Immunochromatography test strip for beta amyloid protein detection and preparation method thereof

Abstract

The invention discloses a beta amyloid protein detection immunochromatography test strip and a preparation method thereof, the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially fixed on a bottom plate, and the nitrocellulose membrane is provided with a quality control line and at least one detection line; a marker obtained by coupling a color reagent and a beta amyloid protein labeled antibody is sprayed on the combination pad, and the detection line and the quality control line are respectively obtained by marking a beta amyloid protein coated antibody and a goat anti-mouse polyclonal antibody. The urine beta amyloid protein immunochromatography detection test strip developed by the invention has various forms, is suitable for various application modes such as single detection and joint detection of A beta 1-40 and A beta 1-42, and has the advantages of simple preparation method, low cost, convenience in operation, no need of complex instruments and equipment, noninvasive sample, high detection efficiency and the like; the method can be applied to various scenes such as primary medical institutions, home self-inspection, large-scale general screening, AD early screening and the like.

Description

Technical Field

[0001] This invention relates to the field of immunochromatography technology, and in particular to an immunochromatographic test strip for detecting β-amyloid protein and its preparation method. Background Technology

[0002] Alzheimer's disease (AD), also known as senile dementia, is a chronic, persistent, and degenerative brain disease that occurs in old age or pre-old age. Related data shows that in 2019, Alzheimer's disease rose to the fifth leading cause of death in my country, with a prevalence rate of 3.21% among the population aged 65 and above.

[0003] Among those newly diagnosed with Alzheimer's disease, the 60-79 age group accounted for the highest proportion, reaching 62.1%. Alzheimer's disease is increasingly affecting younger people in my country, with patients under 60 years old accounting for 21.3% of those surveyed. Only about 30% of respondents sought medical attention within one year of experiencing disease-related symptoms, while 70% sought medical attention within two years. The survival rate for those with AD ranges from 3 to 9 years, depending on the age at symptom onset, resulting in a long-term illness and a significant insidious period. Therefore, early detection, screening, and diagnosis of AD are crucial.

[0004] Currently, the main clinical diagnostic methods for Alzheimer's disease (AD) include the following: (1) Clinical assessment and neuropsychological testing (such as the Mini-Mental State Examination, MMSE): preliminary judgment is made through cognitive function and behavioral assessment, but it is highly subjective and lacks sensitivity for early asymptomatic patients; (2) Cerebrospinal fluid testing: the levels of β-amyloid protein (Aβ) and phosphorylated Tau protein (p-Tau) are analyzed by lumbar puncture, which is highly accurate, but invasive and complicated to operate, and has low patient acceptance; (3) Imaging technology (such as MRI, PET-CT): can directly show brain atrophy or amyloid protein deposition, but it is costly, highly dependent on equipment, and PET-CT is radioactive, making it difficult to use for large-scale screening; (4) Blood biomarker detection (such as plasma GFAP, p-Tau181): has developed rapidly in recent years and has the advantages of convenience and speed, but the concentration of biomarkers in the blood is low and easily affected by other factors. At the same time, sample acquisition is invasive and requires professional sampling, resulting in high testing costs. Therefore, there is an urgent need for a non-invasive product that is highly sensitive and specific, easy to operate, and inexpensive for early AD screening. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an immunochromatographic test strip for the detection of β-amyloid protein and its preparation method, in order to address the shortcomings of the prior art.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: an immunochromatographic test strip for the detection of β-amyloid protein, comprising a sample pad, a conjugation pad, a nitrocellulose membrane and an absorbent pad fixed in sequence on a base plate, wherein the nitrocellulose membrane is provided with a control line and at least one detection line; The conjugation pad is coated with a marker obtained by conjugating a chromogenic reagent with a β-amyloid protein-labeled antibody. The detection line and control line are obtained by streaking with a β-amyloid protein-coated antibody and a goat anti-mouse polyclonal antibody, respectively. The β-amyloid labeled antibody and the β-amyloid coated antibody are monoclonal antibodies that can bind to different epitopes on the same β-amyloid protein; The colorimetric reagent is latex microspheres or colloidal gold.

[0007] Preferably, in this invention, β-amyloid protein is Aβ1-40, β-amyloid protein labeling antibody is Aβ1-40 labeled antibody, and β-amyloid protein coating antibody is Aβ1-40 coated antibody, wherein the Aβ1-40 labeled antibody and the Aβ1-40 coated antibody are monoclonal antibodies that can bind to different epitopes on Aβ1-40. Alternatively, β-amyloid protein may be Aβ1-42, β-amyloid protein labeled antibody may be Aβ1-42 labeled antibody, and β-amyloid protein coated antibody may be Aβ1-42 coated antibody, wherein the Aβ1-42 labeled antibody and the Aβ1-42 coated antibody are monoclonal antibodies that can bind to different epitopes on Aβ1-42.

[0008] Preferably, the nitrocellulose membrane is provided with a quality control line and a detection line; The conjugate pad is coated with a marker obtained by conjugating a chromogenic reagent with an Aβ1-40-labeled antibody, and the detection line and control line are obtained by streaking with an Aβ1-40-coated antibody and a goat anti-mouse polyclonal antibody, respectively; or the conjugate pad is coated with a marker obtained by conjugating a chromogenic reagent with an Aβ1-42-labeled antibody, and the detection line and control line are obtained by streaking with an Aβ1-42-coated antibody and a goat anti-mouse polyclonal antibody, respectively.

[0009] Preferably, the conjugate pad is sprayed with a mixture of two labels obtained by conjugating a colorimetric reagent with an Aβ1-40-labeled antibody and an Aβ1-42-labeled antibody, respectively; the nitrocellulose membrane has two detection lines, which are obtained by streaking with the Aβ1-40-coated antibody and the Aβ1-42-coated antibody, respectively; or the nitrocellulose membrane has one detection line, which is obtained by streaking with a mixture of the Aβ1-40-coated antibody and the Aβ1-42-coated antibody.

[0010] The present invention also provides a method for preparing the β-amyloid protein detection immunochromatographic test strip as described above, comprising the following steps: S1. The chromogenic reagent is coupled with the β-amyloid protein-labeled antibody to obtain the label; S2. The marker is sprayed onto the conjugation pad, and the β-amyloid protein-coated antibody and goat anti-mouse polyclonal antibody are used to draw lines on the nitrocellulose membrane to obtain the detection line and control line, respectively. Finally, the sample pad, conjugation pad, nitrocellulose membrane and absorbent pad are pasted onto the base plate in sequence to obtain the β-amyloid protein detection immunochromatographic test strip.

[0011] Preferably, the colorimetric reagent is latex microspheres, and step S1 specifically includes: The latex microspheres were activated with EDC and NHS, and then the activated latex microspheres were mixed with β-amyloid protein-labeled antibodies and reacted for 2-6 hours. Subsequently, they were blocked with blocking solution containing bovine serum albumin for 1-3 hours. After washing with washing solution, the labeled substance was obtained and stored in preservation solution for later use.

[0012] Preferably, the colorimetric reagent is colloidal gold, and step S1 specifically includes: The colloidal gold solution was adjusted to pH 8.2±0.1 with potassium carbonate aqueous solution, and then mixed with β-amyloid protein-labeled antibody and reacted for 30-60 minutes. Subsequently, blocking solution containing bovine serum albumin was added and blocked for 20-60 minutes. After centrifugation, the labeled substance was obtained, reconstituted with preservation solution, and stored for later use.

[0013] Preferably, in step S2, the sample pad is soaked in the sample pad treatment solution beforehand, and the conjugate pad is soaked in the conjugate pad treatment solution. The sample pad treatment solution comprises the following components: a buffer solution with a pH of 7.0-10.0 and a concentration of 10-500 mM, 0.5-5 wt% bovine serum albumin, 0.1-2% Tween 20 (volume concentration), 0.1-2 wt% S9, and 0.01-1% ProClin 300 (volume concentration). The buffer solution in the sample pad treatment solution is selected from one of Tris-HCl buffer, carbonate buffer, or borate buffer. The conjugate pad treatment solution comprises the following components: 0.1-2% Tween 20 (volume concentration), 0.5-5 wt% sucrose, 0.1-5 wt% bovine serum albumin, 0.01-1% ProClin 300 (volume concentration), and Tris-HCl buffer solution with pH 7.0-9.0 and a concentration of 10-100 mM.

[0014] Preferably, a detection line is provided on the nitrocellulose membrane, in which case: The marker is obtained by conjugating the Aβ1-40-labeled antibody with a chromogenic reagent and then spraying it onto the conjugation pad. The detection line is obtained by streaking the Aβ1-40-coated antibody. Alternatively, a marker can be obtained by conjugating a colorimetric reagent with an Aβ1-42-labeled antibody, which is then sprayed onto the conjugate pad. The detection line is obtained by streaking the Aβ1-42-coated antibody.

[0015] Preferably, a detection line is set on the nitrocellulose membrane, and Aβ1-40 labeled antibody and Aβ1-42 labeled antibody are coupled with a chromogenic reagent respectively. The two labels are mixed and then sprayed onto the conjugate pad. The detection line is obtained by drawing a line through the mixture of Aβ1-40 coated antibody and Aβ1-42 coated antibody.

[0016] Preferably, two detection lines are set on the nitrocellulose membrane. The Aβ1-40 labeled antibody and the Aβ1-42 labeled antibody are coupled with the chromogenic reagent, respectively. The two labels are mixed and then sprayed onto the conjugate pad. The Aβ1-40 coated antibody and the Aβ1-42 coated antibody are respectively drawn on the nitrocellulose membrane to obtain two detection lines.

[0017] The beneficial effects of this invention are: 1. The urine β-amyloid protein immunochromatographic test strip developed in this invention has multiple forms and is suitable for various applications such as single detection and combined detection of Aβ1-40 and Aβ1-42. It has the advantages of simple preparation method, low cost, convenient operation, no need for complicated instruments and equipment, non-invasive sample preparation, and high detection efficiency. It can be applied to various scenarios such as primary medical institutions, home self-testing, large-scale screening, and early AD screening.

[0018] 2. This invention significantly improves the sensitivity of the test strip for urine sample detection by optimizing the formulation of the sample pad and conjugate pad treatment solution during the test strip preparation process. It effectively reduces the adverse effects of complex matrices in urine on the detection results. The prepared Aβ1-40 and Aβ1-42 test strips have good specificity and sensitivity. Within the range of 0.5-100 ng / mL, the color intensity of the T line on the test strip is directly proportional to the antigen concentration, and there is no hook effect. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the structure of the immunochromatographic test strip (two detection lines) for β-amyloid protein detection provided by the present invention; Figure 2 A schematic diagram illustrating the detection effect of the immunochromatographic test strip (one detection line) for β-amyloid protein detection provided by the present invention; Figure 3 Color development effect of red latex microsphere test strips of Aβ1-40 at different concentrations; Figure 4 The color development effect of red latex microsphere test strips of Aβ1-42 at different concentrations is shown in the figure. Figure 5 Color development effect of colloidal gold test strips of different concentrations of Aβ1-40; Figure 6 Color development effect of colloidal gold test strips of different concentrations of Aβ1-42; Figure 7 Colorimetric effects of the Aβ1-40 and Aβ1-42 combined immunochromatographic test strips prepared for Examples 5 and 6: (a) the same color dual-detection line test strip prepared in Example 5, and (b) the dual color single-detection line test strip prepared in Example 6. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0021] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0023] Example 1: Preparation of Aβ1-40 latex microsphere immunochromatographic test strips (1) Solution preparation Sample pad treatment solution: 1% (wt) bovine serum albumin (BSA), 1.5% (v / v) Tween 20, 1.5% (wt) S9, 1.5% (wt) sucrose, 0.1% (v / v) ProClin 300, 50 mM carbonate buffer (pH=8.5); wherein, S9 is a propylene oxide-ethylene oxide-vinyl diamine copolymer, which is a typical amphoteric surfactant; Conjugation pad treatment solution: 1% (v / v) Tween 20, 5% (wt) sucrose, 1% (wt) BSA, 1% (wt) S9, 1% (wt) polyvinylpyrrolidone (PVP), 0.1% (v / v) ProClin 300, 10mM Tris-HCl buffer (pH=8.5). Coating solution: 3% (wt) sucrose, water; Coupling buffer: 10 mM MES (pH = 6.2 ± 0.1), 0.1% (v / v) ProClin 300; EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) solution: 10 mg / mL, prepared with coupling buffer, and used immediately; NHS (N-hydroxysuccinimide) solution: 10 mg / mL, prepared with coupling buffer, and used immediately; Blocking solution: borate buffer (5 mM boric acid, 11.2 mM sodium tetraborate decahydrate, 0.05% (v / v) Tween 20, pH=8.0±0.1), 2% (wt) BSA; Washing solution: 50 mM Tris-HCl (pH = 8.0 ± 0.1), 0.5% (wt) BSA, 0.05% (v / v) Tween 20, 0.03% (v / v) ProClin 300; Preservative solution: 25mM Tris-HCl (pH=7.2±0.1), 150mM NaCl, 0.05% (v / v) Tween20, 1% (wt) BSA, 5% (wt) trehalose, 0.1% (v / v) ProClin 300.

[0024] (2) Conjugation of latex microspheres with Aβ1-40 labeled antibody 1. Take 12.5 μL of latex microspheres (4% solids content, particle size 200 nm, purchased from Suzhou Weidu Biotechnology Co., Ltd., catalog number DR0200CA-1) suspension into a centrifuge tube containing 1 mL of coupling buffer, sonicate to mix, centrifuge, and remove the supernatant; 2. Add 1 mL of coupling buffer, sonicate to mix, centrifuge, and remove the supernatant; 3. Add 1 mL of coupling buffer and sonicate to mix well; 4. Add 3.5 μL of EDC solution, vortex to mix, then add 33 μL of NHS solution and sonicate to mix. 5. Shake the above solution to activate it for 30 minutes; 6. Centrifuge to remove the supernatant; 7. Add 1.5 mL of coupling buffer, sonicate to mix, centrifuge, and remove the supernatant; repeat this step once. 8. Add 1.5 mL of coupling buffer, add 50 μg of Aβ1-40 labeled antibody, vortex to mix, and couple for 3 h. 9. Add 0.5 mL of microsphere blocking solution to the centrifuge tube, vortex to mix, and shake to block for 1.5 h; 10. Centrifuge to remove the supernatant; 11. Add 1.5 mL of microsphere washing buffer, sonicate to mix, centrifuge, and remove the supernatant; repeat this step once. 12. Finally, add 0.5 mL of preservation solution to the centrifuge tube, sonicate to mix, and obtain the labeled substance. Store at 2-8℃ for later use.

[0025] (3) Assembly of Aβ1-40 latex microsphere immunochromatographic test strips 1. Soak the sample pad in the sample pad treatment solution for 20 minutes, then remove it and place it in an oven to dry overnight; the sample pad is made of glass fiber. 2. Immerse the conjugate pad in the conjugate pad treatment solution for 20 minutes, then remove it and place it in an oven to dry overnight; the sample pad is made of glass fiber. 3. Dilute the Aβ1-40 coated antibody and the goat anti-mouse polyclonal antibody to 2 mg / mL using coating buffer. Use a membrane scrubbing instrument to scrub the two antibody solutions onto a nitrocellulose membrane as the detection line (T line) and the control line (C line), respectively. The scrubbing speed is 1 μL / cm. Place the scrubbing nitrocellulose membrane in an oven to dry. 4. Spray the conjugate of latex microspheres and Aβ1-40 labeled antibody (i.e., the label) onto the dried conjugate pad at a spraying speed of 10 μL / cm, and then place the sprayed conjugate pad in an oven to dry. 5. Using a PVC base plate as a fixed base, attach the sample pad, bonding pad, nitrocellulose membrane and absorbent pad in order from left to right, and maintain a 2mm overlap between adjacent components.

[0026] Example 2: Preparation of Aβ1-42 latex microsphere immunochromatographic test strips The method is basically the same as in Example 1, except that the sample pad treatment solution in this example is formulated as follows: 0.5% (wt) bovine serum albumin (BSA), 0.5% (v / v) Tween 20, 0.5% (wt) S9, 1% (wt) sucrose, 0.1% (v / v) ProClin 300, and 500 mM Tris-HCl buffer (pH=8.5); The formulation of the conjugate pad treatment solution is as follows: 0.5% (v / v) Tween 20, 8% (wt) sucrose, 2% (wt) BSA, 2% (wt) S9, 0.5% (wt) polyvinylpyrrolidone (PVP), 0.1% (v / v) ProClin 300, and 50 mM Tris-HCl buffer (pH=8.5).

[0027] Example 3: Preparation of Aβ1-40 colloidal gold immunochromatographic test strips (1) Solution preparation Sample pad preparation solution: 0.5% (wt) bovine serum albumin (BSA), 0.5% (v / v) Tween 20, 0.5% (wt) S9, 0.5% (wt) polyvinylpyrrolidone (PVP), 0.1% (v / v) ProClin 300, 10 mM Tris-HCl buffer (pH=8.5); Constipation treatment solution: 0.5% (wt) Tween 20, 5% (wt) sucrose, 5% (wt) trehalose, 0.1% (wt) BSA, 0.1% (v / v) ProClin 300, 50 mM Tris-HCl buffer (pH=8.5); Coating solution: 2% (wt) sucrose, water; Blocking solution: 10% (wt) BSA, pH=8.5±0.1; Preservative solution: 25mM Tris-HCl (pH=7.2±0.1), 0.5% (v / v) Tween20, 1% (wt) BSA, 5% (wt) trehalose, 10% (wt) sucrose, 0.05% (wt) PEG20000, 0.1% (v / v) ProClin 300.

[0028] (2) The binding of colloidal gold to Aβ1-40 labeled antibody 1. Take 1 mL of colloidal gold solution (particle size 40 nm, purchased from Hangzhou Fenghang Technology Co., Ltd.), and adjust the pH to 8.2 ± 0.1 with 0.2 M potassium carbonate aqueous solution; 2. Add Aβ1-40 labeled antibody to the above solution to make the final concentration of Aβ1-40 labeled antibody in the solution 30 μg / mL, shake, and react for 40 min; 3. Add 20 μL of blocking solution and shake to block for 30 min; 4. Centrifuge and discard the supernatant; 5. Add 500 μL of preservation solution to reconstitute.

[0029] (3) Assembly of Aβ1-40 colloidal gold immunochromatographic test strip: The specific method is the same as in Example 1.

[0030] Example 4: Preparation of Aβ1-42 colloidal gold immunochromatographic test strip The method is basically the same as in Example 1, except that the sample pad treatment solution in this example is formulated as follows: 1% (wt) bovine serum albumin (BSA), 0.1% (v / v) Tween20, 0.5% (wt) S9, 0.5% (wt) polyvinylpyrrolidone (PVP), 0.1% (v / v) ProClin 300, and 10mM Tris-HCl buffer (pH=8.5). The formulation of the conjugate pad treatment solution is as follows: 0.5% (wt) Tween 20, 2% (wt) sucrose, 10% (wt) trehalose, 0.5% (wt) BSA, 0.1% (v / v) ProClin 300, 50 mM Tris-HCl buffer (pH=8.5).

[0031] Example 5: Immunochromatographic test strip for combined detection of Aβ1-40 and Aβ1-42 (same color dual-detection strip) An immunochromatographic test strip capable of simultaneously detecting two indicators (e.g., Aβ1-40 and Aβ1-42) is designed. The antibodies for these two indicators (Aβ1-40-labeled antibody and Aβ1-42-labeled antibody) are labeled separately with colloidal gold chromogenic reagent using the same method described above. The two labels are then mixed in equal volumes and uniformly sprayed onto the conjugate pad. Two detection lines are designed on the nitrocellulose membrane, each coated with a monoclonal antibody for one indicator (Aβ1-40-coated antibody and Aβ1-42-coated antibody), respectively. A shared control line is also included. Following the aforementioned test strip assembly method, a dual-indicator test strip is obtained. During use, if the sample contains both indicators, the corresponding detection lines will develop color. The two labels can be labeled with the same chromogenic reagent or with different types or colors of chromogenic reagents (to reduce interference). The effect is shown in the figure below. Figure 1 As shown.

[0032] Example 6: Immunochromatographic test strip for combined detection of Aβ1-40 and Aβ1-42 (dual-color single-line test strip) An immunochromatographic test strip capable of simultaneously detecting two indicators (e.g., Aβ1-40 and Aβ1-42) was designed. The difference from Example 5 is that the antibodies for these two indicators (Aβ1-40-labeled antibody and Aβ1-42-labeled antibody) are labeled with two different colored latex microspheres (e.g., green and red latex microspheres), following the same procedure as above. The two labels are mixed in equal volumes and then uniformly sprayed onto the conjugate pad. A detection line coated with a mixture of monoclonal antibodies (Aβ1-40-coated antibody and Aβ1-42-coated antibody) for these two indicators is designed on the nitrocellulose membrane. Following the aforementioned test strip assembly method, a dual-indicator test strip is obtained. During use, if the sample contains both indicators, the detection line will display different colors depending on the concentration of each indicator. Therefore, based on the provided standard colorimetric card, not only can the presence and concentration of the two analytes be determined, but also their relative content can be judged; the effect is shown in the figure. Figure 2 As shown.

[0033] Test strip performance testing (1) The performance of the immunochromatographic test strips prepared in Examples 1-4 above was verified. The Aβ1-40 and Aβ1-42 antigen standards were diluted with PBS buffer (pH=7.4) and negative urine to concentrations of 0.05, 0.1, 0.2, 0.3, 0.5, 0.75, 1, 1.25, 1.5, 2, 5, 10, 20, 50, and 100 ng / mL, respectively. 50 μL of each concentration was added to the sample pad of each test strip, and the results were observed after standing for 15 min. The results showed that the Aβ1-40 and Aβ1-42 single-detection immunochromatographic test strips prepared in this invention, whether using latex microsphere or colloidal gold strips, produced consistent results for both urine and PBS buffer samples. Furthermore, within the concentration range of 0.5-100 ng / mL, the color intensity of the T line on the test strip was directly proportional to the concentration of the Aβ1-40 / Aβ1-42 antigen, and no hook effect was observed within this detection range. For both analytes mentioned above, the detection limits of the latex microsphere test strips are around 0.1 ng / mL; the detection line of the colloidal gold test strip is around 0.3 ng / mL, both meeting the detection requirements for the two markers. The color development effects of the test strips after adding different concentrations of the analytes to negative urine samples are shown below. Figure 3-6 As shown.

[0034] (2) Clinical validation: 200 clinical samples (including urine samples from 90 clinically diagnosed AD patients and 110 normal individuals) were collected to evaluate the detection performance of the test strips prepared in Examples 1-4 above. The results are shown in Table 1: Table 1 (3) The color development effect of the Aβ1-40 and Aβ1-42 combined immunochromatographic test strips prepared in Examples 5 and 6 is as follows: Figure 7 As shown, the two target analytes exhibit normal color development within a concentration range of 0.5-10 ng / mL, enabling efficient simultaneous detection of both indicators. This demonstrates advantages such as high sensitivity, high detection efficiency, and convenient and quick operation.

[0035] The test results above show that the β-amyloid protein test strip prepared by this invention is suitable for urine sample detection and has high sensitivity and accuracy. At the same time, the test strip is simple to prepare, low in cost and easy to operate, and can be applied to home self-testing and large-scale screening scenarios.

[0036] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. An immunochromatographic test strip for detecting β-amyloid protein, characterized in that, The sample pad, conjugation pad, nitrocellulose membrane and absorbent pad are fixed sequentially on the base plate. The nitrocellulose membrane is provided with a quality control line and at least one detection line. The conjugation pad is coated with a marker obtained by conjugating a chromogenic reagent with a β-amyloid protein-labeled antibody. The detection line and control line are obtained by streaking with a β-amyloid protein-coated antibody and a goat anti-mouse polyclonal antibody, respectively. The β-amyloid labeled antibody and the β-amyloid coated antibody are monoclonal antibodies that can bind to different epitopes on the same β-amyloid protein; The colorimetric reagent is latex microspheres or colloidal gold.

2. The immunochromatographic test strip for β-amyloid protein detection according to claim 1, characterized in that, The nitrocellulose membrane is provided with a quality control line and a detection line; The conjugate pad is coated with a marker obtained by conjugating a chromogenic reagent with an Aβ1-40-labeled antibody, and the detection line and control line are obtained by streaking with an Aβ1-40-coated antibody and a goat anti-mouse polyclonal antibody, respectively; or the conjugate pad is coated with a marker obtained by conjugating a chromogenic reagent with an Aβ1-42-labeled antibody, and the detection line and control line are obtained by streaking with an Aβ1-42-coated antibody and a goat anti-mouse polyclonal antibody, respectively.

3. The immunochromatographic test strip for β-amyloid protein detection according to claim 1, characterized in that, The conjugation pad is sprayed with a mixture of two labels obtained by conjugating a colorimetric reagent with an Aβ1-40-labeled antibody and an Aβ1-42-labeled antibody, respectively; the nitrocellulose membrane has two detection lines, which are obtained by streaking with the Aβ1-40-coated antibody and the Aβ1-42-coated antibody, respectively; or the nitrocellulose membrane has one detection line, which is obtained by streaking with a mixture of the Aβ1-40-coated antibody and the Aβ1-42-coated antibody.

4. A method for preparing an immunochromatographic test strip for detecting β-amyloid protein as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. The chromogenic reagent is coupled with the β-amyloid protein-labeled antibody to obtain the label; S2. The marker is sprayed onto the conjugation pad, and the β-amyloid protein-coated antibody and goat anti-mouse polyclonal antibody are used to draw lines on the nitrocellulose membrane to obtain the detection line and control line, respectively. Finally, the sample pad, conjugation pad, nitrocellulose membrane and absorbent pad are pasted onto the base plate in sequence to obtain the β-amyloid protein detection immunochromatographic test strip.

5. The method for preparing the immunochromatographic test strip for β-amyloid protein detection according to claim 4, characterized in that, in, The colorimetric reagent is latex microspheres, and step S1 is as follows: The latex microspheres were activated with EDC and NHS, and then the activated latex microspheres were mixed with β-amyloid protein-labeled antibodies and reacted for 2-6 hours. Subsequently, they were blocked with blocking solution containing bovine serum albumin for 1-3 hours. After washing with washing solution, the labeled substance was obtained and stored in preservation solution for later use.

6. The method for preparing the immunochromatographic test strip for β-amyloid protein detection according to claim 4, characterized in that, in, The colorimetric reagent is colloidal gold, and step S1 is as follows: The colloidal gold solution was adjusted to pH 8.2±0.1 with potassium carbonate aqueous solution, and then mixed with β-amyloid protein-labeled antibody and reacted for 30-60 minutes. Subsequently, blocking solution containing bovine serum albumin was added and blocked for 20-60 minutes. After centrifugation, the labeled substance was obtained, reconstituted with preservation solution, and stored for later use.

7. The method for preparing the immunochromatographic test strip for β-amyloid protein detection according to claim 5 or 6, characterized in that, In step S2, the sample pad is soaked in the sample pad treatment solution beforehand, and the conjugate pad is soaked in the conjugate pad treatment solution. The sample pad treatment solution comprises the following components: Buffer solution with pH 7.0-10.0 and a concentration of 10-500 mM, 0.5-5 wt% bovine serum albumin, 0.1-2% Tween 20 (v / v), 0.1-2 wt% S9, and 0.01-1% ProClin 300 (v / v). The buffer solution in the sample pad treatment solution is selected from one of Tris-HCl buffer, carbonate buffer, or borate buffer. The conjugate pad treatment solution comprises the following components: 0.1-2% Tween 20 (volume concentration), 0.5-5 wt% sucrose, 0.1-5 wt% bovine serum albumin, 0.01-1% ProClin 300 (volume concentration), and Tris-HCl buffer solution with pH 7.0-9.0 and a concentration of 10-100 mM.

8. The method for preparing the immunochromatographic test strip for β-amyloid protein detection according to claim 7, characterized in that, in, A detection line is set on the nitrocellulose membrane. At this time: The marker is obtained by conjugating the Aβ1-40-labeled antibody with a chromogenic reagent and then spraying it onto the conjugation pad. The detection line is obtained by streaking the Aβ1-40-coated antibody. Alternatively, a marker can be obtained by conjugating a colorimetric reagent with an Aβ1-42-labeled antibody, which is then sprayed onto the conjugate pad. The detection line is obtained by streaking the Aβ1-42-coated antibody.

9. The method for preparing the immunochromatographic test strip for β-amyloid protein detection according to claim 7, characterized in that, A detection line is set on a nitrocellulose membrane. Aβ1-40 labeled antibody and Aβ1-42 labeled antibody are coupled with a chromogenic reagent, respectively. The two labels are mixed and then sprayed onto the conjugate pad. The detection line is obtained by running the mixture of Aβ1-40 coated antibody and Aβ1-42 coated antibody.

10. The method for preparing the immunochromatographic test strip for β-amyloid protein detection according to claim 7, characterized in that, Two detection lines are set on the nitrocellulose membrane. Aβ1-40 labeled antibody and Aβ1-42 labeled antibody are coupled with a chromogenic reagent, respectively. The two labels are mixed and then sprayed onto the conjugate pad. The Aβ1-40 coated antibody and Aβ1-42 coated antibody are respectively drawn on the nitrocellulose membrane to obtain two detection lines.

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