Method for screening DNA (Deoxyribose Nucleic Acid) antibody by solid
The method of screening DNA antibodies through solid-phase chemical enzymes solves the problems of time-consuming, equipment dependence and specificity of anti-DNA antibodies in the prior art, and achieves efficient and sensitive anti-DNA antibody detection, supporting early diagnosis of disease.
Patent Information
- Application Number
- CN202411642092.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-18
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art has time-consuming, equipment dependence and specificity problems in detecting anti-DNA antibodies, especially in the early stages of the disease, which hinders early diagnosis and intervention.
The method of screening DNA antibodies by solid-phase chemical enzymes is used to collect body fluid samples, extract DNA samples, and bind to solid-phase media to capture DNA or anti-DNA antibodies that bind to the antibody, achieving efficient screening and detection.
This method can improve the sensitivity and specificity of detection, reduce false positives and false negatives, simplify equipment requirements, reduce detection costs, and provide reliable diagnostic support in early stages of the disease.
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Figure CN119614671A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological screening and analysis, and specifically relates to a method for solid phase chemical enzyme screening of DNA antibodies. Background Art
[0002] Current technologies for identifying anti-DNA antibodies (Anti-DNA) have made significant progress, but there are still limitations. Although techniques such as enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) can provide reliable detection, these two methods are not only time-consuming but also require specialized equipment. In addition, the specificity of these tests may vary, resulting in potential false positives or negatives. Another example is flow cytometry and mass spectrometry, which are expected to improve sensitivity and specificity, but they are expensive and require expertise. In addition, detecting anti-DNA antibodies in the early stages of disease progression remains a major problem, hindering early diagnosis and intervention.
[0003] Human body fluids, including blood, urine, saliva, and bronchoalveolar lavage fluid, may contain specific antibodies against disease-associated DNA, or anti-DNA antibodies. These antibodies, called anti-idiotypic antibodies, can bind to the unique molecular structure of disease-specific anti-DNA antibodies, allowing their detection and possible use in diagnostic or therapeutic applications. For example, in autoimmune diseases such as systemic lupus erythematosus (SLE), anti-DNA antibodies play a vital role, and detecting anti-idiotypic antibodies in a patient's blood may serve as a biomarker for disease activity or progression.
[0004] Therefore, it is necessary to study the method of solid phase chemical enzyme screening of DNA antibodies. Summary of the invention
[0005] The object of the present invention is to provide a method for solid phase chemical enzyme screening of DNA antibodies to solve the above problems.
[0006] A technical solution of the present invention is:
[0007] A method for solid phase chemical enzyme screening of DNA antibodies, comprising the steps of:
[0008] (A) Collecting body fluid samples and extracting DNA samples from the body fluid samples;
[0009] (B) Capture of antibody-bound DNA or capture of anti-DNA antibodies.
[0010] Further, in step (B), capturing the DNA bound to the antibody comprises:
[0011] (1) Add the antibody to the reagent DMSO-H 2 O, and then adding galactose oxidase and horseradish peroxidase to react to obtain a galactose oxidized structure with an aldehyde group;
[0012] (2) washing the solid phase medium with amino groups with a buffer solution so that the pH of the surface of the solid phase medium with amino groups is 10-11, reacting the solid phase medium with amino groups with the oxidized structure of galactose with aldehyde groups, adding sodium cyanoborohydride for incubation, removing the supernatant with a filter device, washing with PBS, adding PBS and sodium cyanoborohydride for further incubation to obtain an antibody intermediate bound to the solid phase, and washing the antibody intermediate bound to the solid phase with NaCl, acetonitrile and deionized water in sequence to obtain an antibody bound to the solid phase;
[0013] (3) reacting the DNA sample with the antibody bound to the solid phase, and gently shaking the mixture to obtain a mixture of DNA bound to the antibody;
[0014] (4) First, the antibody-bound DNA mixture is washed with NaCl and deionized water in sequence to remove impurities, and non-specifically bound DNA is eluted to obtain purified antibody-bound DNA.
[0015] Furthermore, in step (1), the volume ratio of the galactose oxidase to the horseradish peroxidase is 1-10:1-10, and the reaction time is 30-60 min.
[0016] Furthermore, in step (2), the reaction and incubation time are both 4 h, the concentration of sodium cyanoborohydride when sodium cyanoborohydride is added for incubation is 50-60 mM, the concentration of sodium cyanoborohydride when PBS and sodium cyanoborohydride are added for continued incubation is 1 M, the concentration of NaCl is 1 M, and the volume percentage of acetonitrile is 10%.
[0017] Furthermore, in step (4), the concentration of NaCl is 1 M, and the elution method is eluting with Tris-EDTA buffer having a pH value of 8.0-9.0, or eluting with 0.1-1.0% SDS, or eluting with 2 M guanidine chloride.
[0018] Further, in step (B), the capturing of anti-DNA antibodies comprises:
[0019] (1) extracting methylated DNA from the DNA sample, and reacting the DNA with TET and 2OG in oxygen to obtain methylated DNA having an aldehyde group;
[0020] (2) washing the amino-containing solid phase medium with a buffer solution so that the surface pH of the amino-containing solid phase medium is between 10 and 11, and then reacting with the methylated DNA having an aldehyde group;
[0021] (3) adding sodium cyanoborohydride to react, removing the supernatant with a filter, washing with PBS, adding PBS and sodium cyanoborohydride to continue the reaction, and obtaining a DNA mixture bound to the solid phase;
[0022] (4) washing the solid-phase bound DNA mixture with NaCl, acetonitrile and deionized water in sequence to remove non-specific binding proteins and obtain pure solid-phase bound DNA for subsequent antibody binding;
[0023] (5) incubating the body fluid sample with the DNA bound to the solid phase, and gently shaking to obtain the anti-DNA antibody bound to the solid phase;
[0024] (6) eluting the anti-DNA antibody bound to the solid phase with a glycine-hydrochloric acid buffer solution, repeating the process multiple times to obtain an anti-DNA antibody.
[0025] Furthermore, in step (2), the reaction time is 4 h; in step (3), the reaction time is 4 h, the concentration of sodium cyanoborohydride when sodium cyanoborohydride is added for reaction is 50-60 mM, and the concentration of sodium cyanoborohydride when PBS and sodium cyanoborohydride are added for further reaction is 1 M.
[0026] Furthermore, in step (4), the concentration of NaCl is 1 M, and the volume percentage of acetonitrile is 10%.
[0027] Furthermore, in step (5), the incubation time is 4 hours and the temperature is 37° C.; in step (6), the concentration of the glycine hydrochloride buffer solution is 0.1 M.
[0028] Furthermore, the method further comprises step (C): detecting DNA bound to the antibody or detecting anti-DNA antibodies.
[0029] The present invention provides a method for solid-phase chemoenzymatic screening of DNA antibodies, which has the advantages that DNA methylation is a crucial epigenetic modification that changes significantly in a variety of diseases. These changes may lead to dysregulated gene expression, leading to the development and progression of the disease. Diseases associated with altered DNA methylation include cancer, neurodegenerative diseases, autoimmune diseases, autism, metabolic disorders, and developmental disorders. Selecting specific antibodies against altered DNA methylation patterns can become a valuable tool for disease diagnosis and treatment. By targeting these specific modifications, researchers and clinicians can develop more accurate diagnostic tests, identify treatment targets, and monitor disease progression and treatment response. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.
[0031] Figure 1 A schematic diagram of capturing DNA bound to an antibody in a method for solid phase chemical enzyme screening of DNA antibodies according to the present invention;
[0032] Figure 2 It is a schematic diagram of capturing anti-DNA antibodies in the method for screening DNA antibodies by solid phase chemical enzyme according to the present invention. DETAILED DESCRIPTION
[0033] In order to make the above-mentioned objects, features and advantages of the present invention more clearly understood, the technical solutions of the present invention are further described below in conjunction with the accompanying drawings and embodiments. However, the present invention is not limited to the embodiments listed, and should also include any other known changes within the scope of the rights claimed by the present invention.
[0034] First, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.
[0035] Secondly, the present invention is described in detail using structural schematic diagrams, etc. When describing the embodiments of the present invention in detail, for the sake of convenience, the schematic diagrams will not be partially enlarged according to the general scale, and the schematic diagrams are only examples, which should not limit the scope of protection of the present invention. In addition, in actual production, the three-dimensional space of length, width and depth should be included.
[0036] Example 1
[0037] This embodiment discloses a method for extracting a DNA sample from body fluid (taking blood as an example).
[0038] 1. Materials include: cell lysis buffer (containing Tris-HCl, EDTA, SDS and proteinase K), phenol-chloroform-isoamyl alcohol mixture (25:24:1), chloroform, anhydrous ethanol, 70% ethanol, microcentrifuge tubes, pipettes, vortex mixers, and spectrophotometer.
[0039] 2. The operation steps are:
[0040] (a) Sample preparation: A known volume of blood (e.g., 200 μL) was transferred to a microcentrifuge tube, and then an equal volume of cell lysis buffer was added and vortexed for 10 s to obtain a sample.
[0041] (b) Lysis and protein digestion: The samples were incubated at 56°C for 30 min to lyse the cells and digest the proteins. The samples were vortexed every 10 min to obtain a lysate.
[0042] (c) Phenol-chloroform extraction: Add an equal volume of phenol-chloroform-isoamyl alcohol mixture to the lysate, vortex for 15 seconds, centrifuge at 13,000 RPM for 5 minutes at 4°C, and transfer the aqueous phase (top layer) to a new tube.
[0043] (d) Chloroform extraction: Add an equal volume of chloroform to the aqueous phase, vortex for 15 seconds, centrifuge at 13,000 RPM for 5 minutes at 4°C, and transfer the aqueous phase to a new tube.
[0044] (e) DNA precipitation: Add 1 / 10 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of anhydrous ethanol to the aqueous phase, gently invert the test tube to mix, and incubate at -20°C for at least 30 min.
[0045] (f) Centrifugation and washing: Centrifuge at 13,000 RPM at 4°C for 10 min, discard the supernatant, wash the precipitate with 70% ethanol, and centrifuge again at 13,000 RPM at 4°C for 5 min.
[0046] (g) Drying and resuspension: Air-dry the pellet at room temperature until the ethanol evaporates. Pellet the DNA and resuspend it in a suitable buffer (e.g., TE buffer) at the desired concentration.
[0047] (h) Quantification and quality assessment: The concentration of the extracted DNA samples was measured using a spectrophotometer. The quality of the DNA samples was assessed by calculating the A260 / A280 ratio. A ratio of approximately 1.8 indicates high DNA quality.
[0048] Example 2
[0049] This embodiment discloses a method for capturing DNA bound to antibodies, and immobilization technology is essential for capturing DNA-bound antibodies prior to next-generation sequencing (NGS) analysis. By solid-phase binding of glycans in the constant region to the enzyme, the variable region, i.e., the binding region, is retained unaffected. This embodiment ensures efficient sequencing and accurate identification of the variable region of the antibody, which is essential for understanding its specificity and potential role in disease.
[0050] See also Figure 1 , Figure 1Schematic diagram of capturing DNA bound to an antibody in a method for solid phase chemical enzyme screening of DNA antibodies according to the present invention. Figure 1 As shown in the figure, the antibody is bound to the stationary phase medium by solid phase binding through oxidation of antibody polysaccharide galactose. The Fc region of the antibody (mAb) is usually composed of polysaccharides with terminal galactose. The modified galactose can be oxidized by galactose oxidase to form an active aldehyde group. The aldehyde group can bind to the amino or hydrazide solid phase medium to form a solid phase-bound antibody. After incubation with body fluid DNA through the solid phase-bound antibody, the antibody-specifically bound anti-DNA is eluted. Figure 1 As shown, the steps for capturing DNA bound to the antibody are:
[0051] 1. Add the antibody to the reagent (DMSO-H 2 O), then add 1-10U galactose oxidase (GAO) and 1-10U horseradish peroxidase (HRP), react for 30-60min, and obtain a galactose oxidation structure with an aldehyde group. In this step, the polysaccharide Galactose or GalNAc is oxidized to form an aldehyde group;
[0052] 2. Rinse 200 μL of the amino-containing solid phase medium with a pH 10-11 buffer solution to make the pH of the surface of the amino-containing solid phase medium around 10-11, and then react with the oxidized structure of galactose with an aldehyde group for 4 h, and add sodium cyanoborohydride (NaCNBH) with a final concentration of 50-60 mM. 3 ), incubate for 4 h, remove the supernatant using a snap-cap spin-column filter, wash 2-3 times with 1x PBS, add about 475 μL of 1x PBS and 25 μL of 1 M NaCNBH 3 , continue incubation for 4 hours to obtain the antibody intermediate bound to the solid phase, wash the antibody intermediate bound to the solid phase with 1M NaCl, 10% ACN (acetonitrile), and deionized water to obtain the antibody bound to the solid phase;
[0053] 3. IC (immunocapture) immune capture step: react the DNA sample obtained in Example 1 with the antibody bound to the solid phase for 4 hours at 37° C. with slight shaking to obtain a mixture of DNA bound to the antibody;
[0054] 4. First, rinse the antibody-bound DNA mixture with 1M NaCl and deionized water to remove impurities, and then use Tris-EDTA (TE) buffer (pH 8.0-9.0) to elute non-specifically bound DNA from the antibody, or elute DNA with 0.1-1.0% SDS, or elute with 2M guanidine chloride to remove non-specifically bound DNA, and obtain purified antibody-bound DNA;
[0055] 5. Use NGS sequencing to identify the purified DNA structure bound to the antibody.
[0056] Advantages of capturing antibody-bound DNA as described above include:
[0057] (1) Antibody heavy chain glycans are oxidized and bound to the solid phase to ensure that the binding properties of the antibody variable region are not affected;
[0058] (2) By immobilizing the antibody on a solid phase, nonspecific binding can be minimized, thereby improving the signal-to-noise ratio and increasing sensitivity.
[0059] (3) Solid-phase technology can be easily adapted to high-throughput formats, allowing for efficient screening of large numbers of samples and automated sample processing.
[0060] Example 3
[0061] This embodiment discloses a method for capturing anti-DNA antibodies. After the anti-DNA antibodies (anti-DNA) are immobilized, the captured anti-DNA antibodies can be eluted and their structures can be identified by mass spectrometry sequencing. This usually involves sequencing the variable regions of the antibody heavy and light chains to determine their unique amino acid sequences and identify potential targets or epitopes.
[0062] See also Figure 2 , Figure 2 Schematic diagram of capturing anti-DNA antibodies in a method for solid phase chemical enzyme screening of DNA antibodies according to the present invention. Figure 2 As shown, methylated DNA first passes through the TET enzyme to convert the methyl group into an aldehyde group, which is then coupled with an amino group or hydrazide. Stable carbon-nitrogen bonds are formed through reduction. The DNA bound to the solid phase binds to the antibody in the body fluid, and after removing the non-specifically bound antibodies, the antibody that specifically binds to the DNA is eluted through omics to obtain the antibody.
[0063] like Figure 2 As shown, the implementation steps of capturing anti-DNA antibodies are:
[0064] 1. The methylated DNA in the DNA sample obtained in Example 1 reacts with TET and 2OG in oxygen to obtain methylated DNA with aldehyde groups. The principle of this step is that only methylated DNA can react to form aldehyde groups, while non-methylated DNA cannot form aldehyde groups;
[0065] 2. Rinse about 200 μL of the amino-containing solid phase medium with a pH 10-11 buffer solution to make the pH on the surface of the amino-containing solid phase medium between 10-11, and then react with the methylated DNA with aldehyde groups for 4 hours;
[0066] 3. Add NaCNBH to a final concentration of 50-60 mM 3 , react for 4 h, remove the supernatant using a snap-cap spin-column filter, wash 2-3 times with 1x PBS, add about 475 μL of 1x PBS and 25 μL of 1 M NaCNBH 3 , the reaction was continued for 4 h to obtain a DNA mixture bound to the solid phase, i.e., the double bonds were changed into single bonds;
[0067] 4. The solid phase-bound DNA mixture was washed with 1 M NaCl, 10% ACN and deionized water to remove non-specific binding proteins and obtain pure solid phase-bound DNA for subsequent antibody binding (not shown);
[0068] 5. Incubate the body fluid sample and the solid-phase bound DNA for 4 h at 37°C with slight shaking to obtain the solid-phase bound anti-DNA antibody;
[0069] 6. Elute the anti-DNA antibody bound to the solid phase with 200-250 μL of 0.1 M glycine hydrochloride buffer solution, repeat this step 2-3 times to ensure maximum antibody recovery, and finally obtain the anti-DNA antibody;
[0070] 7. Proteomic sequencing: After trypsin digestion, mass spectrometry is used to analyze the antibody structural characteristics or anti-DNA antibody structure (not shown).
[0071] Advantages of the above capture anti-DNA antibodies include:
[0072] (1) It specifically binds to methylated DNA, providing a technical means for discovering disease-related antibodies;
[0073] (2) Autoantibody specificity: By simulating disease-associated antigens and using methylated DNA as an antigen, the test can specifically detect antibodies against this modified form of DNA, thereby improving specificity;
[0074] (3) Immobilization can enhance the affinity of methylated DNA for anti-DNA antibodies, thereby achieving more effective binding and improving the sensitivity of detection.
[0075] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.
Claims
1. A method for solid phase chemoenzyme screening of DNA antibodies, characterized in that: Includes steps: (A) Collecting body fluid samples and extracting DNA samples from the body fluid samples; (B) Capture of antibody-bound DNA or capture of anti-DNA antibodies.
2. The method for solid phase chemical enzyme screening of DNA antibodies according to claim 1, characterized in that: In step (B), capturing the DNA bound to the antibody comprises: (1) adding the antibody to a reagent DMSO-H2O, and then adding galactose oxidase and horseradish peroxidase to react to obtain a galactose oxidized structure having an aldehyde group; (2) washing the solid phase medium with amino groups with a buffer solution so that the pH of the surface of the solid phase medium with amino groups is 10-11, reacting the solid phase medium with amino groups with the oxidized structure of galactose with aldehyde groups, adding sodium cyanoborohydride for incubation, removing the supernatant with a filter device, washing with PBS, adding PBS and sodium cyanoborohydride for further incubation to obtain an antibody intermediate bound to the solid phase, and washing the antibody intermediate bound to the solid phase with NaCl, acetonitrile and deionized water in sequence to obtain an antibody bound to the solid phase; (3) reacting the DNA sample with the antibody bound to the solid phase, and gently shaking the mixture to obtain a mixture of DNA bound to the antibody; (4) First, the antibody-bound DNA mixture is washed with NaCl and deionized water in sequence to remove impurities, and non-specifically bound DNA is eluted to obtain purified antibody-bound DNA.
3. The method for solid phase chemical enzyme screening of DNA antibodies according to claim 2, characterized in that: In step (1), the volume ratio of galactose oxidase to horseradish peroxidase is 1-10:1-10, and the reaction time is 30-60 min.
4. The method for solid phase chemical enzyme screening of DNA antibodies according to claim 2, characterized in that: In step (2), the reaction and incubation time are both 4 hours, the concentration of sodium cyanoborohydride when sodium cyanoborohydride is added for incubation is 50-60 mM, the concentration of sodium cyanoborohydride when PBS and sodium cyanoborohydride are added for continued incubation is 1 M, the concentration of NaCl is 1 M, and the volume percentage of acetonitrile is 10%.
5. The method for solid phase chemical enzyme screening of DNA antibodies according to claim 2, characterized in that: In step (4), the concentration of NaCl is 1 M, and the elution method is eluting with Tris-EDTA buffer with a pH value of 8.0-9.0, or eluting with 0.1-1.0% SDS, or eluting with 2 M guanidine chloride.
6. The method for solid phase chemical enzyme screening of DNA antibodies according to claim 1, characterized in that: In step (B), the capturing of anti-DNA antibodies comprises: (1) extracting methylated DNA from the DNA sample, and reacting the DNA with TET and 2OG in oxygen to obtain methylated DNA having an aldehyde group; (2) washing the amino-containing solid phase medium with a buffer solution so that the surface pH of the amino-containing solid phase medium is between 10 and 11, and then reacting with the methylated DNA having an aldehyde group; (3) adding sodium cyanoborohydride to react, removing the supernatant with a filter, washing with PBS, adding PBS and sodium cyanoborohydride to continue the reaction, and obtaining a DNA mixture bound to the solid phase; (4) washing the solid-phase bound DNA mixture with NaCl, acetonitrile and deionized water in sequence to remove non-specific binding proteins and obtain pure solid-phase bound DNA for subsequent antibody binding; (5) incubating the body fluid sample with the DNA bound to the solid phase, and gently shaking to obtain the anti-DNA antibody bound to the solid phase; (6) eluting the anti-DNA antibody bound to the solid phase with a glycine-hydrochloric acid buffer solution, repeating the process multiple times to obtain an anti-DNA antibody.
7. The method for solid phase chemical enzyme screening of DNA antibodies according to claim 6, characterized in that: In step (2), the reaction time is 4 hours; in step (3), the reaction time is 4 hours, the concentration of sodium cyanoborohydride when sodium cyanoborohydride is added for reaction is 50-60 mM, and the concentration of sodium cyanoborohydride when PBS and sodium cyanoborohydride are added for further reaction is 1 M.
8. The method for solid phase chemical enzyme screening of DNA antibodies according to claim 6, characterized in that: In step (4), the concentration of NaCl is 1 M, and the volume percentage of acetonitrile is 10%.
9. The method for solid phase chemical enzyme screening of DNA antibodies according to claim 6, characterized in that: In step (5), the incubation time is 4 hours and the temperature is 37° C.; in step (6), the concentration of the glycine hydrochloride buffer solution is 0.1 M.
10. The method for solid phase chemical enzyme screening of DNA antibodies according to claim 1, characterized in that: The method further comprises the step (C): detecting DNA bound to the antibody or detecting anti-DNA antibodies.