Supplies You Need
1. Microplate reader (450nm)
2. High-precision pipettes and tips: 0.5-10µL, 2-20µL, 20-200µL, 200-1000µL
3. 37°C incubator

Reagent Preparation:
20×Wash Buffer: Dilute 1:20 with distilled water, i.e., add 1 part 20×Wash Buffer to 19 parts distilled water.
Plate Washing Method:
1. Manual Plate Washing: Shake off all liquid from the wells, fill each well with wash buffer, let stand for 1 minute, shake off all liquid from the wells, pat dry on absorbent paper, and wash the plate five times.
2. Automatic Plate Washer: Add 350µL of wash buffer to each well, soak for 1 minute, and wash the plate five times.

Procedure:
1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 20 minutes. Seal the remaining strips in a ziplock bag and return them to 4°C.
2. Arrange the standard and sample wells. Add 50 μL of the standard of different concentrations to each standard well.
3. Add 10 μL of the sample to be tested to the sample wells, followed by 40 μL of sample diluent. Leave blank wells untouched.
4. Add 100 μL of horseradish peroxidase (HRP)-conjugated detection antibody to each standard and sample well, except for the blank wells. Seal the wells with sealing film and incubate at 37°C in a water bath or incubator for 60 minutes.
5. Discard the liquid, pat dry on absorbent paper, fill each well with wash solution, let stand for 1 minute, shake off the wash solution, and pat dry on absorbent paper. Repeat this process five times (a plate washer can also be used). 6. Add 50 μL each of substrates A and B to each well and incubate at 37°C in the dark for 15 minutes. 7. Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 minutes. Result Interpretation: Draw a standard curve: In an Excel worksheet, plot the standard concentration on the horizontal axis and the corresponding OD value on the vertical axis. Calculate the concentration of each sample using the curve equation.

Kit Performance
1. Accuracy: The correlation coefficient (R) between the linear regression of the standard and the expected concentration is greater than or equal to 0.9900.
2. Sensitivity: The minimum detectable concentration is less than 0.1 μg/mL.
3. Specificity: No cross-reaction with other soluble structural analogs.
4. Reproducibility: Both the intra- and inter-assay coefficients of variation are less than 15%.
5. Storage: Store at 2-8°C away from light and moisture.
6. Validity period: 6 months