Kit and method for detecting Alzheimer's disease biomarker p-Tau181 protein
By using magnetic beads and a kit for visualizing non-magnetic microsphere labeled antibodies, combined with magnetic field enrichment and microscopy counting technology, the problem of low detection sensitivity of p-Tau181 protein in AD diagnosis in the prior art was solved, and 0.01 pg/mL was achieved The detection limit of the disease improves the detection rate and reliability of the detection in early stages of Alzheimer's disease.
Patent Information
- Application Number
- CN202411352631.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2024-12-27
AI Technical Summary
The existing AD diagnostic methods have problems such as difficulty in diagnosis, high cost, limited equipment and low detection sensitivity. Especially when detecting the Alzheimer's biomarker p-Tau181 protein, the existing technology is difficult to achieve pg/mL or The detection limit of sub-pg/mL limits its wide application in clinical practice.
Using a kit of visual non-magnetic microspheres including p-Tau181 antibody-labeled magnetic beads and total tau antibody-labeled visual non-magnetic microspheres, a solid-phase antibody-antigen-antibody complex was formed through magnetic field enrichment and incubation, and quantitative analysis was achieved in combination with microscopy counting. Buffer solution and reaction system to improve detection sensitivity.
It significantly reduces the detection limit of detection to 0.01 pg/mL, improves detection sensitivity and specificity, and is expected to improve the detection rate of early Alzheimer's disease, providing a non-invasive and reliable clinical detection method.
Smart Images
Figure CN119199139A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of in vitro detection technology, and specifically relates to a kit and method for detecting phosphorylated Tau-181 protein (p-Tau181 protein), a biomarker of Alzheimer's disease. Background Art
[0002] There are about 50 million dementia patients in the world, and this number is expected to exceed 150 million by 2050. Among all dementias, Alzheimer's disease (AD) is the most common type, accounting for about 60%-80% of all dementia patients. AD diagnosis is difficult to accurately diagnose clinically, and there is currently no specific drug for AD, so clinicians are usually cautious in diagnosing patients.
[0003] At present, AD diagnosis is mainly assisted by several different methods, including: neuropsychological assessment scale, cerebrospinal fluid testing, CT / MRI conventional imaging examinations, and targeted molecular imaging diagnostic methods based on Aβ and tau-PET. However, due to the subjective judgment problem of the assessment scale, it is greatly affected by the clinical experience and judgment ability of the assessor. The difficulty of cerebrospinal fluid collection, the invasive damage of the collection process, and the limitation of domestic clinical resources make cerebrospinal fluid testing difficult to popularize on a large scale; and the detection and diagnosis based on cerebrospinal fluid are currently mainly concentrated in the severe dementia group who need to be hospitalized for treatment, and it is difficult to be effectively used in outpatient clinics. Conventional imaging examinations such as CT / MRI can only reflect the structural changes of the brain, and it is difficult to accurately diagnose AD. PET equipment is expensive and has low equipment operation efficiency. At the same time, it is limited by the application of Aβ and P-tau tracers. Therefore, this method is difficult to be widely popularized in AD diagnosis. Compared with the above four diagnostic methods, blood samples have better stability, are easy to collect, and the concentration of biomarkers in the blood is relatively uniform and stable, making it an ideal test sample.
[0004] AD blood biomarkers, especially Aβ, P-tau and nerve damage-related biomarkers related to the ATN framework, have significantly increased the quality of evidence for blood markers as a clinical aid to AD diagnosis in the past few years. Since AD biomarkers in the blood mainly come from cerebrospinal fluid, these biomarkers usually drop in concentration after crossing the blood-brain barrier into the blood, which significantly increases the difficulty of detection. In addition, due to the limitations at the principle level, existing immunoassay techniques, such as traditional enzyme-linked immunosorbent and chemiluminescence techniques, usually have a detection limit of sub-ng / mL, which is helpless for many biomarkers in the pg / mL or sub-pg / mL concentration range, making a large number of biomarkers unable to be effectively used in clinical practice. The development of a detection technology that exceeds the femtogram sensitivity performance is considered to be a key tool for the development of clinical applications of these low-abundance new biomarkers. Summary of the invention
[0005] In view of this, the present invention aims to develop a new method for detecting the Alzheimer's disease biomarker p-Tau181 protein, so as to reduce the detection limit to pg / mL or sub-pg / mL level, and provide a non-invasive and reliable approach for clinical detection of AD.
[0006] In order to achieve the above object, the present invention specifically adopts the following technical solutions: The first aspect of the present invention provides a kit for detecting the Alzheimer's disease biomarker p-Tau181 protein, which comprises the following components: Magnetic bead working solution, consisting of magnetic beads labeled with p-Tau181 antibody and buffer solution; A visualization non-magnetic microsphere working solution, consisting of visualization non-magnetic microspheres labeled with a total tau antibody and a buffer solution; The pH of the buffer solution is 6.5-8.0, the size of the magnetic beads in the p-Tau181 antibody-labeled magnetic beads is 1-10 μm, and the size of the visualized non-magnetic microspheres in the total tau antibody-labeled visualized non-magnetic microspheres is 1-10 μm.
[0007] In the above kit, the surface of the magnetic beads is modified with any one of carboxyl, amino, tosyl and streptavidin, and the p-Tau181 antibody is labeled on the magnetic beads through the surface modification of the magnetic beads.
[0008] In the above kit, the visualized non-magnetic microspheres are polystyrene microspheres, which can be monodisperse polystyrene microspheres, colored polystyrene microspheres, polystyrene fluorescent microspheres, etc., wherein the colors of the colored polystyrene microspheres include but are not limited to red, blue, yellow, green, black, etc.; the surface of the polystyrene microspheres is also modified with any one of carboxyl groups, amino groups, toluenesulfonyl groups and streptavidin, and the total tau antibody is labeled on the visualized non-magnetic microspheres through surface modification.
[0009] In the above kit, the buffer solution can be selected from phosphate buffered saline (PBS), tris(hydroxymethylaminomethane) buffer (TRIS), 3-marylpropanesulfonic acid buffer (MOPS), 4-hydroxyethylpiperazineethanesulfonic acid buffer (HEPES), and bovine serum albumin, trehalose, Tween 20, preservatives, etc. can also be added to the buffer solution.
[0010] Preferably, in the above kit, the buffer solution is 0.01-0.2 mol / L HEPES (pH 7.4) containing 0.5-5 wt% bovine serum albumin, 0.5-5 wt% trehalose, 0.01-0.1 v / v% Tween 20 and 0.05-0.5 v / v% ProClin 300.
[0011] The above kit may also include a standard substance, namely, p-Tau181 protein.
[0012] Based on the kit provided by the present invention, the second aspect of the present invention provides a method for detecting the Alzheimer's disease biomarker p-Tau181 protein, specifically: Take the sample to be tested, add the magnetic bead working solution and the visual non-magnetic microsphere working solution for incubation, wash and re-dissolve with the buffer solution, take the re-dissolved solution and count the formed complexes under a microscope.
[0013] In the method of the present invention, p-Tau181 protein in the sample to be tested is enriched by magnetic beads labeled with p-Tau181 antibody, and then incubated with visualized non-magnetic microspheres labeled with total tau antibody to form a solid-phase antibody-antigen-antibody complex, and the substance bound to the magnetic beads is adsorbed by the magnetic field, while the unbound substance is washed away. In the method of the present invention, the number of complexes is proportional to the concentration of p-Tau181 protein in the sample to be tested, thereby achieving the purpose of quantitative analysis.
[0014] Preferably, in the above method, the concentration of the magnetic bead working solution is 0.01-0.5 mg / ml, the concentration of the visualized non-magnetic microsphere working solution is 0.01-0.5 mg / mL, and the volume ratio of the magnetic bead working solution to the visualized non-magnetic microsphere working solution is 1:1.
[0015] Preferably, in the above method, the incubation conditions are: incubation at 37° C. for 5-30 min.
[0016] Preferably, in the above method, the sample to be tested is whole blood or serum.
[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses visualized non-magnetic microspheres to label antibodies on the basis of conventional immune reactions. By limiting the relative sizes of magnetic beads and visualized non-magnetic microspheres and the types of visualized non-magnetic microspheres, on the one hand, the generated solid-phase antibody-antigen-antibody complex can be easily found and counted under a microscope. On the other hand, a magnetic bead in the complex is combined with a visualized microsphere, and the structural consistency of the complex is high. Therefore, the number of the complex is proportional to the concentration of the analyte in the sample, thereby achieving quantitative detection.
[0018] By further optimizing the composition of the working solution and the ratio of the magnetic beads labeled with the p-Tau181 antibody to the visualized non-magnetic microspheres labeled with the total tau antibody in the reaction system, the detection limit of the method of the present invention can be as low as 0.01 pg / mL. It can be seen that the present invention significantly improves the detection sensitivity and is expected to increase the detection rate of early Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is a picture of the immune complex observed under a microscope when the kit of Example 1 is used for detection; Figure 2 This is a picture of the immune complex observed under a microscope when the kit of Example 5 is used for detection; Figure 3 This is a picture of the immune complex observed under a microscope when the kit of Example 6 is used for detection; Figure 4 This is a picture of the immune complex observed under a microscope when the kit of Example 7 was used for detection. DETAILED DESCRIPTION
[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The term "comprises" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.
[0021] In order to solve the problem of low concentration of AD biomarker p-Tau181 protein in blood, the present invention has developed a detection kit and detection method with both high sensitivity and high specificity, which can be used for qualitative and quantitative detection of p-Tau181 protein with a detection limit as low as 0.01 pg / mL. The detection kit includes: Magnetic bead working solution, consisting of p-Tau181 antibody-labeled magnetic beads and a buffer solution, wherein the magnetic beads have a size of 1-10 μm; A visualization non-magnetic microsphere working solution, comprising total tau antibody-labeled visualization non-magnetic microspheres and a buffer solution, wherein the visualization non-magnetic microspheres have a size of 1-10 μm; Standard products; The buffer solution is selected from PBS, TRIS, MOPS and HEPES, and is supplemented with bovine serum albumin, trehalose, Tween 20, preservatives and the like.
[0022] Based on the above detection kit, the detection method is: Use the standard to prepare a series of standard solutions of different concentrations; take the standard, add the magnetic bead working solution and the visual non-magnetic microsphere working solution for incubation, wash and re-dissolve with the buffer solution, take the re-dissolved solution under a microscope to count the formed complexes; establish a standard curve of concentration and count; Take the sample to be tested, add the magnetic bead working solution and the visualized non-magnetic microsphere working solution for incubation, re-dissolve with the buffer solution after washing, and take the re-dissolved solution for counting under a microscope; calculate the concentration of p-Tau181 protein in the sample to be tested according to the standard curve.
[0023] The technical solution of the present invention will be described clearly and completely below in conjunction with specific embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.
[0024] In the following examples, rabbit anti-human p-Tau181 antibody and mouse anti-human total tau antibody were purchased from ADX, 2.8 μm carboxyl magnetic beads were purchased from Thermo Fisher Scientific, 5 μm carboxyl magnetic beads were purchased from Suzhou Weidu Biotechnology Co., Ltd., and non-magnetic microspheres were purchased from Suzhou Weidu Biotechnology Co., Ltd., and the antibodies were labeled on magnetic beads or non-magnetic microspheres according to the instructions of the corresponding products; in addition, p-Tau181 protein was purchased from CST, and 4-hydroxyethylpiperazineethanesulfonic acid was purchased from Sigma.
[0025] If no specific techniques or conditions are specified in the following examples, all of them are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.
[0026] Example 1 This example provides a kit for detecting the Alzheimer's disease biomarker p-Tau181 protein, including the following: Magnetic bead working solution, consisting of magnetic beads labeled with rabbit anti-human p-Tau181 antibody and buffer solution, wherein the carboxyl magnetic beads have a diameter of 2.8 μm and a working concentration of 0.2 mg / mL; The visual non-magnetic microsphere working solution is composed of the visual non-magnetic microspheres labeled with mouse anti-human total tau antibody and a buffer solution, wherein the visual non-magnetic microspheres are 2 μm polystyrene carboxyl microspheres, and the working concentration is 0.05 mg / mL; Buffer solution, specifically composed of HEPES (0.025 mol / L, pH=7.4), 3% bovine serum albumin, 5% trehalose, 0.05% Tween 20, and 0.1% ProClin 300; The standard sample was prepared by diluting p-Tau181 protein to 1000 pg / mL, 500 pg / mL, 100 pg / mL, 20 pg / mL, 5 pg / mL, 1 pg / mL, 0.1 pg / mL, 0.01 pg / mL, and 0 pg / mL using HEPES (0.025 mol / L, pH=7.4, containing 2 wt% BSA).
[0027] Example 2 The only difference from Example 1 is that the buffer solution in this example is specifically composed of PBS (0.025M, pH=7.4), 3% bovine serum albumin, 5% trehalose, 0.05% Tween 20, and 0.1% ProClin 300.
[0028] Example 3 The only difference from Example 1 is that the buffer solution in this example is specifically composed of TRIS (0.025M, pH=7.4), 3% bovine serum albumin, 5% trehalose, 0.05% Tween 20, and 0.1% ProClin 300.
[0029] Example 4 The only difference from Example 1 is that the buffer solution in this example specifically consists of MOPS (0.025M, pH=7.4), 3% bovine serum albumin, 5% trehalose, 0.05% Tween 20, and 0.1% ProClin 300.
[0030] Example 5 The difference from Example 1 is that the visual non-magnetic microsphere working fluid used in this example is specifically: The method is composed of mouse anti-human total tau antibody-labeled visualized non-magnetic microspheres and a buffer solution, wherein the visualized non-magnetic microspheres are 2 μm red polystyrene carboxyl microspheres, and the working concentration is 0.04 mg / mL.
[0031] Example 6 The difference from Example 1 is that the magnetic bead working solution and the visual non-magnetic microsphere working solution used in this example are specifically: Magnetic bead working solution, consisting of magnetic beads labeled with rabbit anti-human p-Tau181 antibody and buffer solution, wherein the carboxyl magnetic beads have a diameter of 5 μm and a working concentration of 0.2 mg / mL; The visual non-magnetic microsphere working solution is composed of visual non-magnetic microspheres labeled with mouse anti-human total tau antibody and a buffer solution, wherein the visual non-magnetic microspheres are 5 μm red polystyrene carboxyl microspheres, and the working concentration is 0.1 mg / mL.
[0032] Example 7 The difference from Example 1 is that the magnetic bead working solution and the visual non-magnetic microsphere working solution used in this example are specifically: Magnetic bead working solution, consisting of magnetic beads labeled with rabbit anti-human p-Tau181 antibody and buffer solution, wherein the carboxyl magnetic beads have a diameter of 5 μm and a working concentration of 0.2 mg / mL; The visual non-magnetic microsphere working solution is composed of visual non-magnetic microspheres labeled with mouse anti-human total tau antibody and a buffer solution, wherein the visual non-magnetic microspheres are 5 μm blue polystyrene carboxyl microspheres, and the working concentration is 0.05 mg / mL.
[0033] Example 8 Based on the kit of the present invention, this example provides a method for detecting the Alzheimer's disease biomarker p-Tau181 protein, specifically: Pipette 50 μL of the sample to be tested into the reaction cup, then add 50 μL of magnetic bead working solution and 50 μL of visual non-magnetic microsphere working solution in sequence, and react at 37°C for 10 min; after washing 3 times, add 20 μL of buffer solution to re-dissolve, take 5 μL droplets and load them onto a glass slide, and count them under a microscope.
[0034] According to the method in Example 8, the p-Tau181 protein standard was tested using the kit in Examples 1-4, and the test results are shown in Table 1: Table 1 Summary of immune complex counts of standard products in Examples 1-4
[0035] From the data in Table 1, it can be seen that Examples 1-4 can all achieve high-sensitivity detection of p-Tau181 protein, and HEPES has a better effect than the other three buffers. When detecting 0.01 pg / mL standard, immune complexes can still be observed under the microscope (i.e., detectable). Figure 1 (0.01 pg / mL), the content of free visualized microspheres is small, the number of immune complexes detected in the 0 pg / mL standard is 0, the background is clean, there is no interference, and the sensitivity of the detection is improved.
[0036] In order to verify the precision of the detection kit of Example 1, the following test was performed: samples with concentrations between 20 pg / mL and 1 pg / mL were tested 10 times, and their concentration values were calculated according to the reaction curve, and the mean (M) and standard deviation (S) were calculated, and the coefficient of variation (CV) was calculated, CV = (S / M) × 100%. The repeatability measurement data is shown in Table 2.
[0037] Table 2 Repeatability test results
[0038] According to the data in Table 2, the repeatability CV of 10 tests of samples with a concentration of 20 pg / mL is 2.1%; the repeatability CV of 10 tests of samples with a concentration of 1 pg / mL is 2.8%.
[0039] Figure 3-4 They are respectively the microscopic observation pictures of Examples 5-7. It can be seen from the pictures that the kits in Examples 5-7 can also detect the p-Tau181 protein with a clean background and no interference.
[0040] In summary, the present invention provides a new immunoassay platform for the detection of p-Tau181 protein, a biomarker of Alzheimer's disease. This method has the advantages of clean background, single complex counting, high sensitivity, good specificity, etc.
[0041] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
Claims
1. A kit for detecting p-Tau181 protein, a biomarker of Alzheimer's disease, characterized in that: include: Magnetic bead working solution, consisting of magnetic beads labeled with p-Tau181 antibody and buffer solution; A visualization non-magnetic microsphere working solution, consisting of visualization non-magnetic microspheres labeled with a total tau antibody and a buffer solution; Wherein, the size of the magnetic beads is 1-10 μm, and the size of the visualized non-magnetic microspheres is 1-10 μm.
2. The kit according to claim 1, characterized in that The visualized non-magnetic microspheres are polystyrene microspheres, and the surfaces of the polystyrene microspheres are modified with carboxyl groups, amino groups, toluenesulfonyl groups or streptavidin.
3. The kit according to claim 1, characterized in that The surface of the magnetic beads is modified with carboxyl, amino, toluenesulfonyl or streptavidin.
4. The kit according to claim 1, characterized in that The buffer solution is any one of phosphate buffer, tris(hydroxymethylaminomethane) buffer, 3-malolinepropanesulfonic acid buffer and 4-hydroxyethylpiperazineethanesulfonic acid buffer.
5. The kit according to claim 4, characterized in that The buffer solution also contains bovine serum albumin, trehalose, Tween 20 and a preservative.
6. The kit according to claim 5, characterized in that The buffer solution is a 0.01-0.2 mol / L 4-hydroxyethylpiperazineethanesulfonic acid buffer solution containing 0.5-5% bovine serum albumin, 0.5-5% trehalose, 0.01-0.1% Tween 20 and 0.05-0.5% ProClin 300.
7. A method for detecting p-Tau181 protein, a biomarker of Alzheimer's disease, characterized in that: The detection is performed using the kit described in any one of claims 1 to 6, wherein the method is not intended for the diagnosis and treatment of a disease.
8. The method according to claim 7, characterized in that Specifically, take the sample to be tested, add magnetic bead working solution and visual non-magnetic microsphere working solution for incubation, wash and re-dissolve, take the re-dissolved solution and count the formed complexes under a microscope.
9. The method according to claim 8, characterized in that The concentration of the magnetic bead working solution is 0.01-0.5 mg / ml, the concentration of the visualized non-magnetic microsphere working solution is 0.01-0.5 mg / mL, and the volume ratio of the magnetic bead working solution to the visualized non-magnetic microsphere working solution is 1:
1.
10. The method according to claim 8, characterized in that The incubation conditions are: incubation at 37° C. for 5-30 min.