indicator of response to therapy. The measurement of CA15-3 antigen is more sensitive and specific than the determination of Carcinoemberyonic antigen (CEA), having a lower percentage positivity with benign breast lesions, liver cirrhosis and other carcinomas - 99% of serum donated by healthy volunteers contained less than 40 U/ml of CA15-3. CA15-3 is not elevated during pregnancy . The percentage of raised values found in breast cancer can be as high as 98% but this depends primarily on the tumour stage of the patient population studied. Elevated levels have also been found in patients with lung cancer (63%) and ovarian cancer (80%). 

CA15-3 REFERENCE RANGE 

9    -      51      U/mL

   

TUMOUR MARKERS 

BILE DUCT

CA 19-9

CEA

CF 72-4

 

BLADDER

TPA

TPS

CEA

CA-50

BLADDER/CHORION

HCG

B HCG

   

BONE SARCOMA

AP

HYROXY-

PROLINE

TPA +TPS

CEA

BREAST

CA 15-3

CEA

TPA

TPS

BRONCHIAL/LUNG

NSE

CEA

SCC

CYFRA21-1

CARCINOID

SERATONIN

5-HIAA

   

CERVIX/UTERUS

CEA

SCC

TPA

TPS

COLON/RECTUM

CEA

CA 19-9

CA-50

 

KIDNEY

EPO

RENIN

   

LIVER

AFP

CEA

   

LYMPHATIC SYSTEM

IMMUNO-

GLOBULINS

B-2 MICRO-

GLOBULIN

   

MELANOMA

FERRITIN

MELANIN

   

NECK/HEAD REGION

SCC

EBV

CEA

 

OESOPHAGEAL

CA 19-9

CEA

SCC

 

OVARIAN

CA 12-5

CEA

CA 15-3

CA 72-4

PANCREAS

CA 19-9

CEA

TPA

TPS

PARATHYROID

PTH-INTACT

     

PHEOCHROMO-

CYTOMA

CATECHOL-AMINES

META-

NEPHRINES

HVA

VMA

PITUITARY

HGH

ACTH

PROL.

 

PROSTATE

PSA

PAP

   

STOMACH

CEA

CA 19-9

CA 72-4

 

TESTICULAR

AFP

B HCG

HCG

 

THYROID

THYROGL.

CALCITO.

NSE

 
 
 

Enzyme Immunoassay for the Quantitative Determination of Breast Cancer Antigen CA15-3 in Human Serum

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

PRODUCT NAMES
CA15-3 Enzyme Immunoassay

INTENDED USE
For the quantitative determination of the Cancer Antigen CA15-3 concentration in human serum.

INTRODUCTION
Breast cancer is the most common life-threatening malignant lesion in women of many developed countries today, with approximately 180,000 new cases diagnosed every year. Roughly half of these newly diagnosed patients are node-negative, however 30% of these cases progress to metastatic disease.
There are a number of tumor markers that can help clinicians to identify and diagnose which breast cancer patients will have aggressive disease and which will have an indolent course. These markers include estrogen and progesterone receptors, DNA ploidy and percent-S phase profile, epidermal growth factor receptor, HER-2/neu oncogene, p53 tumor suppressor gene, cathepsin D, proliferation markers and CA15-3. CA15-3 is most useful for monitoring patients post-operatively for recurrence, particularly metastatic diseases. 96% of patients with local and systemic recurrence have elevated CA15-3, which can be used to predict recurrence earlier than radiological and clinical criteria. A 25% increase in the serum CA15-3 is associated with progression of carcinoma. A 50% decrease in serum CA15-3 is associated with response to treatment. CA15-3 are more sensitive than CEA in early detection of breast cancer recurrence. In combination with CA125, CA15-3 has been shown to be useful in early detection of relapse of ovarian cancer. CA15-3 levels are also increased in colon, lung and hepatic tumors. 

PRINCIPLE OF THE TEST
The CA15-3 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 CA15-3 molecule is used for solid phase immobilization (on the microtiter wells). A second monoclonal anti-CA15-3 antibody conjugated to horseradish peroxidase (HRPO) is in the antibody-enzyme conjugate solution. The test sample is allowed to react sequentially with the two antibodies, resulting in 

the CA15-3 molecules being sandwiched between the solid phase and enzyme-linked antibodies. After two separate 1 hour incubation steps 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 stop solution changing the color to yellow. The concentration of CA15-3 is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm. 

REAGENTS
Materials provided with the kit:
* Muriine Monoclonal Anti-CA15-3 coated microtiter plate with 96 wells.
* Sample Diluent, 100 ml.
* Enzyme Conjugate Concentration (22X), 1.0ml.
* Enzyme Conjugate Diluent,, 21ml.
* CA15-3 reference standards, containing 0, 15, 30, 60, 120, and 240 Unit/ml. Liquid. 1 set. These standards have been pre-diluted 51-fold. Please do not dilute them again.
* TMB Reagent (One-Step), 11 ml.
* Stop Solution (1N HCl), 11 ml.

Materials required but not provided:
* Precision pipettes and tips, 0.1 ml, 0.2 ml, 
1 ml, and 5 ml.
* Distilled water.
* Disposable pipette tips.
* Vortex mixer.
* Absorbent paper or paper towel.
* A microtiter plate reader at 450nm wavelength, with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater.
* 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 allowed to reach room temperature (18-25oC ) before use.
2. To prepare working CA 15-3 Conjugate Reagent, add the entire 1.0 ml of Conjugate Concentrate(22x) to 21ml of the Enzyme Conjugate Diluent (1:21 dilution) and mix well. The diluted Enzyme Conjugate Reagent is stable at 4°C for at least 4 months.

ASSAY PROCEDURE
1. Patient serum and control serum should be diluted, 51 fold, before use. Prepare a series of small tubes (such as 1.5 ml microcentrifuge tubes) and mix 20 ml serum with 1.0 ml Sample Diluent. PLEASE DO NOT DILUTE THE STANDARDS.
2. Secure the desired number of coated wells in the holder. Dispense 200 *l of CA15-3 standards, diluted specimens, and diluted controls into the appropriate wells. Gently mix for 10 seconds. 
3. Incubate at 37*C for 1 hour.
4. Remove the incubation mixture by emptying the plate content into a waste container. 
5. Rinse and empty the microtiter plate 5 times with distilled or deionized water. (Please do not use tap water). 
6. Strike the microtiter plate sharply onto absorbent paper or paper towels to remove all residual water droplets.
7. Dispense 200*l of Enzyme Conjugate Reagent into each well. Gently mix for 10 seconds 
8. Incubate at 37*C for 1 hour. 
9. Remove the contents and wash the plate as described in step 6-7 above.
10. Dispense 100 *l TMB substrate reagent into each well. Gently mix for 10 seconds. 
11. Incubate at room temperature in the dark for 20 minutes.
12. Stop the reaction by adding 100*l of Stop Solution to each well.
13. Gently mix for 30 seconds. It is important to make sure that all the blue color changes to yellow color completely.
14. 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 CA15-3 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 CA15-3 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. 

CA15-3Values (U/ml)

Absorbance (450 nm)

0

0.021

15

0.425

30

0.693

60

1.214

120

1.956

240

2.845




 

 

 

 

Expected Values and Sensitivity
Healthy women are expected to have CA15-3 assay values below 35 U/ml.  The minimum detectable concentration of CA15-3 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 Aziz DC. Quantitation of estrogen and progesterone receptors by immunocytochemical and image analyses. A J Clin Pathol 1992;98:105-11
2 Aziz DC, Peter JB. DNA ploidy and cell-cycle analysis. Tools for assessment of cancer prognosis. J Clin Pathol 1991;5:422-38.
3 Clark GM, Dressler LG, Owens MA, Dounds G, Oldaker T, McGuire WL. Prediction of relapse or survival in patients with node-negative breast cancer by DNA flow cytometry. N Engl J Med 1989;320:627-33.
4 Elledge RM, McGuire WL. Prognostic factors and therapeutic decisions in axillary node-negative breast cancer. Annu Rev Med 1993;44:201-10.
5 Foekens JA, Rio C, Seguin P, et al. Prediction of relapse and survival in breast cancer patients by pS2 protein. Cancer Res 1990; 50-3832-7.
6 Isola J, Visakorp T, Holli K, Kallionieml D. Association of p53 expression with other prognostic factors and long term survival in node-negative breast cancer. J Cell Biochem 1992;(Suppl 16D):101.
7 Kute TE, Shao ZM, Snugg NK, Long RT, Russell GB, Case LD. Cathepsin D as a prognostic indicator for node-negative breast cancer patients using both immunoassays and enzymatic assays. Cancer Res 1992;52-198-203.
8 McGuire WL, Tandon AK, Allred D, Chamnes GC, Clark GM. How to use prognostic factors in axillary node negative breast cancer patients. J Natl Cancer Inst 1990;82:1006-7.
9 Nicholson S, Richard J, Sainsbury C, et al. Epidermal growth factor receptor (EGFr): results of a 6 year follow up study in operable breast cancer with emphasis on the node-negative subgroup. Br J Cancer 1991;63:146-50.
10 Somerville JE, Clarke LA, Biggart JD. C-erb B-2 overexpression and histological type of in-situ and invasive breast carcinoma. J Clin Pathol 1992;45-16-20.
11 Ueronese S, Gambacorta M. Detection of Ki-67 rate in breast cancer. Am J Clin Pathol 1991;95:30-4.
12 Lotnicker M, Pavesi F, Scarabelli M. Tumor associated antigens CA15-3 and CA125 in ovarian cancer. Int. J. Biolog Markers 1991; 6:115


 

 

 
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