An Introduction to ELISA Protocol
One of the most common assays used in biotech and biomedical research is the Enzyme-Linked Immunosorbent Assay –– otherwise known as an ELISA. ELISAs are designed as tests to determine the presence of a certain peptide, protein, hormone, or antibody within a liquid sample. By following the relatively simple (in theory) ELISA protocol, scientists can detect and quantify a target substance (analyte) within a complex mixture. Additionally, ELISAs are rapid, sensitive, cost-effective, and can be performed in a high-throughput manner. Here, we’ll outline ELISA protocol in its most basic form and explain how and why this procedure is so effective and popular.
ELISA “Sandwich” Protocol
When antigens cannot be bound directly to the solid surface because of their molecular properties, what is called a “sandwich ELISA” can be employed, because the test acts to bind the target analyte (in this scenario, an antigen) with two antibodies. Here’s how it works:
- First, a “capture” antibody is attached to the microplate being used to carry out the procedure, which is either a high-binding or medium-binding microplate. Depending on the test being performed, the plate will be white, black, or clear but all will have binding properties. This antibody is specifically selected to attach itself to and capture the desired antigen. Remaining binding sites on the plate surface are blocked e.g. with a BSA solution or a blocking reagent.
- Next, the sample (most often a complex liquid mixture) is added to the plate. During this stage, the capture antibody will bind to the target antigen.
- Once the capture antibody binds to the antigen, the next step is to wash the plate in order to remove other substances.
- Then a labelled second antibody –– the “detection” antibody –– is introduced to the plate. This antibody will connect to the antigen as well and “complete the sandwich,” so to speak.
- Once again, wash the plate one last time.
- Finally, a researcher can add a detection substrate, which will then trigger a chemical reaction to produce a measurable signal (colorimetric, fluorescent or luminescent), indicating the presence, or lack thereof, of the target analyte. This way, professionals will be able to see very easily if a given sample contains a certain protein, hormone, antigen, etc.
Though there are other variations of this basic ELISA protocol –– including direct ELISA, indirect ELISA, and competitive ELISA – all ELISAs still operate on the same basic principle. That is, the binding of a selected antigen to an antibody on a solid surface.
What Are ELISAs Used For?
Laboratories use ELISAs for a wide range of tests. For example, ELISAs are used to diagnose serious conditions like HIV, Lyme disease, and Zika virus. ELISAs also have more commercial applications as well. Some businesses use ELISAs to test food products to detect common allergens –– such as peanuts –– within their products. As mentioned above, ELISAs can be tailored to detect a number of different analytes, which makes them both versatile and valuable.
In addition, certain laboratories are currently using ELISA protocols to test for COVID-19 antibodies in the bloodstream. One method developed for this purpose is designed to detect Immunoglobulin G (IgG) antibodies produced as a result of exposure to SARS-CoV-2. This procedure involves the specific interaction of a special protein known as the recombinant Receptor. Binding Domain (RBD) coated to the microplate and SARS-CoV-2 IgG antibodies in a test sample. As with other ELISAs, a Peroxidase-labelled detection antibody and a respective substrate are then added to the plate as well. In this instance a change in color will occur if results are positive. Note, IgG antibodies are generally detectable 10-14 days after initial exposure to COVID-19. It is not clear at this time how long these antibodies will persist within the bloodstream. Lastly, the test is not recommended for use as a sole means of diagnosis of COVID-19.
Optimizing ELISA Protocol
Successful ELISAs are dependent both on professional care as well as the quality of the equipment used. Imperfections in the plates could affect assay outcomes. As such, it’s imperative for all professionals to use only the best ELISA plates to ensure the accuracy of their efforts.
At Greiner Bio-One, we have been manufacturing microplates for diagnostics and immunological research for over 30 years. We offer both high-binding and medium-binding 96 well plates, and they’re perfect for diagnostic and immunological research applications. You can contact us here to learn more about our products and our mission.
Greiner Bio-One has ELISA microplates or ELISA strip well plates.
