The 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 this method, an unknown concentration of antibody or antigen in the sample is detected by using a substance whose concentration is known, usually an enzyme or a radioisotope. This detection is usually based on generation of color by the enzyme acting on a substrate.
Mechanism of ELISA
The basic principle of Enzyme Linked Immunosorbent Assay involves use of antigens or antibodies that are immobilized on a solid surface like the wells of a microtiter plate. The unknown concentration of antigen or antibody present in the sample binds to the immobilized antigen or antibody. After washing away unbound material, an enzyme-conjugated antibody specific to the antigen or sample antibody is added. This enzyme-conjugated antibody will bind to the antigen-antibody complex. Unbound conjugate is washed away and substrate is added. The enzyme conjugate catalyzes a reaction that acts upon the substrate, yielding a colored end-product that can be easily measured with a spectrophotometer or visualized by microscopy. The color intensity is proportional to the amount of antigen or antibody in the original sample.
Types of ELISA
Direct ELISA: In direct ELISA only one antibody is used, either the primary antibody from the test sample or a secondary antibody conjugated to an enzyme. This is used when detecting an antigen or antibody directly without the use of a capture/detector antibody pair.
Indirect ELISA: In indirect ELISA a capture antibody is used to catch the primary antigen/antibody. Then a secondary antibody, specific to the captured antigen/antibody and conjugated to an enzyme, is added to detect the bound primary antigen/antibody. This uses a capture and detection antibody to indirectly detect the analyte.
Sandwich ELISA: Sandwich ELISA involves coating the ELISA plate with a capture antibody that will bind any antigen that is present. After washing, the plate is incubated with the test sample that may contain the target antigen. If present, the antigen will bind to the capture antibody. Next, a detector antibody, which recognizes a different epitope (binding site) on the target antigen, is added. This detector antibody is usually enzyme-labeled. Any antigen that was captured in the first step will now be "sandwiched" between the capture and detector antibodies. Following a wash, a substrate is added. The enzymatic reaction will produce color only in wells where all three components - capture antibody, antigen, and detector antibody - are present.
Competitive ELISA: In competitive ELISA the test antigen competes with an enzyme-conjugated antigen (conjugate) for a limited number of antibody binding sites. As the concentration of test antigen increases, less conjugate will bind to the antibody, resulting in decreased signal. This format is commonly used for small molecules such as hormones, steroids, cytotoxic drugs and pesticides.
Applications of ELISA
ELISA is widely used immunoassay technique as it is very sensitive, specific and versatile. Some key applications of ELISA include:
Detection of infectious agents: Detection of viruses like HIV, Hepatitis B and C; bacteria like Salmonella and Streptococcus; and parasites like Toxoplasma and Giardia.
Detection of antibodies: ELISA is extensively used to detect antibodies produced by the body's immune system in response to infections. This helps diagnose recent or past infections.
Food safety testing: ELISA helps detect allergens, toxins and microbial contamination in food.
Therapeutic drug monitoring: ELISA assists in monitoring drug levels in patients receiving medications like immunosuppressive drugs after organ transplant.
Toxicology screening: ELISA aids in screening for exposure to toxins, poisons, drugs of abuse and their metabolites in urine, blood or tissues.
Pregnancy testing: Most home pregnancy kits employ ELISA technique to detect human chorionic gonadotropin (hCG) hormone in urine.
Veterinary diagnostics: ELISA diagnostics help in screening and management of animal diseases. Applications include detection of heartworm, equine infectious anemia and brucellosis.
Benefits of ELISA
ELISA is an important diagnostic technique due to following benefits:
- Highly sensitive and specific for detection of minute quantities of analytes
- Versatile technique that can detect various molecules like proteins, peptides, antibodies, hormones etc.
- Adaptable to automation allowing high-throughput screening of large number of samples
- Relatively faster, easier to perform and less expensive compared to alternative methods
- No specialized equipment required beyond a microplate reader
- Applicable to analysis of various sample types including serum, plasma, urine, tissues etc.
- Aids in early disease diagnosis and management
- Plays vital role in fields like epidemiology, transplantation, therapeutics and food testing
ELISA represents a powerful diagnostic technique with widespread applications due its versatility, sensitivity, specificity and compatibility with automation. Continued developments aim to improve assay formats, reagents and instruments to enhance routine use of ELISA in various fields.
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Resources:
Understanding the Key Applications of Enzyme Linked Immunosorbent Assay in Diagnostics
How Enzyme Linked Immunosorbent Assay Revolutionizes Immunoassay Techniques
Innovations in Enzyme Linked Immunosorbent Assay Technology: Enhancing Accuracy and Efficiency
Recent development in Enzyme Linked Immunosorbent Assay Market
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