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Enzyme-Linked Immunosorbent Assay: Types and Applications

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ELISA

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2612))

Abstract

Enzyme-linked immunosorbent assay (ELISA) is an immunological assay widely used in basic science research, clinical application studies, and diagnostics. The ELISA technique relies on the interaction between the antigen (i.e., the target protein) versus the primary antibody against the antigen of interest. The presence of the antigen is confirmed through the enzyme-linked antibody catalysis of the added substrate, the products of which are either qualitatively detected by visual inspection or quantitatively using readouts from either a luminometer or a spectrophotometer. ELISA techniques are broadly classified into direct, indirect, sandwich, and competitive ELISA—all of which vary based on the antigens, antibodies, substrates, and experimental conditions. Direct ELISA relies on the binding of the enzyme-conjugated primary antibodies to the antigen-coated plates. Indirect ELISA introduces enzyme-linked secondary antibodies specific to the primary antibodies bound to the antigen-coated plates. Competitive ELISA involves a competition between the sample antigen and the plate-coated antigen for the primary antibody, followed by the binding of enzyme-linked secondary antibodies. Sandwich ELISA technique includes a sample antigen introduced to the antibody-precoated plate, followed by sequential binding of detection and enzyme-linked secondary antibodies to the recognition sites on the antigen. This review describes ELISA methodology, the types of ELISA, their advantages and disadvantages, and a listing of some multifaceted applications both in clinical and research settings, including screening for drug use, pregnancy testing, diagnosing disease, detecting biomarkers, blood typing, and detecting SARS-CoV-2 that causes coronavirus disease 2019 (COVID-19).

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References

  1. Butler JE (2000) Enzyme-linked immunosorbent assay. J Immunoass 21(2–3):165–209

    Article  CAS  Google Scholar 

  2. Crowther JR (1995) ELISA. Theory and practice. Methods Mol Biol (Clifton, N.J.) 42:1–218

    CAS  Google Scholar 

  3. Engvall E (2010) The ELISA, enzyme-linked immunosorbent assay. Clin Chem 56(2):319–320

    Article  CAS  PubMed  Google Scholar 

  4. Engvall E, Perlmann P (1971) Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8(9):871–874

    Article  CAS  PubMed  Google Scholar 

  5. Hornbeck P (1992) Enzyme-Linked Immunosorbent Assays. Curr Protoc Immunol 1:2.1.1–2.1.22

    Article  Google Scholar 

  6. Hornbeck P, Winston SE, Fuller SA (1991) Enzyme-linked immunosorbent assays (ELISA). Curr Protoc Mol Biol 15:11.2.1–11.2.22

    Article  Google Scholar 

  7. Hornbeck PV (2015) Enzyme-linked immunosorbent assays. Curr Protoc Immunol 110:2.1.1–2.1.23

    Article  PubMed  Google Scholar 

  8. Kohl TO, Ascoli CA (2017) Immunoassays. Cold Spring Harbor Protoc 7:pdb.top093690

    Google Scholar 

  9. Lequin RM (2005) Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem 51(12):2415–2418

    Article  CAS  PubMed  Google Scholar 

  10. Reen DJ (1994) Enzyme-linked immunosorbent assay (ELISA). Methods Mol Biol (Clifton, N.J.) 32:461–466

    CAS  Google Scholar 

  11. Aydin S (2015) A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides 72:4–15

    Article  CAS  PubMed  Google Scholar 

  12. Konstantinou GN (2017) Enzyme-linked immunosorbent assay (ELISA). Methods Mol Biol (Clifton, N.J.) 1592:79–94

    Article  CAS  Google Scholar 

  13. Alhajj M, Farhana A (2022) Enzyme linked immunosorbent assay. In StatPearls, Treasure Island (FL)

    Google Scholar 

  14. Grange RD, Thompson JP, Lambert DG (2014) Radioimmunoassay, enzyme and non-enzyme-based immunoassays. Br J Anaesth 112(2):213–216

    Article  CAS  PubMed  Google Scholar 

  15. Kohl TO, Ascoli CA (2017) Direct and indirect cell-based enzyme-linked immunosorbent assay. Cold Spring Harbor Protoc 5:pdb.prot093732

    Google Scholar 

  16. Lin AV (2015) Direct ELISA. Methods Mol Biol (Clifton, N.J.) 1318:61–67

    Article  Google Scholar 

  17. Kohl TO, Ascoli CA (2017) Indirect immunometric ELISA. Cold Spring Harbor Protoc 5:pdb.prot093708

    Google Scholar 

  18. Lin AV (2015) Indirect ELISA. Methods Mol Biol (Clifton, N.J.) 1318:51–59

    Article  Google Scholar 

  19. Gan SD, Patel KR (2013) Enzyme immunoassay and enzyme-linked immunosorbent assay. J Invest Dermatol 133(9):e12

    Article  CAS  PubMed  Google Scholar 

  20. Kohl TO, Ascoli CA (2017) Immunometric antibody Sandwich enzyme-linked immunosorbent assay. Cold Spring Harbor Protoc 6:pdb.prot093716

    Google Scholar 

  21. Kohl TO, Ascoli CA (2017) Immunometric double-antibody Sandwich enzyme-linked immunosorbent assay. Cold Spring Harbor Protoc 6:pdb.prot093724

    Google Scholar 

  22. Stanker LH, Hnasko RM (2015) A double-Sandwich ELISA for identification of monoclonal antibodies suitable for Sandwich immunoassays. Methods Mol Biol (Clifton, N.J.) 1318:69–78

    Article  Google Scholar 

  23. Kohl TO, Ascoli CA (2017) Direct competitive enzyme-linked immunosorbent assay (ELISA). Cold Spring Harbor Protoc 7:pdb.prot093740

    Google Scholar 

  24. Kohl TO, Ascoli CA (2017) Indirect competitive enzyme-linked immunosorbent assay (ELISA). Cold Spring Harbor Protoc 7:pdb.prot093757

    Google Scholar 

  25. Makarananda K, Weir LR, Neal GE (1998) Competitive ELISA. Methods Mol Biol (Clifton, N.J.) 80:155–160

    Article  CAS  Google Scholar 

  26. Plested JS, Coull PA, Gidney MAJ (2003) ELISA. In: Herbert MA, Hood DW, Moxon ER (eds) Haemophilus influenzae protocols. Humana Press, Totowa, pp 243–261

    Google Scholar 

  27. Zhang A, Jin M, Liu F et al (2006) Development and evaluation of a DAS-ELISA for rapid detection of avian influenza viruses. Avian Dis 50(3):325–330

    Article  PubMed  Google Scholar 

  28. Mendoza R, Silver M, Zuretti AR et al (2021) Correlation of automated chemiluminescent method with enzyme-linked immunosorbent assay (ELISA) antibody titers in convalescent COVID-19 plasma samples: development of rapid, cost-effective semi-quantitative diagnostic methods. J Blood Med 12:157–164

    Article  PubMed  PubMed Central  Google Scholar 

  29. Liu STH, Lin HM, Baine I et al (2020) Convalescent plasma treatment of severe COVID-19: a propensity score-matched control study. Nat Med 26(11):1708–1713

    Article  CAS  PubMed  Google Scholar 

  30. Alsaed OS, Alamlih LI, Al-Radideh O et al (2021) Clinical utility of ANA-ELISA vs ANA-immunofluorescence in connective tissue diseases. Sci Rep 11(1):8229

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Dighiero G, Magnac C, de Saint Martin J et al (1987) Detection of anti-mitochondrial antibodies by ELISA and Western-blot techniques and identification by one and two-dimensional gel electrophoresis of M2 target antigens. Clin Exp Immunol 70(3):640–648

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Liu H, Norman GL, Shums Z et al (2010) PBC screen: an IgG/IgA dual isotype ELISA detecting multiple mitochondrial and nuclear autoantibodies specific for primary biliary cirrhosis. J Autoimmun 35(4):436–442

    Article  CAS  PubMed  Google Scholar 

  33. Roth KDR, Wenzel EV, Ruschig M et al (2021) Developing recombinant antibodies by phage display against infectious diseases and toxins for diagnostics and therapy. Front Cell Infect Microbiol 11:697876

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. El-Masry S, El-Sayed IH, Lotfy M et al (2007) Utility of slot-blot-ELISA as a new, fast, and sensitive immunoassay for detection of carcinoembryonic antigen in the urine samples of patients with various gastrointestinal malignancies. J Immunoassay Immunochem 28(2):91–105

    Article  CAS  PubMed  Google Scholar 

  35. Panini NV, Messina GA, Salinas E et al (2008) Integrated microfluidic systems with an immunosensor modified with carbon nanotubes for detection of prostate specific antigen (PSA) in human serum samples. Biosens Bioelectron 23(7):1145–1151

    Article  CAS  PubMed  Google Scholar 

  36. Yin Y, Cao Y, Xu Y et al (2010) Colorimetric immunoassay for detection of tumor markers. Int J Mol Sci 11(12):5077–5094

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Al-Dujaili EA (2006) Development and validation of a simple and direct ELISA method for the determination of conjugated (glucuronide) and non-conjugated testosterone excretion in urine. Clin Chim Acta 364(1–2):172–179

    Article  CAS  PubMed  Google Scholar 

  38. Kuo HT, Yeh JZ, Wu PH et al (2012) Application of immunomagnetic particles to enzyme-linked immunosorbent assay (ELISA) for improvement of detection sensitivity of HCG. J Immunoassay Immunochem 33(4):377–387

    Article  CAS  PubMed  Google Scholar 

  39. Beelaert G, Fransen K (2010) Evaluation of a rapid and simple fourth-generation HIV screening assay for qualitative detection of HIV p24 antigen and/or antibodies to HIV-1 and HIV-2. J Virol Methods 168(1–2):218–222

    Article  CAS  PubMed  Google Scholar 

  40. Workowski KA, Bolan GA (2015) Centers for disease, C.; prevention, sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep 64(RR-03):1–137

    Google Scholar 

  41. Agius R, Nadulski T (2014) Utility of ELISA screening for the monitoring of abstinence from illegal and legal drugs in hair and urine. Drug Test Anal 6(Suppl 1):101–109

    Article  CAS  PubMed  Google Scholar 

  42. Olivieri B, Maric M, Bridge C (2018) Determining the effects of adulterants on drug detection via enzyme-linked immunosorbent assay and adulterant tests strips. Drug Test Anal 10(9):1383–1393

    Article  CAS  PubMed  Google Scholar 

  43. Weng X, Gaur G, Neethirajan S (2016) Rapid detection of food allergens by microfluidics ELISA-Based Optical sensor. Biosensors (Basel) 6(2):24

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. Marshall B. Ketchum University, Fullerton, CA, USA

    Hovhannes Hayrapetyan, Thao Tran, Eglis Tellez-Corrales & Charitha Madiraju

  2. Wayne State University, School of Medicine, Detroit, MI, USA

    Hovhannes Hayrapetyan

Authors
  1. Hovhannes Hayrapetyan
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  2. Thao Tran
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  3. Eglis Tellez-Corrales
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  4. Charitha Madiraju
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Corresponding author

Correspondence to Charitha Madiraju .

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  1. QuantiScientifics LLC, Orange, CA, USA

    Robert S. Matson

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Hayrapetyan, H., Tran, T., Tellez-Corrales, E., Madiraju, C. (2023). Enzyme-Linked Immunosorbent Assay: Types and Applications. In: Matson, R.S. (eds) ELISA. Methods in Molecular Biology, vol 2612. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2903-1_1

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  • DOI: https://doi.org/10.1007/978-1-0716-2903-1_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2902-4

  • Online ISBN: 978-1-0716-2903-1

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