When I'm conducting an ELISA experiment, I don't use up all of the 96-wells at once. So, I cover the other parts that I don't use with tape. However, when I wash the wells with PBS-Tween20 (with wash bottles), sometimes the PBS-Tween20 just drips into the wells that are covered with tape.

When I use the wells that were used to be covered with tape, I sometimes found that after adding the substrate (1-Step Ultra TMB ELISA substrate, ThermoScientific), the wells did not give good results (max OD450 about 0.1-0.2) and the absorbance of the positive control was a lot lower than usual (which should be around 2).

I'm very sure that all of my reagents worked normally e.g. coating buffer, coating Abs, primary Abs and secondary Abs. Could it be that the PBS-Tween20 interfered in the plate coating step?

PS: The type of ELISA that I'm working on is sandwich ELISA/capture ELISA. If you need any more information please let me know. Thanks in advance!

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