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SKU:EK7014
Pack Size:96wells/kit, with removable strips.
Validated Species:Mouse, Rat
Host:No
Application:ELISA
Sensitivity:0.1 ng/ml
Assay Range:0.1-18 ng/ml
Sample Type:Serum
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Introduction

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.

Overview

Product Name Mouse Rat Testosterone ELISA Kit
SKU/Catalog Number EK7014
Description Competitive High Sensitivity ELISA kit for Quantitative Detection of Mouse/Rat Testosterone. 96wells/kit, with removable strips.
Cite This Product Mouse Rat Testosterone ELISA Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK7014)
Validated Species Mouse, Rat
Application ELISA

*Our Boster Guarantee covers the use of this product in the above tested applications.

Cross Reactivity There is no detectable cross-reactivity.
Pack Size 96wells/kit, with removable strips.

Properties

Sensitivity 0.1 ng/ml
*Sensitivity, or Lower Limit of Detection (LLD), is the minimum level of target protein the ELISA assay can detect. We measure 20 blank wells and if the O.D. value is 2 standard deviations higher than the blanks' average O.D. the sample can be deemed positive.
Assay Range 0.1-18 ng/ml
*This assay range is determined using common samples. For samples with low target protein concentrations, users can adjust the standard curve to extend the lower limit of assay range.
Sample Type Serum

*The above listed samples are the ones valided with the assay. If you do not see your sample of interest listed, as long as there is enough level of target protein present in the sample, this Picokine™ ELISA kit should detect it.
**For protocol and tips regarding preparing your sample of interest, please check our ELISA sample preparation guide.
Storage Store the kit at 2°C to 8°C. Keep microwells sealed in a dry bag with desiccants. The reagents are stable until expiration of the kit. Do not expose reagent to heat, sun, or strong light. Avoid multiple freeze-thaw cycles(Shipped with wet ice.)

Kit Components

DescriptionQuantity
Microwells coated with Goat Anti-Rabbit IgG12x8x1 Microwells
Standards6 vials ( ready to use) 0.5 ml
Rabbit Anti-Testosterone Reagent1 vial 7ml
Assay Diluent1 bottle (ready to use) 12ml
Enzyme Conjugate Conc. (20X)1 Vial 0.7ml
TMB Substrate1 bottle (ready to use) 12ml
Stop Solution1 bottle (ready to use) 12ml
20X Wash Buffer1 bottle 25ml

Material Required But Not Provided

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

Background

Testosterone is a steroid hormone that stimulates development of male secondary sexual characteristics, produced mainly in the testes, but also in the ovaries and adrenal cortex.

Mouse Rat Testosterone ELISA Kit Images

Click the images to enlarge.

Mouse Rat Testosterone ELISA Kit
Mouse Rat Testosterone ELISA Kit

WARNINGS AND PRECAUTIONS

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.

SPECIMEN COLLECTION HANDLING

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.

REAGENT PREPARATION

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).

ASSAY PROCEDURE

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.

CALCULATION OF RESULTS

The standard curve is constructed as follows:

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.

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Customer Q&As

Q: How many samples can I test on one ELISA plate?
A: On each 96 well plate, we recommend running an 8-point standard curve with duplicate wells. With the remaining 80 wells, 40 samples can be tested in duplicate.
Q: The volumes of my samples are small, would it be possible to use 50µl/sample?
A: There are two solutions for insufficient sample volume.
1. Dilute the samples with the provided sample diluent buffer into 100ul.
2. If 50ul of sample is added into a well, add 50ul of standard solution.
Q: Will this kit work on tissue samples?
A: For tissue homogenates, endogenous biotin should be considered. Endogenous biotin in tissue homogenates might introduce false positive signal. Please run the test as described below to confirm if there is Endogenous biotin in the tissue lysate samples using the ELISA kit.
• Add tissue homogenates into the wells and then add ABC and TMB without adding any biotinylated detection antibody to see if any signal will be observed.
• If no signal is produced, then you can work on the tissue sample by using the kit.
Q: Is it possible to dilute 10X TBS stock that is 250mM Tris Base/Tris-Hcl, 27 mM KCl, and 1.37M NaCl to be used as the wash buffer?
A: You can dilute the 10X TBS stock to 1XTBS and test the pH value of the 1xTBS.
The 1XTBS can be used if the pH value falls in the range of 7.2-7.6.
Q: Would your kits work normally with samples from Wistar Rat?
A: Our kits would work normally with samples from Wistar Rat.
Q: How can I store the dissolved wash buffer for a longer period of time?
A: You can prepare a concentrated wash buffer (25X) which can be stored at 4°C for 1-3 months.
Q: Does the TMB color developing agent contain H2O2?
A: Yes, the TMB color developing agent contain H2O2.
Q: Does this ELISA kit contain any product produced in humans or primates?
Keywords: component, ingredient
A: None of the components in our ELISA kits are produced in humans or primates.
Q: What is the volume of the recombinant protein control? What is its shelf life?
Keywords: expiration, storage, temperature, size
A: The volume of the control is around 100pg. If unopened, the shelf life of the control is the same as the whole kit - "Store at 4˚C for 6 months, at -20˚C for 12 months." The reconstituted control can be stored at -20˚C for 2 days.
Q: Will it be okay to run the experiment if I accidentally used the wrong buffer (e.g. sample diluent buffer) for antibody dilution instead of the antibody diluent buffer? Keywords: dilution, replace, substitute, sample buffer
A: Using the sample diluent buffer for antibody dilution instead of the antibody diluent buffer will negatively affect the test result to some extent. The values of both standard and sample might be lower than the normal values.
Q: The protocol says to use neutral PBS and provides a recipe. Could I use neutral PBS made with KCl or KH2PO4 (common constituents for most PBS recipes) instead? Keywords: protocol, alternative buffer, applications, reagent recipe
A: Yes, it is ok to use common PBS recipes. We have tried many types of buffers with common constituents, and no significant difference was observed whatsoever.
Q: Why are my O.D. values different than your values on the datasheet? Keyword: troubleshooting, reference
A: We detected the kit in our lab and got our values on the datasheet before delivery, but protein activity will decrease as time goes on, so you may get lower O.D. values than ours. However, it should still be in a reasonable range and the standards can be used to calculate sample values. Good linearity of curve is more important than the actual numerical value.
Q: On the ELISA kit datasheet it says "HRP substrate TMB was used to visualize HRP enzymatic reaction." What is HRP and which part of the kit contains HRP? Keyword: kit components, reagents, protocol
A: It means Avidin-Biotin-Peroxidase Complex(AR1103) and you could find it in Kit Components.
Q: Do you offer any ELISA kits that can work with whole blood samples instead of plasma or urine? Keyword: applications
A: We test serum and plasma routinely, and there is very little difference between serum, plasma and whole blood. The whole blood also contains proteins we need to test. The kit can be used to test whole blood in theory.
Q: Does silicic acid (formula SiOH4) affect the results of the ELISA assay? Keyword: protocol, reagents
A: The acid may affect the binding of antigen to antibody, it is not recommended to use. That being said, it is unlikely to affect the reaction if the solution remains an overall neutral environment.
Q: Is there lot-to-lot variation of the ELISA kits? What are Boster's general services when I have questions about your kits? Keyword: technical support, help, customer service
A: The NIBSC/WHO 1st International Standard is evaluated, we always test our kits before delivery, and customers can find the test result on the protocol. If the customer needs technical support from us to analyze their data, please contact us and we ask that you provide us the following information: the lot# and production date, and when did the customer detect the kit.
Q: The results of my standards do not look correct, what could be the problem? Keyword: troubleshoot, protocol
A: Double check the protocol for plate washing. It should be examined on three aspects: 1) Did you make the washing buffer correctly? 2) How long did you let the buffer stay in wells? 3) What was the volume of the washing buffer added to each well? In addition, pay attention when adding sample to avoid contamination. And we suggest testing the standards again. Values of standards at low concentration are more affected than that at higher concentration, so customer can still get expected values at high concentration even if there is an error. If problems persist, please contact technical support with the catalog and lot number of your product.
Q: What is the well depth of the 96 well plates for the ELISA kits? Keyword: well capacity, product size
A: The well depth is 300ul, and the max capacity is 350ul. The height of the well is 1.1 cm, and the internal size is 1 cm.
Q: The kit does not include wash solution, what should I do? Keyword: wash buffer
A: Our Elisa kits do not come with wash solution, but we have included information about how to make washing buffer on the datasheet. Please refer to the "Material Required But Not Provided" section. We also offer washing buffer for sale separately (Phosphate Buffered Saline Powder SKU: AR0030).
Q: For your ELISA kits, are the capture and detection antibodies polyclonals or monoclonals? Keyword: antibody clonality
A: This information can be found for each kit under the "Properties" section, and you can find the immunogen sequence information in the "Overview" section.
Q: Do your ELISA kits come with sealants or plate covers? Keyword: storage, sequential use
A: The kit can be used within a month sequentially if it's opened and stored at 4 degree. There is no need to use sealants, the plate can be packaged with aluminum foil bag, and for other reagents keep bottle tightly closed.
Q: What is the concentration of Sodium Azide in your Elisa kits? Keyword: preservative
A: Our Elisa kits contain 0.02% Sodium Azide. All of the components except TMB colour developing reagent and stop solution contains 0.02% Sodium azide as the preservative.
Q: Are there positive and negative controls available for my ELISA kit? Keyword: positive control, negative control
A: We can provide a recombinant protein as a control. It costs $50 per control and takes 2 weeks to manufacture. We cannot provide a positive or negative control in serum.
Q: Why do I get positive results for my knockout (KO) model when used as a control?
A: The knockout (KO) model may contain truncated forms of the target protein which can be detected by the ELISA assay.
Q: How long do I soak my plate in the wash buffer?
A: The plate should soak in 0.3mL PBS or TBS wash buffer for at least 1-2 minutes in an automated wash station.
Q: Can I use Tween in my wash buffer?
A: While it is not recommended to use Tween in your wash buffer, small amounts (<0.1% concentration) may decrease background due to insufficient blocking.
Q: What plate type do I use to set up the microplate reader?
A: Our plates are made with the Corning costar plates similar to the DNA-BIND 96 -well plates.
Q: What should I use for negative control?
A: Please contact us for negative control suggestions. You can also check expression databases such as genecards, uniprot etc. Due to logistic reasons, we do not sell serum or lysates that we use internally for positive or negative control.
Q: Where can I find troubleshooting information? What should I do if I have unexpected bands, high background, no signal, weak signal
A: You can find Boster's troubleshoot guides under tech support tab. Please contact us for further assistance on troubleshooting your experiment.
Q: What is the normal level of this protein in my sample of interest?
A: We have reviewed literature and have gathered this information for most of our ELISA kits. You can find this information on the product page or contact us if you cannot find it. However this information is only suggestive and cannot replace pilot studies in determining the optimal sample dilution ratio.
Q: Is the plate separable? Can I use only part of the kit and save the rest for later? How many samples can I run with one kit?
A: Yes the plate is separable if the kit says that there are removable strips. There are 12 strips of 8 wells each, all removable from the plate. The amount of samples you can run depend on a few factors. In the most common ELISA set up, you will use two strips for standards, and 10 strips for samples using duplicates, which let you run up to 40 samples per kit. Contact us if you have questions regarding other situations.
Q: Does the kit contain sample preparation reagents? How do I prepare my samples?
A: Since different sample types require different reagents, we do not include them in the ELISA kit. However we do have each reagent mentioned in the file below available at very reasonable prices. Be sure to check them out. For sample preparation protocols please download the file below: https://www.bosterbio.com/media/pdf/Boster_Sample_Preparation_Protocols.pdf
Q: Can this ELISA kit work on brain tissue homogenate, cell culture supernatant, saliva, urine, serum, whole blood or any other sample type?
A: In theory the ELISA kit will work for all sample types if the target protein is present at a level that falls within the linear range of the ELISA kit detection range. We guarantee the kit to work on the sample types that we have tested. If you want dilution ratio suggestions on these sample types please contact our technical support. For sample types that we have not tested for, we suggest you run pilot experiments to decide the optimal sample dilution.
Q: Can this ELISA kit react with the pro-form of the target protein? Can this ELISA kit react with an isoform of the protein?
A: In general, unless otherwise specified, the ELISA kit is pan-specific, meaning that it will react with all different forms of the target protein if they share the majority of the target protein's sequence. The capture and detection antibodies are reactive to the entire sequence of the standard protein. You can find the sequence information of the standard protein in the "immunogen" section. For more information about the specificity of the kit for your particular experiment, please contact our technical support.
Q: Can this ELISA kit react with human, mouse, rat or other species?
A: If the kit is reactive to another commonly used species (human, mouse, and/or rat), we would have listed it as a separate product. If you want to check cross-reactivity to a species that is not included in the 3 species listed above, please contact our technical support for more information. As a rule of thumb, if the sequences are 90%+ identical, there is a high chance of cross-reactivity for your species of interest.