II.       PRINCIPLES  OF  THE METHOD

The Bio-Line hOST-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, common to two-site IRMA, as well as a need of a shaker or long incubation at 37°C.

III.            REAGENTS PROVIDED

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

2.        The origin of the osteocalcin for the preparation of the calibrators is human.

IV.      SUPPLIES NOT PROVIDED

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

1.        Distilled water

2.        Trasylol® at 10000IU/ml

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

4.        Vortex mixer

5.        Magnetic stirrer

6.        Refrigerated centrifuge

7.        5 ml automatic syringe (Cornwall type) for washing

8.        Aspiration system (optional)

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

V.       REAGENT PREPARATION

A.       Calibrators : Reconstitute the zero calibrator with 1.0 ml calibrator diluent and other calibrators with 0.5 ml calibrator diluent.

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.

VI.                 STORAGE  AND  EXPIRATION  DATING  OF  REAGENTS

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

-         After reconstitution, calibrators and controls are very unstable, use them immediately after reconstitution.   For longer storage periods, aliquots should be made and kept at –20°C for maximally 6 weeks.  Freezing should be performed immediately after use, do not wait for freezing until all the samples are pipetted.

          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.

VII.     SPECIMEN  COLLECTION  AND  PREPARATION

-         Serum or heparin and EDTA plasma provide similar results.

-         Collect blood by venipuncture, taking care to avoid hemolysis, the samples must be kept in an ice bath.  Separate the plasma or serum from the cells within 3 hours, the use of a refrigerated centrifuge is recommended.  Add 100 µl Trasylol® (10000IU/ml) to the plasma or serum immediately after centrifugation (to obtain 1000 IU Trasylol® per ml sample).

With this treatment the samples are stable for 3 days at 2-8°C.  For a longer delay the samples have to be frozen (- 20°C), however the samples can only be thawn once!  For repeat testing freeze the samples in aliquots and discard each sample after the first thawing.

-         Do not use citrate plasma, hemolyzed samples or lipemic samples.

VIII.    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, sample and control.  For determination of total counts, label 2 normal tubes.

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

3.           Dispense 50 μl of anti-OST-¹²⁵I tracer into each tube, including the uncoated tubes for  total counts.

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

5.           Incubate for 2 hours at room temperature

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). Avoid foaming during the addition of the Working Wash solution.

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

9.           Wash tubes again with 2 ml Wash solution (except total counts) and aspirate (or decant).

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

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

IX.      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 OST (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 to be used, a 4-parameter logistic function curve fitting is recommended.

5.       If Trasylol® is added to the samples (100 μl/ml), sample values have to be multiplied by 1.1.

X.       TYPICAL DATA

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

XI.      PERFORMANCE  AND  LIMITATIONS

A.       Detection limit

          Twenty zero calibrators were assayed along with a set of the other calibrators.  The detection limit, defined as the apparent concentration of the average count at zero binding plus two standard deviations, was 0.15 ng/ml.

B.       Specificity

This method detects intact human osteocalcin.  N-terminal and C-terminal fragments, at their maximum levels found in normal and pathological samples, were added to a low and a high value calibrator.  No cross reactivity was observed at these concentrations. 

C.      Precision

SD : Standard Deviation; CV: Coefficient of variation

D.       Accuracy

                                           RECOVERY  TEST

Samples were diluted with zero calibrator.

To the best of our knowledge, no international reference material exists for this parameter.

E.       Time delay between last calibrator and sample dispensing

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

F.       Hook-effect

A sample spiked with OST up to 1000 ng/ml gives higher counts than the last calibrator point.

XII.     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

  XIII.               REFERENCE  INTERVALS

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

Healthy subjects obtained values ranging from 5 to 25 ng/ml (2.5 to 97.5 percentiles).

XIV.   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. 

XV.    BIBLIOGRAPHY

1.       J.P. BROWN, P.D. DELMAS and al.  (May 19, 1984)

"Serum BGP : a specific marker for bone formation in postmenopausal osteoporosis".

The Lancet, 1091-1093.

2.       P.A. PRICE.  (1985)

"Vitamin K-dependent formation of osteocalcin and its function".

Vitamins and hormones, 42,65-108.

3.       R.E. COLEMAN, G. MASHITER and al.  (1988)

"Osteocalcin : a potential marker of metastatic bone disease and response to treatment".

          Eur. J. Cancer Clin. Oncol., 24,1211-1217.

4.       S. MINISOLA and al.  (1988)

"Serum osteocalcin in primary hyperparathyroidism : short-term effect of surgery".

Mineral Electrolyte Metab., 14,201-207.

5.       L.A. COULTON, C.J. PRESTON and al.  (1988)

"An evaluation of serum osteocalcin in Paget's disease of bone and its response to diphosphonate treatment".

Arthritis and Rheumatism, 31,9,1142-1147.

6.       J.S. JOHANSEN, S.B. JENSEN and al.  (1990)

"Serum BGP : a potential marker of GH deficiency and the response to GH therapy".

Journal of Clinical Endocrinology and Metabolism, 71,1,122-126.

7.       M.J. POWER and P.F. FOTTRELL.  (1991)

"Osteocalcin : Diagnostic Methods and Clinical Applications".

Crit. Rev. Clin. Lab. Sci., 28,4,287-335.

8.       B. DEMIAUX, M.E. ARLOT and al.  (1992)

"Serum osteocalcin is increased in patients with biochemical and histomorphometric findings".

Journal of Clinical Endocrinology and Metabolism, 74,5,1146-1151.

9.       J.C. DUMON, H. WANTIER, F. MATHIEU, M. MANTIA and J.J. BODY.  (1996)

Technical and clinical validation of a new immunoradiometric assay for human osteocalcin.

European Journal of Endocrinology, 135,231-237.

XVI.     SUMMARY OF THE PROTOCOL