基于亲和素-生物素 ELISA 的 5hmC 检测

Avidin-Biotin ELISA-Based Detection of 5hmC.

机构信息

Epigenetics ISP, The Babraham Institute, Cambridge, UK.

MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.

出版信息

Methods Mol Biol. 2021;2272:65-76. doi: 10.1007/978-1-0716-1294-1_5.

DOI:10.1007/978-1-0716-1294-1_5

PMID:34009609

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.

摘要

酶联免疫吸附测定（ELISA）技术是半个世纪前发展起来的，但其在分子生物学中的作用仍然很重要。作为最灵敏的免疫测定方法之一，它具有高通量、价格合理和使用方便的特点。本章提供了一种高度可重复的间接三明治 ELISA 方案的步骤，该方案可应用于各种半定量测定，以研究 5-羟甲基胞嘧啶（5hmC）或 TET 酶的分子生物学。该方案有三种变体：5hmC 结合蛋白的评估和验证、抗 5hmC 抗体的筛选和验证，或体外实验中 TET 催化活性的读出。该测定原理基于使用高亲和力的亲和素-生物素系统来有效固定 DNA 片段，以便进一步通过高特异性抗体进行检测。最终通过酶促显色反应进行检测，其强度与附着的抗原量相关。