Abstract
First described by Engvall and Perlmann, the enzyme-linked immunosorbent assay (ELISA) is a rapid and sensitive method for detection and quantitation of an antigen using an enzyme-labeled antibody. Besides routine laboratory usage, ELISA has been utilized in medical field and food industry as diagnostic and quality control tools. Traditionally performed in 96-well or 384-well polystyrene plates, the technology has expanded to other platforms with increase in automation. Depending on the antigen epitope and availability of specific antibody, there are variations in ELISA setup. The four basic formats are direct, indirect, sandwich, and competitive ELISAs. Direct ELISA is the simplest format requiring an antigen and an enzyme-conjugated antibody specific to the antigen. This chapter describes the individual steps for detection of a plate-bound antigen using a horseradish peroxidase (HRP)-conjugated antibody and luminol-based enhanced chemiluminescence (ECL) substrate. The methodological approach to optimize the assay by chessboard titration is also provided.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Engvall E, Perlmann P (1971) Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871–874
Fan A, Cao Z, Li H et al (2009) Chemiluminescence platforms in immunoassay and DNA analyses. Anal Sci 25:5875–5897
Czerkinsky CC, Nilsson LA, Nygren H et al (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65:109–121
Crowther JR (1995) ELISA. Theory and practice. Methods Mol Biol 42:1–218
Stevens PW, Hansberry MR, Kelso DM (1995) Assessment of adsorption and adhesion of proteins to polystyrene microwells by sequential enzyme-linked immunosorbent assay analysis. Anal Biochem 225:197–205
Hornbeck P (2001) Enzyme-linked immunosorbent assays. Curr Protoc Immunol Chapter 2: Unit 2.1. doi: 10.1002/0471142735.im0201s01
Underwood PA, Steele JG (1991) Practical limitations of estimation of protein adsorption to polymer surfaces. J Immunol Methods 142:83–94
Hnasko R, Lin A, McGarvey JA et al (2011) A rapid method to improve protein detection by indirect ELISA. Biochem Biophys Res Commun 410:726–731
Vos Q, Klasen EA, Haaijman JJ (1987) The effect of divalent and univalent binding on antibody titration curves in solid-phase ELISA. J Immunol Methods 103:47–54
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Lin, A.V. (2015). Direct ELISA. In: Hnasko, R. (eds) ELISA. Methods in Molecular Biology, vol 1318. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2742-5_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2742-5_6
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2741-8
Online ISBN: 978-1-4939-2742-5
eBook Packages: Springer Protocols