II.     PRINCIPLES  OF  THE METHOD

The Bio-Line TESTO-ELISA is a solid phase Enzyme Amplified Sensitivity Immunoassay performed on a microtiterplate.    A fixed amount of testosterone labelled with horseradish peroxidase (HRP), compete with unlabelled testosterone present in the calibrators, controls and samples for a limited number of binding sites on a specific antibody.

After 2 hours incubation at room temperature, the microtiterplate is washed to stop the competition reaction.

The revelation solution (TMB – H₂O₂) is added and incubated for 30 min.  The reaction is stopped with the addition of Stop Solution and the microtiterplate is then read at the appropriate wavelength.   The amount of substrate turnover is determined colourimetrically by measuring the absorbance, which is inversely proportional to the TESTO concentration. 

A calibration curve is plotted and TESTO concentration in samples is determined by interpolation from the calibration curve. 

III.      REAGENTS PROVIDED

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

IV.      SUPPLIES NOT PROVIDED

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

1.           High quality distilled water

2.           Pipettes for delivery of: 50 μl, 100 μl, 200 µl, 500 µl and 2 ml (the use of accurate pipettes with disposable plastic tips is recommended)

3.           Vortex mixer

4.           Magnetic stirrer

5.           Horizontal microtiterplate shaker capable of 700 rpm ± 100 rpm

6.           Washer for microtiterplates

7.           Microtiterplate reader capable of reading at 450 nm and 650 nm (or 630 nm)

V.       REAGENT PREPARATION

a.       Calibrators : Reconstitute the zero calibrator with 1.0 ml distilled water and the other calibrators with 0.5 ml distilled water.

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

c.       Working TESTO-HRP conjugate : dilute 0.2 ml of the concentrated TESTO-HRP conjugate into one of the vials of conjugate buffer.  Extemporaneous preparation is recommended.

d.       Working Wash solution : Prepare an adequate volume of Working Wash solution by adding 199 volumes of distilled water to 1 volume of Wash Solution (200x). Use a magnetic stirrer to homogenize. Discard unused Working Wash solution at the end of the day. 

e.        Revelation Solution: pipette 0.1 ml of the chromogen TMB into one of the vials of substrate buffer (H₂O₂ in acetate/citrate buffer).  Extemporaneous preparation is recommended. 

  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.

§             Unused strips must be stored, at 2-8°C, in a sealed bag containing a desiccant until expiration date.

§             After reconstitution, calibrators and controls are stable for 1 week at 2 to 8°C. For longer storage periods, aliquots should be made and kept at ‑20°C.  Avoid successive freeze thaw cycles.

§             The concentrated Wash Solution is stable at room temperature until expiration date.

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

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

§             The Working TESTO-HRP conjugate is stable for 4 hours at room temperature or for 24 hours at 2-8°C, avoid direct sunlight.

§             The freshly prepared revelation solution is stable, before use, for maximum 15 minutes at room temperature and must be discarded afterwards.

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

VII.     SPECIMEN  COLLECTION  AND  PREPARATION

§             Serum must be kept at 2 - 8°C.

§             If the test is not run within 24 hours, storage in aliquots at -20°C is recommended.  Avoid subsequent freeze thaw cycles.

§             Prior to use, all samples should be at room temperature.  It is recommended to vortex the samples before use.

§             Do not use haemolysed 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.

          Perform calibrators, controls and samples in duplicate.  Vertical alignment is recommended.

          Use a clean plastic container to prepare the Wash Solution.

          In order to avoid cross-contamination, use a clean disposable pipette tip for the addition of each reagent and sample.

          For the dispensing of the Revelation Solution and the Stop Solution avoid pipettes with metal parts.

High precision pipettes or automated pipetting equipment will improve the precision. 

          Respect the incubation times. 

          To avoid drift, the time between pipetting of the first calibrator and the last sample must be limited to the time mentioned in section XIII paragraph E (Time delay).

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

The chromogenic solution should be colourless.   If a dark blue colour develops within a few minutes after preparation, this indicates that the preparation is unusable and must be discarded.

Dispense the Revelation Solution within 15 minutes following the washing of the microtiterplate. 

During incubation with Revelation Solution, avoid direct sunlight on the microtiterplate.

B.       Procedure

1.           Select the required number of strips for the run.  The unused strips should be resealed in the bag with a desiccant and stored at 2-8°C.

2.           Secure the strips into the holding frame. 

3.           Pipette 50 µl of each Calibrator, Control and Sample into the appropriate wells.

4.           Pipette 200 µl of TESTO-HRP conjugate into all the wells.

5.           Incubate for 2 hours at room temperature on a horizontal shaker set at 700 rpm ± 100 rpm.

6.           Aspirate the liquid from each well.

7.           Wash the plate 3 times by:

§      Dispensing 0.4 ml of Wash Solution into each well

§      Aspirating the content of each well

8.           Pipette 200 µl of the freshly prepared revelation solution into each well within 15 minutes following the washing step.

9.           Incubate the microtiterplate for 30 minutes at room temperature on a horizontal shaker set at 700 rpm ± 100 rpm, avoid direct sunlight.

10.        Pipette 50 µl of Stop Solution into each well.

11.        Read the absorbencies at 450 nm (reference filter 630 nm or 650 nm) within 1 hour and calculate the results as described in section XI.

IX.      CALCULATION  OF  RESULTS

1.           Read the plate at 450 nm against a reference filter set at 650 nm (or 630 nm).

2.           Calculate the mean of duplicate determinations.

3.           Calculate for each calibrator, control and sample:

6.           By interpolation of the sample (B/B0 (%)) values, determine the TESTO concentrations of the samples from the calibration curve.

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 other calibrators. The detection limit, defined as the apparent concentration two standard deviations below the average OD at zero binding, was 0.05 ± 0.02 ng/ml.

B.       Specificity

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

C.      Precision

SD : Standard Deviation; CV: Coefficient of variation

D.       Accuracy    

                                            RECOVERY  TEST

Sample was diluted with zero calibrator.

Conversion factor :                

          From ng/ml to nmol/L :   x 3.47

          From nmol/L to ng/ml :   x 0.288   

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

The concentrations of the calibrators are determined with the ID-GC/MS reference method.

F.       Correlation with reference method

Serum samples from 21 women and  17 men, ranging from 0.15 to 8.9 ng/ml, were tested with the TESTO-ELISA and a titriated reference method.  The linear regression shows a good correlation between these two methods:

TESTO-ELISA = 1.03 (H³ reference) + 0.19                 r=0.96      n=38

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. Controls which contain azide will interfere with the enzymatic reaction and cannot be used.

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

§             It is recommended that Controls be routinely assayed as unknown samples to measure assay variability.  The performance of the assay should be monitored with quality control charts of the controls.

§             It is good practise to check visually the curve fit selected by the computer.

XIII.               REFERENCE  INTERVALS

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

          Females:       0.46 ng/ml (0.1 – 1.1)

          Males:           6.6 ng/ml (2.6 – 13.5)

XIV.   PRECAUTIONS  AND  WARNINGS

Safety

For in vitro diagnostic use only.

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 all reagents, Stop Solution contains H₂SO₄, the chromogen contains TMB in Dimethylformamide, Substrate buffer contains H₂O₂.   In case of contact, wash thoroughly with water.

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.L. ANDREYKO et al. (1986)

Role of Serum Androgens and Sex Hormone Binding Globulin Capacity in the Evaluation of Hirsutism in Women.

Clin. Biochem, Vol. 19, 58‑61.

2.       A. BIZZARO et al.

Influence of Testosterone Therapy on Clinical and Immunological features of Autoimmune Diseases Associated with Klinefelter's Syndrome

J. Clin. Endocrin. Metab. Vol. 64, N°1: 32‑36

3.       M. CARRABBA et al (1985)

Abnormalities of Sex hormones in Men with Systemic Lupus Erythematosus.

Clin. Rheumatology, N° 4: 422‑425.

4.       P. HILL et al. (1985)

Plasma Testosterone and Breast Cancer.

Eur.J Cancer Clin. Oncol, Vol. 21, N°10, pp. 1265‑1266

5.       CG. MAHLCK et al (1986)

Testosterone, SHBG and Albumin in Patients with ovarian carcinoma.

Acta Obstet. Gynecol. Scand. 65: 533‑S38.

6.       D.M.D PERERA et 31 (1987)

Amniotic Fluid Testosterone and testosterone Glucuronide Levels in the determination of Foetal Sex.

J. Steroid Biochem., Vol. 26,N°2,pp.273‑277.

7.       J. TRACHTENBERG (1987)

Experimental Treatment of Prostatic Cancer by intermittent Hormonal Therapy.

J. Urology, Vol. 137, pp. 785‑788.

8.       R. MARUYAMA et al. (1987)

Sex‑Steroid‑Binding Plasma Protein (SBP), Testosterone, Oestradiol and DHEA in Prepuberty and Puberty.

Acta Endocrinologica, 114: 60‑67

XVI.         SUMMARY OF THE PROTOCOL