DiaMetra Testosterone Elisa Kit

CLINICAL SIGNIFICANCE
Testosterone (17β-OH-4-androstene-3-one) has a molecular weight of 288 Daltons and is considered the principal androgen found in circulation of mature male mammals1 . In males it is synthesised and secreted by the Leydig cells located in the interstitium of the testis; in females, testosterone is produced in various locations such as the ovaries, adrenal gland and peripheral tissues, with additional production due to inter-conversion from other steroid hormones. Testosterone is found in circulation predominantly linked to carrier proteins, the most common of which being sex-hormone binding protein (SHBG). Only 1 – 2% of testosterone is biologically active, i.e. not bound to any plasmatic protein, and is referred to as free testosterone.
Testosterone plays a key part in the development of reproductive tissues and secondary sex characteristics in men. There is an observed and well documented circadian variation of testosterone levels in men with the circulating concentration being higher in the morning and declining throughout the day2 . Testosterone levels also decline in ageing males (andropause) and is often associated with loss of muscle and bone mass, leading to osteoporosis, loss of libido, erectile dysfunction, depression and impaired cognitive function.

PRINCIPLE OF THE METHOD
The Testosterone ELISA is a competitive enzyme immunometric assay (ELISA) where testosterone (antigen) in the sample competes with the antigenic testosterone conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti- testosterone coated on the microplate (solid phase).
After the incubation, the bound/free separation is performed by a simple solid phase washing. Then, the enzyme HRP in the bound fraction reacts with the Substrate (H2O2) and the TMB Substrate and develops a blue colour that changes into yellow when the Stop Solution (H2SO4) is added. The colour intensity is inversely proportional to the testosterone concentration in the sample.
Testosterone concentration in the sample is calculated through a calibration curve.

PRECAUTIONS
Please adhere strictly to the sequence of pipetting steps provided in this protocol. The performance data represented here were obtained using specific reagents listed in this Instruction For Use.
All reagents should be stored refrigerated at 2-8°C in their original container. Any exceptions are clearly indicated.
Allow all kit components and specimens to reach room temperature (22-28°C) and mix well prior to use.
Do not interchange kit components from different lots. The expiry date printed on box and vials labels must be observed. Do not use any kit component beyond their expiry date. 
If you use automated equipment, the user has the responsibility to make sure that the kit has been appropriately validated for its intended use/purpose. 
The incomplete or inaccurate liquid removal from the wells could influence the assay precision and/or increase the background. To improve the performance of the kit on automatic systems is recommended to increase the number of washes.

REAGENT STORAGE AND STABILITY
Store the kit at 2 – 8°C in the dark.
The kit is stable at 2 – 8°C until the expiry date stated on the external kit label. 
Once opened, the kit is stable at 2 – 8°C for 6 months.
Once opened, the calibrators are stable at 2 – 8°C for 6 months. 
The diluted wash solution is stable for 30 days at 2-8°C.
Important note: open the bag containing the Coated Microplate only when it is at room temperature and close it immediately after use.

SAMPLE COLLECTION AND STORAGE
The assay should be performed using serum (standard sampling tubes or tubes containing serum separating gel) or plasma (lithium heparin, sodium heparin or potassium EDTA) samples.

QUALITY CONTROL
Good Laboratory Practice (GLP) requires the use of quality control specimens in each series of assays in order to check the performance of the assay. Controls should be treated as unknown samples, and the results analysed with appropriate statistical methods.
The kit controls provided in the kit should be tested as unknowns and are intended to assist in assessing the validity of results obtained with each assay plate.
The mean concentration of each control level is documented in the QC report included with each kit. These mean concentration levels are determined over several assays which are run in duplicate in multiple locations across each plate.
DiaMetra recommends the users to maintain graphic records of the control values generated with each assay run, including the running means, SDs and %CVs. This information will facilitate the controls trending analysis relating to the performance of current and historical control lots relative to the supplied Quality Control data. The trending will assist in the identification of assays which give control values significantly different from their average range.

CALCULATION OF RESULTS
A variety of data reduction software packages are available, which may be employed to generate the mean calibration curve and to calculate the mean concentrations of unknown samples and controls. A 4-parameter logistic (4PL) curve fit, including Calibrator 0 is required. A smoothed spline fit including Calibrator 0 can be used. Other curve fitting algorithms are not recommended.
Alternatively, a calibration curve may be prepared on semilog graph paper by plotting mean absorbance on the Y-axis against concentration of analyte on the X-axis. Calibrator 0 should be included in the calibration curve. Read the mean absorbance value of each unknown sample off the curve.
In order for the assay results to be considered valid the kit calibrators and control must fall within the specifications detailed in the lot specific certificate of analysis.