3.       PRINCIPLES  OF  THE METHOD

In the present kit, Bio-Line has introduced a pre‑treatment step in order to improve the clinical performance of the assay.  It is well established that the binding proteins interfere with the radioimmunoassay for SM-C.  The pre‑treatment step used by Bio-Line is the acid‑ethanol procedure of Daughaday et al. (8). 

A fixed amount of ¹²⁵I labelled SM‑C competes with the SM‑C to be measured present in the sample or in the calibrator for a fixed amount of antibody sites being immobilized to the wall of a polystyrene tube.  After an overnight incubation at 2‑8°C, an aspiration step stops the competition reaction. The tubes are then washed with 3 ml of wash solution and aspirated again.  A calibration curve is plotted and the SM‑C concentrations of the samples are determined by dose interpolation from the calibration curve.

4.       REAGENTS PROVIDED

Note :      1.             Use the zero calibrator for sample dilutions.

2.   1 ng of the calibrator preparation is equivalent to 1 ng of the 1^st IS 91/554. 

5.       SUPPLIES NOT PROVIDED

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

1.        Distilled water

2.        Pipettes for delivery of: 100 μl, 400 µl, 500 μl, 600 µl, 1 ml  and 3 ml (the use of accurate pipettes with disposable plastic tips is recommended)

3.        Vortex mixer

4.        Magnetic stirrer

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

6.        Aspiration system (optional)

7.        Tube shaker (1200 rpm)

8.        Centrifuge (3000 g)

9.        Incubator at 2-8°C

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

6.       REAGENT PREPARATION

A.       Calibrators: Reconstitute the zero calibrator with 3 ml reconstitution solution and other calibrators with 1 ml reconstitution solution.

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.

7. 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 one week at 2 to 8°C. For longer storage periods, aliquots should be made and kept at ‑20°C for maximum 3 months.

-         Avoid successive freezing and thawing.

-         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.

8.       SPECIMEN  COLLECTION  AND  PREPARATION

-         Serum or plasma samples must be kept at 2‑8°C.

-         If the test is not run within 48 hrs, storage in aliquots at ‑20°C is recommended.

-         Avoid successive freezing and thawing.

-         Serum and heparinized plasma provide similar results.                              

          Y (serum) = 0.95x (hep. plasma) + 28         r = 0.91    n = 28

          Y (serum) = 0.89x (EDTA plasma) + 32       r = 0.88    n = 28

-         After extraction, the samples can be stored at 2-8°C for 7 days.

9.       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.       Pre-treatment step

1.       Label two plastic tubes for each sample and control.

2.       Dispense 100 μl of each sample and control into the first tube.

3.       Add 400 μl of pre‑treatment solution into this tube.

4.       Shake all the tubes at 1200 rpm during 30 minutes.

5.       Centrifuge for 10 minutes at 1500 g.

6.       Take 100 μl of the supernatant and transfer it into the second labelled tube.

7.       Add 600 μl of the neutralisation solution to the second tube.

8.       Vortex each tube.

C.      Modified pre-treatment procedure

In case of renal failure we recommend a modified extraction procedure.

1-8.    See pre-treatment step.

9.       Store the neutralized extract at –20°C for l h, then centrifuge immediately at 3000 g for 30 min at 4°C.

10.     Decant the supernatant into fresh tubes and assay it as described below.

D.       Procedure

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

2.        Briefly vortex calibrators, controls and samples and dispense 100μl of each into respective tubes.

3.        Dispense 500 µl of ¹²⁵Iodine labelled SM-C into each tube, including the uncoated tubes for total counts.

4.        Shake the tube rack gently by hand to liberate any trapped air bubbles.

5.        Incubate overnight at 2-8°C.

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 tubes with 3 ml Working Wash solution (except total counts) and aspirate (or decant). Avoid foaming during the addition of the Working Wash solution.

8.        Let the tubes stand upright for two minutes and aspirate the remaining drop of liquid.

9.        Count tubes in a gamma counter for 60 seconds.

10.     CALCULATION  OF  RESULTS

1.        Calculate the mean of duplicate determinations.

2.        Calculate the bound radioactivity as a percentage of the binding determined at the zero calibrator point (0) according to the following formula 

3.        Using a 3 cycle semi-logarithmic or logit‑log graph paper, plot the (B/B0(%)) values for each calibrator point as a function of the SM-C concentration of each calibrator point.  Reject obvious outliers. 

4.        Computer assisted methods can also be used to construct the calibration curve. If automatic result processing is used, a 4-parameter logistic function curve fitting is recommended.

5.        By interpolation of the sample (B/B0 (%)) values, determine the SM-C concentrations of the samples from the calibration curve.

6.        The concentrations read on the calibration curve must be multiplied by 35 (dilution factor during the pre-treatment step).

7.        For each assay, the percentage of total tracer bound in the absence of unlabelled SM-C (B0/T) must be checked.

11.     TYPICAL DATA

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

12.     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 below the average counts at zero binding, was 0.25 ng/ml (x 35 for neat sample).

B.       Specificity

The percentages of cross-reaction estimated by comparison of the concentration yielding a 50% inhibition are respectively:

Note: this table shows the cross-reactivity for the anti SM-C.

C.      Precision

SD: Standard Deviation; CV: Coefficient of variation

D.       Accuracy    

                                             DILUTION  TEST

Samples were diluted with the zero calibrator.

                                           RECOVERY  TEST

Conversion factor:

From ng/ml to nmol/L:              x 0.1307

From nmol/L to ng/ml:              x 7.649

13.     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.

14.     REFERENCE  INTERVALS

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

Normal subjects

15. PRECAUTIONS  AND  WARNINGS

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. 

16.     BIBLIOGRAPHY

1.       DAUGHADAY W.H. and ROTWEIN P.  (1989)

Insulin-like growth factors I and II.  peptide, messenger ribonucleic acid and gene structures, serum and tissue concentrations.

Endocrine Rev., 10(1) : 68-91.

2.       FROESCH E.R. and ZAPF J.  (1985)

Insulin-like growth factors and insulin : comparative aspects.

Diabetologia, 28 : 485-493.

3.       FURLANETTO R.W., UNDERWOOD L., VAN WYK J.J.., D’ERCOLE A.J.  (1977)

Estimation of spmatomedin-C levels in normals and patients with pituitary disease by radioimmunoassay.

J. Clin. Invest. 60 : 648.

4.       ROSENFELD R.G., WILSON D.M., LEE P.D.K. and  HINTZ R.L.  (1986)

Insulin-like growth factors I and II in evaluation of growth retardation.

J. Pediatr., 109 : 428.

5.       RUDMAN D., KUTNER M.H. and CHAWLA R.K.  (1985)

The short child with subnormal plasma somatomedin-C.

Pediatric Res., 19(10) : 975-980.

6.       ALBERTSSON-WIKLAND K. and HALL K.  (1987)

Growth hormone treatment in short children : relationship between growth and serum insulin-like growth factor I and II levels.

J. Clin. Endocrinol Metab., 65 : 671.

7.       WASS J.A.H., CLEMMONS D.R., UNDERWOOD L.E., BARROW L., BESSER G.M. and VAN WYK J.J.  (1982)

Changes in circulating somatomedin-C levels in bromocriptine treated acromegaly.

Clin. Endocrinol., 17 : 369-377.

8.       DAUGHADAY W.H., MARIZ I.K. and BLETHEN S.L.  (1980)

Inhibition of access of bound somatomedin to membrane receptor and immunobinding sites - a comparison of radioreceptor and radioimmunoassay of somatomedin in native and acid-ethanol extracted serum.

J. Clin. Endocrinol. Metab., 51 : 781.

9.       BREIERB.H., GALLAHER B.W. and GLUCKMAN P.D.  (1991)

Radioimmunoassay for insulin-like growth factor-I : solutions to some potential problems and pitfalls.

Journal of Endocrinology, 128 : 347-357.

10.     SCHOUTEN J.S.A.G. and al.  (1993)

IGF1 : a prognostic factor of knee osteoarthritis.

British J. Rheumatol., 32 : 274-280.

11.     GRONBAEK H., SKJAERBAEK C., NIELSEN B., FRYSTYK J., FOEGH M.L.,  FLYVBJERG A., ORSKOV H.  (1995)

Growth hormone and insulin-like growth factor-I: a suggested role in renal transplantation and graft vessel disease. 

Transplant. Proceed., 27/3 : 2133-2136.

12.     TSITOURAS P.D., ZHONG Y.G., SPUNGEN A.M., BAUMAN W.A.  (1995)

Serum testosterone and growth hormone insulin-like growth factor-I in adults with spinal cord injury.

Hormone and metabolic Research , 27/6 : 287-292.

13.     KOCH A., DORR H.G., GERLING S., BEHRENS R., BOHLES H.J.  (1995)

Effect of growth hormone on IDF-I levels in patients with growth hormone deficiency and Wilson disease.

Hormone Research, 44/1 : 40-44.

17.                           SUMMARY OF THE PROTOCOL