An ELISA is a technique used by laboratories to detect, quantify, and purify proteins from samples. Since it uses antibodies, it can also be expensive since storage conditions and time can affect their performance.
It is, therefore, crucial to optimize ELISA results by optimizing the binding conditions, so you don’t need to buy a new kit or redo the experiment. An ELISA kit contains a capture antibody that binds with the protein of interest to immobilize it on the surface of the well. There is also a detection antibody that will recognize it and generate a signal to detect once you add a substrate solution after incubating.
The more proteins are in our sample, the stronger the signal will be. Here are some ways to get the most out of an ELISA kit when conducting tests.
Dilute your Samples Correctly
It is essential to know how much protein you are loading into each well before beginning an experiment with an ELISA kit. It can affect whether you can detect a signal. If you use a concentrated sample, the antibodies in the kit might not bind with all of them. So, you will have some wells with no captured proteins.
On the other hand, if you dilute your sample a lot (or there is very little protein in each well), the antibodies will bind with all the proteins in the wells, but there won’t be enough to generate a signal. Therefore, you have to be careful when diluting the sample to avoid being too concentrated or too dilute. If you know how to do ELISA sample dilution, it will make your work easy.
Optimize Antibody Binding Conditions
They make antibodies to recognize certain types of proteins. It ensures they have different binding conditions required before being able to bind to it. Varying ELISA kits may need variable incubation time and temperature before you denature the proteins enough for them to bind with the capture antibody. You can optimize binding conditions by using other antibodies tested on your sample at various temperatures or times.
For example, if one antibody needs 1 hour at 37°C, diluting it at 1:100 will need 10 hours and 1:1000 30 hours at the same temperature.
Use a Standard Curve
Before doing an ELISA test, it is essential to know how much protein is in your samples to compare it to the standard curve later. There are standards in ELISA kits, so you can use them to determine the protein concentration in diluted samples.
You must maintain the dynamic range of the apparatus that you are using to read the plates. When preparing the standard curve, the top standard should have an OD below the highest dynamic range. It makes the extrapolation of the curve easy and more accurate.
Use Positive and Negative Controls
It is crucial to include controls when experimenting to know if your method works appropriately or not. Positive control contains the protein that the capture antibody binds with, while negative control does not have any protein.
Doing this will increase the chances of you getting the desired results faster.
Normalize Your Samples for Accurate OD Comparison
Normalizing your samples is a crucial step when you need to compare between different experiments. First, subtract the non-specific background from each well and divide the total signal by its protein concentration.
This way, all of your samples will have the same baseline so you can accurately see the amount of signal from the specific protein of interest.
Optimize Imaging Settings
Setting up your imaging conditions can affect the intensity of the optical density signal. To see all wells clearly on a plate, you must adjust brightness and contrast, regardless of how strong or weak the signal is.
Next, turn off background subtraction and auto exposure. That way, you can control the exposure time best for your sample, especially if it has a powerful signal or a weak one. You can also adjust the gain to ensure that all wells will have a similar exposure time. It ensures that there is no bias when comparing samples on the same plate.
Be Wary of The Limitations of ELISA Assays
There may be some inaccuracies with ELISA kits. However, it will depend on how you use them. If you did not test your samples thoroughly or your antibodies are not working appropriately, you may get the wrong results. Interpreting your results can be challenging when your samples have strong signals, or you have nonspecific binding.
If you are using ELISA kits with polyclonal antibodies, keep in mind that they may not be precise, although they have a broad target range. If you don’t have the target antigen, you won’t be able to use monoclonal antibodies.
ELISA is the most commonly used type of protein quantification assay. Researchers and laboratories prefer to use them as they are easy to use, cost-effective, and readily available. The kits are available for different targets, such as cytokines, growth factors, and immuno-oncology. You can use the kits to get optimal results if you adhere to the principles above.