Single molecule detection method of Alzheimer's disease marker Abeta42

Abstract

The invention discloses a single molecule detection method of an Alzheimer's disease marker A beta 42, and belongs to the technical field of biological detection. A beta 42 is detected by using microspheres, an A beta 42 antibody 1, an A beta 42 antibody 2, a coupling buffer solution, a microsphere washing solution, a microsphere preserving solution, a microsphere confining liquid, an elisa plate, a coating solution and a confining liquid, and finally, an A beta 42 marker is digitally imaged and analyzed by using a fluorescence microscopic imaging technology, so that quantitative detection is performed. The method has the advantages of ultrahigh sensitivity (single molecule level-fg / ml), strong specificity, high sensitivity, low cost, medium and high throughput and the like, and can be used for early screening, early warning and clinical auxiliary diagnosis of AD.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a single molecule detection method for Alzheimer's disease marker Aβ42. Background Art

[0002] Against the backdrop of an aging global population, neurodegenerative diseases such as Alzheimer's disease (AD) are gradually becoming an "invisible killer" threatening public health. Alzheimer's disease is a neurodegenerative disease with an insidious onset and progressive development. It causes patients to experience symptoms such as memory loss, cognitive impairment, and mental and behavioral abnormalities, which seriously affect the quality of life of patients. Early detection and timely intervention and treatment of Alzheimer's disease, such as drug therapy, cognitive training, and life skills training, can effectively improve patients' cognitive functions and living abilities, alleviate discomfort symptoms, and thus improve their quality of life. At the same time, early detection means that intervention can be carried out earlier, and the progression of the disease can be slowed down through drugs, rehabilitation training, and other means. This can not only prolong the patient's autonomous living time, but also maintain the patient's cognitive function and daily living ability to a certain extent. Early detection of Alzheimer's disease can also provide rich data support for scientific research. In-depth research on patients' clinical manifestations, imaging characteristics, biomarkers, etc. will help reveal the pathogenesis of Alzheimer's disease and provide a scientific basis for the development of new treatments and drugs. It is very important to develop AD blood marker detection technology with ultra-high sensitivity (single molecule level ~fg / ml), strong specificity, high sensitivity, low cost, and medium to high throughput for early screening, warning and clinical auxiliary diagnosis of AD. Summary of the invention

[0003] The present invention provides a single molecule detection method for Alzheimer's disease marker Aβ42, and the specific steps are as follows: S1. Activate microspheres. Take 0.05mL of microsphere (1% solid content) suspension into a centrifuge tube with 1mL coupling buffer, mix by ultrasonication, and centrifuge to remove the supernatant; then add 1mL of microsphere coupling buffer, mix by ultrasonication, and centrifuge to remove the supernatant; then add 1mL of microsphere coupling buffer, mix by ultrasonication, and then add 3.5μL of EDC solution, vortex mix, then add 33μL of NHS solution, and mix by ultrasonication; finally, place the centrifuge tube on a turntable to avoid light, activate, centrifuge to remove the supernatant, then add 1.5mL of coupling buffer, mix by ultrasonication, and centrifuge to remove the supernatant; finally, add 1.5mL of coupling buffer to obtain suspension A.

[0004] S2. Coupling microspheres with antibodies. First, add 0.75 mL of coupling buffer to the suspension A in S1 and mix by ultrasonication; add 50 μg of Aβ42 labeled antibody to 0.25 mL of coupling buffer to prepare coupling solution; then add the coupling solution with antibody to the mixed microspheres and vortex to mix; finally, place the centrifuge tube on a turntable for coupling.

[0005] S3. Block and store the microspheres. Add 0.5 mL of microsphere blocking solution to the centrifuge tube and vortex to mix; place the centrifuge tube on a turntable, 37°C, 40 r / min, block for 1 hour in the dark, centrifuge to remove the supernatant; add 1.5 mL of microsphere washing solution, ultrasonically mix, and centrifuge to remove the supernatant; add 1.5 mL of microsphere washing solution and wash once; finally, add 0.5 mL of microsphere preservation solution (microsphere final concentration 1 mg / mL) to the centrifuge tube, ultrasonically mix, and store at 2-8°C in the dark for later use.

[0006] S4. Enzyme-labeled plate coated with antibodies. Coat with Aβ42 antibody at a concentration of 0.1-0.5ug / T, 100ul / T coating solution, static coating at 2-8℃ for 16±1h, wash 5 times with PBS+TWEEN-20 (0.05%wt), and pat dry. Then add 150ul / well blocking solution, block at 37℃ for 2h, wash 5 times with PBS+TWEEN-20 (0.05%wt), and pat dry.

[0007] S5. Prepare immune sandwich. Add 45 µL of calibrator / quality control / serum sample to the ELISA plate coated with S3 in turn. After shaking at 37℃ for 0.5 hours, wash 5 times with PBS+TWEEN-20 (0.05%wt) and pat dry. Dilute the fluorescent microsphere-labeled antibody 20 times with microsphere storage solution and set aside. Then add 100 µL of the fluorescent microsphere-labeled antibody diluted 20 times to the ELISA plate, shake at 37℃ for 0.5 hours, wash 5 times with PBS+TWEEN-20 (0.05%wt) and pat dry.

[0008] S6. Microscopic counting: Place the ELISA plate after patting dry in S5 under a fluorescence microscope and count the fluorescent particles in each hole one by one.

[0009] Preferably, in S1-S5, the coupling buffer formula is: 10 mM MES (pH 6.2±0.05), containing 0.05% ProClin 300.

[0010] Preferably, in S1-S5, the EDC solution has a concentration of 10 mg / mL and is prepared with coupling buffer and used immediately after preparation.

[0011] Preferably, in S1-S5, the concentration of the NHS solution is 10 mg / mL, and it is prepared with coupling buffer and used immediately after preparation.

[0012] Preferably, in S1-S5, the microsphere blocking solution has a formula of: borate buffer (5 mM boric acid, 11.2 mM sodium tetraborate decahydrate, 0.05% Tween-20, pH 9.0±0.05), 1% BSA, and 0.24% ethanolamine.

[0013] Preferably, in S1-S5, the microsphere washing solution has a formula of: 50 mM Tris (PH 8.0±0.05), 0.5% BSA, 0.05% Tween-20, and 0.03% ProClin 300.

[0014] Preferably, in S1-S5, the microsphere preservation solution has a formula of: 25 mM Tris-HCl (PH 7.2±0.05), 150 mM NaCl 0.05% Tween-20, 1% BSA, 5% trehalose, and 0.1% ProClin 300.

[0015] Preferably, the centrifugation conditions in S1-S5 are centrifugation at 15°C and 20,000 g for 10 min.

[0016] Preferably, in S1, the activation conditions are 37° C., 40 r / min, and activation in the dark for 15-30 min.

[0017] Preferably, in S2, the coupling conditions are 37° C., 40 r / min, and light-protected coupling for 2 h.

[0018] Preferably, in S4, the coating solution formula is: sodium carbonate (Na2CO3) 0.1 M, sodium bicarbonate (NaHCO3) 0.1 M, pH 9.6.

[0019] Preferably, in S4, the blocking solution formula is: 0.05M PBS (pH 7.4) + 1% BSA + 0.1% ProClin 300 + 0.1% protein protectant aminopyrine.

[0020] Preferably, the magnification of the fluorescence microscope in S6 is 10-100X.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a highly advanced Alzheimer's disease (AD) blood marker Aβ42 detection technology. The technology achieves detection sensitivity at the single molecule level (fg / ml level), and it can accurately distinguish AD-related biomarkers from markers of other diseases or normal physiological states. The strong specificity reduces the risk of misdiagnosis and improves the accuracy of diagnosis. The technology can detect tiny changes in biomarkers, which is crucial for early detection of the disease. The technology is relatively low-cost, reduces the economic burden on patients, improves the accessibility of medical services, and can process a large number of samples in a short time. This is particularly important for large-scale screening and monitoring projects, improving detection efficiency and shortening the detection cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the detection standard curve of Aβ42 in Example 1. DETAILED DESCRIPTION

[0023] The microspheres used in the following examples are from Beike Nano, catalog number BK2021081104; the Aβ42 antibody linked to the microspheres is from Shanghai Bohu Biotechnology Co., Ltd., product model BH-K016304; the Aβ42 antibody coated with the ELISA plate is from Merck, catalog number ABN1665.

[0024] Example 1 This embodiment provides a single molecule detection technology for the Alzheimer's disease marker Aβ42, the method is as follows: (1) Take 0.05 mL of microsphere (1% solid content) suspension into a 2 mL centrifuge tube with 1 mL of coupling buffer, mix by ultrasonication, centrifuge at 15°C and 20,000 g for 10 min, and remove the supernatant; then add 1 mL of microsphere coupling buffer, mix by ultrasonication, centrifuge at 15°C and 20,000 g for 10 min, and remove the supernatant; then add 1 mL of microsphere coupling buffer, mix by ultrasonication, add 3.5 μL of EDC solution, vortex mix, then add 33 μL of NHS solution, and mix by ultrasonication; finally, place the centrifuge tube on a turntable, incubate at 37°C and 40 r / min, activate for 15-30 min in the dark, centrifuge at 15°C and 20,000 g for 10 min, and remove the supernatant. Then add 1.5 mL of coupling buffer, mix by ultrasonication, centrifuge at 15°C and 20,000 g for 10 min, and remove the supernatant; finally, add 1.5 mL of coupling buffer and wash once; The coupling buffer formula is: 10 mM MES (pH 6.2 ± 0.05), containing 0.05% ProClin 300; The EDC solution has a concentration of 10 mg / mL and is prepared with coupling buffer and used immediately after preparation; The NHS solution has a concentration of 10 mg / mL and is prepared with coupling buffer and used immediately after preparation; The microsphere washing solution formula is: 50mM Tris (pH 8.0±0.05), 0.5% BSA, 0.05% Tween-20, 0.03% ProClin 300; (2) Add 0.75 mL of coupling buffer to the centrifuge tube and mix by ultrasonication. Add 50 μg of the antibody to be labeled to 0.25 mL of coupling buffer. Then add the antibody-containing coupling solution to the mixed microspheres. Vortex mix. Place the centrifuge tube on a turntable and incubate at 37°C, 40 rpm, away from light for 2 h. (3) Add 0.5 mL of microsphere blocking solution to the centrifuge tube and vortex mix; place the centrifuge tube on a turntable, incubate at 37°C, 40 r / min, and block for 1 h in the dark. Centrifuge at 15°C and 20,000 g for 10 min, and remove the supernatant; add 1.5 mL of microsphere washing solution, mix by ultrasonication, centrifuge at 15°C and 20,000 g for 10 min, and remove the supernatant; add 1.5 mL of microsphere washing solution and wash once; finally, add 0.5 mL of microsphere preservation solution (microsphere final concentration 1 mg / mL) to the centrifuge tube, mix by ultrasonication, and store at 2-8°C in the dark for later use; The microsphere blocking solution formula is: borate buffer (5mM boric acid, 11.2mM sodium tetraborate decahydrate, 0.05% Tween-20, pH 9.0±0.05), 1% BSA, 0.24% ethanolamine; The microsphere preservation solution formula is: 25mM Tris (pH 7.2±0.05), 150mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, 0.1% ProClin 300; (4) Coating was performed at an Aβ42 antibody concentration of 0.1-0.5ug / T, 100ul / T coating solution, static coating at 2-8℃ for 16±1h, washed 5 times with PBS+TWEEN-20 (0.05%wt), and patted dry. Then 150ul / well blocking solution was added, blocked at 37℃ for 2h, washed 5 times with PBS+TWEEN-20 (0.05%wt), and patted dry; The coating solution formula is: sodium carbonate (Na2CO3) 0.1 M, sodium bicarbonate (NaHCO3) 0.1 M, pH 9.6; The blocking solution formula is: 0.05M PBS (pH7.4) + 1% BSA + 0.1% ProClin 300 + 0.1% protein protectant; (5) Add 45 µL of calibrator / quality control / serum sample to different wells of the coated ELISA plate. After shaking at 37°C for 0.5 hours, wash 5 times with PBS+TWEEN-20 (0.05%wt) and pat dry. Dilute the fluorescent microsphere-labeled antibody 20 times with microsphere storage solution and set aside. Then add 100 µL of the fluorescent microsphere-labeled antibody diluted 20 times to the ELISA plate, shake at 37°C for 0.5 hours, wash 5 times with PBS+TWEEN-20 (0.05%wt) and pat dry. (6) Place the ELISA plate that has been patted dry under a fluorescence microscope and magnify it at 50X to count the fluorescent particles in each hole. The specific detection curve is shown in Figure 1 , Figure 1 The antigen concentrations corresponding to the 7 points in the graph are 100, 33.33, 11.11, 3.7, 1.23, 0.41, and 0 pg / mL, respectively. Figure 1 It can be seen that the linear range is 0.46-1000 pg / ml (R2 ≥0.95), the sample type is serum, and the detection limit is 460 fg / mL.

[0025] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A product for single-molecule detection of Alzheimer's disease marker Aβ42, characterized in that: The product contains microspheres, Aβ42 antibody 1, Aβ42 antibody 2, coupling buffer, microsphere washing solution, microsphere preservation solution, microsphere blocking solution, ELISA plate, coating solution and blocking solution.

2. The product according to claim 1, characterized in that The Aβ42 antibody 1 is from Shanghai Bohu Biotechnology Co., Ltd., and the product model is BH-K016304.

3. The product according to claim 2, characterized in that The Aβ42 antibody 2 was from Merck, with the catalog number ABN1665.

4. The product according to any one of claims 1 to 3, characterized in that: The product is a test kit.

5. The product according to claim 4, characterized in that The coupling buffer formula is: 10 mM MES, pH 6.2±0.05, 0.05% ProClin 300.

6. The product according to claim 5, characterized in that The formula of the microsphere washing solution is: 50mM Tris, pH8.0±0.05, 0.5% BSA, 0.05% Tween-20, 0.03% ProClin 300.

7. The product according to claim 6, characterized in that The microsphere blocking solution formula is: borate buffer, 1% BSA, 0.24% ethanolamine; the borate buffer is 5mM boric acid, 11.2mM sodium tetraborate decahydrate, 0.05% Tween-20, pH 9.0±0.

05.

8. The product according to claim 7, characterized in that The microsphere preservation solution formula is: 25mM Tris-HCl, pH 7.2±0.05, 150mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, and 0.1% ProClin 300.

9. The product according to claim 8, characterized in that The coating solution formula is: 0.1 M sodium carbonate, 0.1 M sodium bicarbonate, pH 9.6; the blocking solution formula is: 0.05 M PBS, pH 7.4, 1% BSA, 0.1% ProClin 300, 0.1% aminopyrine.

10. Use of the product according to any one of claims 1-3 and 5-9 in the preparation of a product for single-molecule detection of Aβ42, a marker of Alzheimer's disease.

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