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Assay procedure for Sandwich-ELISA


Author:Elabscience
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Bring all reagents and samples to room temperature before use. Centrifuge the sample again after thawing before the assay. All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. It’s recommended that all samples and standards be assayed in duplicate.


1. Add Sample: Add 100μL of Standard, Blank, or Sample per well. The blank well is added with Reference Standard & 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. Incubate for 90 minutes at 37 ℃ .  

2. Biotinylated Detection Ab: Remove the liquid of each well, don't wash. Immediately add 100μL of Biotinylated Detection Ab 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.

3. Wash: Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 350μL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquidat each step is essential. After the last wash, remove remained Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.

4.  HRP Conjugate: Add 100μL of HRP Conjugate working solution to each well. Cover with the Plate sealer. Incubate for 30 minutes at 37°C.

5.  Wash:Repeat the wash process for five times as conducted in step 3.

6.  Substrate: Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for about 15minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes.When apparent gradient appeared in standard wells, user should terminate the reaction.

7.  Stop: Add 50μL of Stop Solution to each well. Then, the color turns to yellow immediately. The order to add stop solution should be the same as the substrate solution.

8.  OD Measurement: Determine the optical density (OD value) of each well at once,using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.

9.  After experiment, put all the unused reagents back into the refrigerator according to the specified storage temperature respectively until their expiry.