Abstract
The enzyme-linked immunosorbent assay is a powerful analytical tool for the assessment of the kind and quantity of specific analytes found within a biological sample. It is based upon the exceptional specificity of antibody recognition of its cognate antigen and the power of enzyme-mediated signal amplification for sensitivity. However, development of the assay is not without challenges. Here, we describe the essential components and features necessary to successfully prepare and carry out the ELISA format.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gibbs J, Vessels M, Rothenberg M (2017) Immobilization principles – selecting the surface for ELISA assays. Corning Incorporated, Life Sciences, Application Note. https://www.corning.com/catalog/cls/documents/application-notes/CLS-DD-AN-454.pdf
Bolstad N, Warren DJ, Nustad K (2013) Heterophilic antibody interference in immunometric assays. Best Pract Res Clin Endocrinol Metab 27:647–661
Juncker D, Bergeron S, Laforte V et al (2014) Cross-reactivity in antibody microarrays and multiplexed sandwich assays: shedding light on the dark side of multiplexing. Curr Opin Chem Biol 18:29–37
Matson RS (2021) Chapter 2: well-based antibody arrays. In: Whitaker KC, Huang R-P (eds) Antibody arrays: methods in molecular biology, 2237: 11–38, Springer protocols. Humana Press, New York
Krainer FW, Glieder A (2015) An updated view on horseradish peroxidases: recombinant production and biotechnological applications. Appl Microbiol Biotechnol 99:1611–1625
Josephy PD, Eling T, Mason RP (1982) The horseradish peroxidase-catalyzed oxidation of 3,5,3′,5′-tetramethylbenzidine. Free radical and charge-transfer complex intermediates. J Biol Chem 257(7):3669–3675
Luis Millan JL (2006) Alkaline phosphatases- structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes. Purinergic Signal 2:335–341. https://doi.org/10.1007/s11302-005-5435-6
Sharma U, Pal D, Prasad R (2014) Alkaline phosphatase: an overview. Ind J Clin Biochem 29(3):269–278. https://doi.org/10.1007/s12291-013-0408-y
Phosphatase, Alkaline from bovine intestinal mucosa https://www.sigmaaldrich.com/US/en/product/sigma/p5521
Tausig F, Wolf FJ (1964) Streptavidin–a substance with avidin-like properties produced by microorganisms. Biochem Biophys Res Commun 14(3):205–209
Avery G (2019) Biotin interference in immunoassay: a review for the laboratory scientist. Ann Clin Biochem 56(4):424–430
Li D, Ferguson A, Cervinski MA et al (2020) AACC guidance document on biotin interference in laboratory tests. [Epub]. J Appl Lab Med 5:575. https://doi.org/10.1373/10.1093/jalm/jfz010
Sequeira S (2019) An overview on interference in clinical immunoassays: a cause for concern. Hamdan Med J 12(4):158–164
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Matson, R.S. (2023). ELISA Essentials: Surfaces, Antibodies, Enzymes, and Substrates. 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_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2903-1_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2902-4
Online ISBN: 978-1-0716-2903-1
eBook Packages: Springer Protocols