Introduction to ELISA
•ELISA = Enzyme-Linked Immunosorbent Assay
• Detects and quantifies antigens or antibodies
• Relies on antigen–antibody specificity
• Applications: diagnostics, research, biotech
Types of ELISA | Minimal academic design
6.
Principle of ELISA
•Immobilize antigen or antibody on microplate
• Bind target with specific antibody
• Add enzyme-labeled antibody for detection
• Add substrate ? color ? analyte concentration
• Read absorbance in microplate reader
Types of ELISA | Minimal academic design
Direct ELISA
• Antigenimmobilized on plate
• Enzyme-labeled primary antibody binds directly
• Substrate ? color development
• Advantages: simple, fast
• Limitations: lower sensitivity, no signal amplification
Types of ELISA | Minimal academic design
Competitive ELISA
• Sampleantigen competes with labeled antigen for binding
• Signal inversely proportional to analyte concentration
• Useful for small antigens (haptens, hormones)
• Limitations: assay setup and analysis more complex
Types of ELISA | Minimal academic design
12.
Comparison of ELISATypes
Type Sensitivity Specificity Typical Use
Direct Low Moderate Rapid screening
Indirect High Moderate Antibody detection
Sandwich Very High High Protein quantification
Competitive Variable High Small molecules,
hormones
Types of ELISA | Minimal academic design
Conclusion
• Choice dependson analyte size and matrix
• Sandwich for proteins; indirect for antibodies
• Competitive for small molecules
• Optimize blocking, washes, and controls to reduce noise
Types of ELISA | Minimal academic design
15.
References
• Lehninger Principlesof Biochemistry
• Molecular Biology of the Cell (Alberts)
• Manufacturer protocols: Thermo Fisher, Bio-Rad
• Review: Crowther, 'ELISA Theory and Practice'
Types of ELISA | Minimal academic design
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