ELISA

By: Kylie Luo


Image Credit: Flickr @ International Maize and Wheat Improvement Center

In the medical field, proper diagnosis of different diseases and disorders are vital to the treatment and recovery of a patient. As some diseases are harder to identify, more tools are needed to be able to help uncover the causation of an illness, leading to better understanding of how to deal with and treat a disease. One important tool that can be used is an immunoassay. This type of test can be used on different substances, but are always used to detect contaminants in a solution. An immunoassay is a procedure designed to be able to detect substances such as proteins through the use of antigens and antibodies. It is usually administered to test water and food samples, but can also examine blood. There are many types of immunoassay tests, but one of the most common is the ELISA test.


ELISA stands for enzyme-linked immunosorbent assay. This test is highly sensitive and is used to detect viruses such as HIV, but has been used under a few circumstances to detect COVID-19. There are 4 main types of ELISA: direct, indirect, sandwich, and competitive. Direct ELISA describes the basis of all 4 types: a sample, typically the blood of a patient, is placed on a plate specially designed for ELISA. From there, a theoretical aspect is applied: each virus is unique from one another due to markers on their exterior called antigens. Since each of these antigens are distinct from one another, they can match with a substance called an antibody. An antibody linked to an enzyme is added to the blood sample, and then filled the rest of the way with a substrate. The substance that the virus has infected is thought to determine which antibody is put in. If the correct antibody is placed with its matching antigen, the antibody, still attached to the enzyme, will react with the substrate, producing color. If a color is produced, that means the test is positive.


Indirect ELISA is very similar, but utilizes 2 different antibodies. The first is called the primary antibody, which is the one that attaches to the antigen. The second antibody, or secondary antibody, is the one attached to the enzyme and will link to the primary antibody, which will then produce the reaction with the substrate. The third type of ELISA is known as the sandwich method. Instead of placing the antigen first, an antibody, called the capture antibody, is placed on the plate, and is then followed by the antigen. As implied by its name, if the antigen and antibody match, the antibody will capture the antigen. From there, the second antibody, which is linked to the enzyme, will be added, creating a sandwich, producing a reaction with the substrate. Lastly, competitive ELISA can be applied to any of the first 3 methods, but instead the secondary antibody is placed first, then the capture, conjugate, and sample. From here, the analyte and conjugate are forced to compete to bind to the secondary antibody, hence its name.


Though all of these methods are relatively similar, there are pros and cons to each. While direct ELISA is the simplest and saves time and supplies, there is no signal boost, and it is sometimes difficult to detect a reaction between the enzyme and substrate. Indirect ELISA takes direct ELISA’s inability to efficiently amplify a signal, and strengthens it through the second antibody. But due to the presence of a secondary antibody, cross reactivity is possible, which can produce false results. Sandwich ELISA, while having a high sensitivity and specificity, depends on the size of the antigen due to its need to bind to two antibodies. Lastly, competitive ELISA is helpful because it can detect antigens in low concentrations, but is also the most complex procedure out of the 4.


Overall, immunoassays are a very important tool for diagnosing different diseases, which can largely decrease time without treatment and in doing so help the recovery of a patient in an efficient manner. It can also provide an example to still developing tests, and prove to be a very useful tool in the understanding of infection, treatment, and how a certain bacteria or virus may function.


Citations:


https://immunochemistry.com/2016/07/07/back-basics-immunoassay/


https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614608/


https://medlineplus.gov/ency/article/003332.htm#:~:text=ELISA%20stands%20for%20enzyme%2Dlinked,detects%20harmful%20substances%2C%20called%20antigens.


https://www.cusabio.com/c-20659.html#:~:text=According%20to%20how%20it%20works,see%20them%20one%20by%20one.&text=In%20direct%20ELISA%2C%20only%20an,secondary%20antibodies%20are%20not%20needed.


Image Credit:

No changes were made to the following image: https://rb.gy/kx5fkb, License: https://creativecommons.org/licenses/by-nc-sa/2.0/, Some Rights Reserved, August 30, 2007.


What Did You Learn?

Questions:

1. What is an immunoassay and what is it used for?


An immunoassay is a procedure that is used to detect contaminants in a sample. It is commonly used to test food and water samples for quality control but can also be applied to other substances. It can detect contaminants as small as bacteria and viruses, depending on the substrate added and serves as a useful diagnosis tool.


2. What is the difference between an antigen and antibody?


An antigen is a substance bound to the exterior of a substance that can elicit an immune response. An antibody is a protein that can bind to an antigen, acting as a cap or flag, preventing the function of the antigen. Each specific antibody can only bind to a specific antigen, for every antigen is unique and has a different shape.


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