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Testosterone ELISA Kit   (DEIA05747)  


Datasheet

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Size
96T
Sample
Saliva
Species Reactivity
Human
Intended Use
This Kit is an Enzyme Immunoassay for the quantitative measurement of free active testosterone in saliva.
Contents of Kit
1. Microtiterwells, 12 x 8 (break apart) strips, 96 wells; Wells coated with a anti-Testosterone antibody (monoclonal).
2. Standard (Standard 0-6), 7 vials, 1 mL each, ready to use; Concentrations: 0.0 - 10 - 50 - 100 - 500 - 1000 - 5000 pg/mL
3. Control, 2 vials, 1.0 mL each, ready to use;
4. Enzyme Conjugate, 1 vial, 26 mL, ready to use;
5. Substrate Solution, 1 vial, 25 mL, ready to use;
6. Stop Solution, 1 vial, 14 mL, ready to use;
Storage
2°C to 8°C
Performance Characteristics
Assay Dynamic Range
The range of the assay is between 1.9 - 5000 pg/mL.

Specificity
The following materials have been evaluated for cross reactivity. The percentage indicates cross reactivity at 50% displacement compared to Testosterone.


Sensitivity
The lowest analytical detectable level of testosterone that can be distinguished from the Zero Standard is 1.9 pg/mL at the 95 % confidence limit. The lowest functional sensitivity of 7.1 pg/mL at the 95% confidence limit was obtained.

Reproducibility
Intra-Assay
The intra-assay variation was determined by 20 replicate measurements of 5 saliva samples within one run. The withinassay variability is shown below:


Inter-Assay
The inter-assay (between-run) variation was determined by duplicate measurements of 5 saliva samples over 10 days.


Inter-Lot
The Inter-Lot (between-lot) variation was determined by triplicate measurements of five saliva samples in three different kit lots. The between lot variability is shown below:


Recovery
Recovery of the CD ELISA was determined by adding increasing amounts of the analyte to six different saliva samples containing different amounts of endogenous analyte. Each sample (native and spiked) was assayed and analyte concentrations of the samples were calculated from the standard curve. The percentage recoveries were determined by comparing expected and measured values of the samples


Linearity
Six saliva samples containing different amounts of analyte were serially diluted with zero standard and assayed with the CD ELISA. Three native samples were serially diluted, and 3 samples were spiked with testosterone and then serially diluted up to 1:128. The percentage recovery was calculated by comparing the expected and measured values for testosterone. An assay linearity of 7.1 - 4500 pg/mL has been identified as the usable range. Samples above this range must be diluted and re-run.


Comparison Studies
A study was performed that evaluated saliva samples from 99 male and female subjects ages 20 to 70 years. The saliva samples were run in duplicate on the test and a commercially available LIA method to determine the concentration of free Testosterone in the samples. A correlation of 0.904 and regression formula of y = 0.9251x - 7.4369 was obtained versus this method. Another study was performed to further evaluate the substantial equivalence of the Testosterone to the LIA saliva test. The concentration of testosterone in 81 additional saliva samples collected from 40 - 65 year old men and women was determined using testosterone kit and the other method. From this study an R2 = 0.9866 was obtained with the following regression.
General Description
Testosterone (17β-hydroxy-4-androstene-3-one) is a C19 steroid with an unsaturated bond between C-4 and C-5, a ketone group in C-3 and a hydroxyl group in the β position at C-17. This steroid hormone has a molecular weight of 288.4 daltons. Testosterone is the most important androgen secreted into the blood. In males, primarily the Leydig cells of the testis secrete testosterone; in females approximately 50% of circulating testosterone is derived from peripheral conversion of androstenedione, approximately 25% from the ovary and 25% from the adrenal glands. Testosterone is responsible for the development of secondary male sex characteristics and its measurements are helpful in evaluating the hypogonadal status. In women high levels of testosterone are generally found in hirsutism and virilisation, polycystic ovaries, ovarian tumors, adrenal tumors and adrenal hyperplasia. In men high levels of testosterone are associated with hypothalamic-pituitary-unit dysfunction, testicular tumors, congenital adrenal hyperplasia and prostate cancer. Low levels of testosterone are encountered in male patients with the following diseases: Klinefelte's syndrome. Hypopituitarism, testicular feminization, orchidectomy, cryptorchidism, enzymatic defects and some autoimmune diseases .
Standard Curve
Standard Optical Units (450 nm)
Standard 0 (0 pg/mL) 2.01
Standard 1 (10 pg/mL) 1.89
Standard 2 (50 pg/mL) 1.57
Standard 3 (100 pg/mL) 1.36
Standard 4 (500 pg/mL) 0.69
Standard 5 (1000 pg/mL) 0.43
Standard 6 (5000 pg/mL) 0.13
Reconstitution And Storage
When stored at 2°C to 8°C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date. Opened reagents must be stored at 2°C to 8°C. Microtiter wells must be stored at 2°C to 8°C. Once the foil bag has been opened, care should be taken to close it tightly again. Opened kits retain activity for two month if stored as described above.
Principles of Testing
The Salivary Testosterone ELISA Kit is based on the competition principle and the microplate separation. An unknown amount of free testosterone present in the sample and a fixed amount of testosterone conjugated with horseradish peroxidase compete for the binding sites of mouse monoclonal testosterone antiserum coated onto the wells. After one-hour incubation the microplate is washed to stop the competition reaction. After addition of the substrate solution the concentration of testosterone is inversely proportional to the optical density measured.
Reagents And Materials Provided
1. Microtiterwells, 12x8 (break apart) strips, 96 wells; Wells coated with a anti-Testosterone antibody (monoclonal).
2. Standard (Standard 0-6), 7 vials, 1 mL each, ready to use; Concentrations: 0.0 - 10 - 50 - 100 - 500 - 1000 - 5000 pg/mL
Conversion: Testosterone (pg/mL) x 3.47 = pmol/L
contain 0.03% Proclin 300, 0.015% BND and 0.010% MIT as preservative.
3. Control, 2 vials, 1.0 mL each, ready to use;
Control values and ranges please refer to vial label or QC-Datasheet.
contain 0.03% Proclin 300, 0.015% BND and 0.010% MIT as preservative.
4. Enzyme Conjugate, 1 vial, 26 mL, ready to use;
Testosterone conjugated to horseradish peroxidase;
contain 0.03% Proclin 300, 0.015% BND and 0.010% MIT as preservative.
5. Substrate Solution, 1 vial, 25 mL, ready to use;
Tetramethylbenzidine (TMB).
6. Stop Solution, 1 vial, 14 mL, ready to use;
contains 0.5M H2SO4. Avoid contact with the stop solution. It may cause skin irritations and burns.
7. Wash Solution, 1 vial, 30 ml (40x concentrated);
BND = 5-bromo-5-nitro-1,3-dioxane
MIT = 2-methyl-2H-isothiazol-3-one

Note: Additional Standard 0 for sample dilution is available upon request.
Materials Required But Not Supplied
1. Calibrated EIA reader adjusted to read at 450 nm
2. Precision pipettes (100 µL and 200 µL)
3. Distilled or Deionized water
4. Timer (60 min. range)
5. Reservoirs (disposable)
6. Test tube or microtube rack in a microplate configuration
7. Semi logarithmic graph paper or software for data reduction
Assay Procedure
General Remarks
All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming. Once the test has been started, all steps should be completed without interruption. Use new disposal plastic pipette tips for each standard, control or sample in order to avoid cross contamination. Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption. As a general rule the enzymatic reaction is linearly proportional to time and temperature.

Test Procedure
Each run must include a standard curve.
1. Secure the desired number of coated in the frame holder.
2. Dispense 100 µL of each Testosterone Standard and Control into appropriate wells.
3. Dispense 100 µL of each sample into selected wells.
4. Dispense 200 µL Enzyme Conjugate into each sample and standard well and Mix the plate thoroughly for 10 seconds.
5. Incubate for 60 minutes at room temperature.
6. Briskly shake out the contents of the wells and rinse the wells 3 times with diluted Wash Solution (400 µL per well). Strike the inverted wells sharply on absorbent paper towel to remove residual droplets.
7. Add 200 µL of Substrate Solution (4) to each well.
8. Incubate for 30 minutes at room temperature.
9. Stop the reaction by adding 100 µL of Stop Solution (5) to each well.
10. Determine the absorbance of each well at 450 ± 10 nm. It is recommended to read the wells within 10 minutes.
Quality Control
Good laboratory practice requires that controls be run with each calibration curve. A statistically significant number of controls should be assayed to establish mean values and acceptable ranges to assure proper performance. It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results. Use controls at both normal and pathological levels. The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to the kit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for direct comparison of the results. It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results. Employ appropriate statistical methods for analysing control values and trends. If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid. In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods.
Precautions
1. All reagents of this test kit which contain human serum or plasma have been tested and confirmed negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should be treated as potential biohazards in use and for disposal.
2. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood.
3. The microplate contains snap-off strips. Unused wells must be stored at 2°C to 8°C in the sealed foil pouch and used in the frame provided.
4. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step.
5. Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
6. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
7. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
8. Allow the reagents to reach room temperature (21-26°C) before starting the test. Temperature will affect the absorbance readings of the assay. However, values for the patient samples will not be affected.
9. Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
10. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
11. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or specimens may give false results.
12. Handling should be done in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.
13. Do not use reagents beyond expiry date as shown on the kit labels.
14. All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes and microtiterplate readers.
15. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of different plates even of the same lot. The kits may have been shipped or stored under different conditions and the binding characteristics of the plates may result slightly different.
16. Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
17. Some reagents contain Proclin, BND and MIT as preservatives. In case of contact with eyes or skin, flush immediately with water.
18. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant volume of water and skin with soap and abundant water. Wash contaminated objects before reusing them. If inhaled, take the person to open air.
19. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national biohazard safety guideline or regulation.
Limitations
Reliable and reproducible results will be obtained when the assay procedure is performed with a complete understanding of the package insert instruction and with adherence to good laboratory practice. Any improper handling of samples or modification of this test might influence the results. The patient should not eat, drink, chew gum or brush teeth for 30 minutes before sampling. Otherwise rinse mouth thoroughly with cold water 5 min prior to sample collection. Do not collect samples when oral diseases, inflammation or lesions exist (blood contamination)

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