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 IGF-I. 

A fixed amount of ¹²⁵I labelled IGF-I competes with the IGF-I to be                measured present in the sample or in the calibrator for a fixed amount of antibody sites immobilized on the wall of a polystyrene tube.  After 2 hours with shaking incubation at room temperature, an aspiration step terminates the competition reaction. The tubes are then washed with 2 ml of washing solution   and aspirated. A calibration curve is plotted and the IGF-I concentrations of the     samples are determined by dose interpolation from the calibration curve.

4.       REAGENTS PROVIDED

Note :    1. Use the dilution buffer for sample dilution                      

               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: 50 μl, 100 µl, 500 μl and 1 ml (the use of accurate pipettes with disposable plastic tips is recommended)

3.        Plastic tubes for dilution of samples

4.        Vortex mixer

5.        Magnetic stirrer

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

7.        Aspiration system (optional)  

8.        Tube shaker (400 rpm)

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

6.       REAGENT PREPARATION

A.       Calibrators: Reconstitute the calibrators with 1ml 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.

7.        STORAGE  AND  EXPIRATION  DATING  OF  REAGENTS

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

-         after econstitution, calibrators and controls are stable for one week at 2 to 8°C. For longer storage periods, aliquots should be made and keep at -20°C for maximum 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.

8.       SPECIMEN  COLLECTION  AND  PREPARATION

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

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

-         Avoid subsequent freeze-thaw cycles.

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.

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

B.       Pre-treatment step

1.              Label one plastic tube for each sample and control.

2.              Dispense 50 μl of each sample and control into the tube.

3.             Add 50 μl of pre‑treatment solution into this tube.

4.              Vortex each tube during 5 seconds.

5.              Incubate 30 minutes at room temperature.

6.             Add 1 ml of the dilution buffer into the tube.

7.              Vortex each tube.

C.     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 200 µl of ¹²⁵Iodine labelled IGF-I 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 for 120 minutes at room temperature on a tube shaker (400 rpm).

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 2 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 IGF-I 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 IGF-I concentrations of the samples from the calibration curve.

6.        For each assay, the percentage of total tracer bound in the absence of unlabelled IGF-I (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 3.4 ng/ml

B.       Specificity

The percentage 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 IGF-I

C.      Precision

SD: Standard Deviation; CV: Coefficient of variation

D.       Accuracy    

                                             DILUTION  TEST

Samples were diluted with Dilution Buffer.

RECOVERY  TEST

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 - Mean and range based on 2.5% & 97.5% percentiles

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