The purpose of washing is to separate bound and unbound ( free / unwanted ) reagents/serum components. This involves the emptying of microwells of reagents followed by the addition of liquid into the wells. Such a process is performed at least 3-6 times for every well. The liquid used to wash wells is usually buffered (PBS) in order to maintain isotonicity, since most Ag-Ab reactions are optimal under such conditions. Tap water is not recommended, since tap water varies greatly in composition (pH, molarity, and so on ). Generally, the mechanical action of flooding wells with a solution is enough to wash wells of unbound reagents. Some workers leave washing solution for a short time (soak time) after each addition (1-5 minutes). Sometimes detergents, notably Tween-20 (0.05%) are added to washing buffers. This can cause problems where excessive frothing takes place producing poor washing conditions, since air is trapped and prevents the washing solution from contacting the well surface. For most cases, this addition does not contribute significantly to the washing procedure. When using detergents, care has to be taken that they do not affect reagents adversely (denature Ag), and greater care is needed to prevent frothing in the wells.
In washing plate manually, the most important factor is that each well receives the washing solution so that, no air bubbles are trapped in the well or a thumb is not placed over corner wells.
Various washing cycles can be programmed. Careful maintenance is essential, since they are prone to machine errors, such as having a particular nozzle being blocked. Further ELISA tips in detail will be recommended in the following aspects.