Application of Anti-DNA Antibody in the Preparation of an Early Diagnosis Kit for Alzheimer's Disease

Abstract

The present invention relates to the application of anti-DNA antibodies in the preparation of an early diagnostic kit for Alzheimer's disease (AD). The present invention is proposed based on the significant discovery that anti-DNA antibodies can specifically capture AD diagnostic biomarkers. Specifically, the present invention first discovers that anti-DNA antibodies can enrich the pathogenic misfolded tau protein in AD. The enriched tau protein can be confirmed by Western blot experiments and can also be amplified and detected by in vitro RT-QuIC. The early diagnostic kit for AD based on anti-DNA antibodies as an enrichment medium proposed by the present invention can improve the clinical sensitivity and specificity of the technology using peripheral body fluids as detection samples, providing technical support for the development of non-invasive peripheral body fluid AD diagnostic technologies.

Description

Technical Field

[0001] The present invention relates to the technical field of biodiagnostic medicine, and particularly to the application of anti-DNA antibodies in the preparation of an early diagnosis kit for Alzheimer's disease. Background Art

[0002] AD is a most common neurodegenerative disease, mainly manifested as memory loss and brain cell death. Currently, there is a lack of effective treatment measures. According to the "Report on Alzheimer's Disease in China 2021", among the people aged 60 and above in China, there are approximately 15.07 million dementia patients, of which 9.83 million are AD patients. With the acceleration of the aging population process, it is estimated that the number of AD patients in China will exceed 30 million in 2050, imposing a heavy burden on families and society. In recent years, due to the development of brain imaging (including CT and magnetic resonance) and body fluid biomarkers, early diagnosis has become possible. However, these new technologies are expensive, or lumbar puncture for cerebrospinal fluid extraction is invasive, CT involves radioactive isotopes, and they are concentrated in large medical units. Therefore, there is an urgent need to develop a new early diagnosis method with high sensitivity, strong specificity, less invasiveness, and low cost.

[0003] Based on two in vitro hypersensitive amplification techniques, real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA), it has been reported that PrP Sc - or αSyn skin biomarkers for the diagnosis of prion disease (PrD) and Parkinson's disease (PD) have been developed using cadaveric and living skin tissues (Sci Transl Med, 2017; JAMA Neurol 2020). The diagnostic method using living skin tissue as the test sample is a minimally invasive method, which not only easily causes psychological burden to patients but also the nursing process at the wound site is troublesome. Therefore, developing a non-invasive, sensitive and specific peripheral body fluid diagnostic technology will be more convenient and have more practical application value. However, the concentration of biomarkers for diagnosis in peripheral body fluids is too low, and it faces the problems of low sensitivity and poor specificity, making it difficult to be popularized and applied. Summary of the Invention

[0004] (I) Technical Problems to be Solved

[0005] In view of the above-mentioned disadvantages and deficiencies of the prior art, the present invention provides the application of anti-DNA antibodies in the preparation of an early diagnosis kit for Alzheimer's disease, using anti-DNA antibodies as an enrichment medium for AD diagnostic biomarkers, thereby improving the clinical sensitivity and specificity of the method using peripheral body fluids as test samples, and providing technical support for the development of a non-invasive peripheral body fluid AD diagnostic technology.

[0006] (II) Technical Solutions

[0007] In a first aspect, the present invention provides the use of anti-DNA antibodies in the preparation of an early diagnostic kit for Alzheimer's disease.

[0008] Preferably, the diagnostic kit further comprises a detection reagent for pathogenic misfolded tau protein.

[0009] Preferably, the detection reagent comprises a detection antibody for pathogenic misfolded tau protein.

[0010] Preferably, the test sample of the diagnostic kit is peripheral body fluid or peripheral tissue, and the peripheral body fluid is peripheral blood, saliva, tears, feces, sweat or urine; the peripheral tissue is skin tissue, submandibular gland, oral mucosa, etc., and the skin tissue can be prepared into a tissue homogenate for detection.

[0011] Preferably, the diagnostic kit comprises magnetic beads, anti-DNA antibodies, tau protein detection antibodies, recombinant tau protein, a fluorescent label (such as thioflavin) and a buffer.

[0012] In a second aspect, the present invention also provides an early diagnostic kit for Alzheimer's disease, which comprises a tau protein enrichment medium and a tau protein detection antibody; the tau protein enrichment medium is an anti-DNA antibody.

[0013] Preferably, the early diagnostic kit for Alzheimer's disease further comprises magnetic beads, recombinant tau protein, a fluorescent label (such as thioflavin) and a buffer.

[0014] In a third aspect, the present invention relates to the use of magnetic beads embedded or coupled with anti-DNA antibodies in the preparation of an early diagnostic kit for Alzheimer's disease.

[0015] In a fourth aspect, the present invention relates to the use of anti-DNA antibodies as an enrichment medium in the preparation of a kit for detecting the expression level of tau protein.

[0016] (III) Beneficial effects

[0017] The present invention is proposed based on the major discovery that anti-DNA antibodies can specifically capture AD diagnostic biomarkers. Specifically, the present invention discovers for the first time that anti-DNA antibodies can enrich pathogenic misfolded tau protein of AD. The enriched tau protein can be confirmed by Western blot experiments, and it can also be amplified and detected by in vitro RT-QuIC. The latter can develop a hypersensitive early diagnostic method for clinical use. The early diagnostic kit for AD based on anti-DNA antibodies as an enrichment medium proposed by the present invention can improve the sensitivity and specificity of the technology using peripheral body fluid as a test sample, and provide technical support for the development of a non-invasive peripheral body fluid AD diagnostic technology.

[0018] An early non-invasive AD diagnosis platform has been developed based on the diagnostic kit proposed by the present invention. The diagnostic detection is more convenient, the sample collection is less difficult, the pain and psychological pressure of patients can be reduced, and it has important practical application value. Description of the Drawings

[0019] Figure 1 It is the result of Western blot analysis of anti-DNA antibody immunoprecipitating pathological tau protein in AD brain homogenate.

[0020] Figure 2 It is the experimental result of RT-QuIC analysis of tau seeding activity immunoprecipitated by anti-DNA antibody from brain tissue and plasma specimens of AD patients. Detailed Implementation Modes

[0021] In order to better explain the present invention for easy understanding, the present invention will be described in detail below in conjunction with the drawings and through specific implementation modes. Example 1

[0022] This example is a detection method that enriches tau protein with anti-DNA antibody and combines with Western blot experiment. The experiment can be operated according to the following method. For the parts not specifically described, refer to the records of relevant literatures (Zou et al., 2004; Yuan et al, 2006) or the conventional operations in the field. The reagents not specifically described are all common commercially available reagent products. The experimental method is as follows:

[0023] (1) Couple two anti-DNA antibodies, Anti-DAN-Ab1 [DNA antibody (ET844.3.1), Lot #: MA1-10611, Thermo, USA] and Anti-DNA-Ab2 [(DNA antibody (ET844.2), Lot #: MA1-10600, Thermo, USA] (100 μg purified IgG) with tosyl-activated superparamagnetic beads (M-280 Dynabeads, Dynal) in 1 ml PBS at 37°C for 20 hours with shaking. Incubate the coupled magnetic beads of anti-DNA antibody with 0.1% BSA and 200 mM Tris-HCl to prevent non-specific binding. The prepared coupled magnetic beads of anti-DNA antibody can be stably stored at 4°C for at least 3 months.

[0024] (2)Prepare brain homogenate (10%) in lysis buffer at 4 °C: Use 100 mM NaCl, 10 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, 10 mM Tris-HCl, pH 7.5, and then centrifuge at 5000 g for 10 min at 4 °C to remove cell debris to obtain brain homogenate. Among them, brain samples are from pathologically confirmed non-AD and AD, and 3 parallels are set for each group.

[0025] (3)Immunoprecipitation IP experiment. Take 10 μL of brain homogenate and conjugate beads of anti-DNA antibody and incubate in 1 mL of lysis buffer (3% Tween-20 and 3% NP-40, PBS, PH 7.5) at room temperature with constant mixing for 18 h (overnight).

[0026] (4)Attract the conjugate magnetic beads of anti-DNA antibody to the side wall of the plastic test tube by external magnetic attraction to remove all unbound antibody solutions. Wash three times in washing buffer (3% Tween-20 and 3% NP-40, PBS, pH 7.5), and elute the immunoprecipitated tau protein mixture from the anti-DNA antibody magnetic beads with 20 μL of eluent, add 3 μL of pH neutralization solution to adjust the pH to 7.5, and store for later use. The eluted tau protein mixture is subjected to Western blotting with anti-phosphorylated tau protein antibody pTau396 (ps396).

[0027] The experimental results are as Figure 1 shown, which are the experimental results of Western blot analysis of immunoprecipitating pathological tau protein in AD and non-AD brain homogenates with anti-DNA antibody. In this experiment, two anti-DNA antibodies, Anti-DAN-Ab1 (denoted as Anti-DAN1 in the figure) and Anti-DAN-Ab2 (denoted as Anti-DAN2 in the figure), were used to immunoprecipitate tau protein from pathologically confirmed non-Alzheimer's disease non-AD (n = 3) and Alzheimer's disease AD (n = 3) brain homogenates respectively. The experimental results show that immunoprecipitated Tau protein was only detected in AD brain homogenates by Western blotting, while tau protein was not detected in non-AD brain homogenates. This indicates that the anti-DNA antibody can bind to pathological tau protein, and the protein blot band has a large and clear gray scale. Example 2

[0028] This example is a method for enriching tau protein with anti-DNA antibody and combining in vitro RT-QuIC for amplified detection. The experiment can be operated according to the following method. For parts not specifically described, refer to the relevant literature records (Wu et al., 2022; Wang et al, 2020) or the conventional operations in this field. Reagents not specifically described are all common commercially available reagent products. The experimental method is as follows:

[0029] (1) Couple two anti-DNA antibodies, Anti-DAN-Ab1 and Anti-DNA-Ab2 (100 μg of purified IgG), to tosyl-activated superparamagnetic beads (M-280 Dynabeads, Dynal) in 1 ml of PBS at 37°C for 20 hours with shaking. Incubate the coupled beads of anti-DNA antibodies with 0.1% BSA and 200 mM Tris-HCl to block non-specific binding. The prepared coupled beads of anti-DNA antibodies can be stably stored at 4°C for at least 3 months.

[0030] (2) Prepare brain tissue homogenate (10%) in lysis buffer at 4°C: Use 100 mM NaCl, 10 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, 10 mM Tris-HCl, pH 7.5, and then centrifuge at 5000g for 10 min at 4°C to remove cell debris to obtain brain homogenate. Among them, brain tissue samples are from pathologically confirmed non-AD and AD, with one parallel set for each group.

[0031] (3) Immunoprecipitation IP experiment. Take 10 μL of brain homogenate or 100 μL of plasma and the coupled beads of anti-DNA antibodies and incubate them in 1 mL of lysis buffer (3% Tween-20 and 3% NP-40, PBS, pH 7.5) at room temperature with constant mixing for 18 h (overnight).

[0032] (4) Attract the coupled magnetic beads of anti-DNA antibodies to the side wall of a plastic test tube by external magnetism to remove all unbound antibody solutions. Wash three times in wash buffer (3% Tween-20 and 3% NP-40, PBS, pH 7.5), and elute the immunoprecipitated tau protein mixture from the anti-DNA antibody magnetic beads with 20 μL of eluent. Add 3 μL of pH neutralization solution to adjust the pH to 7.5 and store for later use. The eluted tau protein mixture is used for RT-QuIC amplification detection.

[0033] (5) Slowly add the tau protein mixture to the RT-QuIC reaction solution to a final concentration of 12 μM to obtain an RT-QuIC reaction mixture containing tau protein. Among them, the RT-QuIC reaction solution contains 10 mM HEPES, 400 mM NaCl, and 10 μM thioflavin T (ThT), and is filtered through a 0.22 μm filter before use. Load 98 µL of the RT-QuIC reaction mixture containing tau protein into each well of a 96-well plate (Nunc).

[0034] (6) Dilute the brain homogenate or plasma 1:200 with a specimen diluent [10 mM HEPES, 0.05% wild-type mouse brain homogenate (as a non-specific binding inhibitor), 1× N2 supplement Gibco]. After centrifugation (5000 g, 4 °C, 5 min), take 2 μL of the supernatant and add it to 98 μL of the RT-QuIC reaction well. Each specimen has four replicate wells. The 96-well plate is sealed with a plastic film (Nalgene Nunc International) and incubated on a BMG FLUOstar microplate reader (BMG Labtech). Each cycle includes 1 min of oscillation (500 rpm) and 1 min of incubation. The microplate reader measures the fluorescence intensity readings every 45 min at an excitation of 450 ± 10 nm and an emission of 480 ± 10 nm. The fluorescence result for each specimen is the average fluorescence value from the four replicate wells. The threshold is the mean fluorescence of the normal control plus four standard deviations of it. If the ThT fluorescence signal of the specimen exceeds the threshold, it is considered positive.

[0035] As Figure 2 shown, immunoprecipitate tau protein from pathologically confirmed non-AD (n = 1) and AD (n = 1) brain homogenates with two anti-DNA antibodies, Anti-DAN-Ab1 and Anti-DNA-Ab2 ( Figure 2 A, 2B). The eluted tau protein complex is diluted at different concentrations (10 -3 to 10 -7 ) and analyzed by ultrasensitive real-time quaking-induced conversion (RT-QuIC). Among them, as shown in Figure 2 A and B, the tau seeding activity of the AD brain homogenate is significantly higher than that of the non-AD control specimen (see 10 -5 to 10 -7 dilution). As shown in Figure 2 C: Immunoprecipitate tau protein from clinically diagnosed non-AD (n = 1) and AD (n = 1) plasma with Anti-DNA1 antibody. The eluted tau protein complex is analyzed by ultrasensitive real-time quaking-induced conversion (RT-QuIC). The tau seeding activity of the AD plasma (blue) is significantly higher than that of the non-AD control specimen (red). Thus, anti-DNA antibodies can be used to enrich tau protein, which can greatly improve the sensitivity and specificity of the AD early diagnosis technology using plasma as the detection sample, providing technical support for non-invasive early diagnosis of AD.

[0036] Finally, 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 foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of anti-DNA antibody in the preparation of an early diagnosis kit for Alzheimer's disease, characterized in that, The anti-DNA antibody serves as an enrichment medium for tau protein; the anti-DNA antibody is the anti-DNA antibody with clone number ET844.3.1 or the anti-DNA antibody with clone number ET844.2 from Thermo Company.

2. The application according to claim 1, characterized in that, The diagnostic kit further includes a detection reagent for pathogenic misfolded tau protein.

3. The application according to claim 2, characterized in that, The detection reagent includes a detection antibody for pathogenic misfolded tau protein.

4. The application according to claim 1, wherein The detection sample of the diagnostic kit is peripheral blood, saliva, tears, feces, sweat or urine.

5. The application according to claim 1, wherein The detection sample of the diagnostic kit is skin tissue, submandibular gland or oral mucosa.

6. The application according to claim 1, characterized in that, The diagnostic kit includes magnetic beads, a tau protein detection antibody, recombinant tau protein, a fluorescent label and a buffer.

7. An early diagnosis kit for Alzheimer's disease, characterized in that, It includes a tau protein enrichment medium and a tau protein detection antibody; the tau protein enrichment medium is an anti-DNA antibody, and the anti-DNA antibody is the anti-DNA antibody with clone number ET844.3.1 or the anti-DNA antibody with clone number ET844.2 from Thermo Company.

8. The early diagnosis kit for Alzheimer's disease according to claim 7, characterized in that, It further includes magnetic beads, a fluorescent label and a buffer.

9. Application of magnetic beads conjugated with anti-DNA antibody in the preparation of an early diagnosis kit for Alzheimer's disease, wherein the anti-DNA antibody serves as an enrichment medium for tau protein; the anti-DNA antibody is the anti-DNA antibody with clone number ET844.3.1 or the anti-DNA antibody with clone number ET844.2 from Thermo Company.

10. Application of anti-DNA antibody as an enrichment medium in the preparation of a kit for detecting tau protein expression level, wherein the anti-DNA antibody is the anti-DNA antibody with clone number ET844.3.1 or the anti-DNA antibody with clone number ET844.2 from Thermo Company.

Images