Testo-easia-090511-1-en.doc

Read entire protocol before use.
TESTO-EASIA

I.
INTENDED USE
Enzyme Immunoassay for the in vitro quantitative measurement of human Testosterone (TESTO) in serum. II.
GENERAL INFORMATION
Proprietary name :
Catalogue number :
Manufactured by :
DIAsource ImmunoAssays S.A. Rue de l'Industrie, 8, B-1400 Nivelles, Belgium. For technical assistance or ordering information contact :
Tel : +32 (0)67 88.99.99
Fax : +32 (0)67 88.99.96

III. CLINICAL BACKGROUND
A.
Biological activity
Testosterone is a C-19 steroid hormone (molecular weight: 288 Da) which is produced from
androstenedione in the testes, adrenals and ovaries. Testosterone is a precursor along with androstenedione
of the estrogen steroids.
Clinical applications
Clinical significance of testosterone level : Source of testosterone :
Women : Ovary, adrenal cortex, peripheral tissues (by conversion of 50-60% other steroids). Men : Testes > 90%, adrenal cortex, peripheral tissues. Clinical diseases with high level of testosterone :
Women : Hirsutism and virilization, polycystic ovary syndrome, congenital adrenal hyperplasia (with 170H-PROG), tumors of adrenal and ovarian origin, breast cancer. Men : Disease of the hypothalamic pituitary unit, some malignant testicular tumors, congenital adrenal hyperplasia, prostate cancer. Clinical diseases with low level of testosterone :
Primary or secondary hypogonadism, Klinefelter's syndrome, other chromosomal alteration,
hypopituitarism, enzymatic defects, orchidectomy and cryptorchidism, testicular feminization, hepatic
cirrhosis, some autoimmune diseases for example : Sjögren's syndrome, systemic lupus.
Other domains for measurement of testosterone level :
In vitro fertilization : the women with high response to gonadotropin have a significant increase in testosterone. Parameter of the prepuberty and puberty. Determination of foetal sex in amniotic fluid. Free testosterone is significantly raised in both male and female acne sufferers. Follow up of cancer and in pathological situations; low testosterone syndrome. IV.
PRINCIPLES OF THE METHOD
VIII. STORAGE AND EXPIRATION DATING OF REAGENTS
The DIAsource TESTO-EASIA is a solid phase Enzyme Amplified Sensitivity Before opening or reconstitution, all kit components are stable until the Immunoassay performed on a microtiterplate breakable wells. A fixed amount expiry date, indicated on the vial label, if kept at 2 to 8°C. of testosterone labelled with horseradish peroxidase (HRP), compete with Unused strips must be stored, at 2-8°C, in a sealed bag containing a unlabelled testosterone present in the calibrators, controls and samples for a limited number of binding sites on a specific antibody. After reconstitution, calibrators and controls are stable for 1 week at 2 to After 1 hour incubation at room temperature, the wells are washed to stop the 8°C. For longer storage periods, aliquots should be made and kept at -20°C. Avoid successive freeze thaw cycles. Chromogenic solution (TMB) is added and incubated for 30 min. The reaction is The concentrated Wash Solution is stable at room temperature until stopped with the addition of Stop Solution and the wells are then read at the appropriate wavelength. The amount of substrate turnover is determined Freshly prepared Working Wash solution should be used on the same day. colourimetrically by measuring the absorbance, which is inversely proportional to After its first use, the conjugate is stable until expiry date, if kept in the A calibration curve is plotted and TESTO concentration in samples is determined The Working TESTO-HRP conjugate is stable for 4 hours at room by interpolation from the calibration curve. Alterations in physical appearance of kit reagents may indicate instability V.
REAGENTS PROVIDED
IX.
SPECIMEN COLLECTION AND PREPARATION
Reagents
Reconstitution
If the test is not run within 24 hours, storage in aliquots at -20°C is Ready for use
recommended. Avoid subsequent freeze thaw cycles. Prior to use, all samples should be at room temperature. It is Dilute 0.1 ml in 1.0 ml
recommended to vortex the samples before use. X.
PROCEDURE
Ready for use
Handling notes
Do not use the kit or components beyond expiry date. Add 1.0 ml distilled
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. Add 0.5 ml distilled
Perform calibrators, controls and samples in duplicate. Vertical alignment Use a clean plastic container to prepare the Wash Solution. In order to avoid cross-contamination, use a clean disposable pipette tip Dilute 200 x with
for the addition of each reagent and sample. distilled water (use a magnetic stirrer). For the dispensing of the Revelation Solution and the Stop Solution avoid pipettes with metal parts. Add 0.5 ml distilled
High precision pipettes or automated pipetting equipment will improve the Ready for use
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 Ready for use
Prepare a calibration curve for each run, do not use data from previous Note: 1. Use the zero calibrator for sample dilutions.
Dispense the Revelation Solution within 15 minutes following the washing VI.
SUPPLIES NOT PROVIDED
During incubation with Revelation Solution, avoid direct sunlight on the The following material is required but not provided in the kit: Procedure
Pipettes for delivery of: 50 μl, 100 μl, , 500 µl, 1 ml and 2 ml (the use of Select the required number of wells for the run. The unused wells should accurate pipettes with disposable plastic tips is recommended) be resealed in the bag with a desiccant and stored at 2-8°C. Secure the wells into the holding frame. Pipette 50 µl of each Calibrator, Control and Sample into the appropriate Microtiterplate reader capable of reading at 450 nm and 650 nm (or 630 Pipette 100 µl of TESTO-HRP conjugate solution into all the wells. Incubate for 1 hour at room temperature. VII.
REAGENT PREPARATION
§ Dispensing 0.4 ml of Wash Solution into each well Calibrators : Reconstitute the zero calibrator with 1.0 ml distilled water
and the other calibrators with 0.5 ml distilled water. Pipette 100 µl of the Chromogenic solution into each well within 15 minutes Controls : Reconstitute the controls with 0.5 ml distilled water.
Working TESTO-HRP conjugate : Prepare an adequate volume of
Incubate the microtiterplate for 30 minutes at room temperature, avoid conjugate solution by adding 100 µl of the concentrated TESTO-HRP conjugate to 1 ml of conjugate buffer. Use a vortex to homogenize. Pipette 100 µl of Stop Solution into each well. Extemporaneous preparation is recommended. Read the absorbencies at 450 nm (reference filter 630 nm or 650 nm) Working Wash solution : Prepare an adequate volume of Working Wash
within 1 hour and calculate the results as described in section XI. 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. XI.
CALCULATION OF RESULTS
Accuracy
Read the plate at 450 nm against a reference filter set at 650 nm (or 630 Added TESTO
Recovered TESTO
Recovery
Calculate the mean of duplicate determinations. Calculate for each calibrator, control and sample: Using either linear-linear of semi-logarithmic graph paper, plot the (B/B0(%)) values for each calibrator point as a function of the TESTO concentration of each calibrator point. Reject obvious outliers. Computer assisted methods can also be used to construct the calibration curve. If automatic result processing is used, a 4-parameter logistic By interpolation of the sample (B/B0 (%)) values, determine the TESTO concentrations of the samples from the calibration curve. Dilution
Theoretical Concent.
Measured Concent.
XII.
TYPICAL DATA
The following data are for illustration only and should never be used instead of TESTO-EASIA
B/B0 (%) values
Sample was diluted with zero calibrator. To the best of our knowledge, no international reference material exists for this XIII. PERFORMANCE AND LIMITATIONS
Time delay between last calibrator and sample dispensing
As shown hereafter, assay results remain accurate even when a sample is Detection Limit
dispensed 60 minutes after the calibrators have been added to the coated 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 ng/ml. Specificity
The percentage of cross-reaction estimated by comparison of the concentration yielding a 50% inhibition are respectively: Compound
Cross-Reactivity
XIV. 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. Precision
XV.
REFERENCE INTERVALS
These values are given only for guidance; each laboratory should establish its <X> ± SD
<X> ± SD
Concentration range
Number of subjects
SD : Standard Deviation; CV: Coefficient of variation (*) The range is based on 2.5 % and 97.5 % percentiles XVI. PRECAUTIONS AND WARNINGS
Experimental Treatment of Prostatic Cancer by intermittent
Safety
Hormonal Therapy.
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 Sex-Steroid-Binding Plasma Protein (SBP), Testosterone, Oestradiol
human blood derivatives will not transmit hepatitis, AIDS or other infections. and DHEA in Prepuberty and Puberty.
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. XVIII.
SUMMARY OF THE PROTOCOL
Nevertheless, components containing animal substances should be treated as Avoid any skin contact with all reagents, Stop Solution contains HCl. In case of SAMPLE(S)
CALIBRATORS
CONTROLS
Do not smoke, drink, eat or apply cosmetics in the working area. Do not pipette by mouth. Use protective clothing and disposable gloves. XVII. BIBLIOGRAPHY
Incubate for 1 hour at room temperature. Role of Serum Androgens and Sex Hormone Binding Globulin
Capacity in the Evaluation of Hirsutism in Women.
Wash 3 times with 400 µl of Wash Solution and aspirate. Influence of Testosterone Therapy on Clinical and Immunological
features of Autoimmune Diseases Associated with Klinefelter's
Incubate for 30 min at room temperature. Syndrome
J. Clin. Endocrin. Metab. Vol. 64, N°1: 32-36 M. CARRABBA et al (1985)
Abnormalities of Sex hormones in Men with Systemic Lupus
Read on a microtiterplate reader and record the absorbance of each well at 450 nm Erythematosus.
Plasma Testosterone and Breast Cancer.
Eur.J Cancer Clin. Oncol, Vol. 21, N°10, pp. 1265-1266 Testosterone, SHBG and Albumin in Patients with ovarian carcinoma.
Acta Obstet. Gynecol. Scand. 65: 533-S38.
Amniotic Fluid Testosterone and testosterone Glucuronide Levels in
the determination of Foetal Sex.
J. Steroid Biochem., Vol. 26,N°2,pp.273-277.

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