Enzyme-linked immunosorbent assay (ELISA) is also known as an enzyme immunoassay (EIA). ELISA is defined as a biochemical technique used in many applications including, microbiology, blood screening, veterinary andimmunology for the detection of antigens and antibodies present in a sample.
ELISA technology is considered as an important diagnostic tool in plant and medicine pathology. It is also used for quality control checks in food industries. ELISAs depend on characteristic antibodies to fix the target antigen. A detection system is then used to mark the existence and the number of antigen binding.
Types of ELISA
ELISA can be classified into three main types. Sandwich, Indirect, and Competitive.
First, a ‘capture’ antibody is bound to a well. Then, after the addition of the sample, only the proteins recognized by the antibody are ‘captured’. Finally, the detection of the bound protein is carried out using a second detection antibody.
The detection and capture antibodies are also known as the matched antibody pairs or matched-pairs. In the end, the detection antibody is detected using enzyme labeled secondary antibodies.
In this type of ELISA, the protein sample is directly bound to the well through absorption. Then, the presence of proteins in the sample is detected using an antibody, known as an antigen. In this type sensitivity is increased due to numerous epitomes in each primary antibody that allow signal amplification.
Here, the presence of a primary antibody is produced with the sample resulting in a complex. When the complex settles down, a secondary antibody is added to the wells. The primary antibody is recognized only if the antigen is not bound to it. Thus, the secondary antibody is seen to compete with the antigen.
The Principle of ELISA
Enzyme-linked Immunosorbent Assays (ELISA) add the sensitivity of general enzyme assays with the specificity of antibodies. This is carried out using easily performed assays to detect the antibodies or antigens. An antibody or antigen concentration measurement is provided by ELISAs. Enzyme-conjugated antibodies are the most flexible for detection.
There are differences in this method:
- ELISA can detect the presence of antigens that are recognized by an antibody.
- They are used for testing antibodies that recognize an antigen in the sample.
- There are also some combined antibody, antigen detection kits.
The five-step procedure of an ELISA:
1. Primary Antigen (or antibody)is used to coat microtiter plate wells
2. The plate is washed to remove unbound antibody. False-positive results are minimisedby blocking unbound sites (usually with bovine serum albumen or other animal proteins)
3. Addition of a selected enzyme-conjugatedprimary detection antibody.
4. Addition of secondary antibody conjugated to an enzyme (such as anti-mouse IgG) Reaction of this enzyme with any substrate present to produce a colored product indicating a positive reaction.
Results are measured with a spectrophotometer. Some assays include a Sample Addition Monitor system that shows a colour change at each step of the process. This can be important when automated liquid handling systems are used, as it can indicate at which stage the process was interrupted or if reagent was not added.
Advantages of ELISA
ELISA has a number of benefits compared to the other immunoassay techniques. It is often preferred because it has high sensitivity and specificity. ELISA also offers more accuracy compared to other techniques such as radioimmunoassay (RIA) tests.
ELISA assays are usually in 96 well microplate format. Most ELISA microplates have break apart wells, so smaller runs can be performed if required. The key advantage is that 96 determinations can be performed in a single run, providing results in usually less than3 hours. This is both quick and cost effective.
ELISA does not require radioisotopes or costly radiation counters. They do
however require accurate micropipettes, an incubator, a washing system and a microplate spectrophotometer reader.
Nowadays many labs use fully automated ELISA processing systems, on which a wide variety of assays can be tested. These robotic machines are liquid handling systems capable of performing the entire assay from beginning to end. They can often process up to 15 assays on each day shift.
The major advantage of ELISA tests is their accuracy and ability to provide quick results. Serum or plasma samples are required to perform an ELISA test so centrifugation of the patient’s sample is often required.
Because ELISA tests are able to test antigens and antibodies, they are considered as flexible tools by medical professionals, and laboratories from all over the world.
ELISA technology is convenient for blood screening to detect markers of infection including HIV. Thisis mandatory in most countries in order to prevent the release of infected blood and toassure the safety of blood units and blood components for transfusion. Applications of ELISA tests also include the food industry for detecting allergens, and toxicology to screen for specific classes of drugs. .
The ELISA procedure is simple to use. It offers excellent sensitivity and high specificity due to the reaction between an antibody and antigen.
ELISA is also highly efficient because it is easy to perform simultaneous analysis without any complex sample pre-treatment.
ELISA tests are eco-friendly and safe thanks to the avoided use of radioactive substances and major amounts of organic solvents.
Finally, ELISA tests are cost-effective as the process can be easily automated and 96 tests performed in a single assay, with little hands on time.
Sensitivity is high
It is observed that ELISA has high sensitivity due to an enzyme as a reporting group. An enzyme is known as an organic catalyst. This is a small portion of which could cause reactions for the production of the chromogenic reaction phenomenon.
Thus, this system is considered as an enzyme amplification system. It is possible to produce a tracer of an antibody with ELISA. Quantification of antibody or antigen can be carried out using the nanogram or microgram levels. To increase sensitivity of the assay, the ELISA plate should be covered using high-affinity antibodies.
The selectivity of an antigen or antibody results in specificity. The antibody or antigen-binding is produced only in the epitope of an antigen-binding or antigen site of an antibody. A complementary relationship between an antigen-binding site and epitope both, in spatial configuration and chemical structure is observed. There’s a strong specificity due to the reaction between antibody and antigen.
These advantages of ELISA kits make it a more preferred biotechnical tool for a majority of applications. It is useful for both the clinical diagnosis of various conditions or diseases and scientific research.