Model Answer
0 min readIntroduction
Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used biochemical technique primarily employed to detect and quantify the presence of a specific substance, typically an antigen, in a biological sample. Developed by Eva Engvall and Gunnar Perlmann in the 1970s, ELISA has revolutionized diagnostic testing in various fields, including medicine, immunology, and food safety. Its high sensitivity, specificity, and relatively low cost have made it a cornerstone of many research and clinical laboratories. Understanding the principles and procedures of ELISA is crucial for comprehending modern immunological assays and their applications.
Principle of ELISA
The fundamental principle behind ELISA relies on the highly specific interaction between an antigen and its corresponding antibody. This interaction is then detected and quantified using an enzyme-linked antibody, which catalyzes a reaction producing a measurable signal, usually a color change. The intensity of the signal is directly proportional to the amount of antigen present in the sample.
Procedure of ELISA
There are several variations of ELISA, but the core steps remain consistent. Here's a breakdown of the common procedures:
1. Coating/Adsorption
The wells of a microplate are coated with either the antigen or the antibody. This is achieved by passively adsorbing the protein to the plastic surface. The plate is incubated to allow the protein to bind.
2. Blocking
After coating, the plate is blocked with a protein-rich solution (e.g., bovine serum albumin - BSA, or casein) to prevent non-specific binding of antibodies to the plastic surface. This reduces background noise.
3. Incubation with Primary Antibody
The sample containing the antigen (or potentially the antigen) is added to the wells. If the antigen is present, it binds to the immobilized antibody (or vice versa). A primary antibody specific to the antigen is then added and incubated, allowing it to bind to the antigen.
4. Washing Steps
Between each incubation step, the plate is washed several times with a buffer solution (typically PBS-Tween) to remove unbound proteins and reagents.
5. Incubation with Secondary Antibody (for Indirect & Sandwich ELISA)
In indirect ELISA, an enzyme-conjugated secondary antibody, specific to the primary antibody, is added. In sandwich ELISA, an enzyme-conjugated detection antibody is added, which binds to a different epitope on the antigen than the capture antibody. This step amplifies the signal.
6. Substrate Addition and Signal Detection
A substrate specific to the enzyme conjugated to the secondary antibody is added. The enzyme catalyzes a reaction that produces a detectable signal, such as a color change. The intensity of the color is measured using a spectrophotometer.
Types of ELISA
- Direct ELISA: Antigen is immobilized, and an enzyme-conjugated antibody directly binds to it.
- Indirect ELISA: Antigen is immobilized, a primary antibody binds, followed by an enzyme-conjugated secondary antibody.
- Sandwich ELISA: Capture antibody is immobilized, antigen binds, then a detection antibody (enzyme-conjugated) binds to the antigen.
- Competitive ELISA: A known amount of antigen competes with the sample antigen for binding to an immobilized antibody.
Applications of ELISA
- Disease Diagnosis: Detecting antibodies or antigens associated with infectious diseases like HIV, Hepatitis, Lyme disease, and COVID-19.
- Hormone Quantification: Measuring hormone levels in serum or other bodily fluids (e.g., thyroid hormones, cortisol).
- Food Safety: Detecting allergens or toxins in food samples.
- Drug Screening: Identifying the presence of drugs or their metabolites in biological samples.
- Immunological Research: Quantifying antibody levels, studying immune responses, and characterizing antigens.
- Veterinary Diagnostics: Diagnosing diseases in animals.
ELISA is also used in quality control processes, monitoring environmental samples, and in various research applications requiring sensitive and specific detection of biomolecules.
Conclusion
ELISA remains a vital tool in biomedical research and diagnostics due to its versatility, sensitivity, and cost-effectiveness. The different formats of ELISA allow for adaptation to a wide range of applications, from detecting infectious diseases to quantifying hormones. Continued advancements in ELISA technology, such as multiplexing and automation, are further expanding its capabilities and solidifying its role in modern biological sciences. Its ability to provide quantitative data makes it invaluable for both research and clinical settings.
Answer Length
This is a comprehensive model answer for learning purposes and may exceed the word limit. In the exam, always adhere to the prescribed word count.