ELISA: Principles, Types, and Applications

• ELISA, or Enzyme-Linked Immunosorbent Assay, is an immunoassay technique used to detect and quantify biomolecules via enzyme linkage
• ELISA's applications include medicine, research, and the food industry due to its high sensitivity

Principle of ELISA

• ELISA relies on the antigen-antibody interaction
• Either an enzyme-linked antibody or an enzyme-linked antigen produces a colorimetric reaction when a target analyte is present
• A spectrophotometer measures the color change intensity to determine the analyte concentration

Types of ELISA

• There are four main types of ELISA: direct, indirect, sandwich, and competitive

Direct ELISA

• The antigen is directly immobilized on a plate
• An enzyme-conjugated antibody detects the immobilized antigen
• Direct ELISA is simple, quick, and involves fewer steps
• It has low sensitivity and is prone to background noise

Indirect ELISA

• Indirect ELISA is similar to direct, but uses a secondary antibody to amplify the signal
• It has higher sensitivity and allows for multiple detection systems
• It is more time-consuming with a higher risk of cross-reactivity

Sandwich ELISA

• Sandwich ELISA uses two antibodies: one for capturing and one for detection
• The use of two antibodies ensures high specificity
• It is highly specific and suitable for complex samples
• Sandwich ELISA requires paired antibodies and is more expensive

Competitive ELISA

• In competitive ELISA, the sample antigen competes with a pre-coated antigen for binding to a limited amount of antibody
• It is highly precise and useful for detecting toxins, hormones, and drugs
• Competitive ELISA is more complex and difficult to optimize

ELISA Types and Summary

• Direct and sandwich ELISAs are used to detect antigens in clinical samples
• Indirect ELISA is used to detect antibodies in clinical samples
• Competitive ELISA is useful for low-concentration samples or when precise quantification is needed

Applications of ELISA

• ELISA is used for disease diagnosis, detecting diseases like HIV, hepatitis, dengue, and COVID-19
• It is utilized in hormone analysis, measuring hormones like HCG, insulin, and cortisol
• ELISA is used for allergy testing, specifically detecting IgE-specific antibodies
• ELISA is used for food safety testing, detecting toxins, mold, and antibiotic residues
• It plays a role in immunology research and vaccine development

Advantages of ELISA

• High sensitivity and specificity are among its key advantages
• ELISA enables simultaneous analysis of multiple samples
• It allows for accurate quantification

Limitations of ELISA

• Skilled technicians are required to perform ELISA
• There is a risk of cross-contamination if not properly handled
• Environmental conditions can affect the results

Standard ELISA Protocol

• Collect biological samples, such as blood, urine, or serum during sample preparation
• Coat the microplate by immobilizing the antigen or antibody
• Washing is performed to remove unbound components
• Enzyme-conjugated antibody/antigen is added for detection
• Introduce the substrate to trigger a colorimetric reaction via Substrate addition
• Measure absorbance using a spectrophotometer to get results

Recent Advancements in ELISA

• Electrochemical ELISA enhances sensitivity
• Magnetic particle-based ELISA allows for faster reaction times
• Microfluidic ELISA reduces reagent consumption and costs

Conclusion

• ELISA is a powerful immunoassay technique widely used in medical diagnostics, food safety, and biological research
• Ongoing advancements are making ELISA more accurate, faster, and cost-effective

Key Terminology

• Antigen: The substance that triggers an immune response
• Antibody: A protein produced by the immune system to neutralize antigens
• Enzyme-linked: Attached to an enzyme
• Spectrophotometer: An instrument that measures the absorbance of light
• Microplate: A multi-welled plate used in ELISA
• Colorimetric reaction: A chemical reaction that produces a color change
• Sensitivity: The ability to detect low levels of a substance
• Specificity: The ability to detect only the target substance
• Cross-reactivity: When a test reacts with unintended substances