Washing

The washing step in ELISA is to wash out any unbound antigens, antibodies, and other molecules that may interfere with the results. Hence proper washing is important and the use of an appropriate buffer will also ensure accurate results. Improper washing can lead to false positives or false negatives.

Insufficient washing will result in high background noise, while excessive washing might result in decreased sensitivity caused by elution of the antibody and/or antigen from the well. Washing is performed in a physiologic buffer such as Tris-buffered saline (TBS) or phosphate-buffered saline (PBS). A detergent such as 0.05% Tween-20 can also be added to the buffer to help remove non-specific binders. A high signal:noise ratio allows for easy detection.