Detection method of flow fluorescent single molecule pTau217

Abstract

The invention discloses a flow fluorescent single molecule pTau217 detection method, which comprises the following steps: reacting aminated polystyrene microspheres with diphenyl cyclooctyne-disulfide bond-active ester and azide functionalized streptavidin in sequence to prepare a composite microsphere dispersion liquid; the method comprises the following steps: reacting an elisa plate with 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, and coupling and capturing an antibody; sequentially adding a protein sample and a biotinylation detection antibody into the enzyme plate coupled with the capture antibody for reaction, and cleaning; adding composite microsphere dispersion liquid, oscillating and cleaning; adding a disulfide bond reducing agent for room-temperature reaction; and collecting the reacted solution, and counting the microspheres in the solution to realize the detection of the content of the flow fluorescent single molecule pTau217. According to the detection method disclosed by the invention, the detection on the quantity of the pTau217 protein can be realized, and the detection sensitivity is relatively high; the related operation method is simple, the detection cost is low, and the method is suitable for popularization and application.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to a flow cytometry fluorescence single-molecule pTau217 detection method. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disorder characterized by the accumulation of β-amyloid (Aβ) and neurofibrillary tangles (NFTs) in the brain. Tau protein is a major component of NFTs, and its abnormal phosphorylation is considered a key factor in neurodegeneration.

[0003] Among the various phosphorylated forms of tau protein, pTau217 is considered to be of great value in the early diagnosis of Alzheimer's disease. Studies have shown that the positive diagnostic value of pTau217 for AD is 450 fg / ml or 160 fg / ml. However, existing chemiluminescence, electrochemiluminescence, and flow cytometry detection methods are sensitive enough to achieve a detection limit of only 1000 fg / ml, limiting their clinical application.

[0004] Currently, single-molecule immunoassay arrays (SimoA) are commonly used to detect proteins at femtomolar (fg / ml) levels. SimoA technology is a single-molecule immunoassay based on microarray chips. Thousands of micron-scale wells, each approximately 40 fl, are carved (or cast) on a millimeter-scale chip. Magnetic immune complex beads are then distributed within the wells. Fluorescent spots are counted using a high-resolution fluorescence microscope. Based on Poisson distribution theory, the ratio of the number of wells containing both beads and fluorescent product to the total number of wells containing beads is calculated to determine the protein concentration in the test sample. However, SimoA technology is plagued by high reagent and consumable costs, extremely high specialized equipment costs, and complex detection procedures, limiting its clinical application. Summary of the Invention

[0005] The main purpose of the present invention is to provide a simple, efficient and highly sensitive single-molecule pTau217 detection method for detecting femtomolar (fg / ml) pTau217 in a sample.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: A flow cytometry fluorescence single-molecule pTau217 detection method comprises the following steps: 1) Preparation of microsphere-SS-Streptavidin complex; Aminated polystyrene microspheres are dispersed in a first buffer, diphenylcyclooctyne-disulfide bond-active ester (DBCO-SS-NHS) is added, reacted at room temperature, and centrifuged to obtain microsphere-SS-DBCO; the obtained microspheres are then ultrasonically dispersed in a second buffer, azide-functionalized streptavidin (Streptavidin-Azido) is added, incubated, centrifuged, and the second buffer is added for ultrasonic dispersion to obtain a composite microsphere dispersion (microsphere-SS-Streptavidin composite dispersion); 2) Coupling the carboxylase-labeled plate with the pTau217 capture antibody; After washing, add acidic buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) to the plate and react at room temperature. Then add the capture antibody and let it stand at room temperature. After removing the liquid, add the blocking agent (ethanolamine or glycine, etc.), block the plate, and remove the liquid to obtain the enzyme plate coupled with the capture antibody. 3) Add buffer and pTau217 protein sample to the microwells of the enzyme plate coupled with the capture antibody, incubate with shaking, and wash; then add biotinylated detection antibody (biotinylated pTau217 detection antibody), incubate with shaking, and wash; 4) adding the microsphere-SS-Streptavidin composite dispersion to the microwells obtained in step 3), shaking, and washing the resulting enzyme plate; then adding a disulfide bond reducing agent and reacting at room temperature; 5) Collect all the solutions in the microwells of the ELISA plate and count the microspheres in the solution, thereby realizing the flow cytometry fluorescence detection of single-molecule pTau217 content.

[0007] In the above solution, the particle size of the amino polystyrene microspheres is 3-8 μm.

[0008] In the above scheme, the mass ratio of the amino-modified polystyrene microspheres, diphenylcyclooctyne-disulfide bond-active ester, and azide-functionalized streptavidin is (90-110) mg: (34-40) mg: (2.7-3.3) g.

[0009] In the above scheme, the room temperature reaction time in step 1) is 2 to 3 hours; and the incubation time is 2 to 3 hours.

[0010] In the above scheme, the centrifugal force used in the centrifugal separation step is 12000~14000g, and the time is 20~40min.

[0011] In the above scheme, in step 1), the first buffer is MES buffer (pH 5.0-6.0); the second buffer can be PBS buffer (pH 7.0-7.6) or TBS buffer (pH 7.4-8.0).

[0012] In the above scheme, the acidic buffer in step 2) is MES buffer, and its pH value is 5.0-6.0.

[0013] In the above scheme, the capture antibody is pTau217 antibody.

[0014] Furthermore, 20-100 μg of antibody is added to each microwell (single) of the ELISA plate.

[0015] In the above scheme, in step 2), the reaction time at room temperature is 30-40 minutes, and the standing time at room temperature is 2-3 hours.

[0016] In the above scheme, the shaking incubation step in step 3) adopts a temperature of 30-40°C, an shaking speed of 700-1000 rpm, and a time of 30-40 min.

[0017] Furthermore, in step 3), when the sample contains pTau217 protein, the capture antibody captures the pTau217 protein, and the biotinylated detection antibody binds to it to form an antigen-antibody sandwich structure complex.

[0018] In the above scheme, the oscillation rate in step 3) is 700-1000 rpm and the temperature is 30-40°C.

[0019] In the above scheme, the content of microspheres in the dispersion of microsphere-SS-Streptavidin complex is 0.06~0.3 mg / mL.

[0020] In the above scheme, the oscillation rate used in step 4) is 700~1000rpm.

[0021] In the above scheme, the oscillation treatment time in step 4) is 30-40 min, and the reaction time at room temperature is 10-20 min.

[0022] Furthermore, microsphere-SS-Streptavidin was reacted with the antigen-biotinylated antibody complex to obtain a microsphere-SS-Streptavidin-biotin-antibody-pTau217 protein complex.

[0023] Furthermore, a disulfide bond reducing agent including but not limited to tris(2-carboxyethyl)phosphine hydrochloride (TCEP), β-mercaptoethanol (BME) or dithiothreitol (DTT) is used to reduce the disulfide bonds of the above complex and cut the disulfide bonds.

[0024] Furthermore, the fluorescent polystyrene microspheres were collected by washing the ELISA plate with a buffer solution.

[0025] Furthermore, the microspheres were counted using a flow cytometer to obtain the number of pTau217 molecules.

[0026] In the present invention, capture antibodies are coupled to the microwells of the ELISA plate. When the sample contains pTau217 protein, the capture antibody captures the pTau217 protein, which is then bound by a biotinylated detection antibody to form an antigen-antibody sandwich complex. This sandwich complex contains biotin groups. When avidinized microspheres containing disulfide bonds are added, the avidin binds to the biotin. After washing, each microsphere corresponds to one pTau217 protein molecule. When a disulfide bond reducing agent is added, the disulfide bonds are severed, freeing the microspheres. Counting the microspheres in the solution yields the number of pTau217 protein molecules.

[0027] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention uses single-particle counting in flow cytometry to detect single-molecule pTau217 proteins in samples, greatly improving the detection sensitivity of flow cytometry and significantly outperforming existing fluorescence-based detection methods. 2) The detection method described in the present invention uses conventional consumables, is low-cost, and does not require special equipment, which greatly reduces the detection cost and clinical applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Prepare a reaction structure diagram for the "microsphere-SS-Streptavidin" structural complex.

[0029] Figure 2 Schematic diagram of the reaction process of the present invention.

[0030] Figure 3 This is the calibration curve obtained in Example.

[0031] Figure 4 The figure shows the fitting equation curve of the linear range test for the embodiment.

[0032] Figure 5 The results of 10 samples were tested in the embodiment. DETAILED DESCRIPTION

[0033] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to the accompanying drawings. It should be noted that if there are any processes not specifically described below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0034] In the following examples, the detection reagents for single molecules of pTau217 (phosphorylated tau protein 217) include: 5 μm amino-labeled fluorescent polystyrene microspheres; DBCO-SS-NHS (diphenylcyclooctyne-disulfide-active ester); Streptavidin-Azido (azide-functionalized streptavidin); biotinylated pTau217 detection antibody; carboxylase-labeled plate; pTau217 capture antibody; calibrator prepared from pTau217 protein; MES buffer; and PBS buffer.

[0035] The preparation steps of the microsphere-SS-Streptavidin complex used are as follows: 100 mg of amino-modified polystyrene microspheres (Nanjing Jike Biotechnology Co., Ltd., particle size 3-8 μm) were washed with 25 mM MES buffer, centrifuged at 13,000 g for 30 minutes, and then sonicated in 25 mM MES; 1.23 mL of DBCO-SS-NHS (concentration 30 g / L) was added; after reacting at room temperature for 2 hours, the microspheres were centrifuged at 13,000 g for 30 minutes, and sonicated in PBS buffer for reconstitution; 3 g of Streptavidin-Azido was added and incubated at 4°C overnight, then centrifuged at 13,000 g for 30 minutes, and sonicated in PBS buffer for reconstitution.

[0036] The steps for coupling the capture antibody to the ELISA plate are as follows: Wash the ELISA plate twice with MES buffer and remove the liquid; add 25mM MES buffer (pH 5.0), 1% (w / v) EDC solution and 3% (w / v) NHS solution, and react at room temperature for 30 minutes; add 20-100ug capture antibody and mix at room temperature for 2 hours; remove the liquid, add a blocking agent such as ethanolamine or glycine, block for 30 minutes, and remove the liquid.

[0037] Example 1

[0038] A flow cytometry fluorescence single-molecule pTau217 detection method comprises the following steps: 1) Add 100 μL of PBS buffer (pH 7.4) to the microwells of the ELISA plate coupled with the capture antibody, followed by 10 μL of calibrator or sample. 2) Incubate at 37°C with shaking for 30 min; wash the plate three times with PBS buffer; 3) Add 50 μL of biotinylated pTau217 detection antibody and shake at 37°C for 30 minutes; wash the plate three times with PBS; 4) Add 50 μL of the prepared composite microsphere dispersion and mix thoroughly at 37°C for 30 minutes; wash the plate three times with PBS; 5) Add 50 μL of TECP and react at room temperature for 10 minutes; 6) Aspirate all the solution in the microwells of the ELISA plate and count the microspheres using a flow cytometer.

[0039] Using the counting detection method of the present invention, pTau217 protein was taken and a calibration curve was drawn in the concentration range of 10~2560fg / mL. The results are as follows: Figure 3 shown.

[0040] According to the counting detection method of the present invention, two clinical samples with high (H) and low (L) concentrations are diluted proportionally, with the dilution as the X-axis and the mean of the test results as the Y-axis, and a linear equation fitting is performed, with R²=0.9999; the dilution is substituted into the equation to calculate the regression concentration, and the relative deviation between the test mean and the regression concentration is within 15% for the low value and within 5% for the high value, indicating that the detection method has good linearity. Figure 4 As shown in Table 1.

[0041] Table 1 Linearity test results

[0042] According to the counting detection method of the present invention, one clinical sample was added with pTau217 protein to prepare samples at three concentration levels. The samples were tested 20 times and the repeatability of the test samples was calculated, as shown in Table 2.

[0043] Table 2 Test sample repeatability

[0044] According to the counting detection method of the present invention, 10 clinical samples were taken for testing. Figure 5 shown.

[0045] Table 3 Test results of 10 clinical samples

[0046] The above description is only an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent transformation made by using the contents of the present invention description and drawings, or directly or indirectly applied in the relevant technical field, is also included in the patent protection scope of the present invention.

Claims

1. A flow cytometry fluorescence single-molecule pTau217 detection method, characterized in that: The steps include: 1) Dispersing the amino-modified polystyrene microspheres in a first buffer, adding diphenylcyclooctyne-disulfide bond-active ester, reacting at room temperature, and separating to obtain microsphere-SS-DBCO; ultrasonically dispersing the microspheres in a second buffer, adding azide-functionalized streptavidin, incubating, separating, and dispersing the microspheres in a second buffer to obtain a composite microsphere dispersion; 2) Add acidic buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide to the ELISA plate and react at room temperature; add the capture antibody and let it stand at room temperature; remove the liquid and then add the blocking agent, block the plate, and remove the liquid to obtain the enzyme plate coupled with the capture antibody; 3) Adding buffer and pTau217 protein sample to the microwells of the resulting enzyme plate, incubating with shaking, and washing; adding biotinylated detection antibody, incubating with shaking, and washing; 4) adding the composite microsphere dispersion to the micropores obtained in step 3), shaking, and washing; adding a disulfide bond reducing agent to react at room temperature; 5) Collect the solution in the microwells of the ELISA plate, count the microspheres, and detect the content of single-molecule pTau217 by flow cytometry.

2. The detection method according to claim 1, wherein The particle size of the amino polystyrene microspheres is 3-8 μm.

3. The detection method according to claim 1, wherein The mass ratio of the amino polystyrene microspheres, diphenylcyclooctyne-disulfide bond-active ester, and azide-functionalized streptavidin is (0.090-0.11): (0.034-0.04): (2.7-3.3).

4. The detection method according to claim 1, wherein Step 1) The room temperature reaction time is 2-3 hours; the incubation time is 2-3 hours.

5. The detection method according to claim 1, wherein In step 1), the first buffer is MES buffer with a pH of 5.0-6.0; the second buffer is PBS buffer with a pH of 7.0-7.6; or TBS buffer with a pH of 7.4-8.0; and in step 2), the acidic buffer is MES buffer with a pH of 5.0-6.

0.

6. The detection method according to claim 1, characterized in that In step 2), the reaction time at room temperature is 30-40 minutes, and the standing time at room temperature is 2-3 hours.

7. The detection method according to claim 1, characterized in that Step 3) The shaking incubation step adopts a temperature of 30-40°C, an shaking rate of 700-1000 rpm, and a time of 30-40 min.

8. The detection method according to claim 1, wherein Step 4) The shaking treatment time is 30-40 min, and the reaction time at room temperature is 10-20 min.

9. The detection method according to claim 1, wherein The disulfide bond reducing agent is tris(2-carboxyethyl)phosphine hydrochloride, β-mercaptoethanol or dithiothreitol.

10. The detection method according to claim 1, characterized in that The microspheres were counted using flow cytometry to obtain the number of pTau217 molecules.

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