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Mouse/Rat Testosterone ELISA is based on the principle of competitive binding between Testosterone in the test specimen and Testosterone-HRP conjugate for a constant amount of rabbit anti-Testosterone. In the incubation, goat anti-rabbit IgG-coated wells are incubated with 25ml of Testosterone standards, controls, patient samples, 100 ml Testosterone-HRP conjugate reagent and 50ml rabbit anti-Testosterone reagent at room temperature 60 minutes. During the incubation, a fixed amount of HRP-labeled Testosterone competes with the endogenous Testosterone in the standard, sample, or quality control serum for a fixed number of binding sites of the specific Testosterone antibody. Thus, the amount of Testosterone peroxidase conjugate immunologically bound to the well progressively decreases as the concentration of Testosterone in the specimen increases. Unbound Testosterone peroxidase conjugate is then removed and the wells washed. Next, a solution of TMB Reagent is then added and incubated at room temperature for 15 minutes, resulting in the development of blue color. The color development is stopped with the addition of stop solution, and the absorbance is measured spectrophotometrically at 450nm.
*Our Boster Guarantee covers the use of this product in the above tested applications.
1. Distilled or deionized water
2. Precision pipettes
3. Disposable pipette tips. Multichannel pipettes are recommended in the condition of large amount of samples in the detection.
4. ELISA reader capable of reading absorbance at 450nm
5. Absorbance paper or paper towel
6. Graph paper
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1. For Research Use Only. Not for use in diagnostic procedures.
2. Potential biohazardous materials: The calibrator and controls contain human source components, which have been tested and found non-reactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However, there is no test method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent. These reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories" 1984.
3. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are handled.
4. The components in this kit are intended for use as an integral unit. The components of different lots should not be mixed.
5. It is recommended that standards, control and serum samples be run in duplicate.
6. Optimal results will be obtained by strict adherence to this protocol. Accurate and precise pipetting, as well as following the exact time and temperature requirements prescribed are essential. Any deviation from this may yield invalid data.
7. Do not use sodium azide as preservative. Sodium azide inhibits HRP enzyme activities.
1. Collect blood specimens and separate the serum immediately.
2. Typically, specimens may be stored refrigerated at (2°C to 8°C) for 5 days. If storage time exceeds 5 days, store frozen at (-20°C) for up to one month.
3. Avoid multiple freeze-thaw cycles.
4. Prior to assay, frozen sera should be completely thawed and mixed well.
5. Do not use grossly lipemic specimens.
20X Enzyme conjugate: Prepare 1X working solution at 1:20 with assay diluent (e.g. Add 0.1ml of the Testosterone enzyme conjugate concentrate to 1.9ml of assay diluent)
20X Wash Buffer: Prepare 1X Wash buffer by adding the contents of the bottle (25 ml, 20X) to 475 ml of distilled or deionized water. Store at room temperature (18-26°C).
1. Secure the desired number of coated wells in the holder.
2. Dispense 25 µl of standards, specimens and controls into appropriate wells.
3. Dispense 100 µl of working dilution of Testosterone-HRP Conjugate Reagent into each well.
4. Dispense 50 µl of rabbit anti-Testosterone reagent to each well. Thoroughly mix for 30 seconds. It is very important to mix completely.
5. Incubate at room temperature 60 minutes.
6. Rinse and flick the microwells 3 times with 1x wash buffer water.
7. Dispense 100 µl of TMB Reagent into each well. Gently mix for 5 seconds.
8. Incubate at room temperature (20-25°C) for 15 minutes.
9. Stop the reaction by adding 50µl of Stop Solution to each well.
10. Gently mix 30 seconds. It is important to make sure that all the blue color changes to yellow color completely.
11. Read absorbance at 450 nm with a microtiter well reader within 15 minutes.
1. Calculate the mean absorbance value (A450) for each set of reference standards, controls and samples.
2. Construct a standard curve by plotting the mean absorbance obtained for each reference standard against its concentration in ng/ml on a linear-linear graph paper, with absorbance values on the vertical or Y axis, and concentrations on the horizontal or X axis.
3. Use the mean absorbance values for each specimen to determine the corresponding concentration of Testosterone in ng/ml from the standard curve.
4. Any values obtained for diluted samples must be further converted by applying the appropriate dilution factor in the calculations.
*EK7014 bulk discount: 20% off for 4+ of same kits, 30% off for 20+ of same kits, applicable only to USA and Canada.
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