ELISA protocol
Incubate for 120 minutes at 37 ⁰C.
Invert the plate and pat it against thick clean absorbent paper.
Incubate for 60 minutes at 37°C.
(wash 1/5) Aspirate each well and wash by filling each well with Wash Buffer (approximately 400µL).  
(wash 2/5) Aspirate each well and wash by filling each well with Wash Buffer (approximately 400µL).  
(wash 3/5) Aspirate each well and wash by filling each well with Wash Buffer (approximately 400µL).  
(wash 4/5) Aspirate each well and wash by filling each well with Wash Buffer (approximately 400µL). 
(wash 5/5) Aspirate each well and wash by filling each well with Wash Buffer (approximately 400µL).  
Complete removal of liquid at each step is essential.
Add 50µL of Stop Solution to each well.
If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
Determine the optical density (OD value) of each well at once, using a microplatereader set to 450 nm.
User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
Add 100µL of Standard, Blank, or Sample per well.
The blank well is added with Sample diluent.
Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible.
Mix it gently.
Cover the plate with sealer we provided.
Remove the liquid from each well, don't wash. 
Add 100µL of Detection Reagent A working solution to each well.
Cover with the Plate sealer.
Gently tap the plate to ensure thorough mixing.
Incubate for 1 hour at 37°C.
(wash 1/3) Aspirate each well and wash by filling each well with Wash Buffer (approximately 400µL)
(wash 2/3) Aspirate each well and wash by filling each well with Wash Buffer (approximately 400µL).  
(wash 3/3) Aspirate each well and wash by filling each well with Wash Buffer (approximately 400µL).  
 (a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer are needed).
Complete removal of liquid at each step is essential.
After the last wash, completely remove remaining Wash Buffer by aspirating or decanting.
Add 100µL of Detection Reagent B working solution to each well.
Cover withthe Plate sealer.
Add 90µL of Substrate Solution to each well.
Cover with a new Plate sealer and incubatefor 15-30 minutes at 37°C.
Protect the plate from light.
The reaction time can be shortened orextended according to the actual color change, but this should not exceed more than 30 minutes.
When apparent gradient appears in standard wells, user should terminate the reaction.
After the last wash, completely remove remaining Wash Buffer by aspirating or decanting.
Invert the plate and pat it against thick clean absorbent paper.
