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 duplicates.
1. Add Sample: Add Standard working solution of different concentrations to the first two columns: Each concentration of the solution is added into two wells side by side (100 uL for each well). Add samples to other wells (100 uL for each well). Cover the plate with sealer provided in the kit. Incubate for 90 min at 37℃. (Note: solutions should be added to the bottom of micro ELISA plate well, avoid touching the inside wall and foaming as possible.)
2. Biotinylated Detection Ab/Ag: Remove the liquid of each well, do not wash. Immediately add 100 μL of Biotinylated Detection Ab/Ag working solution to each well. Cover with the Plate sealer. Gently mix up. Incubate for 1 hour at 37°C.
3. Wash: Aspirate or decant the solution from each well，add 350 uL of wash buffer to each well. Soak for 1~2 min and aspirate or decant the solution from each well and pat it dry against clean absorbent paper. Repeat this wash step 3 times. Note: a microplate washer can be used in this step and other wash steps. Complete removal of liquid at each step is essential. After the last washing, remove the remained Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4. HRP Conjugate: Add 100 μL of HRP Conjugate working solution to each well. Cover with the Plate sealer. Incubate for 30 min at 37°C.
5. Wash: Aspirate or decant the solution from each well, repeat the wash process for five times as conducted in step 3.
6. Substrate: Add 90 μL of Substrate Reagent to each well. Cover with a new plate sealer. Incubate for about 15 min at 37°C. Protect the plate from light. (Note: the reaction time can be shortened or extended according to the actual color change, but not more than 30min.)
7. Stop: Add 50 μL of Stop Solution to each well. (Note: 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 with a micro-plate reader set to 450 nm. The micro-plate reader should be preheated and set in advance.
9. After experiment, put all the unused reagents back into the refrigerator according to the specified storage condition respectively until their expiry.