2. Principles of the method

A.       Key feature

The Free βhCG IRMA has been developed in order to provide medical laboratories with an assay characterized by two dominant features :

·          A 100 % recognition with free β chain to guarantee detection of ectopic tumour secreting predominantly free β chain rather than native hCG.

·          A pregnancy test that provides a precise quantitative measurement of βhCG Free level in serum when using 1 hour incubation protocol without cross reaction with dimeric hCG.

B.       Principle of the test

The Bio-Line βhCG-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. Calibrators 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. The use of several distinct Mabs avoids hyperspecificity.

3. Reagents Provided

Note:  1.   Use the specimen diluent for sera dilutions.

2.   1 mIU of the calibrator preparation is equivalent to 1 mIU of the 3^rd IRP 75/551.

4. Supplies not provided

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

1.           Distilled water

2.           Pipettes for delivery of: 50 μl, 200 μl, 500 µl and 1 ml. (the use of accurate pipettes with disposable plastic tips is recommended)

3.           Vortex mixer

4.           Magnetic stirrer

5.           Tube shaker (600 rpm)

6.           5 ml automatic syringe (Cornwall type) for washing

7.           Aspiration system (optional).

8.           Any gamma counter capable of measuring ¹²⁵I may be used (minimal yield 70%).

5. reagents preparation

A.       Calibrators: Reconstitute the calibrators with 0.5 ml distilled water.. 

B.       Controls: Reconstitute the controls with 0.5 ml distilled water.

C.       Working Wash solution: Prepare an adequate volume of Working Wash solution by adding 69 volumes of distilled water to 1 volume of Wash Solution (70x). Use a magnetic stirrer to homogenize. Discard unused Working Wash solution at the end of the day.

6. Storage and expiration dating of reagents

-         Before opening or reconstitution, all kit components are stable until the expiry date, indicated on the label, if kept at 2 to 8°C.

-                 After reconstitution, calibrators and controls are stable for 7 days at 2-8°C.  

          For longer storage periods, aliquots should be made and kept at –20°C for 3 months.  Avoid subsequent freeze-thaw cycles.

-         Freshly prepared Working Wash solution should be used on the same day.

-         After its first use, tracer is stable until expiry date, if kept in the original well-closed vial at 2 to 8°C.

-         Alterations in physical appearance of kit reagents may indicate instability or deterioration.

7. Specimens collection and preparation

         Serum must be kept at 2 – 8°C.

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

         Avoid subsequent freeze-thaw cycles.

8. Procedure

A.       Handling notes

          Do not use the kit or components beyond expiry date.  Do not mix materials from different kit lots. 

Bring all the reagents to room temperature prior to use.

          Thoroughly mix all reagents and samples by gentle agitation or swirling. In order to avoid cross-contamination, use a clean disposable pipette tip for the addition of each reagent and sample.

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

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

B.       Procedure

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

2.           Briefly vortex calibrators, samples and controls and dispense 50 μl of each into the respective tubes.

3.           Dispense 200 µl of Incubation Buffer into each tube except total counts.

4.           Incubate for 30 min at room temperature with continuous shaking (600 rpm).   

5.           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.       

6.           Wash the tubes twice with 3 ml Wash Solution (except total counts).  Avoid foaming during the addition of the Working Wash Solution.

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

8.           After the last washing, let the tubes standing upright for two minutes and aspirate the remaining drop of  liquid.

9.           Dispense 200 μl of anti-βhCG-¹²⁵I tracer into each tube, including the uncoated tubes for total counts.

10.        Shake the rack containing the tubes gently by hand to liberate any trapped air bubbles.

11.        Incubate for 30 min at room temperature with continuous shaking (600 rpm).           

12.        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.

13.        Wash the tubes with 3 ml Wash Solution (except total counts).  Avoid foaming during the addition of the Working Wash Solution.

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

15.        Let the tubes standing upright for two minutes and aspirate the remaining drop of  liquid.

16.        Count the tubes in a gamma counter for 60 seconds.

9. Calculation of results

1.           Calculate the mean of duplicate determinations.

2.           On semi-logarithmic or linear graph paper plot the c.p.m. (ordinate) for each calibrator against the corresponding concentration of βhCG (abscissa) and draw a calibration curve through the calibrator points, reject the obvious outliers.

3.           Read the concentration for each control and sample by interpolation on the calibration curve. 

4.           Computer assisted data reduction will simplify these calculations.  If automatic result processing is used, a 4-parameter logistic function curve fitting is recommended.

10. Typical data

The following data are for illustration only and should never be used instead of the real time calibration curve.

11. Performance and limitations

A.      Detection Limit

Twenty zero calibrators were assayed along with a set of other calibrators. The detection limit, defined as the apparent concentration two standard deviations above the average counts at zero binding, was 0.03 mIU/ml.

B.       Specificity

Cross-reactive hormones were added to a low and to a high βhCG value calibrator.  The apparent βhCG response was measured.

C.      Precision

D.       Accuracy

                                           RECOVERY  TEST

Samples were diluted with specimen diluent.

E.       Time Delay

As shown below, assay results remain accurate even when a sample is dispensed up to 30 minutes after the calibrator has been added to the coated tubes.

F.       Hook effect

A serum sample with a concentration of 12800 mIU/ml βhCG gives a signal above the highest calibrator concentration.

12. Internal Quality control

-         If the results obtained for Control 1 and/or Control 2 are not within the range specified on the vial label, the results cannot be used unless a satisfactory explanation for the discrepancy has been given.

-         If desirable, each laboratory can make its own pools of control samples, which should be kept frozen in aliquots.

-         Acceptance criteria for the difference between the duplicate results of the samples should rely on Good Laboratory Practises

13. Reference intervals

The values provided below are given only for guidance; each laboratory should establish its own normal range of values. 

14. Precautions and warning

Safety

For in vitro diagnostic use only.

This radioactive product can be transferred to and used only by authorized persons; purchase, storage, use and exchange of radioactive products are subject to the legislation of the end user's country.  In no case the product must be administered to humans or animals.

All radioactive handling should be executed in a designated area, away from regular passage.  A logbook for receipt and storage of radioactive materials must be kept in the lab.  Laboratory equipment and glassware, which could be contaminated with radioactive substances, should be segregated to prevent cross contamination of different radioisotopes.

Any radioactive spills must be cleaned immediately in accordance with the radiation safety procedures.  The radioactive waste must be disposed of following the local regulations and guidelines of the authorities holding jurisdiction over the laboratory.  Adherence to the basic rules of radiation safety provides adequate protection.

The human blood components included in this kit have been tested by European approved and/or FDA approved methods and found negative for HBsAg, anti-HCV, anti-HIV-1 and 2.  No known method can offer complete assurance that human blood derivatives will not transmit hepatitis, AIDS or other infections.  Therefore, handling of reagents, serum or plasma specimens should be in accordance with local safety procedures.

All animal products and derivatives have been collected from healthy animals. Bovine components originate from countries where BSE has not been reported. Nevertheless, components containing animal substances should be treated as potentially infectious.

Avoid any skin contact with reagents (sodium azide as preservative).  Azide in this kit may react with lead and copper in the plumbing and in this way form highly explosive metal azides.  During the washing step, flush the drain with a large amount of water to prevent azide build-up.

Do not smoke, drink, eat or apply cosmetics in the working area.  Do not pipette by mouth.  Use protective clothing and disposable gloves. 

15. Bibliography

1.       CHEN  F. et al., (1987)

Radioimmunoassay of the serum free β-subunit of human chorionic gonadotropin in trophoblastic disease.

J. Clin. Endocrinol. Metab., 64:313.

2.       DAWOOD  M.Y. et al., (1977)

Human chorionic gonadotropin and its subunit in hydatidiform mole and choriocarcinoma.

Obstet. Gynecol., 50:172.

3.       GASPARD  V.J. et al., (1980)

Serum concentration of human chorionic gonadotropin and its alpha and beta subunit in Trophoblastic tumours.

Clin. Endocrinol. (Oxf.), 13:219.

4.       PIERCE  J.G. et al., (1981)

Glycoprotein hormones : structure and function.

Annu.  Rev. Biochem., 50:465.

5.       KARDANO A. et al., (1994)

Human Chorionic Gonadotropin β-Subunit Nicking enzymes in Pregnancy and Cancer Patient Serum.

J. Clin. Endocr. Metab., 79:761-767.

16. Summary of the protocol