ELISAs are often used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones in research and diagnostics. 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 my top 5 top simple tips that will help you avoid making these errors.
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 around for vital information in the manual in the middle of performing the 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.
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 I suggest you take things 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, lessening the chances of an error.
3. Add a contrasting background
While adding samples or any colorless reagent to the wells, it can be difficult to see whether or not you have added it when the plate is sitting on your bench. I recommend using a sheet of black paper or something similar to create a dark background under your plate. This will make it simpler to detect 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 data, making your results less usable. For instance, 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. Also, using the same tip for each well can lead to an accumulation of bubbles, which will throw of the ELISA reading. Tiny 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. Using the same tip can thus result in faulty pipetting, which will affect your results. Therefore, in this case it is advisable to change the tips between wells to ensure consistency. While this attention to detail may seem overly picky, it can make a big difference in the accuracy and repeatability of your results.
5. Use the spectrophotometer as a back up
Finally, a great tip for the worst case scenario: you’ve finished setting up your assay and realize that the ELISA reader is not working. Don’t panic! You can still 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, since the spec won’t automatically analyze it for you. Next, read all of your samples well by well, again using cuvettes on a standard spec, and plot the readings against your standard curve. If you don’t have minimum quantity cuvettes, try diluting all of your standards and samples equally and use regular volume cuvettes.
Lastly, I would like to suggest storing the leftover reagents from the kit such as substrates, stop solution etc. (at least until their expiration date). They may come handy someday in case you run out of reagents or feel like experimenting with the standard protocol.
These suggestions may sound like common sense, but I know that when I was a beginner, I ended up making many of these mistakes. What are your tips for improving ELISAs?