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ELISA stands for enzyme-linked immunosorbent assay. It is a test used in biochemistry. ELISA was first introduced in 1971. This test was developed by Peter Perlmann and Eva Engvall. This test is a type of enzyme immunoassay. It is also called EIA. ELISA helps to find a special substance in a liquid. This substance is often a small protein, like an amino acid.
What is ELISA?
ELISA uses antibodies to catch the substance. These antibodies are made to find the substance we want to measure. ELISA is very useful in medicine. It helps to detect diseases. It is also used in plant studies. Biotechnology uses ELISA too. Many industries use ELISA for quality control. This test helps to check if products are safe and correct.
ELISA History
Before ELISA, radioimmunoassay was the common method. Radioimmunoassay uses radioactive materials to detect substances. Radioactive methods had health risks. Scientists wanted a safer alternative.
In 1966, Wide and Jerker Porath found a way to attach antibodies to solid surfaces. Later, Eva Engvall and Peter Perlmann introduced ELISA in 1971. Their work replaced radioactive tests with safer enzyme-linked tests.
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How ELISA Works?
ELISA works using antibodies. Antibodies can bind to a specific target. This target is called the antigen. In a typical ELISA test, the antigen is placed on a solid surface. Usually, this surface is a plastic plate. A specific antibody is then added. This antibody can bind only to the antigen.
The antibody is connected to an enzyme. When a certain chemical is added, the enzyme reacts. This reaction creates a color change. The color shows that binding has happened.
The antigen is first applied to the well’s surface. Next, a special protein is added to block extra empty spaces. The enzyme-associated antibody is then added. If there is an antigen, it binds to it. After binding, the well is washed. This step removes any unbound antibodies. Finally, the enzyme’s substrate is added. If the antibody has bound to the antigen, a color change will appear.
ELISA Principle
ELISA is a test used in biochemistry. It is called a “wet lab” method because it uses liquid during the test. ELISA helps to find a special substance in a liquid sample. This special substance is called an analyte. The analyte can be measured by quantity or just by presence.
In this method, liquids are kept inside small wells or chambers. These wells keep the liquids from mixing or spilling. This is different from “dry lab” methods like paper strips. Instead of reading liquids, dry labs frequently read dried strips. In ELISA, the liquid reacts with other liquids step-by-step. This reaction gives a signal that shows if the substance is present.
The signal is often shown by a color change. This is caused by an enzyme reaction. The liquid sample is placed on a solid surface. This surface helps to catch and hold the analyte. The solid surface stays in place and does not move.
This surface is usually part of an ELISA plate. The plate has many small wells in rows. Special chemicals are added after the sample. These chemicals help the reaction happen. After each step, the plate is washed to remove unwanted things. At the end, a color shows if the analyte is present.
A machine measures how much light passes through the liquid. This helps to know how much analyte is there. More color usually means more analyte. The enzymes used in ELISA make the signal stronger. This helps make small amounts easy to detect. Another name for the material we test is a ligand. It binds to a unique reagent, which explains this. ELISA is known as a ligand-binding assay for this reason.
The solid surface is coated with a chemical that binds the ligand. This coating is clear and fixed at the bottom of the wells. Sometimes the coating is also on the sides of the wells. The wells help keep everything in place. Usually, antibodies are used to catch the substance.
Antibodies are good at finding and holding one specific substance. Sometimes, the substance we are testing is an antibody itself. In that case, we use its matching antigen for the test. This process helps find very small amounts of substances.
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Different Types of ELISA
There are several types of ELISA. Each type works a little differently. One common type is the Direct ELISA. In this method, the antibody linked to the enzyme directly binds to the antigen. Another type is the Indirect ELISA. Here, a primary antibody binds to the antigen. A secondary antibody that contains an enzyme then binds to the primary antibody.
There is also the Sandwich ELISA. This uses two antibodies. One captures the antigen, and the other helps detect it. The Competitive ELISA is another type. This method measures how well antibodies can bind in competition with the antigen.
Direct ELISA
The antigen is first applied to the plate in Direct ELISA. An enzyme-linked antibody is added after the extra spaces have been blocked. The antigen is directly bound by this antibody. The substrate is then added to produce a noticeable color. The color tells us if the antigen was present.
Indirect ELISA
Two antibodies are used in indirect ELISA. First, the antigen is applied to the plate. The primary antibody binds to this antigen. The first antibody is then bound by a secondary antibody that is attached to an enzyme. The result is a color change when the substrate is added. The signal from this method is stronger.
Sandwich ELISA
Sandwich ELISA uses a capture antibody first. This antibody is fixed on the plate. When the sample is added, the antigen binds to the capture antibody. After that, a second antibody, known as the detection antibody, is added. This detection antibody also binds to the antigen.
The detection antibody is then bound by an enzyme-linked secondary antibody. When the substrate is finally added, the outcome is displayed by the color. This method is very specific and sensitive.
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Competitive ELISA
Competitive ELISA works differently. The antibody and antigen first mix together. This mixture is added to a plate coated with more antigen. The more antigen in the sample, the fewer free antibodies will bind to the plate. A secondary antibody with an enzyme is then added. The amount of antigen produced is inversely correlated with its color.
ELISA Applications
ELISA is used in many fields. Numerous diseases, including dengue, Chagas disease, malaria, Johne’s disease, and others, can be identified using ELISA tests. In medical laboratories, ELISA tests are also widely used for in vitro diagnostics. ELISA can also be used for:
- In medicine, ELISA helps detect antibodies for diseases. It is very useful in HIV testing and hepatitis diagnosis.
- Doctors also use ELISA to detect food allergies. It finds allergens in food like milk, nuts, and eggs.
- In toxicology, ELISA helps screen for drugs. It is a fast method to test for certain substances.
- ELISA is also used in agriculture. It helps detect plant diseases early.
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ELISA Modern Developments
Today, ELISA is still improving. New methods use fluorescent or chemiluminescent signals. These signals are even more sensitive than color changes. They help detect very small amounts of substances. Some ELISA methods now use nanoparticles. These nanoparticles change color when the antigen is present. Modern ELISA can detect tiny amounts of substances in samples.
The Role of Enzymes in ELISA
ELISA uses special substances called enzymatic markers. These markers help show the final result of the test. They work by changing color. The color change shows if the substance being tested is present.
- One common marker is called OPD. OPD means o-phenylenediamine dihydrochloride. When OPD reacts, it turns amber. It is used to detect an enzyme called horseradish peroxidase (HRP). The amount of antigen produced is inversely correlated with its color.
- Another common marker is TMB. TMB means 3,3′,5,5′-tetramethylbenzidine. When TMB reacts with HRP, it turns blue. If acid is added, the blue color turns yellow. The acid can be sulfuric acid or phosphoric acid.
- Another marker is ABTS. ABTS means 2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt. ABTS turns green when HRP is present.
- One more marker is PNPP. PNPP means p-Nitrophenyl Phosphate, Disodium Salt. PNPP reacts with alkaline phosphatase. When it reacts, it turns yellow. These color changes help scientists.
ELISA in COVID-19 Testing
In COVID-19, ELISA testing was essential during the COVID-19 outbreak. It made it easier to find coronavirus antibodies. ELISA was essential to vaccine research. It showed how well the bodies made antibodies after being vaccinated. Many labs used ELISA to track the virus’s spread.
ELISA eSimoa Technology
eSimoa is a new version of ELISA. It stands for Enzyme-Linked Single Molecule Array. eSimoa is more sensitive than normal ELISA. It can detect extremely small amounts of substances.
This method uses magnetic beads instead of plastic plates. It measures reactions at the single molecule level. eSimoa helps in early diagnosis of diseases. It is very useful in drug research and development. There is some debate about who first developed eSimoa. Some credit Chi-An Cheng’s team in Taiwan. Others credit David Walt’s team at Harvard.
ELISA Limitations
ELISA is not perfect. Sometimes it gives false positives or false negatives. It needs careful handling to avoid errors. Also, the reaction plates are for one-time use only. They cannot be reused.
FAQs on ELISA Full Form
What is the purpose of an ELISA test?
ELISA is a common lab test. It is used to find antibodies in the blood. It helps check if the body has fought an infection.
What is the common ELISA range?
ELISA is a very sensitive test. It can detect very small amounts. The range is usually from 0.01 ng to 0.1 ng. It can also measure 0.1 to 1 femtomole (fmole).
What is the ELISA principle?
ELISA works on a simple idea. It uses the binding between antibodies and antigens. Antibodies stick only to specific antigens. This reaction helps detect the target substance. A color change shows if the substance is present.
What is ELISA and how does it work?
ELISA (Enzyme-Linked Immunosorbent Assay) is a lab test used to detect proteins, antibodies, or hormones. It works by using antibodies and color change to identify a substance in a sample.
What does ELISA stand for?
ELISA stands for Enzyme-Linked Immunosorbent Assay, a test used in medical labs to detect antigens or antibodies in blood or other body fluids.
What is an example of an ELISA test?
A common example is the HIV test, where ELISA is used to detect HIV antibodies in the blood, helping in early diagnosis of the infection.
