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!
Displaying your problem I can think of several possibilities:
1. hermetically seal all positions of the plate and open only those that are to be used.
After use, empty them, wash them, seal them again and mark them as used.
2. Using a plate reader that allows to select the positions should be read.
3 Be careful nucho stability unused reagents (if you use intervals are long, could deteriorate).
and if that fails, use plates formed by strips, so that each single test strips that are needed are used .:
As an example:
http://www.brand.de/es/productos/productos-life-science/brandplatesr-microplacas/placas-de-tiras-12xf8/
The Tween-20 detergent probably dissolves the adhesive on the tape, allowing the Tween-20 to get into the covered wells. Once in there, it coats the surface of the plate and prevents proteins from sticking to it. You might try using foil sealers instead of tape, but they might also leak.
thank you very much that was of great help! :)
I guess I'll have to be more careful in the washing step...
better you use detachable elisa plate if you have to use limited wells . By using this there will be less contamination.
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