3.        Principles of the method 

The Bio-Line hPTH-120 min-IRMA is a two-step immunoradiometric assay based on coated-tube separation.  It allows the determination of intact human PTH (hPTH) in serum.  Goat antibodies specific to the 1-34 hPTH fragment (N-terminal fragment) are attached to the lower and inner surface of the plastic tubes.  Calibrators or samples are added to the tubes.  After 1 hour incubation, washing removes the occasional excess of antigen, mid-regional and C-terminal fragments.

¹²⁵I labelled monoclonal antibodies specific to the 44-68 hPTH fragment are added.  After 1 hour incubation and washing the remaining radioactivity bound to the tube reflects the intact h-PTH concentration.  This two-step IRMA is highly specific of the intact h-PTH and does not cross react with active and inactive fragments even at high concentrations as suggested by HACKENG and al.

4.          Reagents provided 

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

2.   1 pg of the calibrator preparation is equivalent to 1 µIU of 1^st IRP 79/500. -> calibration necessary

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, 300 μl, 1 ml, 2 ml and 3 ml. (the use of accurate pipettes with disposable plastic tips is recommended)

3.           Vortex mixer

4.           Magnetic stirrer

5.           Tube shaker (700 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%).

6.       Reagents preparation

A.       Calibrators : Reconstitute the zero calibrator with 3 ml reconstitution solution and the other calibrators with 2 ml reconstitution solution. 

B.       Controls : Reconstitute the controls with 2 ml reconstitution solution.

C.       Working Wash solution : Prepare an adequate volume of Working Wash solution by adding 27 volumes of distilled water to 1 volume of Wash Solution (28x). 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.

-         The calibrators and controls are very unstable, use them immediately after reconstitution, freeze immediately in aliquots and keep them at –20°C for maximally 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 and plasma must be kept at 2 – 8°C.

§          If the test is not run within 8 hours, storage at –20°C is recommended.

§          Avoid subsequent freeze-thaw cycles.

§          Serum or plasma (heparin and EDTA) provides similar results.  -> still to establish

Y(serum) = 1.01 x (EDTA plasma) – 21.54     r=0.91      n=6

Y(serum) = 0.99 x (heparin plasma) – 6.14    r=0.94      n=10

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.       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, controls and samples and dispense 300 μl of each into the respective tubes.

3.           Dispense 100 µl Incubation Buffer in each tube except those for total counts.

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

5.           Incubate for 1 hour at room temperature on a tube shaker (700 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 the tubes with 2 ml 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 again the tubes with 2 ml Wash Solution (except total counts) and aspirate (or decant).

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

11.        Dispense 100 μl of anti-PTH-¹²⁵I tracer into each tube, including the uncoated tubes for total counts.

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

13.        Incubate for 1 hour at room temperature on a tube shaker (700 rpm).           

14.        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.

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

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

17.        Wash again the tubes with 2 ml Wash Solution (except total counts) and aspirate (or decant).

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

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

10.       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 PTH (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.

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 limitation 

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 4.1 pg/ml.

B.       Specificity

Possible interfering peptides were added to a low and to a high PTH level serum.  The apparent PTH response was measured.

This demonstrates that the hPTH-120 min-IRMA does not cross react with hPTH fragments and hPTH-related protein.

C.      Precision

SD : Standard Deviation; CV: Coefficient of variation

D.       Accuracy               

                                           RECOVERY  TEST

Samples were diluted with zero calibrator.

E.       Time Delay

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

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 interval

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

The range of PTH levels in 117 normal patients, expressed as 2.5% to 97.5% percentiles, was 6.2 to 29 pg/ml.

15. 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. 

16.     Bibliography 

1.       HABENER J.F., and POTTE J.T., Jr.  (1978)

"Biosynthesis of parathyroid hormone".

New Engl. J. Med., 299, 11:580 and 299, 12:635.

2.       MARTIN K.J., HRUSKA K.A., FREITAG J.J., KLAH S. and SLOTOPOLSKY E.  (1979)

"The peripheral metabolism of parathyroid hormone".

New Engl. J. Med., 301, 20:1092.

3.       GOLTZMAN D., HENDERSON B. and LOVERIDGE N.  "Cytochemical bioassay of PTH.  (1980)

Characteristics of the assay and analysis of circulating hormone forms".

J. Clin. Invest., 65:1309.

4.       POTTS J.T. Jr., KRONENBERG H.M., ROSENBLATT M.  (1982)

"Parathyroid hormone : Chemistry, biosynthesis and mode of action".

Adv. Protein Chem., 323.

5.       HACKENG W.H.L., LIPS P., NETELENBOS J.C. and LIPS C.J.M.  (1986)

"Clinical implication of estimation of intact parathyroid hormone (PTH) versus total immunoreactive PTH in normal subjects and hyperparathyroid patients".

J. Clin. Endocrinol. Metab., 63:447.

6.       BOUILLON R., COOPMANS W., DE GROOTE D.E.H., RADOUX D., ELIARD P.H.  (1990)

"Immunoradiometric assay of Parathyrin with polyclonal and monoclonal region specific antibodies".

Clin. Chem., 36/2:271-276.

17. Summary of the protocol