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One of the defining features of an ELISA assay is the ability to quickly and effectively wash away unbound molecules. This allows ELISA assays to capture specific antigens or antibodies from crude samples, enabling researchers to identify, isolate, and quantify nearly any molecule. Optimizing the parameters of the wash steps is critical to obtaining the best results from your ELISA assay. Use this guide to help decide how you will optimize the parameters of your washes:
One of the most significant parameters to optimize is the wash volume. The amount of wash solution that is used to wash the ELISA wells influences the stringency of the wash: too little and unbound molecules may remain, greatly increasing background signal; too much, and you risk stripping specifically bound molecules.
As a rule of thumb, the wash volume should be at least as high as the coating volume; manufacturers will list the coating volume on the datasheet, providing a starting point for your optimization. A commonly used industry standard is 200 µl, and manufactures will often recommend 300 µl wash volumes.
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Another major parameter you might wish to consider is the number of wash cycles used. As with wash volume, too few wash cycles cause high background, while too many washes reduce signal strength. The typical number of wash cycles is three; however, you must consider the manufacturers suggestions and the type of plate you’re using: plates coated by the manufacturer typically require fewer washes than in-house coated plates.
Aspiration of the wash buffer is important to optimize. There are several variables to consider, including aspiration height, and aspiration method, and well shape.
Aspiration height, the distance from the bottom of the well to the aspiration head, contributes significantly to the residual volume of the wells. Residual volume of wash buffer includes unbound molecules, and greater volumes cause higher background. The optimal height of a rigid aspiration head has a range of just 0.1mm; even slightly too high or too low will drastically increase the residual volume. Precise calibration of the washing step is crucial for rigid aspiration heads. Floating heads can move up and down the well according to the volume, so adjusting the height is much less important.
The shape of the well itself can also affect residual volume. Wells with sharp corners tend to retain the most residual volume, while v-wells retain the least. If you experience high background that’s hard to troubleshoot, investigate this parameter.
Given the significance of the wash step, you may wish to perform tests to calibrate your washes before performing sensitive experiments.
Keywords: ELISA, optimization, plate washing, wash parametersClick for more optimization tips