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 .
