200ng/L

Standard No. 5

150μl of the original standard was added to 150μl of the standard diluent

100ng/L

Standard No. 4

150μl of the original standard was added to 150μl of the standard diluent

50ng/L

To 150 μl of standard No. 4, add 150 μl of standard diluent

25 ng/L

Standard No. 2

To 150 μl of standard No. 3, add 150 μl of standard diluent

12.5 ng/L

Standard No. 1

To 150 μl of standard No. 2, add 150 μl of standard diluent 2. Sample Addition: Set up blank wells (blank control wells without sample or enzyme-linked reagent; all other steps remain the same), standard wells, and test sample wells. Accurately add 50 μl of standard to the enzyme-linked coated plate. Add 40 μl of sample diluent to the test sample wells, followed by 10 μl of the test sample (final sample dilution is 5x). Add the sample to the bottom of the plate, avoiding contact with the well walls. Gently shake to mix. 3. Incubation: Seal the plate with sealing film and incubate at 37°C for 30 minutes. 4. Preparation: Dilute the 30x concentrated wash buffer 30x with distilled water and set aside. 5. Wash: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with wash buffer, let it sit for 30 seconds, then discard. Repeat this process 5 times and pat dry. 6. Add enzyme: Add 50µl of enzyme-labeled reagent to each well, excluding the blank well. 7. Incubation: Same as in 3. 8. Wash: Same as in 5. 9. Color development: First add 50µl of Color Developer A to each well, then add 50µl of Color Developer B. Gently shake to mix. Develop at 37°C in the dark for 10 minutes. 10. Stop: Add 50µl of Stop Buffer to each well to terminate the reaction (the blue color will immediately turn yellow). 11. Measurement: Use the blank well as the zero setting and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. Measurements should be performed within 15 minutes after adding the stop solution. Calculation: Use the concentration of the standard as the horizontal axis and the OD value as the vertical axis to draw a standard curve on graph paper. Based on the sample's OD value, find the corresponding concentration from the standard curve; then multiply by the dilution factor. Alternatively, use the standard concentration and OD value to calculate the linear regression equation for the standard curve. Substitute the sample's OD value into the equation to calculate the sample concentration. Multiply by the dilution factor to obtain the actual sample concentration.

1. Specimens should be extracted as soon as possible after collection, according to relevant literature, and experiments should be performed as soon as possible after extraction. If experiments cannot be performed immediately, specimens can be stored at -20°C, but repeated freezing and thawing should be avoided.

2. Samples containing NaN3 cannot be tested because NaN3 inhibits the activity of horseradish peroxidase (HRP).

5. The sealing film is for single use only to avoid cross contamination.

6. Please store the substrate in a dark place.

7. Strictly follow the instructions for operation. The test results must be determined based on the readings of the microplate reader.

8. All samples, washing solutions and various wastes should be treated as infectious materials.

9. Components of different batches of this reagent must not be mixed.