Digital detection method for Alzheimer's disease protein marker Abeta40 based on fluorescence immunoadsorption

Abstract

The invention discloses a digital detection method for an Alzheimer's disease protein marker A beta 40 based on fluorescence immunoadsorption, and belongs to the technical field of biological detection. According to the present invention, the improved detection antibody fluorescence coding technology is adopted, the plate-type platform is combined, the original operation habit is retained, the fluorescence coding and the digital amplification are performed, the fluorescence microscopic system is adopted to perform digital imaging, and the digital analysis is combined so as to achieve the single molecule level detection of the AD marker A beta 40; compared with a biotin-streptavidin reaction system, the technology has the advantages that the operation steps are reduced, the operation time is shortened, meanwhile, the technology has the advantages of being economical, convenient to operate, high in sensitivity, medium and high in throughput and the like, can be used for detecting Abeta40 in body fluid (such as blood and saliva) except cerebrospinal fluid, and shows huge application potential in early screening of AD.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a digital detection method for the Alzheimer's disease protein marker Aβ40 based on fluorescence immunosorption. Background Art

[0002] The "Clinical Application Guidelines for Humoral Markers of Alzheimer's Disease (2024 Edition)" clearly states that AD has become a huge challenge to our society. Its blood markers are of great significance for large-scale early screening and long-term follow-up. However, the concentration of AD blood markers is much lower than that in cerebrospinal fluid. Therefore, it is imperative to develop ultra-sensitive, highly specific, and medium-high throughput AD blood marker detection technologies. Summary of the Invention

[0003] The technology of the present invention is based on a plate platform design. Through an open detection platform, an improved detection antibody fluorescence encoding technology is combined with a fluorescence microscopy imaging system to directly achieve digital imaging, analysis, and detection of the AD marker Aβ40.

[0004] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0005] A digital detection method for the Alzheimer's disease protein marker Aβ40 based on fluorescence immunosorption, the method comprising the following steps:

[0006] S1: Coating an ELISA plate with an Aβ40 capture antibody;

[0007] S2: Adding a quality control product or body fluid sample containing Aβ40;

[0008] S3: Improved fluorescence encoding technology for the Aβ40 detection antibody;

[0009] S4: Digital imaging and analysis of the ELISA plate.

[0010] Further, S1 is specifically:

[0011] Preparing an ELISA plate coating solution: Sodium carbonate (Na 2 CO 3 ): 0.1 M, Sodium bicarbonate (NaHCO 3 ): 0.1 M, pH 9 - 10.

[0012] Preparing an ELISA plate blocking solution: 0.05 M PBS (pH 7.4) + 1% BSA + 0.1% ProClin300.

[0013] Coat according to the antibody concentration of 0.1 - 0.5 μg / T, with 40 - 100 μL / T of coating solution, and let it stand still at 2 - 8°C for 16 ± 1 h. Wash 5 times with PBS + TWEEN - 20 (0.05% wt), and pat dry; add 150 μL / well of blocking solution, block at 37°C for 2 h, wash 5 times with PBS + TWEEN - 20 (0.05% wt), and pat dry.

[0014] Further, S2 is specifically as follows:

[0015] Add 20 - 70 μL of Aβ40 standard / quality control / humoral sample into different wells of the pre - coated ELISA plate respectively; after reacting on a shaker at 37°C for 0.5 h, wash 5 times with PBS + TWEEN - 20 (0.05% wt), and pat dry.

[0016] Further, S3 is specifically as follows:

[0017] The fluorescence coding technology refers to:

[0018] Prepare the coupling buffer: 10 mM MES (pH 6.2 ± 0.1), containing 0.05% ProClin 300.

[0019] Prepare the EDC solution: 10 mg / mL, prepared with the coupling buffer, and use it immediately after preparation.

[0020] Prepare the NHS solution: 10 mg / mL, prepared with the coupling buffer, and use it immediately after preparation.

[0021] Prepare the microsphere blocking solution: borate buffer (5 mM boric acid, 11.2 mM sodium tetraborate decahydrate, 0.05% Tween - 20, pH 9.0 ± 0.1), 1% BSA, 0.24% ethanolamine.

[0022] Prepare the microsphere washing solution: 50 mM Tris (pH 8.0 ± 0.1), 0.5% BSA, 0.05% Tween - 20, 0.03% ProClin 300.

[0023] Prepare the microsphere storage solution: 25 mM Tris (pH 7.2 ± 0.1), 150 mM NaCl, 0.05% Tween - 20, 1% BSA, 5% trehalose, 0.1% ProClin 300.

[0024] (1) Take 0.05 mL of the microsphere (1% solid content) suspension into a 2 mL centrifuge tube containing 1 mL of coupling buffer, mix well by ultrasonic treatment, centrifuge at 15 °C and 10000 - 20000 g for 10 min, and remove the supernatant; add 1 mL of microsphere coupling buffer, mix well by ultrasonic treatment, centrifuge at 15 °C and 10000 - 20000 g for 10 min, and remove the supernatant; add 1 mL of microsphere coupling buffer, mix well by ultrasonic treatment; add 3.5 μL of EDC solution, mix well by vortex, then add 33 μL of NHS solution, mix well by ultrasonic treatment; place the centrifuge tube on a turntable, activate at 37 °C, 40 r / min, protected from light for 15 - 30 min; centrifuge at 15 °C and 10000 - 20000 g for 10 min, and remove the supernatant; add 1.5 mL of coupling buffer, mix well by ultrasonic treatment, centrifuge at 15 °C and 10000 - 20000 g for 10 min, and remove the supernatant; add 1.5 mL of coupling buffer, and repeat the washing step once.

[0025] (2) Add 0.75 mL of coupling buffer, mix well by ultrasonic treatment; add 30 - 60 μg of the antibody to be labeled to 0.25 mL of coupling buffer to obtain the coupling solution; add the coupling solution containing the antibody to the mixed microspheres, mix well by vortex; place the centrifuge tube on a turntable, couple at 37 °C, 40 r / min, protected from light for 2 h; add 0.5 mL of microsphere blocking solution to the centrifuge tube, mix well by vortex; place the centrifuge tube on a turntable, block at 37 °C, 40 r / min, protected from light for 1 h; centrifuge at 15 °C and 10000 - 20000 g for 10 min, and remove the supernatant; add 1.5 mL of microsphere washing solution, mix well by ultrasonic treatment, centrifuge at 15 °C and 10000 - 20000 g for 10 min, and remove the supernatant; add 1.5 mL of microsphere washing solution, and repeat the washing step once; finally, add 0.5 mL of microsphere storage solution (final microsphere concentration 1 mg / mL) to the centrifuge tube, mix well by ultrasonic treatment. After diluting the antibody-labeled microspheres 20 times with the microsphere storage solution, take 100 μL and add it to an enzyme-linked immunosorbent assay (ELISA) plate containing quality control products, body fluid samples, etc. of Aβ40. After reacting on a shaker at 37 °C for 1 h, wash 5 times with PBS + TWEEN-20 (0.05% wt), and pat dry.

[0026] The improved fluorescence coding technology refers to:

[0027] Biotinylated Aβ40 detection antibody: Dilute the concentration of the Aβ40 detection antibody to 0.5 to 3 mg / mL using PBS buffer, then sequentially add 5 μL of a pure aqueous solution of biotin with a concentration of 5 mg / mL and 100 μL of NaHCO with a concentration of 84 mg / mL 3A pure aqueous solution was placed in a shaker and reacted at room temperature for 1 h. After the reaction ended, 130 μL of the reaction solution was added to a centrifuge tube containing a pre-equilibrated desalting column. The desalting column adsorbed the sample, and it was centrifuged at a centrifugal force of 1000 g for 2 min. Then the adsorption column was removed. After diluting the biotinylated Aβ40 detection antibody 400-fold with PBS buffer, 50 μL was taken and added to an enzyme-linked immunosorbent assay (ELISA) plate containing Aβ40 quality control product, body fluid sample, etc. After reacting on a shaker at 37 °C for 1 h, immediately 50 μL of streptavidin-conjugated fluorescent particles was added. After reacting on a shaker at 37 °C for 1 h, it was washed 5 times with PBS + TWEEN-20 (0.05% wt) and patted dry.

[0028] Specifically, S4 is as follows:

[0029] The ELISA plate was placed on the sample stage of a fluorescence microscopy system which was not equipped with any liquid path detection module. Using a 20x, 40x, or 50x objective lens, digital imaging was performed through transmission or reflection. The standard curve was plotted using image recognition counting and data processing software. The signal values of the quality control product and body fluid sample were substituted into the standard curve to calculate the Aβ40 concentration in the quality control product and body fluid sample.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention adopts an improved fluorescence coding technology for detection antibodies, combined with a plate platform, retaining the original operation habits. Through fluorescence coding and digital amplification, digital imaging is performed using a fluorescence microscopy system, combined with digital analysis, to achieve single-molecule level detection of the AD biomarker Aβ40. Compared with the biotin-streptavidin reaction system, this technology reduces the operation steps and operation time, and at the same time has the advantages of economy, convenient operation, high sensitivity, medium-high throughput, etc. It can be used for the detection of Aβ40 in body fluids other than cerebrospinal fluid (such as blood, saliva, etc.), showing great application potential in the early screening of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the standard curve for the detection of human Aβ40 protein by the protein digital detection technology based on fluorescence immunosorption in Example 1.

[0033] Figure 2 It is the standard curve for the detection of human Aβ40 protein by the protein digital detection technology based on fluorescence immunosorption in Comparative Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0034] Example 1

[0035] (1) Coat the ELISA plate with Aβ40 capture antibody (manufacturer: Thermo Fisher Scientific, catalog number: 44-348A) at a concentration of 0.5 μg / T, 40 μL / T coating solution, and let it stand at 2°C for 15 h. Wash it 5 times with PBS + TWEEN-20 (0.05% wt), and pat it dry; add 150 μL / well blocking solution, block it at 37°C for 2 h, wash it 5 times with PBS + TWEEN-20 (0.05% wt), and pat it dry for later use.

[0036] (2) Add 45 μL of Aβ40 standards at concentrations of 0.46, 1.37, 4.12, 12.35, 37.04, 111.11, 333.33, 1000 pg / mL to the coated ELISA plate respectively; after reacting on a shaker at 37°C for 0.5 h, wash it 5 times with PBS + TWEEN-20 (0.05% wt), and pat it dry.

[0037] (3) Dilute the concentration of Aβ40 detection antibody (manufacturer: Thermo Fisher Scientific, catalog number: 600-401-J10) to 2 mg / mL with PBS buffer, then sequentially add 5 μL of biotin pure aqueous solution at a concentration of 5 mg / mL and 100 μL of NaHCO 3 pure aqueous solution; place it on a shaker and react at room temperature for 1 h. After the reaction, take 130 μL of the reaction solution and add it to a centrifuge tube containing a pre-equilibrated desalting column (Thermo Fisher Scientific, catalog number: 89882) to allow the desalting column to adsorb the sample, centrifuge it at a centrifugal force of 1000 g for 2 min, and remove the adsorption column. After diluting the biotinylated Aβ40 detection antibody 400-fold with PBS buffer, take 50 μL and add it to the ELISA plate containing Aβ40 quality control product or body fluid sample. After reacting on a shaker at 37°C for 1 h, immediately add 50 μL of streptavidin-conjugated fluorescent particles (Sigma-Aldrich, catalog number: 49532), react on a shaker at 37°C for 1 h, wash it 5 times with PBS + TWEEN-20 (0.05% wt), and pat it dry.

[0038] (4) Place the ELISA plate on the sample stage of the fluorescence microscopy system, use a 40x objective lens, and perform digital imaging by reflection. Use counting and data processing software to establish the correlation equation of the standard curve. Substitute the signal values of the Aβ40 quality control product and body fluid sample into the standard curve equation to calculate the Aβ40 concentration in the quality control product and body fluid sample.

[0039] The results of the counting and data processing software show that through four-parameter fitting, the concentration of the Aβ40 standard product and the signal value have a good correlation, R 2 is 0.994(Figure 1 )。

[0040] Comparative Example 1

[0041] (1) Coat the ELISA plate with Aβ40 capture antibody at a concentration of 0.5 μg / T, 40 μL / T coating solution, and incubate statically at 2°C for 15 h. Wash 5 times with PBS + TWEEN-20 (0.05% wt), and pat dry. Add 150 μL / well of blocking solution, block at 37°C for 2 h, wash 5 times with PBS + TWEEN-20 (0.05% wt), and pat dry for later use.

[0042] (2) Add 45 μL of Aβ40 standards at concentrations of 0.46, 1.37, 4.12, 12.35, 37.04, 111.11, 333.33, 1000 pg / mL to the coated ELISA plate respectively; after reacting on a shaker at 37°C for 0.5 h, wash 5 times with PBS + TWEEN-20 (0.05% wt), and pat dry.

[0043] (3) Take 0.05 mL of microsphere (1% solid content, Sigma-Aldrich, product number: 49532) suspension into a 2 mL centrifuge tube containing 1 mL of coupling buffer, mix well by sonication, centrifuge at 15°C and 20000 g for 10 min, and remove the supernatant; add 1 mL of microsphere coupling buffer, mix well by sonication, centrifuge at 15°C and 20000 g for 10 min, and remove the supernatant; add 1 mL of microsphere coupling buffer, mix well by sonication; add 3.5 μL of EDC solution, mix well by vortex, then add 33 μL of NHS solution, mix well by sonication; place the centrifuge tube on a turntable, activate at 37°C, 40 r / min, in the dark for 15 - 30 min; centrifuge at 15°C and 20000 g for 10 min, and remove the supernatant; add 1.5 mL of coupling buffer, mix well by sonication, centrifuge at 15°C and 20000 g for 10 min, and remove the supernatant; add 1.5 mL of coupling buffer, and repeat the washing step once.

[0044] (4) Add 0.75 mL of coupling buffer and mix well by sonication; add 50 μg of the antibody to be labeled to 0.25 mL of coupling buffer to obtain the coupling solution; add the coupling solution containing the antibody to the mixed microspheres and mix well by vortexing; place the centrifuge tube on the turntable, couple at 37 °C, 40 r / min in the dark for 2 h; add 0.5 mL of microsphere blocking solution to the centrifuge tube and mix well by vortexing; place the centrifuge tube on the turntable, block at 37 °C, 40 r / min in the dark for 1 h; centrifuge at 15 °C, 20000 g for 10 min to remove the supernatant; add 1.5 mL of microsphere washing solution, mix well by sonication, centrifuge at 15 °C, 20000 g for 10 min to remove the supernatant; add 1.5 mL of microsphere washing solution and repeat the washing step once; finally, add 0.5 mL of microsphere storage solution (final microsphere concentration 1 mg / mL) to the centrifuge tube, mix well by sonication, and store at 2 - 8 °C in the dark for later use.

[0045] (5) Dilute the fluorescent microsphere-labeled antibody 20-fold with microsphere storage solution for later use; then add 100 μL of the fluorescent microsphere-labeled antibody diluted 20-fold to the enzyme-linked immunosorbent assay (ELISA) plate, react on a shaker at 37 °C for 0.5 h, and then wash 5 times with PBS + TWEEN-20 (0.05% wt) and pat dry.

[0046] (6) Place the ELISA plate on the sample stage of the fluorescence microscopy system, use a 40× objective lens to perform digital imaging by reflection, and establish the correlation equation of the standard curve using counting and data processing software. Substitute the signal values of the Aβ40 quality control product and the body fluid sample into the standard curve equation to calculate the Aβ40 concentrations in the quality control product and the body fluid sample.

[0047] The results of the counting and data processing software show that there is no correlation between the Aβ40 standard product concentration and the signal value, and the recognition and detection of Aβ40 cannot be achieved ( Figure 2 ).

[0048] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A digital detection product for Alzheimer's disease protein marker Aβ40 based on fluorescent immunoadsorption, characterized in that: The product contains streptavidin-conjugated fluorescent particles, an ELISA plate, an ELISA plate coating solution, an ELISA plate blocking solution, an Aβ40 capture antibody, and a biotinylated Aβ40 detection antibody.

2. The product according to claim 1, characterized in that The product is a test kit.

3. The product according to claim 1 or 2, characterized in that The Aβ40 capture antibody was from Thermo Fisher Scientific, catalog number 44-348A.

4. The product according to claim 3, characterized in that The preparation method of the biotinylated Aβ40 detection antibody is as follows: the concentration of the Aβ40 detection antibody is diluted to 0.5-3 mg / mL using PBS buffer, and then 4-6 uL of a 4-6 mg / mL biotin pure aqueous solution and 90-110 uL of a 82-86 mg / mL NaHCO3 pure aqueous solution are added in sequence, and reacted at room temperature.

5. The product according to claim 4, characterized in that The concentration of Aβ40 detection antibody was diluted to 2 mg / mL using PBS buffer.

6. The product according to claim 4 or 5, characterized in that The Aβ40 detection antibody is from Thermo Fisher Scientific, with the catalog number being 600-401-J10.

7. The product according to any one of claims 1-2, 4-5, characterized in that: The ELISA plate coating solution is formulated as 0.1 M sodium carbonate, 0.1 M sodium bicarbonate, pH 9-10.

8. The product according to claim 7, characterized in that The ELISA plate blocking solution is 0.05M PBS pH7.4, 1% BSA, and 0.1% ProClin 300.

9. Use of the product according to claim 1 or 2 in the preparation of a product for detecting Aβ40, a protein marker of Alzheimer's disease.

10. Use of the product according to claim 7 in the preparation of a product for detecting Aβ40, a protein marker of Alzheimer's disease.

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