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采用解吸增强镧系元素荧光免疫分析检测和定量 γ-H2AX。

Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay.

机构信息

Department of Biology, Jackson State University, Jackson, MS, USA.

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA.

出版信息

Sci Rep. 2021 Apr 26;11(1):8945. doi: 10.1038/s41598-021-88296-3.

DOI:10.1038/s41598-021-88296-3
PMID:33903655
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8076281/
Abstract

Phosphorylation of the histone protein H2AX to form γ-H2AX foci directly represents DNA double-strand break formation. Traditional γ-H2AX detection involves counting individual foci within individual nuclei. The novelty of this work is the application of a time-resolved fluorescence assay using dissociation-enhanced lanthanide fluorescence immunoassay for quantitative measurements of γ-H2AX. For comparison, standard fluorescence detection was employed and analyzed either by bulk fluorescent measurements or by direct foci counting using BioTek Spot Count algorithm and Gen 5 software. Etoposide induced DNA damage in A549 carcinoma cells was compared across all test platforms. Time resolved fluorescence detection of europium as a chelated complex enabled quantitative measurement of γ-H2AX foci with nanomolar resolution. Comparative bulk fluorescent signals achieved only micromolar sensitivity. Lanthanide based immunodetection of γ-H2AX offers superior detection and a user-friendly workflow. These approaches have the potential to improve screening of compounds that either enhance DNA damage or protect against its deleterious effects.

摘要

组蛋白 H2AX 的磷酸化形成 γ-H2AX 焦点直接代表 DNA 双链断裂的形成。传统的 γ-H2AX 检测涉及在单个核内单独计算焦点的数量。这项工作的新颖之处在于应用了一种时间分辨荧光分析，使用解吸增强的镧系荧光免疫分析进行 γ-H2AX 的定量测量。为了进行比较，采用了标准荧光检测，并通过使用 BioTek Spot Count 算法和 Gen 5 软件进行的体荧光测量或直接焦点计数进行了分析。在所有测试平台上都比较了依托泊苷诱导的 A549 癌细胞中的 DNA 损伤。铕作为螯合物的时间分辨荧光检测使 γ-H2AX 焦点的定量测量达到纳摩尔分辨率。比较体荧光信号仅达到微摩尔灵敏度。基于镧系元素的 γ-H2AX 免疫检测提供了优越的检测和用户友好的工作流程。这些方法有可能改进对增强 DNA 损伤或防止其有害影响的化合物的筛选。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/c4fd184eaccd/41598_2021_88296_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/4b4a320e8548/41598_2021_88296_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/5598cbeffcfb/41598_2021_88296_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/40b211690c57/41598_2021_88296_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/90a838e53c5f/41598_2021_88296_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/686878edd6f5/41598_2021_88296_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/c4fd184eaccd/41598_2021_88296_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/4b4a320e8548/41598_2021_88296_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/5598cbeffcfb/41598_2021_88296_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/40b211690c57/41598_2021_88296_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/90a838e53c5f/41598_2021_88296_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/686878edd6f5/41598_2021_88296_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/daab/8076281/c4fd184eaccd/41598_2021_88296_Fig6_HTML.jpg

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