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      “An overview of ELISA: A review and update on best laboratory practices for quantifying peptides and proteins in biological fluids”

(Journal of International Medical Research, 2025)

DOI: 10.1177/03000605251315913

The article provides a comprehensive overview of ELISA (Enzyme-Linked Immunosorbent Assay) — its history, technical formats, laboratory protocols, and best practices for accurate quantification of peptides and proteins in biological fluids (blood, urine, saliva, cerebrospinal fluid, etc.).

🏗️ Main Topics Covered

  1. Historical Background
    • ELISA was developed in the 1960s based on enzyme-substrate detection principles.
    • The use of microplates since the 1980s improved reproducibility and standardization.
  2. ELISA Formats
    • Direct, Indirect, Sandwich, and Competitive ELISA are described.
    • Also discusses Cell-based ELISA, both direct and indirect.
  3. Laboratory Protocols & Optimization
    • Covers key steps and workflows for each ELISA format.
    • Recommends techniques to improve precision, sensitivity, repeatability, and minimize analytical errors.
  4. Best Laboratory Practices
    • Emphasizes use of standards, positive/negative controls, and strict control of variables (temperature, pH, incubation time).
    • Suggests using technical replicates (duplicates or triplicates) to ensure data quality.
  5. Required Equipment
    • Lists essential materials: ELISA microplates, plate readers, buffers, enzyme-labeled antibodies, substrates.
  6. Analytical Applications
    • Quantification of peptides (<50 amino acids) for conditions like diabetes, IBD, PCOS, and drug testing.
    • Used in diagnostics (pregnancy tests, infectious diseases), blood typing, biomarker discovery, biotech quality control.
  7. Practical Insights
    • Backed by over 30 years of laboratory experience from the authors.
    • Offers detailed advice for setting up and running a reliable ELISA lab.

✅ Conclusion

This review serves as a practical guide for scientists and lab technicians, offering updated recommendations for improving the accuracy, reproducibility, and standardization of ELISA assays.

It is especially useful for those working in clinical diagnostics, biomedical research, and biotech production.

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