A method for single molecule detection of Alzheimer's disease marker Aβ42

Abstract

The application discloses a kind of single molecule detection methods of Alzheimer's disease marker A beta 42, belong to biological detection technical field.The application utilizes microsphere, A beta 42 antibody 1, A beta 42 antibody 2, coupling buffer, microsphere washing liquid, microsphere preservative, microsphere blocking liquid, enzyme label plate, coating fluid and blocking fluid to detect A beta 42, finally through fluorescence microscopic imaging technology, A beta 42 marker is digitally imaged and analyzed, and then quantitative detection is carried out, the method has ultra-high sensitivity (single molecule level ~ fg / ml), strong specificity, high sensitivity, low cost, medium-high throughput and the like advantages, can be used for early screening, early warning and clinical auxiliary diagnosis of AD.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and in particular relates to a single-molecule detection method for the Alzheimer's disease biomarker Aβ42. Background Technology

[0002] Against the backdrop of a global aging population, neurodegenerative diseases such as Alzheimer's disease (AD) are gradually becoming "silent killers" threatening public health. Alzheimer's disease is an insidious, progressive neurodegenerative disease that causes symptoms such as memory loss, cognitive impairment, and abnormal mental and behavioral states, severely impacting patients' quality of life. Early detection and timely intervention, such as medication, cognitive training, and daily living skills training, can effectively improve patients' cognitive function and daily living abilities, alleviate symptoms, and thus improve their quality of life. Early detection also means earlier intervention, slowing the progression of the disease through medication and rehabilitation training. This not only extends the patient's independent living time but also, to some extent, maintains their cognitive function and daily living abilities. Early detection of Alzheimer's disease also provides rich data support for scientific research. In-depth studies of patients' clinical manifestations, imaging characteristics, and biomarkers help to reveal the pathogenesis of Alzheimer's disease, providing a scientific basis for developing new treatments and drugs. Developing blood biomarker detection technologies for Alzheimer's disease (AD) with ultra-high sensitivity (single-molecule level ~fg / ml), high specificity, high sensitivity, low cost, and medium to high throughput is of great importance for the early screening, early warning, and clinical auxiliary diagnosis of AD. Summary of the Invention

[0003] This invention provides a single-molecule detection method for the Alzheimer's disease biomarker Aβ42, the specific steps of which are as follows:

[0004] S1. Activation of microspheres. Take 0.05 mL of microsphere (1% solids) suspension into a centrifuge tube containing 1 mL of coupling buffer, sonicate to mix, and centrifuge to remove the supernatant; then add 1 mL of microsphere coupling buffer, sonicate to mix, and centrifuge to remove the supernatant; add another 1 mL of microsphere coupling buffer, sonicate to mix, add 3.5 μL of EDC solution, vortex to mix, then add 33 μL of NHS solution, and sonicate to mix; finally, place the centrifuge tube on a turntable to activate in the dark, centrifuge to remove the supernatant, add another 1.5 mL of coupling buffer, sonicate to mix, and centrifuge to remove the supernatant; finally, add another 1.5 mL of coupling buffer to obtain suspension A.

[0005] S2. Conjugation of microspheres and antibodies. First, add suspension A from S1 to 0.75 mL of conjugation buffer and sonicate to mix. Add 50 μg of Aβ42-labeled antibody to 0.25 mL of conjugation buffer to prepare the conjugation solution. Then, add the conjugation solution containing the antibody to the mixed microspheres and vortex to mix. Finally, place the centrifuge tube on a turntable for conjugation.

[0006] S3. Blocking and preserving microspheres. Add 0.5 mL of microsphere blocking solution to a centrifuge tube and vortex to mix. Place the centrifuge tube on a turntable and incubate at 37°C, 40 rpm, and in the dark for 1 hour, then centrifuge to remove the supernatant. Add 1.5 mL of microsphere washing solution, sonicate to 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 (final microsphere concentration 1 mg / mL) to the centrifuge tube, sonicate to mix, and store at 2-8°C in the dark for later use.

[0007] S4. Coating the microplate with antibody. Coat with 0.1-0.5 μg / T of Aβ42 antibody using 100 μL / T of coating buffer. Incubate at 2-8°C for 16 ± 1 h. Wash 5 times with PBS + TWEEN-20 (0.05% wt) and blot dry. Then add 150 μL / well blocking buffer and block at 37°C for 2 h. Wash 5 times with PBS + TWEEN-20 (0.05% wt) and blot dry.

[0008] S5. Preparation of the immunosandwich. Add 45 µL of calibrator / control sample / serum sample sequentially to the ELISA plate coated in S3. Incubate at 37°C with shaking for 0.5 hours, then wash 5 times with PBS + TWEEN-20 (0.05% wt) and pat dry. Dilute the fluorescent microsphere-labeled antibody 20-fold with microsphere preservation solution and set aside. Then add 100 µL of the 20-fold diluted fluorescent microsphere-labeled antibody to the ELISA plate, incubate at 37°C with shaking for 0.5 hours, then wash 5 times with PBS + TWEEN-20 (0.05% wt) and pat dry.

[0009] S6. Microscopic Counting. After patting the S5 plate dry, place it under a fluorescence microscope and count the fluorescent particles in each well in turn.

[0010] Preferably, in S1-S5, the coupling buffer formulation is: 10mM MES (pH 6.2±0.05) containing 0.05% ProClin 300.

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

[0012] Preferably, in S1-S5, the concentration of the NHS solution is 10 mg / mL, prepared with coupling buffer, and used immediately after preparation.

[0013] Preferably, in S1-S5, the microsphere blocking solution is formulated as follows: borate buffer (5mM boric acid, 11.2mM sodium tetraborate decahydrate, 0.05% Tween-20, pH 9.0±0.05), 1% BSA, and 0.24% ethanolamine.

[0014] Preferably, in S1-S5, the microsphere washing solution formulation is: 50mM Tris (pH 8.0±0.05), 0.5% BSA, 0.05% Tween-20, and 0.03% ProClin 300.

[0015] Preferably, in S1-S5, the microsphere preservation solution is formulated as follows: 25mM Tris-HCl (pH 7.2±0.05), 150mM NaCl 0.05% Tween-20, 1% BSA, 5% trehalose, and 0.1% ProClin 300.

[0016] Preferably, the centrifugation conditions in S1-S5 are centrifugation at 15℃ and 20000g for 10 min.

[0017] Preferably, in S1, the activation conditions are 37°C, 40 r / min, and activation in the dark for 15-30 min.

[0018] Preferably, in S2, the coupling conditions are 37°C, 40 r / min, and coupling in the dark for 2 hours.

[0019] Preferably, in S4, the coating solution is formulated as follows: sodium carbonate (Na2CO3) 0.1 M, sodium bicarbonate (NaHCO3) 0.1 M, pH 9.6.

[0020] Preferably, in S4, the blocking solution is formulated as follows: 0.05M PBS (pH 7.4) + 1% BSA + 0.1% ProClin 300 + 0.1% protein protectant aminopyrine.

[0021] Preferably, the magnification of the fluorescence microscope in S6 is 10-100X.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention discloses a highly advanced blood biomarker detection technology for Alzheimer's disease (AD), Aβ42. This technology achieves detection sensitivity at the single-molecule level (fg / ml), and can accurately distinguish AD-related biomarkers from those of other diseases or normal physiological states. Its high specificity reduces the risk of misdiagnosis and improves diagnostic accuracy. This technology can detect minute changes in biomarkers, which is crucial for early disease detection. The technology is relatively low-cost, reducing the financial burden on patients and improving accessibility to healthcare services. Furthermore, it can process large numbers of samples in a short time. This is particularly important for large-scale screening and monitoring programs, improving detection efficiency and shortening the testing cycle. Attached Figure Description

[0024] Figure 1 This is the detection standard curve for Aβ42 in Example 1. Detailed Implementation

[0025] 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; and the Aβ42 antibody coating the ELISA plate is from Merck, catalog number ABN1665.

[0026] Example 1

[0027] This embodiment provides a single-molecule detection technique for the Alzheimer's disease biomarker Aβ42, the method of which is as follows:

[0028] (1) Take 0.05 mL of microsphere (1% solid content) suspension into a 2 mL centrifuge tube containing 1 mL of coupling buffer, sonicate to mix, centrifuge at 15℃ and 20000 g for 10 min, and remove the supernatant; then add 1 mL of microsphere coupling buffer, sonicate to mix, centrifuge at 15℃ and 20000 g for 10 min, and remove the supernatant; then add 1 mL of microsphere coupling buffer, sonicate to mix, then add 3.5 μL of EDC solution, vortex to mix, then add 33 μL of NHS solution, sonicate to mix; finally place the centrifuge tube on a turntable, activate at 37℃ and 40 r / min in the dark for 15-30 min, then centrifuge at 15℃ and 20000 g for 10 min, and remove the supernatant. Add 1.5 mL of coupling buffer, sonicate to mix, centrifuge at 15 °C and 20000 g for 10 min, and remove the supernatant; finally, add 1.5 mL of coupling buffer and wash once.

[0029] The coupling buffer formulation is: 10 mM MES (pH 6.2 ± 0.05), containing 0.05% ProClin 300;

[0030] The EDC solution has a concentration of 10 mg / mL, is prepared with coupling buffer, and should be used immediately after preparation.

[0031] The NHS solution concentration is 10 mg / mL, prepared with coupling buffer, and used immediately after preparation.

[0032] The microsphere washing solution formulation is as follows: 50 mM Tris (pH 8.0 ± 0.05), 0.5% BSA, 0.05% Tween-20, and 0.03% ProClin 300;

[0033] (2) Add 0.75 mL of coupling buffer to the centrifuge tube, sonicate to mix, 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 to mix, place the centrifuge tube on a turntable, and couple at 37℃, 40 r / min, in the dark for 2 h.

[0034] (3) Add 0.5 mL of microsphere blocking solution to the centrifuge tube and vortex to mix. Place the centrifuge tube on a turntable and seal it at 37℃, 40 r / min, and in the dark for 1 h. Centrifuge at 15℃ and 20000 g for 10 min and remove the supernatant. Add 1.5 mL of microsphere washing solution, sonicate to mix, and centrifuge at 15℃ and 20000 g for 10 min to remove the supernatant. Add 1.5 mL of microsphere washing solution and wash once. Finally, add 0.5 mL of microsphere preservation solution (final concentration of microspheres 1 mg / mL) to the centrifuge tube, sonicate to mix, and store at 2-8℃ in the dark for later use.

[0035] The microsphere blocking solution is formulated as follows: borate buffer (5mM boric acid, 11.2mM sodium tetraborate decahydrate, 0.05% Tween-20, pH 9.0±0.05), 1% BSA, 0.24% ethanolamine;

[0036] The microsphere preservation solution was formulated as follows: 25 mM Tris (pH 7.2 ± 0.05), 150 mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, and 0.1% ProClin 300.

[0037] (4) Coating was performed at an Aβ42 antibody concentration of 0.1-0.5 μg / T, using 100 μL / T coating buffer. The coating was allowed to stand at 2-8°C for 16 ± 1 h. The wells were then washed 5 times with PBS + TWEEN-20 (0.05 % wt) and patted dry. Then, 150 μL / well blocking buffer was added, and the wells were blocked at 37°C for 2 h. The wells were then washed 5 times with PBS + TWEEN-20 (0.05 % wt) and patted dry.

[0038] The coating solution formulation is as follows: sodium carbonate (Na2CO3) 0.1 M, sodium bicarbonate (NaHCO3) 0.1 M, pH 9.6;

[0039] The blocking solution formulation is: 0.05M PBS (pH 7.4) + 1% BSA + 0.1% ProClin 300 + 0.1% protein protectant;

[0040] (5) Add 45 µL of calibrator / quality control / serum sample to different wells of the coated ELISA plate. After incubating at 37°C with shaking 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 preservation solution and set aside. Then add 100 µL of the 20-fold diluted fluorescent microsphere-labeled antibody to the ELISA plate, incubate at 37°C with shaking for 0.5 hours, wash 5 times with PBS + TWEEN-20 (0.05 %wt), and pat dry.

[0041] (6) Place the dried ELISA plate under a fluorescence microscope at 50X magnification and count the fluorescent particles in each well. See the specific detection curve for details. Figure 1 , Figure 1 The antigen concentrations corresponding to the seven points were 100, 33.33, 11.11, 3.7, 1.23, 0.41, and 0 pg / mL, respectively. Figure 1 The linear range is 0.46-1000 pg / ml (R2 ≥0.95), the sample type is serum, and the limit of detection is 460 fg / mL.

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a product for detecting Aβ42 in the preparation of a single-molecule product for detecting Alzheimer's disease, characterized in that, The product contains microspheres, Aβ42 antibody 1, Aβ42 antibody 2, conjugation buffer, microsphere washing solution, microsphere preservation solution, microsphere blocking solution, ELISA plate, coating solution, and blocking solution; The detection includes the following steps: S1. Activated microspheres: Take 0.05 mL of microspheres, 1% solids content, and suspend them in a centrifuge tube containing 1 mL of coupling buffer. Mix well by sonication and centrifuge to remove the supernatant. Then add 1 mL of microsphere coupling buffer, mix well by sonication and centrifuge to remove the supernatant. Add another 1 mL of microsphere coupling buffer, mix well by sonication, add 3.5 μL of EDC solution, vortex to mix, and then add 33 μL of NHS solution and sonicate to mix. Finally, place the centrifuge tubes on a turntable to activate them in the dark, centrifuge to remove the supernatant, add 1.5 mL of coupling buffer, sonicate to mix, centrifuge to remove the supernatant, and finally add 1.5 mL of coupling buffer to obtain suspension A. S2. Conjugated microspheres and antibodies: First, add suspension A from S1 to 0.75 mL of coupling buffer and sonicate to mix. Then, add 150 μg of Aβ42 antibody to 0.25 mL of coupling buffer to make the coupling solution. Next, add the coupling solution containing the antibody to the mixed microspheres and vortex to mix. Finally, place the centrifuge tube on a turntable for coupling. S3. Sealed preservation of microspheres: Add 0.5 mL of microsphere blocking solution to the centrifuge tube and vortex to mix. Place the centrifuge tube on a turntable and incubate at 37°C, 40 r / min, and in the dark for 1 hour. Centrifuge to remove the supernatant. Add 1.5 mL of microsphere washing solution, sonicate to 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 to the centrifuge tube to achieve a final microsphere concentration of 1 mg / mL. Sonicate to mix and store at 2-8°C in the dark for later use. S4. Antibody coating on ELISA plate: Coating was performed using Aβ42 antibody at a concentration of 0.1-0.5 μg / T, with 100 μl / T coating buffer. The coating was allowed to stand at 2-8℃ for 16±1 h, followed by washing 5 times with PBS + 0.05% wt TWEEN-20 and patting dry. Then, 150 μL / well blocking buffer was added, and the mixture was blocked at 37℃ for 2 h. The wells were then washed 5 times with PBS + 0.05% wt TWEEN-20 and patted dry. S5. Preparation of the immune sandwich: Add 45µL of calibrator / quality control / serum sample sequentially to the S3-coated ELISA plate; after incubation at 37°C with shaking for 0.5 hours, wash 5 times with PBS + 0.05%wt TWEEN-20 and pat dry; dilute the fluorescent microsphere-labeled antibody 1 20-fold with microsphere preservation solution and set aside; then add 100µL of the 20-fold diluted fluorescent microsphere-labeled antibody 1 to the ELISA plate, incubate at 37°C with shaking for 0.5 hours, wash 5 times with PBS + 0.05%wt TWEEN-20 and pat dry; S6. Microscopic Counting: After the S5 plate was dried, it was placed under a fluorescence microscope, and the fluorescent particles in each well were counted in turn. In S1~S5, the formulation of the microsphere blocking liquid is as follows: Borate buffer, 1% BSA, 0.24% ethanolamine; The borate buffer solution is composed of: 5 mM boric acid, 11.2 mM sodium tetraborate decahydrate, 0.05% Tween-20, pH 9.0 ± 0.05; In S1~S5, the microsphere preservation solution is formulated as follows: 25mM Tris-HCl, 150mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, and 0.1% ProClin 300. The coupling buffer formulation is: 10 mM MES, pH 6.2 ± 0.05, 0.05% ProClin 300; The microsphere washing solution formulation is as follows: 50 mM Tris, pH 8.0 ± 0.05, 0.5% BSA, 0.05% Tween-20, 0.03% ProClin 300; The coating solution is formulated as follows: 0.1 M sodium carbonate, 0.1 M sodium bicarbonate, pH 9.6; the blocking solution is formulated as follows: 0.05 M PBS, pH 7.4, 1% BSA, 0.1% ProClin 300, 0.1% aminopyrine.

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