[ ASSAY PROCEDURE ]

1. Determine wells for diluted standard, blank and sample. Prepare 7 wells for standard, 1 well for blank.  Add 100µL each of dilutions of standard （read Reagent Preparation）, blank and samples into the appropriate wells. Cover with the Plate sealer. Incubate for 2 hours at 37oC. 

2.  Remove the liquid of each well, don’t wash. 

3.  Add 100µL of Detection Reagent A working solution to each well. Incubate for 1 hour at 37oC after covering it with the Plate sealer.

4.  Aspirate the solution and wash with 350µL of 1× Wash Solution to each well using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1~2 minutes. Remove the remaining liquid from all wells compley by snapping the plate onto absorbent paper. Totally wash 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper. 

5.  Add 100µL of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37oC after covering it with the Plate sealer. 

6.  Repeat the aspiration/wash process for total 5 times as conducted in step 4. 

7.  Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 25 minutes at 37oC （Don't exceed 30 minutes）. Protect from light. The liquid will turn blue by the addition of Substrate 

Solution. 

8.   Add 50µL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 

9.  Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediay. 

Note:

1.   Assay preparation: Keep appropriate numbers of wells for 1 experiment and remove extra wells from microplate. Rest wells should be resealed and stored at -20oC. 

2.   Samples or reagents addition Please use the freshly prepared Standard. Please carefully add samples to wells and mix gently to avoid foaming. Do not touch the well wall. For each step in the procedure, totaldispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips between additions of standards, samples, and reagents. Also, use separated reservoirs for each reagent. 

3.   Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents are added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be controlled. 

4.   Washing: The wash procedure is critical. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor precision and false elevated absorbance reading. 

5.  Controlling of reaction time: Observe the change of color after adding TMB Substrate （e.g. observation once every 10 minutes）, if the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading. 

6.  TMB Substrate is easily contaminated. Please protect it from light. 

7.  The environment humidity which is less than 60% might have some effects on the final performance, therefore, a humidifier is recommended to be used at that condition.