ELISAs (enzyme-linked immunosorbent assays) are often used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones in research and diagnostics. Today, a wide range of ELISA formats exist to suit your needs e.g. indirect ELISA, direct ELISA, competitive ELISA and sandwich ELISA. While it has become easier to perform ELISAs, thanks to the various commercially available kits, there are still a number of common mistakes that can ruin your experiment. Here are our top 5 top simple tips that will help you avoid these errors and improve your ELISA.

1. Personalize the protocol

After reading the entire set of instructions provided in the ELISA kit manual, it is helpful to type out a small, personalized, stepwise protocol. This will help you avoid having to search for vital information during your experiment. It’s also a good way to do a mental run-through of the protocol, so you don’t encounter any surprises along the way. While you perform the ELISA, make sure to write down any small deviations that you make from the protocol. This is important for both planned and unplanned deviations (i.e. mistakes). It may just be that you accidentally find a way to improve your ELISAs!

2. Arrange Your Samples in the Order You’ll Use Them

It is common (and good) practice to make a template that shows where all of your samples are on the 96-well plate, to save having to label the plate itself. But we suggest taking things even further by actually arranging the samples in the order you have them on the template before you add them to the plate. This makes it much easier to keep track of what you’ve added, reducing the chances of an error.

3. Add a Contrasting Background

It can be difficult to see colorless reagent or samples in your plate when it’s sitting on your bench. You can help yourself by placing a sheet of black paper or something similar to create a dark background under your plate. This will make it much easier to see whether or not you have already added a reagent to a particular well.

4. Be Consistent With Your Pipetting

Small differences in pipetting can lead to large variations in ELISA data. To avoid pipetting-related inconsistencies in your results, take the following pointers on board:

  • When adding the same volume of solution to multiple wells or making serial dilutions, it can be hard to keep track of whether or not you have dispensed the last drop in the pipette tip. Make sure your lab mates know not to disturb you during this process!
  • Using the same tip for each well can lead to an accumulation of bubbles. This will throw off the ELISA reading, so use fresh tips for each well.
  • Minute differences in volume delivery, due to different levels of wetting inside the tips with each successive use, can also noticeably affect the standard curve, which makes your results less accurate. This is another reason to change your tips after each use.

While this attention to detail may seem excessive, it can make a big difference to your ELISA results.

5. Use the Spectrophotometer as a Back Up

Here’s a great tip for the worst-case scenario! You’ve finished setting up your assay and realize that the ELISA plate reader is not working. Don’t panic! You can rescue your data by using a spectrophotometer to quantitate standards and samples. Use minimum quantity cuvettes to read your series of standards. You’ll have to plot the standard curve by hand (or in Excel), since the spectrophotometer won’t automatically generate it for you. Next, read all of your samples well by well, again using cuvettes on a standard spectrophotometer, and plot the readings against your standard curve. If you don’t have minimum quantity cuvettes, dilute your standards and samples equally and use regular volume cuvettes.

Lastly, we suggest storing the leftover reagents from your ELISA kit (e.g. as substrates, stop solution etc. at least until their expiration date). They may come in handy someday in case you run out of reagents or feel like experimenting with the standard protocol.

What are your tips for improving ELISAs?

Originally published in 2012. Updated and republished in May 2017.

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