Avidin-Biotin ELISA-Based Detection of 5hmC

Nelly N. Olova*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract / Description of output

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.

Original languageEnglish
Title of host publicationTET Proteins and DNA Demethylation
Subtitle of host publicationMethods and Protocols
EditorsOzren Bogdanovic, Michiel Vermeulen
Place of PublicationNew York, NY
PublisherHumana, New York, NY
Number of pages12
ISBN (Electronic)9781071612941
ISBN (Print)9781071612934
Publication statusPublished - 20 May 2021

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords / Materials (for Non-textual outputs)

  • 5-Hydroxymethylcytosine
  • Antibody validation
  • Biotin-labeled DNA
  • HRP conjugate
  • Immunoassay
  • modified DNA readers
  • Neutravidin
  • Sandwich ELISA
  • Spectroscopy
  • TET oxidation


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