ELISA Kit

Usage	I. Sample Handling and Requirements: 1. The detection range of the kit is not equivalent to the concentration range of the analyte in the sample. Before the experiment, it is recommended to estimate the analyte concentration in the sample based on relevant literature and conduct preliminary experiments to determine the actual concentration. If the analyte concentration in the sample is too high or too low, please dilute or concentrate the sample appropriately. 2. If the sample type to be tested is not listed in the instructions, it is recommended to conduct preliminary experiments to verify the validity of the assay. 3. Serum: Collect whole blood in a serum separator tube and incubate at room temperature for 2 hours or at 2-8°C overnight. Then, centrifuge at 1000×g for 20 minutes. The supernatant can be collected or stored at -20°C or -80°C. Avoid repeated freezing and thawing. 4. Plasma: Collect the sample using EDTA or heparin as an anticoagulant. Centrifuge the sample at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. 5. Tissue Homogenization: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate may affect test results). Weigh the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL of PBS. The specific volume can be adjusted based on experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and thoroughly grind on ice or in a homogenizer. To further lyse tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes, and remove the supernatant for testing. 6. Cell culture supernatant: Centrifuge at 1000×g for 20 minutes, remove the supernatant, and test immediately. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freezing and thawing. 7. Other biological samples: Centrifuge at 1000×g for 20 minutes, remove the supernatant, and test immediately. 8. Sample appearance: The sample should be clear and transparent, and any suspended matter should be removed by centrifugation. 9. Sample storage: Samples collected for testing within one week can be stored at 4°C. If testing is not possible, aliquot the sample into a single-use aliquot and freeze at -20°C (for testing within one month) or -80°C (for testing within six months). Avoid repeated freezing and thawing. Hemolysis of the sample can affect the final test results, so hemolyzed samples are not suitable for this test. 2. Equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipettes and tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37°C constant temperature box 4. Distilled or deionized water III. Preparation before testing: 1. Please take out the test kit from the refrigerator 10 minutes in advance and equilibrate it to room temperature. 2. Preparation of Gradient Standard Working Solution: Add 1 mL of Universal Diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 10 ng/mL). Then dilute to the following concentrations: 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.312 ng/mL, 0.156 ng/mL, and 0 ng/mL. Serial Dilution Method: Take 7 EP tubes and add 500 μL of Universal Diluent to each tube. Pipette 500 μL of the 10 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 5 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 3. Preparation of Biotin-antibody working solution: Centrifuge the concentrated Biotin-antibody at 1000×g for 1 minute 15 minutes before use. Dilute 100×concentrated Biotin-antibody to 1×working concentration with universal diluent (e.g. 10×μL concentrate + 990×μL universal diluent) and use on the same day. 4. Preparation of Enzyme Conjugate Working Solution: 15 minutes before use, centrifuge the 100x concentrated enzyme conjugate at 1000 × g for 1 minute. Dilute the 100 μL concentrated HRP enzyme conjugate with universal diluent to a 1 × working concentration (e.g., 10 μL concentrate + 990 μL universal diluent). Use the same day. 5. Preparation of 1 × Wash Solution: Add 10 mL of 20 × Wash Solution to 190 mL of distilled water. (Concentrated wash solution removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing.) IV. Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return them to 4°C. 2. Sample Addition: Add 50 μL of sample or standard of varying concentrations to the corresponding wells. Add 50 μL of universal diluent to the blank wells, followed by 50 μL of Biotin-Antibody Working Solution to each well. Cover with a sealer and incubate at 37°C for 1 hour. (Recommendation: Dilute the sample to be tested at least 1-fold with universal diluent before adding it to the ELISA plate to minimize matrix effects. When calculating sample concentration, multiply the dilution factor. It is recommended to run replicates for all samples and standards.) 3. Wash: Discard the remaining liquid and add 300 μL of 1x wash buffer to each well. Let stand for 1 minute, then shake off the wash buffer and pat dry on absorbent paper. Repeat this process three times (a microplate washer is also an option). 4. Add Enzyme Conjugate Working Solution: Add 100 μL of enzyme conjugate working solution to each well, cover with a film, and incubate at 37°C for 30 minutes. 5. Wash: Discard the solution and wash the plate five times according to the procedure in step 3. 6. Add Substrate: Add 90 μL of substrate (TMB) to each well, cover with a film, and incubate at 37°C in the dark for 15 minutes. 7. Add Stop Solution: Remove the plate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at 450 nm. V. Calculation of Experimental Results: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as a correction value. Plot a standard curve for the four-parameter logistic function on double-logarithmic graph paper with concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is higher than the upper limit of the standard curve, it should be diluted appropriately and re-measured. When calculating the sample concentration, multiply the dilution factor by the corresponding dilution factor. Typical Data and Reference Curves: The following data and curves are for reference only. Experimenters should establish a standard curve based on their own experiments. Concentration (ng/mL) 10 5 2.5 1.25 0.625 0.312 0.156 0 OD  value 0.216 0.298 0.417 0.669 0.955 1.405 1.79 1.865
Sample Type	Range (%)	Average Recovery (%)
Serum (n=8)
Dilution ratio	Recovery rate (%)	Serum	Plasma
1:2	Range	83-95	88-96
	Average recovery rate	91	93
1:4	Range	89-104	87-108
	Average recycling rate	93	97

Theory This kit uses a competitive enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled antibody, and HRP conjugate are sequentially added to microwells pre-coated with a universal testosterone (T) antigen. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and then to yellow by acid. The intensity of the color is negatively correlated with the universal testosterone (T) content in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. Source All Synonym Testosterone (T) ELISA Kit Detection Type Competition Law Description Testosterone, also known as testosterone, testosterone, or testosterone, has a molecular formula of C19H28O2. Testosterone is an androgenic steroid containing a keto group at the third and a hydroxyl group at the seventeenth position, respectively. It is biosynthesized from cholesterol in several steps and converted into inactive metabolites in the liver. Testosterone exerts its effects by binding to and activating androgen receptors. In most vertebrates, testosterone is secreted primarily by the testicles of males and, to a lesser extent, by the ovaries of females. Composition

Name	9 6 T Configuration  style="height: 10px; width: 29.98%; text-align: center;" width="29.9800%">Remarks
Pre-coated 96-well enzyme plate	8 holes×12 strips	None
Standard	2 bottles	Dilution according to the instructions
Universal diluent	2×20mL	None
Concentrated Biotin-Antibody (100×)	60uL	Dilution according to the instructions
Concentrated enzyme conjugate (100×)	120uL	Dilution according to the instructions
20× Wash solution	2×10mL	Dilute according to instructions
Substrate (TMB)	10mL	None
Stop solution	6mL	none
Sealing film	4 sheets	None
Instructions	1 serving	None

General Notes 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use.
2. Improper plate washing may result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly before adding substrate. Do not allow the wells to dry out excessively during the entire process.
3. Clean the bottom of the plate of any residual liquid and fingerprints, as this may affect the OD value.
4. The substrate developer solution should be colorless; substrate solution that has turned blue should not be used.
5. Avoid cross-contamination of reagents and samples to prevent erroneous results.
6. Avoid direct exposure to strong light during storage and incubation.
7. Do not expose any reagents to bleaching agents or the strong fumes emitted by bleaching agents. Any bleaching agent will destroy the biological activity of the reagents in the kit.
8. Do not use expired products, and do not mix components from different product numbers and batches.
9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized.
10. If there is a possibility of disease transmission, all samples should be managed properly and the samples and testing devices should be handled according to the prescribed procedures. Storage Temp. Store at 2-8°C. Test Range 0.156-10 ng/mL