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Assay procedure

Step 1: Number: determine the number of well to be used and store unused wells in 4 ℃. Set a blank well without any solution.

Step 2: Prepare sample: pipette Positive control and Negative control 50μl to the well respectively. Controls need test in duplicate. Pipette sample dilution 40μl and testing sample 10μl to testing sample well. Pipette sample to the bottom of well, don’t touch the wall as far as possible, and mix gently.

Step 3: Incubate: Cover with the adhesive strip provided, incubate for 30 min at 37℃.

Step 4: Configurate liquid: Dilute wash solution 30-fold (or 20-fold) with distilled water.

Step 5: Washing: Uncover the adhesive strip, discard liquid, pipette washing buffer to every well, still for 30s then drain, repeat 5 times.

Step 6: Add enzyme: Pipette HRP-Conjugate reagent 50μl to each well, except blank well.

Step 7: Incubate: Operation with 3.

Step 8: Washing: Operation with 5.

Step 9: Color: Pipette Chromogen Solution A 50ul and Chromogen Solution B to each well, avoid the light preservation for 15 min at 37℃

Step 10: Stop the reaction: Pipette Stop Solution 50μl to each well, stop the reaction (the blue change to yellow).

Step 11: Calculate: take blank well as zero. Read absorbance at 450nm after pipette Stop Solution within 15min.