5. Urine: Collect the first morning urine (midstream) or 24-hour urine, centrifuge at 2000×g for 15 minutes, collect the supernatant, and store the sample at -20°C. Avoid repeated freezing and thawing.

6. Saliva: Collect the sample using a saliva sample 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.

7. Other biological samples: Centrifuge at 1000×g for 20 minutes, remove the supernatant, and store at -20°C. Avoid repeated freezing and thawing.

Notes:

1. The sample should be clear and transparent, and suspended matter should be removed by centrifugation. Hemolysis of the sample will affect the results, so hemolyzed samples should not be used.

2. If the sample is to be tested within one week of collection, it can be stored at 4°C. 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 the sample to room temperature before the experiment.

3. If the concentration of the test substance in your sample is higher than the highest value of the standard, please dilute it appropriately based on the actual situation (it is recommended to conduct a pilot experiment to determine the dilution factor).

II.Preparation for the Test

1. Remove the test kit from the refrigerator 30 minutes in advance and equilibrate to room temperature. 2. Dilute 25 μg/mL of concentrated wash buffer to 1 μg/mL of working solution with double-distilled water. Return any unused solution to 4°C. 3. Standard: Add 1.0 mL of Universal Standard & Sample Diluent to the lyophilized standard. Tighten the cap and let stand for 10 minutes until fully dissolved. Then gently mix (concentration: 20,000 pg/mL). Subsequently, serially dilute the solution to 20,000 pg/mL, 10,000 pg/mL, 5,000 pg/mL, 2,500 pg/mL, 1,250 pg/mL, 625 pg/mL, and 312.5 pg/mL. The standard diluent (0 pg/mL) serves as a blank well. Prepare the required amount of standard solution and set aside. It is recommended that the prepared standard solution be added to the sample within 15 minutes; do not allow it to sit for extended periods. 4. Biotin Conjugate Working Solution (1x): Centrifuge before opening the bottle. Dilute with Biotin Conjugate Diluent immediately before use. Prepare the solution based on the pre-calculated total volume required for each experiment (50 μL per well). Add 0.1-0.2 mL more than the original volume. Prepare the solution at a ratio of 10 μL of biotin conjugate to 990 μL of biotin conjugate diluent. Mix gently and prepare within one hour before use.

5. Streptomycin-Horseradish Peroxidase Conjugate Working Solution (1x): Centrifuge before opening the bottle. Dilute with enzyme conjugate diluent immediately before use. Prepare the solution based on the pre-calculated total volume required for each experiment (100 μL per well). Add 0.1-0.2 mL more.

6. TMB Substrate - Pipette the required amount of solution. Do not pour any remaining solution back into the reagent bottle.

Note:

1. Ensure all components of the kit are dissolved and mixed thoroughly before use. Discard any unused standard after reconstitution. 2. Concentrated biotin conjugates and concentrated enzyme conjugates are relatively small and may disperse throughout the tube during transportation. Centrifuge at 1000 × g for 1 minute before use 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. 3. 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. 4. Sample addition should be rapid, ideally within 10 minutes for each addition. To ensure experimental accuracy, replicate wells are recommended. When pipetting reagents, maintain a consistent order of addition from one well to the next. This will ensure consistent incubation times for all wells.

5. 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.

6. The chromogen TMB should be protected from direct sunlight during storage and use. After adding the substrate, carefully observe the color changes in the reaction wells. If a gradient is already evident, terminate the reaction early to avoid excessive color changes that may affect the microplate reader reading.

7. All test tubes and reagents used in the experiment are disposable. Reuse is strictly prohibited, as this will affect the experimental results.

8. 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.

9. Kit components from different batches must not be mixed (except for the wash solution and the reaction stop solution). 10. The ELISA strips in the kit are removable. Please use them in batches according to experimental needs. III. Procedure 1. Before beginning the experiment, all reagents should be equilibrated to room temperature and prepared in advance. When diluting reagents or samples, mix thoroughly and avoid foaming. If the sample concentration is too high, dilute with sample diluent to bring the sample within the detection range of the kit. 2. Sample Addition: Set up separate wells for standards and wells for the sample to be tested. Add 50 μL of the standard or sample to be tested, being careful not to create bubbles. Place the sample at the bottom of the well, avoiding contact with the sides. Next, add 50 μL of biotin conjugate (1x) to each well. Gently shake to mix. Cover the plate with a film or film and incubate at 37°C for 1 hour.

3. To ensure the validity of the experimental results, use a fresh standard solution for each experiment.

4. After incubation for 1 hour, discard the liquid in the wells, spin dry, and wash the plate three times. Add 200 μL of washing solution (1 x) to each well, soak for 1-2 minutes each time, and spin dry.

5. 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.

6. Discard the liquid in the wells, spin dry, and wash the plate five times. Add 200 μL of washing solution (1 x) to each well, soak for 1-2 minutes each time, and spin dry. 7. Add 90 μL of TMB colorimetric reagent to each well in sequence and develop at 37°C in the dark for 15-20 minutes (shorten or extend the time depending on the actual color development, but do not exceed 30 minutes). 8. Add 50 μL of stop solution to each well in sequence to terminate the reaction (the blue color will immediately turn yellow). The stop solution should be added in the same order as the substrate solution as much as possible. To ensure accurate results, add the stop solution as soon as possible after the substrate reaction time expires. 9. Measure the optical density (OD) of each well in sequence using a microplate reader at a wavelength of 450 nm. Measure within 5 minutes of adding the stop solution. 10. *Samples may require dilution. Please refer to the Sample Preparation section. Calculation of Results 1. The OD values of the competition standards and samples can be directly substituted into the calculation. If duplicate wells are set up, the average value should be taken for calculation.

2. For the convenience of calculation, although the concentration is the independent variable and the OD value is the dependent variable, we still 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) when drawing. At the same time, for the intuitiveness of the experimental results, the figure provides the original data rather than the logarithmic value. Due to different experimental operating conditions (such as operators, pipetting techniques, plate washing techniques and temperature conditions, etc.), the OD values of the standard curve will vary. The standard curve provided is for reference only, and the experimenter needs to establish a standard curve based on his own experiment. The OD value of the used sample can be used to calculate the sample concentration on the standard curve, and then multiplied by the dilution factor to obtain the actual concentration of the sample. It is recommended to use professional curve drawing software, such as Curve Expert.

1. If the entire kit is stored at -20°C, please place the kit at 4°C the night before the experiment.

2. Salt precipitation may occur when the concentrated wash solution is stored at low temperatures. When diluting, warm it in a water bath to help dissolve it.

3. A small amount of water-like substance may be present in the wells of a newly opened ELISA plate. This is normal and will not affect the experimental results.

4. This kit is for laboratory research and development use only and is not intended for use on humans or animals.

5. Reagents should be treated as hazardous substances and should be handled with care and disposed of properly.

6. Always wear gloves, lab coats, and protective glasses to avoid contact between skin and eyes with the stop solution and TMB. If contact occurs, rinse thoroughly with water.