BL-20CT : 100 determinations

HCG is a glycoprotein synthesised by the syncytiotrophoblast of the placenta throughout pregnancy. HCG-molecular weight 37,900 - comprises two subunits.

The hCG a subunit – molecular weight 14,900 – is chemically similar to the a subunits of FSH, LH and TSH hormones. The hCG b subunit – molecular 23,000 – has a structure similar to that of the LH b subunit, differing by only a few epitopes. HCG has biologicals characteristics similar to LH. During pregnancy, hCG stimulates the remaining corpus luteum and the placenta tissue to secrete the various steroid hormones. In addition to its stimulating action on the luteal and placental tissue, hCG, by crossing the placenta, is essential to differentiate the genital tractus of the fetus, which occurs around the 7^th week of pregnancy.

The Bio-Line hCG+b -Irma is an immunoradiometnc assay based on coated-tube separation in which several monoclonal antibodies directed against distinct epitopes of hCG have been used.. Mabs1, the capture antibodies, are attached to the lower and inner surface of the plastic tube. Standards or samples added to the tubes will 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.

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-Monoclonal-anti-b hCG tracer: 1 vial (red solution) containing 5.5 ml. Activity < 18µCi or 700 kBq.

3. Coated tubes: 2 x 50 tubes, coated with Anti-b -hCG 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 and 500 µl (the use of accurate pipettes with disposable plastic tips is recommended)

3. 2 ml automatic syringe (Cornwall type) for washing

4. Aspiration system (optional)

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

 

A. Serum or plasma : Serum and plasma must be kept at 2 – 8°C.

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

Serum, heparinized or EDTA plasma provide similar results.

                    Y (serum) = 1.03x (hep. plasma) + 0.07 r = 0.99 n = 30

                    Y (serum) = 1.00x (EDTA plasma) + 0.47 r = 0.99 n = 30

B. Urine : Urine samples can be tested in the b hCG qualitative procedure only. Samples must be kept at 2 to 8°C for up to 2 days.

If the test is not run within 24 h, storage at –20°C is recommended.

Do not use the kit or components beyond expiration date. Do not mix materials from differents kit lots. Bring all the reagents to room temperature prior to use. Thoroughly mix all reagents and samples by gentle agitation or swirling.

Use a clean disposable pipette tip for addition of each different reagent and sample in order to avoid cross-contamination.

High precision pipettes or automated pipetting equipment will improve the precision. Respect the incubation times.

Prepare a standard curve for each run, do not use data from previous runs.

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

2. Vortex mix briefly standards, samples, controls and dispense 50 µl of each into the respective tubes.

            3. Dispense 50 µl of tracer into each tube, including the ones for Total count.

4. Shake the rack containing the tubes gently by hand.

5. Incubate for 30 min at 37°C. (or alternative : 30 min at room temperature on a tube shaker).

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

7. Wash the tubes with 2 ml Wash Solution (except total counts). Avoid foaming during the addition of the Wash Solution.

8. Aspirate (or decant) the content of each tube (except total counts).

9. After the washing let the tubes standing upright for 2 minutes and aspirate the remaining drop of liquid.

            10. 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 hCG (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.

                            INTRA ASSAY                                                                     INTER ASSAY

C. Speciflcity

Cross-reactive hormones were added to a low b hCG value serum and to a high value standard (150 mIU/ml). The apparent b hCG response was measured.