ELISA is a powerful technique for studying interactions between proteins. It's a simple process, but there are certain steps that you need to follow in order to get good data. In this article I'll explain how to set up an ELISA plate so that your experiment goes smoothly and gives you accurate results!
Use a sterile pipette to dispense the sample.
Use the same sterile pipette to dispense the buffer.
Use a different sterile pipette to dispense the conjugated secondary antibodies, if needed.
Use yet another sterile pipette to dispense primary antibodies, if needed.
Sample preparation is an important step in the ELISA protocol. The sample must be prepared before it can be tested, and many factors affect the quality of results. For example, if you fail to prepare your sample properly, you may get an inaccurate result that can negatively impact your experiment.
First things first: check if your kit includes a sample preparation reagent kit or not (it will say on its packaging). If so, use this kit to prepare your samples; otherwise follow these steps:
Note: You may also want to add Tween 20® when preparing RBCs or FFPE tissue sections as they tend not to settle as easily due to their high protein content relative compared with other types of cells which tend have more fat content than proteins making them easier suspended in solution longer term storage requirements prior
Add primary antibody to plate. Primary antibodies are used to detect antigens in your sample, and can be purchased from a range of suppliers. It is important that you store these at 4 degrees Celsius and dilute them according to the manufacturer's instructions (usually 1:50).
The secondary antibody is specific for the primary antibody, and should be conjugated to a fluorescent molecule. Examples of common secondary antibodies include Alexa Fluor™ 488 goat anti-rabbit (for use with Alexa Fluor® 488 donkey anti-mouse), Alexa Fluor® 647 goat anti-rabbit (for use with Alexa Fluor® 647 donkey anti-mouse), and Cy3 goat anti-mouse (for use with Cy3 donkey anti-goat).
Secondary antibodies that react with both primary antibodies can also be used; however, if you are using this type of secondary antibody instead of one that is appropriate for your experiment, it's important to verify that your detection method will work with this particular set up before proceeding.
After the plate has been washed and dried, you're ready to read it. There are two different ways to read your ELISA plate: absorbance or fluorescence.
ELISA is a technique that uses antibodies to detect and measure the presence of a specific protein. ELISAs can be used as either qualitative or quantitative assays. In general, an ELISA is used to detect antigens (for example, antibodies), but ELISAs are also used for detection of other molecules (such as enzymes) by conjugating them with antibodies that recognize the molecule of interest.
ELISAs are very sensitive because they use a secondary antibody that has been labeled with an enzyme such as horseradish peroxidase or alkaline phosphatase (AP).
ELISA is a really useful technique for looking at protein interactions. It allows us to see how much of each protein is present, and then work out whether this correlates with disease states or not. The protocol outlined here should be followed when performing an ELISA experiment in order for it to be successful.