Quantifying the Level of 8-oxo-dG Using ELISA Assay to Evaluate Oxidative DNA Damage in MCF-7 Cells

Published: May 24, 2024

doi:

Liu Nian*^1,2, Xiaohua Li*³, Jiahui Du, Song-Bai Liu²

¹Suzhou Key Laboratory of Medical Biotechnology,Suzhou Vocational Health College, ²Department of Chemistry and Life Sciences,Suzhou University of Science and Technology, ³Department of Thyroid and Breast Surgery,Wuzhong People’s Hospital of Suzhou City

Summary

This protocol describes an efficient method for quantitatively detecting DNA oxidative damage in MCF-7 cells by ELISA technology.

Abstract

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) base is the predominant form of commonly observed DNA oxidative damage. DNA impairment profoundly impacts gene expression and serves as a pivotal factor in stimulating neurodegenerative disorders, cancer, and aging. Therefore, precise quantification of 8-oxoG has clinical significance in the investigation of DNA damage detection methodologies. However, at present, the existing approaches for 8-oxoG detection pose challenges in terms of convenience, expediency, affordability, and heightened sensitivity. We employed the sandwich enzyme-linked immunosorbent assay (ELISA) technique, a highly efficient and swift colorimetric method, to detect variations in 8-oxo-dG content in MCF-7 cell samples stimulated with different concentrations of hydrogen peroxide (H₂O₂). We determined the concentration of H₂O₂ that induced oxidative damage in MCF-7 cells by detecting its IC₅₀value in MCF-7 cells. Subsequently, we treated MCF-7 cells with 0, 0.25, and 0.75 mM H₂O₂ for 12 h and extracted 8-oxo-dG from the cells. Finally, the samples were subjected to ELISA. Following a series of steps, including plate spreading, washing, incubation, color development, termination of the reaction, and data collection, we successfully detected changes in the 8-oxo-dG content in MCF-7 cells induced by H₂O₂. Through such endeavors, we aim to establish a method to evaluate the degree of DNA oxidative damage within cell samples and, in doing so, advance the development of more expedient and convenient approaches for DNA damage detection. This endeavor is poised to make a meaningful contribution to the exploration of associative analyses between DNA oxidative damage and various domains, including clinical research on diseases and the detection of toxic substances.

Introduction

DNA oxidative damage is a consequence of an imbalance between the generation of reactive oxygen species (ROS) and the cellular antioxidant defense system¹. It primarily involves the oxidation of DNA purine and pyrimidine bases²^,³. This oxidative modification of DNA bases not only compromises the integrity of the genome but also encompasses a wide range of pathological issues, including cancer, neurodegenerative diseases, and cardiovascular diseases⁴^,⁵. The guanine base in DNA has the lowest reduction potential and is the most susceptible to oxidation⁶. Therefore, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) serves as a primary marker for assessing the extent of DNA oxidative damage⁷^,⁸. The accurate quantification of 8-oxo-dG has become a critical issue in addressing various aspects of disease occurrence, progression, and the assessment of multifactorial oxidative burden⁹.

The traditional methods for detecting 8-oxo-dG, such as high-performance liquid chromatography with electrochemical detection (HPLC-ECD), mass spectrometry, and related hyphenated techniques, exhibit high sensitivity and specificity¹⁰^,¹¹^,¹². However, these techniques often have complex operational requirements and high costs, which hinder their widespread applicability and practicality in high-throughput sample analysis. With the continuous advancement of science and technology, a variety of new, efficient, and accurate methods have emerged. The application of these new technologies enables us to quantify the level of 8-oxo-dG more accurately and provides more powerful tools for an in-depth study of the association between oxidative stress and disease. For instance, researchers have applied nanopore technology to quantitatively detect and sequence DNA¹³, identify DNA damage types using a single-click code-sequencing strategy¹⁴, develop high-throughput sequencing methods, and create 8-oxoG-based biosensors by integrating biotin-streptavidin with ELISA¹⁵. Among them, ELISA, with its recognized advantages in terms of specificity, high-throughput screening, and cost, is one of the ideal solutions for 8-oxo-dG detection. Therefore, it is crucial to develop a high-throughput, highly sensitive, convenient, and rapid method for detecting 8-oxo-dG.

The enzyme-linked immunosorbent assay (ELISA) technique, developed in 1971¹⁶, has rapidly advanced over the past 50 years and has now become one of the most commonly used detection methods in the fields of biology and medicine¹⁷^,¹⁸^,¹⁹. ELISA technology exhibits high sensitivity and specificity, possesses a short reaction time, and is easy to use, making it a widely recognized choice for large-scale sample testing and high-throughput analysis²⁰. As a result, ELISA has been widely used for quantitative or semiquantitative analysis of compounds, proteins, antibodies, or molecules within cells²¹^,²²^,²³. For example, it has been utilized in the detection of biomarkers associated with various diseases, drug residues, and biomolecules²⁴. ELISAs can be categorized into four main types based on experimental design and principles²⁵. These methods include direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA²⁶^,²⁷. Among these, sandwich ELISA, which utilizes two specific antibodies, one for capturing the target molecule and the other for detection, was chosen for the study in this paper. The experimental principle of sandwich ELISA is as follows: First, a specific antibody is immobilized in the wells of a microplate to capture the analyte of interest. After the standard or sample is added, the target analyte binds to the immobilized antibody. Subsequently, a labeled antibody that recognizes a different epitope on the antigen is added, forming a sandwich structure. Following the removal of unbound antibodies, a substrate is added. Under the catalytic action of the secondary antibody, a color reaction occurs, and the intensity of the color is positively correlated with the concentration of the target analyte in the sample. Finally, the optical density (OD) was measured to determine the concentration of the sample. Sandwich ELISA has the advantages of increased sensitivity and specificity for target samples, which makes it suitable for detecting low concentrations of target analytes and complex samples²⁸. Additionally, the results obtained can be quantified for further analysis. These factors make sandwich ELISA a commonly used detection method in both scientific research and clinical laboratories²⁹.

This study aimed to quantitatively detect 8-oxo-dG in MCF-7 cells to determine the degree of DNA oxidative damage in the cells. This study consists of two main parts: constructing an MCF-7 cell DNA oxidative damage model and detecting 8-oxo-dG using ELISA. First, MCF-7 cells were cultured in vitro and treated with different concentrations of H₂O₂ for different durations. Cell viability was evaluated using a CCK-8 assay to determine the half-maximal inhibitory concentration (IC₅₀) of H₂O₂ in MCF-7 cells. Based on the IC₅₀ values, an appropriate H₂O₂ treatment time and induction concentration were chosen. To extract samples of MCF-7 cells damaged by oxidation, cell samples, and supernatants were obtained and added to enzyme-linked wells previously coated with 8-oxo-dG antibodies. The 8-oxo-dG present in the sample will bind to the antibodies bound to the solid-phase carrier. Then, 8-oxo-dG antibodies labeled with horseradish peroxidase were added. The reaction mixture was incubated at a constant temperature to ensure complete binding of the sample and the antibody. The unbound enzyme was removed by washing, and then the colorimetric substrate was added, which produced a blue color. Under the action of acid, the solution turned yellow. Finally, the OD value of the reaction well samples was measured at 450 nm, and the concentration of 8-oxo-dG in the sample was proportional to the OD value. By generating a standard curve, the concentration of 8-oxo-dG in the sample can be calculated.

Protocol

1. Construction of an H2O2 -induced DNA oxidative damage model in MCF-7 cells MCF-7 cell recovery Transfer the cell culture cryogenic tube, which contains 3.5 x 106 MCF-7 cells and is stored in a -80°C refrigerator, rapidly to a foam box containing liquid nitrogen. Retrieve the tube with forceps and place it in a 37 °Cconstant temperature water bath for approximately 1 min to thaw the preserved cells. NOTE: During…

Representative Results

As illustrated in Figure 3, the X-axis denotes the concentration of H2O2 applied to MCF-7 cells, while the Y-axis indicates cell viability. Treatment with 0.75 mM for 12 h reduced the viability of MCF-7 cells to 67%. Concomitant with the increase in concentration, there was a significant decrease in the viability of MCF-7 cells, particularly at a concentration of 1.5 mM, where the viability decreased to below 3% (Table 1). The experimental results sugge…

Discussion

The development of ELISA methods holds great importance for both existing and new DNA damage detection methodologies. In comparison to traditional HPLC and mass spectrometry techniques, this approach not only is user-friendly but also exhibits high sensitivity and meets the demands of high-throughput screening³⁰. This enables the monitoring of 8-oxo-dG in large-scale disease screening studies, facilitating a deeper understanding of the correlation between this biomarker and various diseases.

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Offenlegungen

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Jiangsu Higher Education Institution Innovative Research Team for Science and Technology (2021), Program of Jiangsu Vocational College Engineering Technology Research Center (2023), Key Technology Programme of Suzhou People's Livelihood Technology Projects (SKY2021029), Open Project of Jiangsu Biobank of Clinical Resources (TC2021B009), Project of State Key Laboratory of Radiation Medicine and Protection, Soochow University (GZK12023013), Programs of the Suzhou Vocational Health College (SZWZYTD202201), and Qing-Lan Project of Jiangsu Province in China (2021, 2022).

Materials

0.25% Trypsin-EDTA(1x)	Gibco	25200-072
Cell Counting Kit-8	Dojindo	CK04
Cell Counting Plate	QiuJing	XB-K-25
CO₂ incubator	Thermo	51032872
DMEM basic(1X)	Gibco	C11995500BT
FBS	PAN	ST30-3302
GraphPad Prism X9	GraphPad Software		statistical analysis software
H₂O₂(3%)	Jiangxi Caoshanhu Disinfection Co.,Ltd.	1028348
high-speed centrifuge	Thermo	9AQ2861
Human 8-oxo-dG ELISA Kit	Zcibio	ZC-55410
L-1000XLS+ Pipettes	Rainin	17014382
L-20XLS+ Pipettes	Rainin	17014392
liquid nitrogen tank	Mvecryoge	YDS-175-216
MCF-7 CELL	BNCC	BNCC100137
Multiskan FC microplate photometer	Thermo	1410101
PBS	Solarbio	P1020
Penicillin-Streptomycin Solution, 100X	Beyotime	C0222
Trinocular live cell microscope	Motic	1.1001E+12
Ultra-low temperature freezer	Haire	V118574

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