This protocol describes an efficient method for quantitatively detecting DNA oxidative damage in MCF-7 cells by ELISA technology.
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 IC50 value 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.
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.
1. Construction of an H2O2 -induced DNA oxidative damage model in MCF-7 cells
2. Preparation of cellular samples and ELISA reagents preparation
3. ELISA experiment
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 suggested an IC50 of 0.7655 mM for the MCF-7 cells.
In this series of experiments, we employed an enhanced ELISA technique for the quantification of 8-oxo-dG levels. Through data analysis, the regression equation of the standard curve is:
Y = 23.66 × X + 0.07038.
As depicted in Figure 4A, the calibration curve demonstrated high linearity (R²=0.978), and Figure 4B shows that the relationships between the measured concentrations of 8-oxo-dG in the samples and their corresponding added amounts were strongly correlated, indicating good precision (the coefficient of variation was less than 5%). Moreover, the consistency of the obtained results across multiple replicates indicates the reliability and reproducibility of the experimental method.
In summary, the experimental data indicate that our optimized ELISA method is capable of successfully detecting fluctuations in 8-oxo-dG levels under well-controlled conditions. This has significant implications for future applications of this technique in a wider range of sample types to monitor DNA damage caused by oxidative stress. Despite certain limitations, the method's prospects for application appear promising upon optimization of the experimental conditions.
Figure 1: Preparing H2O2 dilutions. The schematic diagram delineates the method for preparing hydrogen peroxide (H2O2) dilutions, involving the meticulous combination of solutions and the precise transfer of varied H2O2 concentrations into tubes labeled as 1.0 mM, 0.5 mM, and 0.25 mM correspondingly. The targeted concentration is attained by transferring 1.5 mM H2O2 into the tube designated as 0.75 mM. Please click here to view a larger version of this figure.
Figure 2: Enzyme-linked immunosorbent Assay (ELISA) for antigen detection. This schematic illustration depicts the sequential steps of an Enzyme-Linked Immunosorbent Assay (ELISA) for antigen detection: Sample addition and blank preparation; HRP-labeled antigen incubation; washing and plate preparation; substrate addition and incubation; reaction termination and optical density (OD) measurement. Please click here to view a larger version of this figure.
Figure 3: H2O2 inhibited MCF-7 cell proliferation. Cell viability was evaluated by CCK-8 assay treated with various concentrations of H2O2 (0, 0.25, 0.50, 0.75, 1.0, 1.5, or 2.0 mM) for 12 h and IC50 was calculated for the indicated condition. Please click here to view a larger version of this figure.
Figure 4: Quantitative analysis of 8-oxo-dG in MCF-7 cells following H2O2 treatment. (A) ELISA standard curve for 8-oxo-dG. Depicts the relationship between known concentrations of 8-oxo-dG (1.25, 2.5, 5, 10, 20, and 40 ng/mL) and their respective absorbance values. The regression equation of the standard curve is Y = 23.66 × X + 0.07038. (n=3, R2=0.978). (B) 8-oxo-dG concentration in H2O2-treated MCF-7 cells. The graph displays the levels of 8-oxo-dG in MCF-7 cells subjected to different concentrations of H2O2 (0, 0.25, and 0.75 mM). Each data point represents the mean ± SE (standard error) from three independent experiments (n=3), illustrating the impact of oxidative stress on DNA damage. Please click here to view a larger version of this figure.
Parameter | ||||||
H2O2 concentration, mM | 0 | 0.25 | 0.5 | 0.75 | 1 | 2 |
MCF-7 cell viability, % | 100 | 82.8 | 81.37 | 61.07 | 13.4 | 2.77 |
Table 1: Cell viability of MCF-7 cells treated with H2O2.
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.
During experimental procedures, several critical steps are essential to ensure the reliability and reproducibility of the results. Initially, the experiment proficiently assessed the sensitivity of MCF-7 cells to H2O2, with an IC50 value of 0.7655 mM at 12 h, highlighting the tolerance level of MCF-7 cells. This outcome is of great scientific significance for understanding the antioxidant defense mechanisms of MCF-7 cells and their survival under oxidative stress while providing an important reference indicator for future screening of antioxidant drugs or related treatment mechanisms31. Furthermore, the selection and pairing of 8-oxo-dG antibodies is vital for ELISA detection and directly affects the specificity and sensitivity of the ELISA detection system. In addition, optimizing the signal amplification strategy and sample processing steps are important factors that affect the accuracy of detection. We found that using a stable substrate reaction system can reduce background errors and increase the sensitivity range of detection.
During the experimental process, technical issues such as unstable antibodies, signal fluctuations, and cross-reactivity of reactants may occur. To address these issues, we adopted a more stable experimental protocol, which included the use of stable antibody solvents to improve antibody stability and the optimization and adjustment of experimental conditions. Additionally, to address the error caused by signal fluctuations, we standardized the experiment using standard 8-oxo-dG samples to ensure consistency of the results.
Regarding the limitations of the experimental method, although ELISA provides a high-throughput approach, it may still be limited by the specific antibody coverage and inevitable substrate cross-reactivity32. Therefore, additional sample purification or preprocessing steps may be required for complex samples. Moreover, the detection of samples with lower concentrations may necessitate further optimization of antibody activity to meet the research requirements.
In conclusion, the ELISA detection method for 8-oxo-dG has important implications for specific research areas. It has potential application value in environmental monitoring, disease risk assessment, and early diagnosis and provides a new detection approach for 8-oxo-dG in agriculture, food safety, and drug screening33. For example, in environmental biomonitoring, the detection of DNA oxidative damage stress caused by environmental toxins in organisms can help identify potential risks in a timely manner34. In the future, with further optimization of detection technology and its application in multiple disciplines, this method is expected to play a more critical role in life science research.
The authors have nothing to disclose.
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).
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 |