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 freezing and thawing should be avoided. 2. Samples containing NaN3 cannot be tested, as NaN3 inhibits horseradish peroxidase (HRP) activity.
Procedure 1. Dilution of Standard: This kit provides one standard at full strength. Users can dilute the standard in a small test tube according to the chart below. 2. Sample Addition: Set up a blank well (no sample or enzyme-labeled reagent is added to the blank control well; all other steps remain the same), a standard well, and a well for the sample to be tested. Accurately add 50 μl of the standard sample to the ELISA-coated plate. First, add 40 μl of the sample diluent to the sample wells to be tested, followed by 10 μl of the sample to be tested (final sample dilution is 5-fold). Add the sample to the bottom of the plate well, avoiding contact with the well walls. Gently shake to mix. 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, and spin dry. Fill each well with wash solution. Let it sit for 30 seconds before discarding. Repeat this process 5 times and pat dry. 6. Enzyme Addition: Add 50 μl of enzyme-labeled reagent to each well, except for the blank well. 7. Incubation: Same as in step 3. 8. Washing: Same 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. 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 the zero setting and measure the absorbance (OD) of each well at 450 nm. Measurement should be performed within 15 minutes after adding the Stop Solution.
Calculation
Draw a standard curve on graph paper with the standard concentration as the horizontal axis and the OD value as the vertical axis. Use the standard curve to find the corresponding concentration based on the sample's OD value; then multiply it 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, and then multiply it by the dilution factor to obtain the actual sample concentration. | 
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