Enzyme-linked+immunosorbent+assay+(ELISA)

__**Enzyme-linked immunosorbent assay (ELISA) **__


 * Basic Description **

Detection of specific proteins may be achieved by use of various methods. Key components in most of these methods are antibodies. Antibodies (produced by B-cells in the immune system) are Y-shaped proteins that recognize foreign invaders, called antigens, using structural components called paratopes (located at the tip of the ‘Y’) that are specific to epitopes found on the antigen. The binding mechanism is thought of using a “lock and key” metaphor, and it allows the two structures to bind together with precision. Secondary antibodies bind to specific primary antibodies, allowing for detection of the protein of interest. There is a direct relationship between target protein concentration and the number of antibodies that bind.

The primary antibody is typically developed by injecting an animal with the antigen to be studied and harvesting produced antibodies. Secondary antibodies are obtained by injecting a different species of animal with the antibodies from the animal used to obtain the primary. This is advantageous because an enzyme used for catalysis of a detectable substrate, or other means of detection, can be attached to these secondary antibodies which will bind to any primary antibody derived from the first animal. This results in a more universal detector that can be used for a wide range of primary antibodies, as long as they were derived from that first animal species.

Among the aforementioned protein detection methods is enzyme-linked immunosorbant assay (ELISA), in which an unknown amount of antigen is fixed to an adsorbent surface. A specific antibody is added to the surface, allowing it to bind to the antigen. After the antigen is immobilized, the detection antibody is added. A complex is formed between the detection antibody and the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody that is linked to an enzyme. Between each step, the plate is typically washed with a mild detergent or wash solution to remove any proteins or antibodies that are not specifically bound. After the final wash step, the plate is developed by adding substrate specific to the enzyme to produce a visible signal (in this case, a color change), which, following determination of absorbance, indicates the concentration of antigen in the sample.1

When the antibodies are labeled with a fluorescent dye, they are a useful tool for the detection of specific antigens or proteins using fluorescence microscopy. One of the big advantages of ELISA is that it can also be used to quantify proteins (Alberts et al. 2008).

The diagram below depicts the ELISA test mechanism, both a positive and negative result if testing for an antigen.


 * http://www.plantpath.wisc.edu/soyhealth/virus/virus_images/elisasteps.gif **


 * Purpose of Technique **

Radioimmunoassay was the among the first techniques for labeling the antigen or antibodies. RIA was first introduced in 1960 for the measurement of endogenous plasma insulin by Solomon Berson and Ro- salyn Yalow. In 1968, for the first time an antibody was labeled by Miles and Hales. Avrameas and colleagues used the enzyme-labeled antigen and antibodies. The other group, Pierce and colleagues established the same project in Los Angeles. Finally, Engvall and Perlmann published the first article on ELISA in 1971 (Lequin, 2005).


 * Origin and History **

The origin of this technique stems from scientists looking for a safer and more environmentally friendly immunoassay technique in the 1960’s since radioactive labeling was the only current method available at the time. Therefore, in the 1960’s, the ELISA technique was conceptualized and developed by Peter Perlmann, and Eva Engvall at Stockholm University in Sweden. In 1971, Engvall and Perlmann published their first paper on ELISA and demonstrated its uses by making a quantitative measurement of IgG in rabbit serum with alkaline phosphatase as the label (Lequin, 2005).


 * Recent Research **

A recent research article that utilized this technique is “Prevalence of feline immunodeficiency virus and feline leukemia virus among client-owned cats and risk factors for infection in Germany.” The ELISA test allowed investigators to determine frequency and risk factors for infection of cats in a large population in Germany by evaluating 17,462 owned cats that were tested for the presence of feline immunodeficiency virus antibodies or feline leukemia virus antigen. The ELISA test revealed that the prevalence of FIV did not change significantly between 1993 and 2002, but that the prevalence of FeLV has decreased notably. Therefore, this technique has allowed investigators in Germany to show that the prevalence of FeLV and FIV have changed and therefore protocols for testing and vaccinating should also be modified in correlation to eradicate these two diseases (Gleich, Krieger, & Hartmann, 2009).

A second article that uses the ELISA technique is “Detection of hazelnuts and almonds using commercial ELISA test kits.” In this article, the investigators used three commercial sandwiched ELISA test kits to detect hazelnuts and almonds in foods and compare different forms of processing as well as those foods known to be problematic. The investigators used cooked oatmeal, dipping chocolate and baked muffins. This technique was important to the study because it proved that all three ELISA test kits were useful in detecting traces of hazelnut and almonds in foods, which is important because it allows foods to be properly labeled as containing or not containing these ingredients in order to help people with allergies avoid those foods (Garber & Perry, 2009).

<span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">Lastly, a third example of a research article that utilizes the ELISA test technique is “Evaluation of ELISA for detection of rabies antibodies in domestic carnivores.” In this study, researchers were investigating the specificity, sensitivity, and reliability of a commercial rabies ELISA (BioPro) for serological testing of cats and dogs in order to allow free travel throughout Europe. Currently, only two neutralisation tests (fluorescent antibody virus neutralisation test) or the rapid fluorescent focus inhibition test (RFFIT)) are recommended for serological testing of pets in Europe. However, these are time-consuming, expensive, and require knowledgable technicians and special facilities. The study using the ELISA Bio Pro tests concluded that specificity was 100% and agreed with the FAVN tests 86%. Therefore, the ELISA technique has been shown to be an effective, cost-friendly, and valuable method for evaluating the levels of rabies antibodies in domesticated vaccinated dogs and cats (Wasniewski & Cliquet, 2012).


 * <span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">References **

<span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2008). Molecular <span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;"> Biology Of The Cell (fifth ed.). New York, NY: Garland Science.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">Lequin, R. M. (2005). Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent <span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;"> Assay (ELISA). Clinical Chemistry, 51(12), 2415-2418.