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Assay procedure for Competitive-ELISA


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 and Biotinylated Detection Ab: Add 50μl of Standard, Blank, or Sample per well. The blank well is added with Reference Standard &Sample Diluent. Immediately add 50 μl of Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer we provided. Gently tap the plate to ensure thorough mixing. Incubate for 45minutes at 37°C. (Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible.)

2. Wash: Aspirate each well and wash, repeating the process three times Wash by filling each well with Wash Buffer (approximately 350μl) using a squirt bottle, multi-channel pipette, manifold dispenser or automated washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.

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

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

5.  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.

6.  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.

7.  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.

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