@inbook{782b52f48bfe475b9766f493f8fade62,
title = "Avidin-Biotin ELISA-Based Detection of 5hmC",
abstract = "The enzyme-linked immunosorbent assay (ELISA) technique has been developed half a century ago, and yet its role in molecular biology remains significant. Among the most sensitive of immunoassays, it offers high throughput, combined with affordability and ease of use. This chapter provides the procedure of a highly reproducible indirect sandwich ELISA protocol, which can be applied to a variety of semi-quantitative assays for the investigation of the molecular biology of 5-hydroxymethylcytosine (5hmC) or TET enzymes. Three variations of this protocol are described: assessment and validation of 5hmC-binding proteins, screening and validation of anti-5hmC antibodies, or a readout of TET catalytic activity in in vitro experiments. The assay principle is based on the use of a high affinity avidin-biotin system for efficient immobilization of DNA fragments for further detection by high specificity antibodies. A colorimetric enzymatic reaction is ultimately developed with intensity correlating with the amount of attached antigen.",
keywords = "5-Hydroxymethylcytosine, Antibody validation, Biotin-labeled DNA, HRP conjugate, Immunoassay, modified DNA readers, Neutravidin, Sandwich ELISA, Spectroscopy, TET oxidation",
author = "Olova, {Nelly N.}",
note = "Funding Information: This work was supported by the Knowledge Exchange and Commercialisation (KEC) Fund at the Babraham Institute, Cambridge. Publisher Copyright: {\textcopyright} 2021, Springer Science+Business Media, LLC, part of Springer Nature.",
year = "2021",
month = may,
day = "20",
doi = "10.1007/978-1-0716-1294-1_5",
language = "English",
isbn = "9781071612934",
series = "Methods in Molecular Biology",
publisher = "Humana, New York, NY",
pages = "65--76",
editor = "Ozren Bogdanovic and Michiel Vermeulen",
booktitle = "TET Proteins and DNA Demethylation",
}