Testosterone, also known as testis hormone, molecular formula: C₁₉H₂₈O₂. Testosterone is an androgen steroid, bearing a ketone group at position 3 and a hydroxyl group at position 17. It is biosynthesized from cholesterol via several steps and converted into inactive metabolites in the liver. Testosterone exerts its biological effects by binding to and activating androgen receptors. In most vertebrates, testosterone is mainly secreted by the testes in males, and to a lesser extent, by the ovaries in females.
Assay Principle
This kit adopts competitive enzyme-linked immunosorbent assay (ELISA). Into microplates pre-coated with canine testosterone (T) antigen, samples, standards, Biotin-labeled antibody and HRP conjugate are added sequentially. After incubation and washing steps, substrate TMB is applied for color development. TMB is catalyzed by horseradish peroxidase (HRP) to produce a blue color, which turns yellow upon acid termination. The color intensity is negatively correlated with the concentration of canine testosterone (T) in the sample. The absorbance (OD value) is read at 450 nm using a microplate reader to calculate sample concentration.
Precautions
1. Strictly follow the specified incubation time and temperature to guarantee accurate results. All reagents must be equilibrated to room temperature (20–25°C) before use. Refrigerate reagents immediately after use.
2. Improper plate washing may lead to inaccurate readings. Ensure complete removal of residual liquid in wells prior to substrate addition. Avoid prolonged drying of microwells throughout the procedure.
3. Wipe residual liquid and fingerprints from the bottom of the microplate, as contaminants will interfere with OD values.
4. The TMB substrate solution shall remain colorless; discard substrate solution that has turned blue.
5. Prevent cross-contamination between reagents and samples to avoid erroneous test results.
6. Avoid direct intense light exposure during reagent storage and incubation.
7. All reaction reagents must not come into contact with bleach solvents or their volatile fumes; bleach components will destroy the biological activity of kit reagents.
8. Do not use expired products; components from different lot numbers or batch codes cannot be mixed.
9. Recombinant proteins from external sources may fail to be recognized due to mismatched antibody affinity with kit capture antibodies.
10. Handle all potentially infectious samples properly, and dispose of specimens and testing devices in accordance with standardized biosafety protocols.
Kit Components:
| Component No. | Component Name | Appearance | 48T | 96T | Storage | | EJ1515283A | Pre-coated Assay Plate | — | 48wells | 96wells | 2-8℃. | | EJ1515283B | Standard | Solid | 1vial | 2 vials | 2-8℃. | | EJ1515283C | Universal Diluent | Liquid | 1×20mL | 2×20mL | 2-8℃. | | EJ1515283D | Biotin-antibody (100×) | Liquid | 30μL | 60μL | 2-8℃. | | EJ1515283E | Streptavidin-HRP (100×) | Liquid | 60μL | 120μL | 2-8℃. | | EJ1515283F | Wash Buffer (20×) | Liquid | 1×10mL | 2×10mL | 2-8℃. | | EJ1515283G | TMB Substrate | Liquid | 5mL | 10mL | 2-8℃. | | EJ1515283H | Stop Solution | Liquid | 3mL | 6mL | 2-8℃. | | EJ1515283I | Plate Sealer | — | 4 pieces | 4 pieces | 2-8℃. |
Note: EJ1515283B、EJ1515283D、EJ1515283E and EJ1515283F shall be diluted in accordance with the instructions. |
Sample Handling & Requirements
1. The detectable concentration range of the kit does not equal the analyte concentration range in raw samples. It is recommended to estimate sample analyte concentrations via relevant literature and conduct pre-tests to confirm actual concentrations before formal experiments. Dilute or concentrate samples appropriately if analyte levels fall outside the linear range.
2. If your sample matrix is not listed in this manual, perform a pre-test to verify assay compatibility.
3. Serum: Collect whole blood in serum separation tubes, incubate at room temperature for 2 hours or store overnight at 2–8°C, then centrifuge at 1000×g for 20 minutes. Harvest supernatant for immediate testing, or aliquot and store at -20°C / -80°C; avoid repeated freeze-thaw cycles.
4. Plasma: Collect blood with EDTA or heparin as anticoagulant. Centrifuge specimens within 30 minutes post-collection at 1000×g, 2–8°C for 15 minutes. Collect supernatant for immediate testing, or store at -20°C / -80°C; avoid repeated freeze-thaw cycles.
5 Tissue Homogenate: Rinse tissue with pre-cooled PBS (0.01 M, pH=7.4) to remove residual blood (lysed erythrocytes in homogenate interfere with detection results). Weigh and mince tissue, then mix tissue with PBS at a standard weight-to-volume ratio of 1:9 (e.g., 1 g tissue corresponds to 9 mL PBS; adjust volume as required and record details. Addition of protease inhibitor cocktail to PBS is recommended). Transfer the mixture to a glass homogenizer and grind thoroughly on ice, or use a mechanical homogenizer. For complete cell lysis, sonicate the homogenate or perform repeated freeze-thaw cycles. Finally, centrifuge the homogenate at 5000×g for 5–10 minutes and collect supernatant for testing.
6. Cell Culture Supernatant: Centrifuge culture medium at 1000×g for 20 minutes, collect supernatant for immediate testing, or store at -20°C / -80°C; avoid repeated freeze-thaw cycles.
7. Cell Lysate: For adherent cells, wash gently with pre-cooled PBS, digest with trypsin, then harvest cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be pelleted directly by centrifugation. Wash harvested cells 3 times with pre-cooled PBS, resuspend 1×10⁶ cells in 150–200 μL PBS (protease inhibitors are recommended; reduce PBS volume if analyte abundance is extremely low). Lyse cells via repeated freeze-thaw or sonication. Centrifuge the lysate at 1500×g, 2–8°C for 10 minutes and retain the supernatant for detection.
8. Other Biological Matrices: Centrifuge samples at 1000×g for 20 minutes and collect supernatant for testing.
9. Sample Appearance: Samples must be clear and transparent; all suspended particulates must be removed by centrifugation.
10. Sample Storage: Samples intended for testing within 1 week can be stored at 4°C. For delayed testing, aliquot samples into single-use volumes and freeze at -20°C (stable for 1 month) or -80°C (stable for 6 months). Avoid repeated freeze-thaw cycles. Hemolyzed samples are unsuitable for this assay, as hemolysis will distort final detection results.
Sample Dilution Protocols
Estimate analyte concentration in advance. Follow the dilution schemes below if sample dilution is required:
100-fold dilution (single step): Mix 5 μL sample with 495 μL Assay Diluent.
1000-fold dilution (two-step): Step 1: Dilute 5 μL sample into 95 μL diluent (20×). Step 2: Transfer 5 μL of the 20× diluted sample into 245 μL diluent (50×), achieving total 1000-fold dilution.
100000-fold dilution (three-step): Step 1: Mix 5 μL sample with 195 μL diluent (40×). Step 2: Transfer 5 μL 40× sample into 245 μL diluent (50×). Step 3: Transfer 5 μL 2000× sample into 245 μL diluent (50×), total dilution factor = 100000×.
Guidelines for dilution: Transfer volume per step ≥ 3 μL; single-step dilution factor ≤ 100×. Mix thoroughly after each dilution and avoid bubble formation.
Required Lab Equipment (User-Supplied)
1. Microplate reader (capable of 450 nm absorbance measurement)
2. High-precision pipettes and matching tips: 0.5–10 μL, 5–50 μL, 20–200 μL, 200–1000 μL
3. 37°C incubator
4. Distilled or deionized water
Pre-Assay Reagent Preparation
1. Remove the kit from refrigeration 10 minutes in advance and equilibrate to room temperature.
2. Preparation of standard serial working solutions: Add 1 mL of universal diluent to the lyophilized standard. Let it stand for 15 minutes for complete dissolution, then mix gently (the concentration is 10 ng/mL). Prepare serial dilutions to obtain concentrations of 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, add 500 μL of universal diluent to each tube. Pipette 500 μL from the 10 ng/mL standard working solution into the first EP tube and mix to prepare the 5 ng/mL standard working solution. Repeat this pipetting and mixing step sequentially for subsequent tubes. The last tube serves directly as the blank well; no liquid is transferred from the second-to-last tube. See figure below for details.
3. Preparation of Biotin-antibody Working Solution: Centrifuge the 100× concentrated Biotin-antibody at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated Biotin-antibody to 1× working concentration with assay diluent (Example: 10 μL concentrate + 990 μL assay diluent). Prepare fresh right before use.
4. Preparation of Streptavidin-HRP Working Solution: Centrifuge the 100× concentrated Streptavidin-HRP at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated Streptavidin-HRP to 1× working concentration with assay diluent (Example: 10 μL concentrate + 990 μL assay diluent). Prepare fresh right before use.
5. Preparation of 1× Wash Buffer: Add 10 mL of 20× concentrated wash buffer into 190 mL distilled water. (Crystallization may occur in concentrated wash buffer stored in the refrigerator, which is a normal phenomenon. Place the buffer at room temperature and wait for all crystals to dissolve completely before preparation.)
Assay Procedure
1. Take the required strips from the aluminum foil pouch equilibrated to room temperature for 10 minutes. Seal the remaining strips in a self-sealing bag and store at 4℃.
2. Sample loading: Add 50 μL of sample or standards of different concentrations into corresponding wells. Add 50 μL of Universal Diluent to blank wells, followed by 50 μL of Biotin-antibody working solution to each well. Cover with Plate Sealer and incubate at 37℃ for 60 minutes. (Recommendation: Dilute test samples at least 1-fold with Universal Diluent before loading onto the microplate to reduce matrix interference on test results. Multiply the final calculated sample concentration by the corresponding dilution factor. It is recommended to set up replicate wells for all test samples and standards.)
3. Washing: Discard the liquid. Add 300 μL of 1× Wash Buffer to each well, let stand for 1 minute, flick out the wash buffer and pat dry on absorbent paper. Repeat the washing process 3 times (a microplate washer may also be used).
4. Addition of conjugate working solution: Add 100 μL of conjugate working solution to each well. Cover with Plate Sealer and incubate at 37℃ for 30 minutes.
5. Washing: Discard the liquid and wash the plate 5 times following the washing procedure described in Step 3.
6. Substrate addition: Add 90 μL of TMB Substrate to each well. Cover with Plate Sealer and incubate at 37℃ in the dark for 15 minutes.
7. Stop solution addition: Take out the microplate. Add 50 μL of Stop Solution directly into each well, then immediately measure the OD value of each well at 450 nm.
Data Analysis
Representative Data and Reference Curve
1. Calculate the average OD values of replicate wells for standards and samples. Plot the standard curve using a 4-parameter logistic function on log-log graph paper, with concentration as the X-axis and OD value as the Y-axis (exclude blank well values during plotting).
2. If the sample OD value exceeds the upper limit of the standard curve, dilute the sample appropriately and re-test. Multiply the calculated sample concentration by the corresponding dilution factor.
The data and curve shown below are for reference only. Operators shall generate the standard curve based on their own experimental data.
| 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 |
|
Note: This figure is for reference only. Sample concentrations shall be calculated using the standard curve generated from data of each individual experiment.
Kit Performance
1. Precision: Intra-assay coefficient of variation (CV) < 10%, Inter-assay coefficient of variation (CV) < 10%.
2. Recovery: Three concentration levels of canine Testosterone (T) were spiked into selected healthy canine serum and plasma samples for recovery calculation.
| Sample Type | Range (%) | Mean Recovery (%) |
|---|
| Serum (n=8) | 81–101 | 96 | | Plasma (n=8) | 92–105 | 101 |
|
3. Linearity of Dilution: High concentration canine T was spiked into 4 selected healthy canine serum and plasma samples. Samples were diluted within the dynamic range of the standard curve to evaluate linearity.
| Dilution Ratio | Recovery (%) | Serum | Plasma |
|---|
| 1:2 | Range | 83–95 | 88–96 | | 1:2 | Mean Recovery | 91 | 93 | | 1:4 | Range | 89–104 | 87–108 | | 1:4 | Mean Recovery | 93 | 98 |
|
Troubleshooting Guide
If unsatisfactory assay results occur, take photos of color development, save experimental data, retain used microplate strips and unused reagents, then contact our technical support for troubleshooting. Refer to the troubleshooting items below:
1. Poor standard curve linearity
① Incorrect standard dilution: Reconstitute and dilute standard strictly following recommended protocol.
② Inaccurate pipetting: Calibrate pipettes regularly and verify tip tightness/sealing performance.
③ Evaporation of reaction solution: Seal microplate with plate sealer film.
④ Incomplete washing: Perform sufficient wash cycles with adequate volume of wash buffer.
⑤ Foreign residues at well bottom: Wipe microplate bottom prior to absorbance measurement.
2. Faint or absent color development
① Insufficient incubation duration: Adhere to specified incubation time.
② Incorrect incubation temperature: Incubate at recommended temperature.
③ Insufficient reagent addition volume: Verify pipette accuracy and follow operating procedure strictly.
④ Improper reagent dilution: Double-check dilution steps for all reagents.
⑤ Inactivated Streptavidin-HRP: Mix Streptavidin-HRP with substrate and verify activity via color reaction test.
3. Low OD readings
① Wrong microplate reader parameter: Confirm detection wavelength setting on instrument.
② Missing stop solution addition: Add appropriate volume of stop solution.
③ Overlong delay before plate reading: Measure absorbance immediately after termination.
④ Excessively high analyte concentration in sample: Determine optimal dilution factor via preliminary pre-test.
⑤ Too low analyte concentration in sample: Determine optimal dilution factor via preliminary pre-test.
4. High background signal
① Contaminated TMB substrate: Replace with fresh substrate solution.
② Excess substrate incubation time: Strictly control color development duration.
③ Wrong dilution for detection antibody or Streptavidin-HRP: Dilute reagents in accordance with official recommended protocol.
④ Incomplete plate washing: Carry out adequate wash cycles with sufficient wash buffer volume.
ZJ26F0939394