BL-27-CT: 100 determinations

CEA is a 200,000 Daltons oncofetal glycoprotein expressed by normal tissues during the first 6 months of fetal life. Lateron The expression of CEA by normal cells becomes largely repressed except in cancer tissues of various cell types which may secrete large amounts of this oncofetal protein into the circulaion. Widely accepted as a useful adjunct for monitoring the course of cancer diseases, CEA should not be regarded as a tumor-specific marker because it is still secreted in small amounts by certain normal tissues during adult life, with small serum level increases in case of begnin diseases such as cirrhosis, hepatitis, inflammatory bowel diseases, renal failure and in heavy smokers. Therefore, the measurement of CEA serum concentration for diagnostic purposes must be considered with great care.

The Bio-Line CEA-Irma is an immunoradiometric assay based on coated-tube separation. . Mabs1, the capture antibodies, are attached to the lower and inner surface of the plastic tube. Standards or samples added to the tubes with at first show low affinity for Mabs1. Addition of Mab2, the signal antibody labelled with ¹²⁵I, will complete the system and trigger the immunological reaction. After washing, the remaining radioactivity bound to the tube reflects the antigen concentration. The use of several distinct Mabs avoids hyperspecificity-common the 2-site IRMA-, as well as the need of a shaker or long incubation at 37°C

1. Radioactive material: Radioactive material may be received, acquired, possessed and used only by physicians, clinical laboratories, or hospitals for "In-Vitro" clinical or laboratory tests not involving internal or external administration of the material, or the radiation therefrom, to human beings or animals.

Compliance with these basic rules of radiation safety should provide adequate protection:

1. Do not eat, drink, smoke, or apply cosmetics in areas where radioactive material is used.

2. Do not pipet by mouth reagents containing radioactive materials.

3. Wear protective clothing; i.e., lab coats and disposable gloves, in order to avoid direct contact with radioactive reagents.

4. Work with radioactive materials should be performed in a designed area.

5. Radioactive materials should be stored in an acceptable location.

6. A log should be kept for receipt and disposal of radioactive materials.

7. Radioactive spills or accidents should be taken care of immediately according to established procedures.

8. Disposal of radioactive materials must comply with prevailing regulations and guidelines of the agencies holding jurisdiction over the laboratory.

2. Sodium azide: Sodium Azide, used as a bacteriostatic agent, is toxic in acid medium. In addition, it may form potentially explosive lead or copper azides. To avoid dangerous deposits, waste solutions should be flushed away with large volumes of water.

3. Hepatitis and Acquired Immune Deficiency Syndrome (HTLV-III): All Bio-Line reagents included in this kit have been tested and found to be non reactive for hepatitis B surface antigen. They have also been screened and determined to be non-reactive for HTLV-III antibody. However, human serum products should be handled as if potentially capable of transmitting hepatitis, Acquired Immune Deficiency Syndrome, or other infectious agents.

Kit contains sufficient reagents for 100 determinations.

2. ¹²⁵I Anti-CEA tracer: 1 vial (red solution) containing 5.5 ml. Activity < 9µCi or 440 kBq.

3. Coated tubes: 2 x 50 tubes, coated with Anti-CEA monoclonal antibodies.

4. Wash solution concentrate: 1 vial of 10 ml of concentrate, to be diluted into 700 ml distilled water and stored at 4° C.

The following material is required but not provided in the kit:

1. Distilled water

2. Pipettes for delivery of: 50 µl, 100 µl, and 500 µl (the use of accurate pipettes with disposable plastic tips is recommended)

3. 5 ml automatic syringe (Cornwall type) for washing

4. Aspiration system (optional)

5. Gamma counter, set for ¹²⁵I counting and Vortex mixer and magnetic stirrer.

Serum and plasma must be kept at 2-8°C.

If the test is not within 24 hours, storage at –20°C is recommanded and will not result in loss of immuno-reactivity for at least 6 months.

Serum, heparinized or EDTA plasma provide similar result.

1. Label coated tubes in duplicate for each standard, sample, control. For the determination of the total counts, label 2 normal tubes.

2. Vortex mix briefly standards, samples, controls and dispense 100 µl of each into the respective tubes.

             3. Dispense 50 µl of tracer into eachl tube.

4. Shake the tube rack gently to remove any bubbles.

5. Incubate for 2 hours at 37°C.

6. Aspirate (or decant) the contents of the coated tubes.(except total counts). Be sure than the plastic tip of the aspirator reaches the bottom of the coated tube in order to remove all the liquid.

Wash the tubes twice with Wash Solution and aspirate (or decant). Avoid foaming during the addition of the Wash Solution.

Count the tubes in a gamma counter for 60 seconds.

 

On semilogarithmic or linear graph paper plot the c.p.m. (ordinale) for each standard against the corresponding concentration of CEA (abscissa) and draw a standard curve through the standard points, reject the obvious outliers. Read the concentration for each control and sample by interpolation on the standard curve. Computer assisted data reduction will simplify these calculations.

B/BO x 100 = Counts (Std or sample) / Counts (Zero Standard) x 100

          INTRA ASSAY                         INTER ASSAY

                            RECOVERY TEST

                            DILUTION TEST

            1. BURTIN P., and GOLD P., 1978Carcinoembryonic antigen.Scand. J., Immunol. 8 (suppl.8) : 27-38.

BOOTH S.N., JAMIESON G.C., KING J.P.G. et al., 1974Carcinoembryonic antigen in management of colorectal carcinoma.Br. Med. J. iv, 183-187

GOLD P., FREEDMAN S.G., 1965Specific carcinoembryonic antigens of the guman digestive system.J. Exp.Med.122,467-481

HERLYN M., SEARS H.F., STEPLEWSKI Z., and KOPROWSKI H., 1982Monoclonal antibodiy detection of a circulating tumor-associated antigen I.Presence of antigen in sera of patients with colorectal, gastric and pancreatic carcinoma.J.Clin. Immunol. 2,165-140

LAURENCE D.J.R., STEVENS V., BETTELHEIM R., et al. 1972Role of plasma carcinoembryonic antigen in diagnosis of gastrointestinal, mammary and bronchial carcinoma.Br. Med. J. iii, 605-609

National Institute of Health Consensus Statement, 1981Carcinoembryonic antigen ; its role as a marker in the management of cancer.Br. Med. J. ;282,373-375

RODGERS G.T., 1976Heterogeinity of carcinoembryonic antigen. Implications on its role as a tumor marker substance.Biochem. Biophys. Acta 458, 355-373