• Prepare Reagents: Dilute 1 volume of Concentrated Washing Buffer (20×)with 19 volumes of distilled water, mix well.
• Add Samples: Open the foil pouch and remove the Microplate. Set up 1 well as Blank, 2 wells as negative control, 2 wells as positive control. After dispensing 50μL of Sample Diluent , dispense 50μL of sample or negative control or positive control to the respective wells. Gently vibrating the plate.
• Incubate: Cover the Microplate with plate cover and incubate the Coated Microplate in a thermostat-controlled water-bath or microplate incubator at 37℃ for 60 minutes.
• Wash the Plate: Remove the plate cover. Aspirate the contents of all wells. Fill the wells with the diluted washing buffer ( 10~20 seconds to soak) then aspirate again. Repeat the procedure for 5 times. Make sure that the rest volume is minimal, by tapping plate onto absorbent paper.
• Add Conjugate: Add 100μL of conjugate to each well (except for the blank well).
• Incubate: Cover the Microplate and incubate the plate at 37℃ for 30 minutes.
• Wash the Plate: Repeat the wash procedure as in step 4.
• Add Substrate: Add 50μL of Substrate Solution A and 50μL of Substrate Solution B to each well, mix well. Cover and incubate at 37℃ for 30 minutes.
• Stop reaction: Add 50μL Stop Solution to each well, mix well.
• Read the absorbance at 450 nm. If a dual wavelength measurement is used, the reference wavelength should be selected from 620nm to 690nm.

PRECAUTIONS

• Allow all kit components to reach room temperature before use.
• Follow the direction insert to control the reaction temperature and time strictly.
• Do not mix components of different lot numbers to use.
• The kit should be store at 2-8℃. Do not use kit components beyond their expiration date.