| Usage |
I. Specimen Collection, Preparation, and Storage
- Serum: After placing whole blood samples at room temperature for 2 hours or at 4°C overnight, centrifuge at 1000×g for 20 minutes. Remove the supernatant for testing. Blood collection tubes should be disposable, pyrogen-free, and endotoxin-free. Store at -20°C or -80°C and avoid repeated freezing and thawing.
- Plasma: Within 30 minutes of collection, centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for testing. EDTA-Na2 is recommended as an anticoagulant to avoid hemolysis or high-lipidemia samples. Store at -20°C or -80°C and avoid repeated freezing and thawing. Tissue homogenization: Take an appropriate amount of tissue and wash it in pre-chilled PBS (0.01M, pH 7.0-7.2) to remove blood (lysed red blood cells in the homogenate will affect the measurement results). After weighing, mince the tissue and mix it with the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio; the specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS). Pulse thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or freeze-thawed repeatedly (keep the sonication in an ice bath and repeat the freeze-thaw cycle twice). Finally, centrifuge the homogenate at 5000×g for 5-10 minutes. Remove the supernatant for analysis. Cell culture supernatant: Centrifuge the cell supernatant at 1000×g for 20 minutes to remove impurities and cell debris. Remove the supernatant for testing and store at -20°C or -80°C, but avoid repeated freezing and thawing. Urine: Collect the first morning urine (midstream) or 24-hour urine collection, centrifuge at 2000×g for 15 minutes, collect the supernatant, and store the sample at -20°C. Avoid repeated freezing and thawing. Saliva: Collect the sample using a saliva collection tube, then centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for testing, or aliquot and store at -20°C. Avoid repeated freezing and thawing. Other biological samples: Centrifuge at 1000×g for 20 minutes, remove the supernatant, and store at -20°C. Avoid repeated freezing and thawing.
Note:
- Samples should be clear and transparent, and suspended matter should be removed by centrifugation. Hemolysis of the sample can affect the results, so hemolyzed samples should not be used.
Samples can be stored at 4°C if tested within one week of collection. If testing cannot be performed promptly, aliquot the sample into single-use portions and freeze at -20°C (for testing within one month) or -80°C (for testing within three to six months). Avoid repeated freeze-thaw cycles. Bring samples to room temperature before experimenting.
If the concentration of the test substance in your sample is higher than the highest concentration of the standard, dilute it appropriately based on the actual concentration (it is recommended to conduct a pilot experiment to determine the dilution factor).
II.Pre-Test Preparation
- Remove the test kit from the refrigerator 30 minutes in advance and equilibrate to room temperature. Dilute 25 μg of concentrated wash buffer to 1 μg of working solution with double-distilled water. Return any unused solution to 4°C. Standards: Add 1.0 mL of Universal Standard & Sample Diluent to the lyophilized standard. Tighten the cap and let stand for 10 minutes to fully dissolve. Then gently mix (concentration: 900 ng/mL). Subsequently, perform serial dilutions to 900 ng/mL, 450 ng/mL, 225 ng/mL, 112.5 ng/mL, 56.25 ng/mL, 28.13 ng/mL, and 14.07 ng/mL. Use the standard diluent (0 ng/mL) as a blank well. Prepare the required amount of standard and set aside. It is recommended that the prepared standard be added to the sample within 15 minutes; it is not recommended to allow the sample to sit for extended periods. Biotin Conjugate Working Solution (1x): Centrifuge before opening the bottle. Dilute with Biotin Conjugate Diluent immediately before use. Prepare 1x Biotin Conjugate Working Solution (50 μL per well) based on the pre-calculated total volume required for each experiment. Prepare an additional 100-200 μL. For example, prepare 10 μL of Biotin Conjugate to 990 μL of Biotin Conjugate Diluent. Mix gently and mix thoroughly. Prepare within one hour of use. Streptomycin-Horseradish Peroxidase Conjugate Working Solution (1x): Centrifuge before opening the bottle. Dilute with enzyme conjugate diluent immediately before use. Prepare the pre-calculated total volume required for each experiment (100 μL per well). Prepare an additional 100-200 μL. For example, prepare 10 μL of enzyme conjugate to 990 μL of enzyme conjugate diluent. Mix gently. Prepare within one hour of use. TMB Substrate—Pipette the required volume of solution. Do not pour any remaining solution back into the reagent bottle. Note: Ensure all kit components are dissolved and mixed thoroughly before use. Discard any remaining standard after reconstitution. Concentrated biotin conjugates and concentrated enzyme conjugates are relatively small and may disperse throughout the tube during transportation. Before use, centrifuge at 1000 × g for 1 minute to allow any liquid on the tube walls or cap to settle to the bottom. Mix the solution by carefully pipetting 4-5 times before use. Prepare the standard, biotin conjugate working solution, and enzyme conjugate working solution according to the required volume and use the corresponding diluents. Do not mix them. Concentrated wash solution may crystallize after removal from the refrigerator. This is normal. Dissolve the crystals completely in a water bath or incubator before preparing the wash solution (do not heat above 40°C). The wash solution should be at room temperature before use. Samples should be added quickly, ideally within 10 minutes for each addition. To ensure accuracy, replicate wells are recommended. When pipetting reagents, maintain a consistent order of addition from well to well. This will ensure consistent incubation times for all wells. During the wash process, any remaining wash solution in the reaction wells should be patted dry on absorbent paper. Do not place filter paper directly into the reaction wells to absorb water. Before reading, be sure to remove any remaining liquid and fingerprints from the bottom of the wells to avoid affecting the microplate reader reading. The color developer, TMB, should be protected from direct sunlight during storage and use. After adding the substrate, carefully observe the color change in the reaction wells. If a gradient is already evident, terminate the reaction early to prevent excessive color from affecting the microplate reader reading. The test tubes and reagents used in the experiment are disposable. Reuse is strictly prohibited, as this will affect the experimental results. Wear a lab coat and latex gloves for proper protection during the experiment, especially when testing blood or other body fluid samples. Please follow the national biological laboratory safety regulations. Components from different batches of the kit should not be mixed (except for the wash solution and the reaction stop solution). The enzyme labeling strips in the kit are removable; please use them in batches according to your experimental needs.
III.Procedure
- Before beginning the experiment, all reagents should be equilibrated to room temperature. Prepare all reagents in advance. When diluting reagents or samples, mix thoroughly, avoiding foaming as much as possible. If the sample concentration is too high, dilute with sample diluent to bring the sample within the detection range of the kit.
Sample Addition: Set up separate wells for standards and wells for test samples. Add 50 μL of the standard or test sample, being careful not to create bubbles. Pour the sample onto the bottom of the ELISA plate well, minimizing contact with the well walls. Next, add 50 μL of biotin conjugate (1x) to each well, gently shake to mix. Cover the plate or place it on a film and incubate at 37°C for 1 hour.
To ensure the validity of your results, use a fresh standard solution for each experiment. After 1 hour of incubation, discard the liquid from the wells, spin dry, and wash the plate three times, adding 200 μL of washing solution (1 x) to each well, soaking for 1-2 minutes each time, and spin dry. Then, add 100 μL of streptomycin-HRP (1 x) to each well, gently shake to mix, cover the plate or cover with film, and incubate at 37°C for 1 hour. Discard the liquid from the wells, spin dry, and wash the plate five times, adding 200 μL of washing solution (1 x) to each well, soaking for 1-2 minutes each time, and spin dry. Then, add 90 μL of TMB colorimetric reagent to each well and develop at 37°C in the dark for 15-20 minutes (this time may be shortened or extended depending on the actual color development time, but should not exceed 30 minutes). Add 50 μL of stop solution to each well to stop the reaction (the blue color immediately turns yellow). The order of adding the stop solution should be as consistent as possible with the order of adding the substrate solution. To ensure accurate results, add the stop solution as soon as possible after the substrate reaction time expires. Measure the optical density (OD) of each well at 450 nm using a microplate reader. Perform the assay within 5 minutes after adding the stop solution. Samples may need to be diluted. Please refer to the sample preparation section. Calculation of Results: The OD values of the competition standards and samples can be directly substituted into the calculations. If replicates are used, the average value should be used for calculations. For ease of calculation, although concentration is the independent variable and OD value is the dependent variable, the graphs use the OD value of the standard as the horizontal axis (X-axis) and the concentration of the standard as the vertical axis (Y-axis). For intuitive visualization of the results, the graphs present raw data rather than logarithmic values. Due to differences in experimental conditions (such as operator, pipetting technique, plate washing technique, and temperature), the OD values of the standard curve may vary. The provided standard curve is for reference only. Experimenters should establish a standard curve based on their own experiments. The OD value of the sample used can be used to calculate the sample concentration on the standard curve and then multiply it by the dilution factor to obtain the actual sample concentration. It is recommended to use professional curve drawing software, such as curve expert.
Concentration (ng/mL) |
OD |
900 |
0.183 |
450 |
0.385 |
225 |
0.575 |
112.5 |
0.823 |
56.25 |
1.157 |
28.13 |
1.592 |
14.07 |
1.624 |
0 |
2.256 |
Precision
Intra-assay precision (within-assay precision): CV% <8%
Three samples of known concentrations were tested 20 times on a single ELISA plate to assess intra-assay precision.
Inter-assay precision (between-assay precision): CV% <10%
Three samples of known concentrations were tested 40 times on three different ELISA plates to assess inter-assay precision.
Recovery
Recovery experiments were performed by spiking different samples with known concentrations of 5-HT to determine the recovery range and average recovery.
Sample type |
Recovery range |
Average recovery |
Serum (n=5) |
88-103% |
95% |
EDTA plasma(n=5) |
95-107% |
101% |
heparin Plasma (n=5) 80-95% 93% |
93% |
Linearity
The samples spiked with 5-HT were diluted 2-fold, 4-fold, 8-fold, and 16-fold for recovery experiments, and the recovery rate range was obtained.
Sample type |
1:2 |
1:4 |
1:8 |
1:16 |
Serum(n=5) |
82-93% |
95-104% |
87-98% |
92-99% |
EDTA Plasma (n=5) |
89-99% |
83-96% |
98-104% |
88-101% |
heparin plasma (n=5) |
87-96% |
87-96% |
96-105% |
95-103% |
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