Understanding ELISA: The Science Behind Enzyme-Linked Immunosorbent Assays
Exploring the Principles, Types, and Applications of ELISA in Diagnostics and Research
An enzyme-linked immunosorbent assay (ELISA) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. In the ELISA technique, an antibody or antigen is immobilized onto a solid surface, typically a polystyrene 96-well microtiter plate. Then, a biochemically conjugated enzyme is added, followed by a substrate to produce a quantifiable colorimetric readout or fluorescent signal depending on the presence of the antibody-antigen interaction. ELISA assays are very sensitive and can be semi-quantitatively or quantitatively measuring the presence and concentration of antibodies, antigens or other proteins.
Discovery and Development
Enzyme Linked Immunosorbent Assay was discovered independently in 1971 by Engvall and Perlmann at Stockholm University and by Van Weemen and Schuurs at the University of Utrecht. Both research groups described techniques to detect specific antibodies using an enzyme coupled to antigens and were labeled enzyme-linked immunosorbent assays. Since then, the ELISA technique has evolved significantly and is now commercially available as diagnostic kits for measuring a wide range of clinical analytes, biomarkers, autoantibodies and antibodies produced against infectious agents like viruses and bacteria.
Principle and Procedure
The key principle behind ELISA is the use of an enzyme to amplify the detection signal, allowing for sensitive detection of molecules present in low concentrations. The basic procedure involves coating the wells of a microtiter plate with an antibody specific to the target antigen. This is called the capture antibody. Samples containing the target antigen are then added to the wells along with a secondary antibody coupled to an enzyme (typically horseradish peroxidase or alkaline phosphatase), which binds to a different site on the antigen if present. Unbound components are washed away and the enzyme is allowed to convert a colorless substrate to a colored product, directly proportional to the amount of target antigen present. The color reaction is quantified using a spectrophotometer or colorimeter.
Types of ELISA
There are several variations of ELISA techniques depending on the analyte and detection method used:
- Direct ELISA - Uses a single antibody for antigen capture and detection. Best for detecting antigens.
- Indirect ELISA - Uses two antibodies, one to capture and another enzyme-conjugated antibody for detection. Used to detect antigens and antibodies.
- Sandwich ELISA - Uses two antibodies that bind to different sites on the target antigen to 'sandwich' it for detection. Highly specific for detecting antigens.
- Competitive ELISA - Uses two forms of the analyte, one bound to the plate and another enzyme-conjugated form to compete for antigen binding. Used to quantify small molecules.
Besides the above, ELISA can also be performed as microtiter plate-based assays or using lateral flow immunoassays like immunochromatography. Fluorescence and chemiluminescence are also detector alternatives to colorimetric ELISAs.
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Applications of ELISA**
Given its sensitivity, reproducibility and availability as commercial kits, ELISA has widespread applications in research, clinical diagnosis, food analysis and other settings:
- Diagnose infectious diseases like HIV, hepatitis, influenza by detecting antibodies
- Measure serum antibodies after vaccination or toxin exposure
- Detect tumor markers for cancer screening
- Quantitative analysis of hormones, cytokines, enzymes and other biomarkers
- Detection of food allergens and contaminants in food industry compliance testing
- Confirmation of therapeutic drug levels in patients
- Quantify biomarkers and protein therapeutics in pharmaceutical R&D
- Detection of veterinary disease pathogens and diagnosis
ELISA is considered the gold standard technique for protein detection due to its convenience, specificity, ability to scale and increasing miniaturization and automation of the assay procedure. With widespread commercialization, it has transformed clinical diagnostics and research applications globally.
ELISA provides a robust and reproducible biochemical method for sensitive detection and quantification of proteins, antibodies and biomarkers. Since its inception about five decades ago, it has evolved significantly and plays a key role in clinical diagnostics, pharmaceutical development and food analysis due to availability of commercial kits, reducing costs and technical improvements. The versatility, scalability and precision afforded by ELISA techniques will continue supporting advances in science, healthcare and industry.
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