A DNA methylation detection kit based on HSA-enhanced chemiluminescence and application thereof

Abstract

This invention discloses a DNA methylation detection kit based on HSA-enhanced chemiluminescence and its application, belonging to the field of biomedical detection technology. The kit includes: a solid-phase carrier coated with an anti-5-methylcytosine monoclonal antibody, an alkaline phosphatase-labeled anti-5-methylcytosine monoclonal antibody, and a chemiluminescent substrate solution. The chemiluminescent substrate solution contains a 1,2-dioxane compound and human serum albumin, wherein human serum albumin acts as a luminescence enhancer. This invention achieves the detection of the overall DNA methylation level by immunografting of 5-methylcytosine on DNA, combined with an alkaline phosphatase-catalyzed chemiluminescent reaction. This method is independent of gene sequence, simple to operate, highly sensitive, and provides stable signals, making it suitable for early lung cancer screening.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a DNA methylation detection kit, detection method, and application based on human serum albumin (HSA)-enhanced chemiluminescence. Background Technology

[0002] DNA methylation (5-methylcytosine, 5mC) is one of the most significant epigenetic modifications in the mammalian genome. In the early stages of tumor development, the methylation pattern of genomic DNA undergoes global changes, exhibiting characteristics of overall hypomethylation and localized hypermethylation. Circulating free DNA (cfDNA), originating from apoptotic and necrotic cells, carries genetic and epigenetic information from its source tissues. Therefore, cfDNA methylation profiling has become one of the most promising early tumor screening methods in the field of liquid biopsy.

[0003] Currently, the mainstream technologies for detecting cfDNA methylation include:

[0004] I. Gene Sequence-Dependent Detection Methods

[0005] Methods such as bisulfite sequencing and methylation-specific PCR are representative. While these methods can provide methylation information at single-base resolution, they have significant drawbacks: they are complex to operate, time-consuming, costly, and highly dependent on known gene sequence information. More importantly, these methods mainly target the local methylation status of specific gene loci and cannot quickly reflect changes in the overall methylation level of DNA in a sample, while abnormal overall methylation levels are one of the important markers of early tumor development.

[0006] II. Immunoassay based on anti-5mC antibodies

[0007] This type of method utilizes anti-5-methylcytosine antibodies to specifically recognize methylation modifications on single-stranded DNA. It does not rely on gene sequence information and can directly assess the overall methylation level of total cfDNA, with the potential advantages of simple operation and high throughput.

[0008] However, existing immunoassays based on anti-5mC antibodies mainly use colorimetric methods (such as ELISA) for signal readout. Because the concentration of cfDNA in plasma is extremely low (usually only at the ng / mL level), and the methylation level of cfDNA in early-stage tumor patients varies relatively little, the sensitivity of traditional colorimetric methods is insufficient to meet the clinical needs of early screening, and false negative results are easily obtained.

[0009] Chemiluminescence immunoassay is widely used to improve the sensitivity of immunoassays. Among them, chemiluminescence systems based on alkaline phosphatase (ALP) and 1,2-dioxane compounds (such as CDP-Star and AMPPD) are favored due to their stable signal and low background. Existing studies (such as patent US5145772A) have shown that adding macromolecules such as bovine serum albumin (BSA) or human serum albumin (HSA) to the chemiluminescence substrate solution can enhance the chemiluminescence signal generated by ALP-catalyzed decomposition of 1,2-dioxane to a certain extent. The principle is that the hydrophobic cavity of serum albumin can provide a protective microenvironment for the luminescent intermediate.

[0010] However, the above findings only address the general improvements to chemiluminescence systems and do not provide optimization for the specific application of DNA methylation immunoassay. Existing detection protocols based on anti-5mC antibodies typically use conventional chemiluminescence substrates (such as CDP-Star without enhancers) or simple BSA blocking solutions. Their signal intensity and signal-to-noise ratio are still insufficient to stably and sensitively detect the overall methylation differences of trace amounts of cfDNA in clinical plasma samples. For example, when using conventional CDP-Star substrates to detect low concentrations of methylated DNA, the luminescence signal is weak and the signal-to-noise ratio is low, making it difficult to effectively distinguish between early-stage lung cancer patients and healthy individuals.

[0011] In conclusion, developing a cfDNA methylation detection method that is independent of gene sequence, simple to operate, highly sensitive, stable, and can reflect the overall methylation level is of significant clinical importance and application value for early tumor screening. Summary of the Invention

[0012] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a DNA methylation detection kit based on HSA-enhanced chemiluminescence. This kit uses alkaline phosphatase (ALP) as the labeling enzyme and a 1,2-dioxane compound as the chemiluminescent substrate, specifically CDP-Star. HSA is introduced as a luminescence enhancer in DNA methylation detection, achieving ultrasensitive, highly stable, and rapid detection of the overall methylation level of plasma cfDNA. The CAS number of the HSA is 70024-90-7, and the CAS number of the CDP-Star is 160081-62-9.

[0013] The technical principle of this invention includes: magnetic beads (capture units) coated with anti-5-methylcytosine antibody specifically recognize 5mC modifications on single-stranded DNA and capture them from the sample; subsequently, alkaline phosphatase-labeled anti-5mC antibody (detection unit) binds to the captured DNA, forming a double-antibody sandwich complex; finally, CDP-Star chemiluminescent substrate is added, and ALP catalyzes the dephosphorylation of CDP-Star to generate an unstable intermediate. This intermediate decomposes within the hydrophobic cavity of the HSA and emits photons, which are read by a chemiluminescence detector. The intensity of the luminescence signal is proportional to the overall methylation level of cfDNA in the sample.

[0014] The enhancement mechanism of HSA lies in the fact that the dephosphorylation intermediate generated after ALP-catalyzed dephosphorylation of CDP-Star is hydrophobic and can diffuse to the binding site I (Sudlow's site I) in the HSA molecular structure. In the hydrophobic cavity of HSA, the decomposition rate of the intermediate is accelerated and the fluorescence quantum yield is significantly improved, thereby achieving exponential amplification of the signal.

[0015] On one hand, the present invention provides a DNA methylation detection kit based on HSA-enhanced chemiluminescence, comprising the following units:

[0016] (a) Capture unit: a solid-phase carrier coated with anti-5-methylcytosine monoclonal antibody;

[0017] (b) Detection unit: Alkaline phosphatase-labeled anti-5-methylcytosine monoclonal antibody;

[0018] (c) Chemiluminescent substrate solution: containing 1,2-dioxane compounds and human serum albumin (HSA), wherein HSA acts as a luminescence enhancer;

[0019] (d) Buffer and washing solution: including binding buffer, washing buffer and substrate dilution solution.

[0020] Furthermore, the 1,2-dioxane compound is CDP-Star.

[0021] Furthermore, the 1,2-dioxane compound is CDP-Star, with a concentration of 0.1-1.0 mM.

[0022] Furthermore, the concentration of the human serum albumin is 0.05-1.0 mg / mL.

[0023] Furthermore, the chemiluminescent substrate solution also contains Tris-HCl buffer (pH 9.5), MgCl2, and NaCl.

[0024] Furthermore, the solid-phase support is magnetic microparticles or enzyme-labeled plate wells.

[0025] On the other hand, the present invention also provides a method for detecting DNA methylation using the above-mentioned kit, comprising the following steps:

[0026] (1) Extract free DNA from the sample and perform heat denaturation treatment to make it into single-stranded DNA;

[0027] (2) The denatured single-stranded DNA was mixed with the solid-phase carrier described in (a) and incubated to allow the 5-methylcytosine on the DNA to specifically bind to the anti-5-methylcytosine antibody.

[0028] (3) After washing, incubate with the alkaline phosphatase-labeled anti-5-methylcytosine antibody described in (b);

[0029] (4) After washing, add the chemiluminescent substrate solution described in (c), incubate for 5-15 minutes, and then read the luminescence value using a chemiluminescence detector;

[0030] The luminescence value is directly proportional to the overall methylation level of DNA in the sample.

[0031] Furthermore, the sample was cell-free DNA from blood plasma.

[0032] On the other hand, the present invention also provides the application of the above-described kit in the preparation of diagnostic reagents for early screening of lung cancer.

[0033] Furthermore, the early lung cancer screening includes screening for stage I-II lung adenocarcinoma.

[0034] The present invention has the following beneficial effects: (1) It does not depend on gene sequence and directly detects 5-methylcytosine chemical modification, avoiding the need for PCR primers and sequencing; (2) Using HSA as a luminescence enhancer for DNA methylation detection increases the chemiluminescence intensity of CDP-Star by 10.7 times; (3) The ALP-dioxane system has a long signal stability window, far exceeding the 25 minutes of the HRP-luminol system, making it suitable for high-throughput detection; (4) Clinical verification shows that it can effectively distinguish between early lung cancer patients and healthy controls, and the detection rate of stage I lung cancer reaches 88.9%, providing a simple, sensitive and stable new method for early lung cancer screening. Attached Figure Description

[0035] Figure 1 A bar chart comparing the luminescence intensity of different chemiluminescent substrate systems. Detailed Implementation

[0036] The technical solutions described in this invention will now be clearly and completely described with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only a part of the feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0037] Example 1: Preparation of a chemiluminescent substrate solution

[0038] 1. Main experimental materials

[0039] CDP-Star (Catalog No.: MT0009, purity ≥98%) was purchased from Dalian Meilun Biotechnology Co., Ltd.; HSA (Catalog No.: A1653-5G, purity ≥96%) was purchased from Sigma-Aldrich; BSA (Catalog No.: V900933-100G, purity ≥98%) was purchased from Sigma-Aldrich; ALP (Catalog No.: MB2849-1, ≥3000 U / mg) was purchased from Dalian Meilun Biotechnology Co., Ltd.; Tris-base, hydrochloric acid, MgCl2·6H2O, and NaCl were all of analytical grade.

[0040] 2. Preparation of substrate solution

[0041] Prepare 100 mL of CDP-Star chemiluminescent substrate solution: Weigh 15.8 mg of CDP-Star and dissolve it in 50 mL of Tris-HCl buffer (0.2 M, pH 9.5); add 20.3 mg of MgCl2·6H2O; add 584 mg of NaCl; add 50 mg of HSA; and bring the volume to 100 mL with 0.1 M Tris-HCl buffer (pH 9.5). Store in the dark at 4°C for later use. The concentration of CDP-Star in the chemiluminescent substrate solution is 0.25 mM, the concentration of MgCl2·6H2O is 1.0 mM, the concentration of NaCl is 0.1 M, and the concentration of HSA is 0.5 mg / mL.

[0042] Comparative Example 1: Preparation of a chemiluminescent substrate solution

[0043] The difference between this comparative example and Example 1 is that the substrate solution was prepared using a Tris-HCl buffer (pH 9.5) containing only 0.25 mM CDP-Star, 1.0 mM MgCl2 and 0.1 M NaCl, and did not contain HSA.

[0044] Comparative Example 2: Preparation of a Chemiluminescent Substrate Solution

[0045] The difference between this comparative example and Example 1 is that BSA is used instead of HSA in the preparation of the substrate solution.

[0046] Experimental Example 1: Luminous Intensity Test

[0047] 1. Experimental Methods

[0048] A 96-well microplate (Corning, white opaque plate) coated with anti-5mC antibody (catalog number AF5722, Beyotime) was used. 100 μL of methylated DNA standard (10 ng / mL, single-stranded) was added to each well, and the plate was incubated at 37°C for 1 hour. After washing, 100 μL of ALP-labeled anti-5mC antibody (1:1000 dilution) was added, and the plate was incubated at 37°C for 30 minutes. After washing five times, 100 μL of the substrate solution from Example 1 and Comparative Examples 1-2 were added respectively. The fluorescence intensity (RLU) was continuously read using a chemiluminescence analyzer (SpectraMax L), and the peak RLU was recorded. Each group had three replicates, and the mean ± standard deviation was used. Signal-to-noise ratio (S / N) = Sample well RLU / Blank well RLU (using DNA-free PBS instead of samples).

[0049] 2. Experimental Results:

[0050]

[0051] The experimental results are shown in Table 1 and Figure 1 As shown, a comparison between Comparative Example 1 and Example 1 reveals that the addition of HSA increased the luminescence intensity of the CDP-Star by approximately 10.7 times and the signal-to-noise ratio by approximately 10.6 times, confirming that HSA has a significant luminescence enhancement effect. A comparison between Comparative Example 2 and Example 1 shows that the enhancement effect of HSA is significantly better than that of BSA, with its luminescence intensity increasing by approximately 3.8 times, indicating that HSA has a specific enhancement advantage, which is related to the unique hydrophobic cavity structure of HSA.

[0052] Experiment 2: Clinical Sample Testing and Validation of Early Lung Cancer Screening Effectiveness

[0053] 1. Sample collection

[0054] Plasma samples were collected from 40 patients with pathologically confirmed early-stage (stage I-II) lung adenocarcinoma and 40 age- and sex-matched healthy individuals undergoing physical examinations. All samples were collected in the morning on an empty stomach in EDTA anticoagulant tubes, and plasma was separated within 2 hours (1600×g, 10 minutes), aliquoted, and stored at -80℃.

[0055] 2. Preparation of ALP-anti-5mC antibody labeling

[0056] One-step labeling with glutaraldehyde: Dissolve 2 mg ALP (10 mg / mL) in 0.5 mL PBS buffer (0.1 M, pH 7.2); add 2 mg anti-5-methylcytosine monoclonal antibody (mouse-derived, 2 mg / mL); slowly add 25% glutaraldehyde solution to a final concentration of 0.2%, and stir at room temperature for 2 hours; add Tris-HCl buffer (0.05 M, pH 8.0) to a final concentration of 50 mM to terminate the reaction; remove unbound glutaraldehyde and small molecule impurities using a Sephadex G-25 gel column; collect the elution peak containing the ALP-antibody conjugate, add an equal volume of glycerol, and store at -20℃ for later use.

[0057] 3. Testing Process

[0058] Step 1: cfDNA extraction and quantification: cfDNA was extracted from 1 mL of plasma using the QIAamp Circulating Nucleic Acid Kit and quantified using a Qubit 4.0 fluorometer.

[0059] Step 2: Heat denaturation: Take 20 μL of cfDNA solution, heat at 95℃ for 5 minutes, and immediately place on ice for 5 minutes.

[0060] Step 3: Immunocapture: (1) Add 10 μL of anti-5mC antibody-coated magnetic beads (1 mg / mL) to an EP tube; (2) Add 50 μL of binding buffer, which contains PBS buffer, 0.1% BSA and 0.05% Tween-20; (3) Add 20 μL of denatured cfDNA; (4) Incubate at 37°C with shaking for 45 minutes.

[0061] Step 4: Washing: After magnetic separation, wash 3 times with 200 μL of washing buffer containing PBS buffer and 0.05% Tween-20.

[0062] Step 5: ALP-labeled antibody binding: (1) Add 100 μL of ALP-labeled anti-5mC antibody (1:1000 dilution); (2) Incubate at 37°C with shaking for 30 minutes; (3) Wash 3 times.

[0063] Step 6: Chemiluminescence detection: (1) Add 100 μL of the chemiluminescence substrate solution (CDP-Star+ HSA) prepared in Example 1; (2) Incubate at room temperature in the dark for 10 minutes; (3) Read the luminescence value (RLU) using a chemiluminescence detector.

[0064] 4. Experimental Results

[0065] (1) Distinguishing ability

[0066]

[0067] (2) Diagnostic efficacy (ROC analysis)

[0068]

[0069] (3) Detection results of different lung cancer stages

[0070]

[0071] The experimental results are shown in Tables 2-4. The method of the present invention can effectively distinguish between early-stage lung cancer patients and healthy individuals, with a detection rate of 88.9% for stage I lung cancer, demonstrating that the present invention has extremely high clinical application value in early lung cancer screening.

[0072] Experimental Example 3: Specificity Verification

[0073] To verify the specificity of the method of the present invention for 5mC, the inventors conducted a specificity verification experiment.

[0074] 1. Experimental Methods

[0075] The DNA standards shown in Table 5 were synthesized and prepared by Sangon Biotech (Shanghai) Co., Ltd., and purified by HPLC to a purity ≥95%. The sequence design followed these principles: 50 bp in length, with a modification site (C, 5mC, or 5hmC) approximately every 5 bp, and the G+C content in the base composition controlled between 40% and 60%. Each standard had a concentration of 10 ng / mL and was detected according to the detection procedure in Example 2.

[0076]

[0077] 2. Experimental Results

[0078] The experimental results are shown in Table 6. The method of the present invention has high specificity for 5-methylcytosine, while the signal for unmethylated DNA is only 1.3% of that for methylated DNA, and it has a good linear response.

[0079]

[0080] Experiment 4: Reagent Kit Stability Test

[0081] 1. Experimental Methods: The components of the kit of this invention (anti-5mC magnetic beads, ALP-labeled antibody, CDP-Star+HSA substrate solution) were stored at 4°C for 0, 3, 6, 12, and 18 months. Samples were taken at each time point, and the same batch of positive lung cancer plasma samples and negative control samples were tested according to the method in Example 2. The positive lung cancer plasma samples were plasma samples from patients with stage II lung adenocarcinoma confirmed in Example 2.

[0082] 2. Experimental Results

[0083]

[0084] The test results are shown in Table 7. After being stored at 4°C for 18 months, the relative activity of the kit of this invention remained above 90%, and the signal-to-noise ratio was >1200, which meets the requirements for clinical testing.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A DNA methylation detection kit based on HSA-enhanced chemiluminescence, characterized in that, include: (a) Capture unit: a solid-phase carrier coated with anti-5-methylcytosine monoclonal antibody; (b) Detection unit: Alkaline phosphatase-labeled anti-5-methylcytosine monoclonal antibody; (c) Chemiluminescent substrate solution: containing 1,2-dioxane compounds and human serum albumin; The human serum albumin is used as a luminescence enhancer to enhance the chemiluminescence signal of 1,2-dioxane compounds.

2. The reagent kit according to claim 1, characterized in that, The 1,2-dioxane compound is CDP-Star, with a concentration of 0.1-1.0 mM.

3. The reagent kit according to claim 1, characterized in that, The concentration of the human serum albumin is 0.05-1.0 mg / mL.

4. The reagent kit according to claim 1, characterized in that, The chemiluminescent substrate solution also contains Tris-HCl buffer, MgCl2 and NaCl, wherein the buffer has a pH of 9.

5.

5. The reagent kit according to claim 1, characterized in that, The solid-phase carrier is the well of an enzyme-labeled plate.

6. A method for detecting DNA methylation using the kit according to any one of claims 1-5, characterized in that... Includes the following steps: (1) Extract free DNA from the sample and perform heat denaturation treatment to make it into single-stranded DNA; (2) The denatured single-stranded DNA was mixed with the solid-phase carrier described in (a) and incubated to allow the 5-methylcytosine on the DNA to specifically bind to the anti-5-methylcytosine antibody. (3) After washing, incubate with the alkaline phosphatase-labeled anti-5-methylcytosine antibody described in (b); (4) After washing, add the chemiluminescent substrate solution described in (c), incubate for 5-15 minutes, and then read the luminescence value using a chemiluminescence detector; The luminescence value is directly proportional to the overall methylation level of DNA in the sample.

7. The method according to claim 6, characterized in that, The sample was cell-free DNA from blood plasma.

8. The use of the kit according to any one of claims 1-5 in the preparation of diagnostic reagents for early screening of lung cancer.

9. The application according to claim 8, characterized in that, The early lung cancer screening includes screening for stage I-II lung adenocarcinoma.

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