Specimen Requirements

1. Specimens should be extracted as soon as possible after collection, following the relevant literature. 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 freeze-thaw cycles should be avoided.
2. Samples containing sodium nitrate (NaN3) cannot be assayed, as NaN3 inhibits horseradish peroxidase (HRP) activity.

Procedure

1. Dilution of Standard: This kit provides one standard sample at full strength. Users can dilute the sample in a small test tube according to the following chart. 10μg/L

Standard No. 5

Add 150μl of the original standard to 150μl of the standard diluent

5μg/L

Standard No. 4

Add 150μl of the original standard to 150μl of the standard diluent

Standard No. 3

Add 150μl of Standard No. 4 to 150μl of Standard Diluent

1.25μg/L

Standard No. 2

Add 150μl of Standard No. 3 to 150μl of Standard Diluent

3. Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

4. Prepare the solution: Dilute the 30x concentrated wash solution 30x with distilled water and set aside.

5. Wash: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with wash solution, let it sit for 30 seconds, then discard. Repeat this process 5 times and pat dry.

6. Add enzyme: Add 50 μl of enzyme-labeled reagent to each well, excluding the blank well.

7. Incubation: Perform the same procedure as in step 3.

8. Wash: Perform the same procedure as in step 5. 9. Color Development: Add 50 μl of Color Developing Reagent A to each well, followed by 50 μl of Color Developing Reagent B. Gently shake to mix thoroughly. Incubate at 37°C in the dark for 10 minutes. 10. Stop: Add 50 μl of Stop Solution to each well to terminate the reaction (the blue color will immediately turn yellow). 11. Measurement: Use the blank well as a zero value and measure the absorbance (OD) of each well at 450 nm. Measurements should be performed within 15 minutes after adding the Stop Solution.

Calculation

Draw a standard curve on graph paper with the concentration of the standard as the horizontal axis and the OD value as the vertical axis. Find the corresponding concentration of the sample from the standard curve based on the OD value of the sample; then multiply it by the dilution factor. Alternatively, use the concentration of the standard and the OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation to calculate the sample concentration, and then multiply it by the dilution factor to obtain the actual concentration of the sample.

Standard Curve

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.