A kit for detecting a marker of alzheimer's disease and a method for preparing the same

Abstract

The application discloses a kit for detecting Alzheimer's disease markers and a preparation method thereof, and relates to the technical field of immune detection. The kit for detecting Alzheimer's disease markers comprises a shell and a test strip. The test strip comprises a bottom plate extending along a first direction, and a sample pad, a combination pad, a coated pad and a water absorption paper which are arranged on the bottom plate and are spaced apart along the first direction. The combination pad is coated with A beta 1-40 labeled first antibody, A beta 1-42 labeled first antibody and quality control labeled first antibody. The coated pad is sequentially provided with a first detection piece, a second detection piece and a quality control piece along the first direction. The first detection piece is coated with one of A beta 1-40 second antibody and A beta 1-42 second antibody. The second detection piece is coated with the other one of A beta 1-40 second antibody and A beta 1-42 second antibody. The quality control piece is coated with quality control second antibody. The technical scheme of the application can realize simultaneous detection of A beta 1-40 and A beta 1-42.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunodetection technology, in particular to a kit for detecting Alzheimer's disease markers and a preparation method thereof. BACKGROUND

[0002] Alzheimer's disease (AD) has a huge impact on the lives of patients and their families due to impaired thinking, memory, and independence, affecting the quality of life. Abnormal levels of amyloid-beta (Aβ) formed between neurons in the brain form plaques that are neurotoxic, leading to neuronal degeneration. Currently, in vitro quantitative detection of beta-amyloid 1-40 (Aβ1-40) and beta-amyloid 1-42 (Aβ1-42) is one of the tools recognized clinically as an aid in diagnosing Alzheimer's disease (AD), and by detecting the content of Aβ1-42 in plasma and the ratio of Aβ1-42 / Aβ1-40, the Aβ pathology in the brain can be reflected, which can be used for early assessment of the risk of AD and mild cognitive impairment.

[0003] Common methods for detecting Aβ1-40 and Aβ1-42 include chemiluminescence and enzyme-linked immunoassay, etc., among which, the enzyme-linked immunoassay detection operation step is cumbersome, time-consuming, and has poor precision; the chemiluminescence method is high in cost. Although some immunofluorescence rapid detection kits have appeared for large-scale screening of Alzheimer's disease content, most of these immunofluorescence rapid detection kits for detecting Aβ1-40 and Aβ1-42 are kits or test strips for detecting a single specific myocardial marker, i.e., the content of Aβ1-42 and the content of Aβ1-40 are first detected separately, and then the ratio between the two is calculated. The kit using the separate detection mode not only causes waste of detection samples but also has high use cost.

[0004] Therefore, the existing kit for detecting Alzheimer's disease markers has the defect that Aβ1-40 and Aβ1-42 cannot be detected simultaneously. SUMMARY

[0005] The main purpose of the present application is to provide a kit for detecting Alzheimer's disease markers and a preparation method thereof, aiming to solve the problem that the existing kit for detecting Alzheimer's disease markers cannot simultaneously detect Aβ1-40 and Aβ1-42.

[0006] To achieve the above-mentioned purpose, the present application provides a kit for detecting Alzheimer's disease markers, comprising a shell and a test strip, the test strip comprising a bottom plate extending in a first direction and a sample pad, a conjugate pad, a coating pad and a water-absorbing paper spaced apart on the bottom plate in the first direction;

[0007] The binding pad is coated with an Aβ1-40 labeled primary antibody, an Aβ1-42 labeled primary antibody and a quality control labeled primary antibody; the coated pad is sequentially provided with a first detection element, a second detection element and a quality control element in a first direction, the first detection element is coated with one of an Aβ1-40 secondary antibody and an Aβ1-42 secondary antibody, the second detection element is coated with the other of the Aβ1-40 secondary antibody and the Aβ1-42 secondary antibody, and the quality control element is coated with a quality control secondary antibody.

[0008] In an embodiment, the labels in the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody are all fluorescent microspheres.

[0009] In an embodiment, the fluorescent microspheres are time-resolved fluorescent microspheres, and the particle size of the fluorescent microspheres is 150-300 nm microspheres; and / or,

[0010] The Aβ1-40 labeled primary antibody is obtained by coupling fluorescent microspheres and Aβ1-40 primary antibodies at 0.2-0.4 mg / mL Aβ1-40 primary antibodies per 1 mL fluorescent microspheres; and / or,

[0011] The Aβ1-42 labeled primary antibody is obtained by coupling fluorescent microspheres and Aβ1-42 primary antibodies at 0.2-0.4 mg / mL Aβ1-42 primary antibodies per 1 mL fluorescent microspheres.

[0012] In an embodiment, the antibody in the quality control labeled primary antibody is a chicken IgY antibody, the quality control secondary antibody is a goat anti-chicken IgY antibody, and the IgY labeled primary antibody is obtained by coupling fluorescent microspheres and chicken IgY antibodies at 0.2-0.4 mg / mL chicken IgY antibodies per 1 mL fluorescent microspheres.

[0013] The present application provides a preparation method of the kit for detecting Alzheimer's disease markers, comprising the following steps:

[0014] A substrate is provided, and Aβ1-40 labeled primary antibodies, Aβ1-42 labeled primary antibodies and quality control labeled primary antibodies are placed on the substrate, and then dried to obtain a binding pad;

[0015] A base film is provided, and a first detection element, a second detection element and a quality control element are sequentially arranged on the base film in a first direction, and then dried to obtain a coated pad;

[0016] A base plate and an absorbent paper are provided, and the base plate, a sample pad, the absorbent paper, the binding pad and the coated pad are assembled to obtain a test strip;

[0017] An outer shell is provided, and the outer shell and the test strip are assembled to obtain the kit for detecting Alzheimer's disease markers.

[0018] In an embodiment, the step of providing a substrate, disposing the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody on the substrate, and drying to obtain a binding pad further comprises:

[0019] mixing the fluorescent microspheres and the activating agent, and activating to obtain activated fluorescent microspheres;

[0020] mixing the activated fluorescent microspheres with the Aβ1-40 primary antibody, the Aβ1-42 primary antibody and the chicken IgY antibody respectively, and coupling to obtain fluorescent microsphere-coupled Aβ1-40 primary antibody, fluorescent microsphere-coupled Aβ1-42 primary antibody and fluorescent microsphere-coupled chicken IgY antibody;

[0021] mixing the fluorescent microsphere-coupled Aβ1-40 primary antibody, the fluorescent microsphere-coupled Aβ1-42 primary antibody and the fluorescent microsphere-coupled chicken IgY antibody with a blocking agent respectively, and blocking to obtain the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody.

[0022] In an embodiment, the step of providing a substrate, disposing the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody on the substrate, and drying to obtain a binding pad further comprises:

[0023] providing a substrate, mixing the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody, the quality control labeled primary antibody and a marker diluent to obtain a binding pad coating solution, spraying the binding pad coating solution on the substrate, and drying to obtain a binding pad.

[0024] In an embodiment, the marker diluent is a mixed solution of 0.5-1% bovine serum albumin, 5-10% trehalose and 0.05M Tris-HCl buffer; and / or,

[0025] the pH value of the marker diluent is 8.0-9.0; and / or,

[0026] the mass ratio of the Aβ1-40 labeled primary antibody to the marker diluent is 1:(5-10); and / or,

[0027] the mass ratio of the Aβ1-42 labeled primary antibody to the marker diluent is 1:(5-10); and / or,

[0028] the mass ratio of the quality control labeled primary antibody to the marker diluent is 1:(30-40); and / or,

[0029] the substrate is a glass cellulose membrane, the width of the substrate is 6-7mm, the thickness of the substrate is 335-405μm, and the spraying amount of the binding pad coating solution is 8-10μL / cm.

[0030] In an embodiment, the step of providing a base membrane, sequentially arranging a first detection member, a second detection member and a quality control member along a first direction on the base membrane, and drying to obtain a coated pad comprises:

[0031] The coating solution is mixed with Aβ1-40 secondary antibody, Aβ1-42 secondary antibody and goat anti-chicken IgY antibody respectively to obtain Aβ1-40 secondary antibody coating solution, Aβ1-42 secondary antibody coating solution and goat anti-chicken IgY antibody coating solution.

[0032] A base membrane is provided, and the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the goat anti-chicken IgY antibody coating solution are respectively blotted on the base membrane, and dried to obtain a coated pad.

[0033] In an embodiment, the coating solution is a mixed solution of 0.01M phosphate buffer and 1-3% trehalose; and / or,

[0034] The concentration of the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the goat anti-chicken IgY antibody coating solution is 1.5-2.5 mg / mL; and / or,

[0035] The base membrane is a nitrocellulose membrane, the pore size of the base membrane is 8-15 μm, the width is 24.5-25 mm, the thickness is 225-255 μm, and the amount of the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the goat anti-chicken IgY antibody coating solution used when blotted is 1-1.5 μL / cm.

[0036] The technical scheme of the present application adopts the principle of immunofluorescence chromatography and the double antibody sandwich method to detect Aβ1-40 and Aβ1-42. The Aβ1-40 labeled first antibody and the Aβ1-42 labeled first antibody are coated on the binding pad, and the Aβ1-40 second antibody and the Aβ1-42 second antibody are coated on the coating pad. When the sample to be tested is added, the Aβ1-40 and Aβ2-40 in the sample bind to the Aβ1-40 labeled first antibody and the Aβ1-42 labeled first antibody in the binding pad area to form the Aβ1-40 labeled first antibody and the Aβ1-42 labeled first antibody. The complex of Aβ1-40 and Aβ1-40 labeled first antibody and the complex of Aβ1-42 and Aβ1-42 labeled first antibody will diffuse along the coating pad and bind to the Aβ1-40 second antibody or the Aβ1-42 second antibody on the first detection piece and the second detection piece to form the corresponding complex of Aβ1-40, Aβ1-40 labeled first antibody, Aβ1-40 second antibody and Aβ1-42, Aβ1-42 labeled first antibody, Aβ1-42 second antibody. The higher the concentration of Aβ1-40 and Aβ2-40 in the sample to be tested, the more the corresponding labeled first antibody-antigen-second antibody will be gathered on the detection line, and the stronger the fluorescence signal at a specific wavelength will be. By analyzing the fluorescence signal, the content of Aβ1-40 and Aβ1-42 can be obtained, and the simultaneous detection of Aβ1-40 and Aβ1-42 can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained according to the structures shown in the drawings without creative labor for those skilled in the art.

[0038] Figure 1 The plan view of an embodiment of the kit for detecting Alzheimer's disease markers provided by the present application;

[0039] Figure 2 The plan view of the test strip in Figure 1 ;

[0040] Figure 3 The linear range result graph of the kit for detecting Alzheimer's disease markers provided by the present application in Example 1 for detecting Aβ1-40 and Aβ1-42;

[0041] Figure 4 The linear range result graph of the kit for detecting Alzheimer's disease markers provided by the present application in Example 2 for detecting Aβ1-40 and Aβ1-42;

[0042] Figure 5 Figure of linear range result of the kit for detecting Alzheimer's disease marker provided in Embodiment 3 of the present application for detecting Aβ1-40 and Aβ1-42;

[0043] Figure 6 Figure of linear range result of the kit for detecting Alzheimer's disease marker provided in Embodiment 4 of the present application for detecting Aβ1-40 and Aβ1-42.

[0044] BRIEF DESCRIPTION OF DRAWINGS

[0045] 100, kit for detecting Alzheimer's disease marker; 1, shell; 11, sample hole 2, test strip; 21, bottom plate; 22, sample pad; 23, binding pad; 24, coated pad; 241, first detection piece; 242, second detection piece; 243, quality control piece; 25, absorbent paper.

[0046] The implementation, functional features and advantages of the present application will be further described with reference to the embodiments in combination with the accompanying drawings. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0048] It should be noted that if the embodiments of the present application involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement condition, etc. between the components in a certain posture, and if the certain posture changes, the directional indications also change accordingly.

[0049] In addition, if the description of "first", "second" and the like is involved in the embodiments of the present application, the description of "first", "second" and the like is only for the purpose of description, and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features. Therefore, the features limited by "first", "second" can be explicitly or implicitly included at least one of the features. In addition, if "and / or" or "and / or" appears throughout the text, it means that the three parallel schemes are included, for example, "A and / or B" includes A scheme, or B scheme, or A and B scheme. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the realization of the ordinary skilled in the art, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, and is not within the protection scope required by the present application.

[0050] Alzheimer's disease (AD) is the most common type of senile dementia, which affects the quality of life due to impaired thinking, memory and independence, and has a huge impact on patients and their families. At present, in vitro quantitative detection of beta-amyloid 1-40 (Aβ1-40) and beta-amyloid 1-42 (Aβ1-42) is one of the clinically recognized tools for assisting the diagnosis of Alzheimer's disease (AD), which can be used for early assessment of the risk of AD dementia and mild cognitive impairment by detecting the content of Aβ1-42 in plasma and the ratio of Aβ1-42 / Aβ1-40 to reflect the Aβ pathology in the brain.

[0051] Common methods for detecting Aβ1-40 and Aβ1-42 include chemiluminescence and enzyme-linked immunoassay, etc. Among them, the enzyme-linked immunoassay has complicated operation steps, time-consuming and poor precision; the chemiluminescence method has high cost. Although some immunofluorescence rapid detection kits have appeared for large-scale screening of Alzheimer's disease content, most of these immunofluorescence rapid detection kits for detecting Aβ1-40 and Aβ1-42 are kits or test strips for detecting a single specific myocardial marker, that is, the content of Aβ1-42 and the content of Aβ1-40 are detected separately, and then the ratio between the two is calculated. The kit using the separate detection mode not only causes waste of detection samples but also has high use cost. Therefore, the existing kit for detecting Alzheimer's disease markers has the defect of being unable to simultaneously detect Aβ1-40 and Aβ1-42.

[0052] In view of this, the present application provides a kit 100 for detecting Alzheimer's disease markers.

[0053] Please refer to Figure 1 and Figure 2In an embodiment of the present application, the kit 100 for detecting Alzheimer's disease markers comprises a housing 1 and a test strip 2, the test strip 2 comprising a base plate 21 extending along a first direction and a sample pad 22, a conjugate pad 23, a coated pad 24 and an absorbent paper 25 arranged on the base plate 21 along the first direction;

[0054] The conjugate pad 23 is coated with an Aβ1-40 primary antibody, an Aβ1-42 primary antibody and a quality control primary antibody; the coated pad 24 comprises a first detection member 241, a second detection member 242 and a quality control member 243 arranged along the first direction in sequence, the first detection member 241 is coated with one of an Aβ1-40 secondary antibody and an Aβ1-42 secondary antibody, the second detection member 242 is coated with the other of the Aβ1-40 secondary antibody and the Aβ1-42 secondary antibody, and the quality control member is coated with a quality control secondary antibody.

[0055] In the technical scheme of the present application, the Aβ1-40 primary antibody specifically recognizes Aβ1-40 in the sample to be tested, the Aβ1-40 secondary antibody specifically recognizes Aβ1-40 in the sample to be tested, and the Aβ1-40 primary antibody and the Aβ1-40 secondary antibody do not recognize the same site of Aβ1-40; the Aβ1-42 primary antibody specifically recognizes Aβ1-42 in the sample to be tested, the Aβ1-42 secondary antibody specifically recognizes Aβ1-42 in the sample to be tested, and the Aβ1-42 primary antibody and the Aβ1-42 secondary antibody do not recognize the same site of Aβ1-42; and the quality control secondary antibody is used to capture the quality control primary antibody.

[0056] The technical scheme of the present application adopts the immunofluorescence chromatography principle and double antibody sandwich method to detect Alzheimer's disease markers Aβ1-40 and Aβ1-42. The kit provided by the present application coats Aβ1-40 labeled primary antibody and Aβ1-42 labeled primary antibody on the binding pad 23 respectively, and coats Aβ1-40 secondary antibody and Aβ1-42 secondary antibody on the coating pad 24 respectively. After adding the sample to be tested through the sample hole 11, the Aβ1-40 labeled primary antibody in the binding pad 23 region specifically recognizes Aβ1-40 in the sample to be tested, forming a complex of Aβ1-40 and Aβ1-40 labeled primary antibody, and the Aβ1-42 labeled primary antibody in the binding pad 23 region specifically recognizes Aβ1-42 in the sample to be tested, forming a complex of Aβ1-42 and Aβ1-42 labeled primary antibody. Due to the chromatography effect, the complex of Aβ1-40 and Aβ1-40 labeled primary antibody, and the complex of Aβ1-42 and Aβ1-42 labeled primary antibody will diffuse along the coating pad 24, and combine with Aβ1-40 secondary antibody or Aβ1-42 secondary antibody coated on the first detection piece 241 and the second detection piece 242 to form a complex of Aβ1-40, Aβ1-40 labeled primary antibody and Aβ1-40 secondary antibody, and a complex of Aβ1-42, Aβ1-42 labeled primary antibody and Aβ1-42 secondary antibody. The higher the concentration of Aβ1-40 and Aβ2-40 in the sample to be tested, the more the labeled primary antibody-antigen-secondary antibody complex will gather on the detection line, and the stronger the fluorescence signal under a specific wavelength. By analyzing the fluorescence signal, the content of Aβ1-40 and Aβ1-42 can be obtained, and the simultaneous detection of Aβ1-40 and Aβ1-42 can be realized.

[0057] Compared with a single Aβ1-40 detection kit or a single Aβ1-42 detection kit, the kit 100 for detecting Alzheimer's disease markers provided by the technical scheme of the present application can simultaneously obtain the content of Aβ1-40 and Aβ1-42 using the same sample to be tested, without the need to replace the sample for two detections, which can reduce the sample amount and improve the reliability of the detection result. Moreover, the kit provided by the present application has been proved by interference test to have less interference when simultaneously detecting Aβ1-40 and Aβ1-42.

[0058] It should be noted that the first detection piece 241 is coated with one of Aβ1-40 secondary antibody and Aβ1-42 secondary antibody, and the second detection piece 242 is coated with the other of Aβ1-40 secondary antibody and Aβ1-42 secondary antibody, which means that the positions of Aβ1-40 secondary antibody and Aβ1-42 secondary antibody are not limited. The first detection piece 241 can be coated with Aβ1-40 secondary antibody, and the second detection piece 242 can be coated with Aβ1-42 secondary antibody, or the first detection piece 241 can be coated with Aβ1-42 secondary antibody, and the second detection piece 242 can be coated with Aβ1-40 secondary antibody. Both of the above two setting modes belong to the protection scope of the present application, and both of them can realize rapid detection with high sensitivity.

[0059] In the embodiments of the present application, the labels in the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody are all fluorescent microspheres.

[0060] The fluorescent microspheres have good stability and dispersibility, high sensitivity, and the technical scheme of the present application uses fluorescent microspheres as labels, which helps to improve the stability and sensitivity of the kit.

[0061] In the embodiments of the present application, the fluorescent microspheres are time-resolved fluorescent microspheres, and the particle size of the fluorescent microspheres is 150-300 nm.

[0062] The time-resolved fluorescent microspheres have higher sensitivity and specificity, good stability and repeatability, and are not easy to quench. The technical scheme of the present application selects time-resolved fluorescent microspheres to label antibodies, which can obtain higher detection sensitivity and good batch consistency, so that the prepared kit is suitable for long-term storage and application.

[0063] In the embodiments of the present application, the fluorescent microspheres are time-resolved fluorescent microspheres, and the particle size of the fluorescent microspheres is 150-300 nm. The time-resolved fluorescent microspheres with a particle size of 150-300 nm are selected, which can ensure a faster chromatographic rate, shorten the detection time, and have higher detection sensitivity and accuracy, and can improve the detection range of Aβ1-40 and Aβ1-42. Preferably, time-resolved fluorescent microspheres with a particle size of 200-300 nm are selected to label antibodies.

[0064] In the embodiments of the present application, the Aβ1-40 labeled primary antibody is obtained by coupling 0.2-0.4 mg / mL Aβ1-40 primary antibody per 1 mL fluorescent microspheres. The use amount of fluorescent microspheres and Aβ1-40 primary antibody is set in the above range, the coupling efficiency of fluorescent microspheres and Aβ1-40 primary antibody is high, which can ensure the reliability and sensitivity of the detection results, and at the same time control the production cost. In this range, the addition concentration of the Aβ1-40 primary antibody can be 0.2 mg / mL, 0.3 mg / mL and 0.4 mg / mL. Preferably, the addition concentration of the Aβ1-40 primary antibody is set to 0.3 mg / mL.

[0065] In the embodiment of the present application, the Aβ1-42 marker primary antibody is coupled by the fluorescent microspheres and the Aβ1-42 primary antibody at 0.2-0.4 mg / mL of the Aβ1-42 primary antibody per 1 mL of the fluorescent microspheres. The use amount of the fluorescent microspheres and the Aβ1-42 primary antibody is set in the above range, the coupling efficiency of the fluorescent microspheres and the Aβ1-42 primary antibody is high, the reliability and sensitivity of the detection result can be ensured, and the production cost is controlled. In this range, the addition concentration of the Aβ1-42 primary antibody can be 0.2 mg / mL, 0.3 mg / mL and 0.4 mg / mL. Preferably, the addition concentration of the Aβ1-42 primary antibody is set to 0.3 mg / mL.

[0066] In the embodiment of the present application, the antibody in the quality control marker primary antibody is chicken IgY antibody, and the quality control secondary antibody is goat anti-chicken IgY antibody. The IgY antibody has high specificity, affinity and stability, can reduce the possibility of non-specific binding, and is easy to mass-produce.

[0067] In the embodiment of the present application, the IgY marker primary antibody is coupled by the fluorescent microspheres and the chicken IgY antibody at 0.2-0.4 mg / mL of the chicken IgY antibody per 1 mL of the fluorescent microspheres. The IgY marker primary antibody is prepared by coupling the fluorescent microspheres and the chicken IgY antibody. The use amount of the fluorescent microspheres and the chicken IgY antibody is set in the above range, the effectiveness requirement of the kit can be met, and the production cost is controlled. In this range, the addition concentration of the chicken IgY antibody can be 0.2 mg / mL, 0.3 mg / mL and 0.4 mg / mL.

[0068] The present application provides a preparation method of the kit 100 for detecting Alzheimer's disease markers, comprising the following steps:

[0069] A substrate is provided, the Aβ1-40 marker primary antibody, the Aβ1-42 marker primary antibody and the quality control marker primary antibody are arranged on the substrate, and drying is performed to obtain a binding pad 23;

[0070] A base film is provided, a first detection member 241, a second detection member 242 and a quality control member 243 are sequentially arranged on the base film along a first direction, and drying is performed to obtain a coating pad 24;

[0071] A bottom plate 21 and an absorbent paper 25 are provided, and the bottom plate 21, the sample pad 22, the absorbent paper 25, the binding pad 23 and the coating pad 24 are assembled to obtain a test strip 2;

[0072] An outer shell 1 is provided, and the outer shell 1 and the test strip 2 are assembled to obtain the kit 100 for detecting Alzheimer's disease markers.

[0073] The preparation method provided by the application sets the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody on the combination pad 23 respectively, and sequentially sets a first detection piece 241, a second detection piece 242 and a quality control piece 243 on the coating pad 24 in a first direction, wherein the first detection piece 241 is coated with one of the Aβ1-40 secondary antibody and the Aβ1-42 secondary antibody, and the second detection piece 242 is coated with the other one of the Aβ1-40 secondary antibody and the Aβ1-42 secondary antibody; then, the kit is assembled according to the conventional method for preparing the kit, the bottom plate 21, the sample pad 22, the combination pad 23 processed above, the coating pad 24 processed above and the water absorption paper 25 are assembled to obtain the test strip 2, and then the shell 101 and the test strip 2 are assembled to obtain the kit 100 for detecting the Alzheimer's disease marker. The kit 100 for detecting the Alzheimer's disease marker prepared by the above preparation method has a simple structure, is convenient to operate and can realize the simultaneous detection of Aβ1-40 and Aβ1-42.

[0074] In the embodiment of the application, the sample pad 22 is a sample pad treated by soaking in a sample pad treatment liquid, and the preparation method of the sample pad treated by soaking in the sample pad treatment liquid comprises the following steps:

[0075] The sample pad is treated by soaking in the sample pad treatment liquid and is dried at 40-50℃;

[0076] The sample pad 22 is made of glass cellulose membrane.

[0077] The sample pad treatment liquid is a mixed solution of 0.01M phosphate buffer, 1% bovine serum albumin, 4% trehalose, 1.0% Tween 20 and 1% EDTA-2Na. The sample pad treatment liquid with the above components can adjust the sample flow characteristics and promote the uniform flow of the sample along the membrane.

[0078] In the embodiment of the application, the combination pad 23 is a combination pad treated by soaking in a combination pad treatment liquid, and the preparation method of the combination pad treated by soaking in the combination pad treatment liquid comprises the following steps:

[0079] The combination pad is treated by soaking in the combination pad treatment liquid and is dried at 40-50℃;

[0080] The combination pad treatment liquid is a mixed solution of 0.01M phosphate buffer, 5% trehalose and 0.5% casein. The combination pad treatment liquid with the above components is beneficial to ensure the uniform release of the microspheres and protect the labeled antibody to prolong the shelf life of the kit.

[0081] In the embodiment of the present application, the step of providing a substrate, disposing Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody and quality control labeled primary antibody on the substrate, and drying to obtain a binding pad 23 comprises the following steps:

[0082] Mixing the fluorescent microspheres and the activating agent, and activating to obtain activated fluorescent microspheres;

[0083] Mixing the activated fluorescent microspheres with Aβ1-40 primary antibody, Aβ1-42 primary antibody and chicken IgY antibody respectively, and coupling to obtain fluorescent microsphere-coupled Aβ1-40 primary antibody, fluorescent microsphere-coupled Aβ1-42 primary antibody and fluorescent microsphere-coupled chicken IgY antibody;

[0084] Mixing the fluorescent microsphere-coupled Aβ1-40 primary antibody, the fluorescent microsphere-coupled Aβ1-42 primary antibody and the fluorescent microsphere-coupled chicken IgY antibody with blocking agent respectively, and blocking to obtain Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody and quality control labeled primary antibody.

[0085] In the embodiment of the present application, the step of mixing the fluorescent microspheres and the activating agent, and activating to obtain activated fluorescent microspheres comprises the following steps:

[0086] Mixing the fluorescent microspheres with first boric acid-borax buffer, 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and shaking in a shaking table at room temperature for 30 min in the dark, centrifuging at 14000 r / m for 25 min, removing the supernatant, re-dissolving with second boric acid-borax buffer, and washing by centrifuging at 14000 r / m for 25 min, and removing the supernatant to obtain activated fluorescent microspheres;

[0087] The mass ratio of the fluorescent microspheres, 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is (12-18):(10-17):(10-17).

[0088] The first boric acid-borax buffer has a molar concentration of 0.05 M and a pH of 6.0.

[0089] The second boric acid-borax buffer has a molar concentration of 0.05 M and a pH of 7.5.

[0090] In the embodiment of the present application, the step of blocking comprises adding bovine serum albumin (BSA) with a mass concentration of 10%, and blocking in a shaking table at room temperature for 30 min, and after blocking is completed, centrifuging at 14000 r / m for 25 min to remove the supernatant, and re-dissolving with second boric acid-borax buffer, and washing by centrifuging at 14000 r / m for 25 min to remove the supernatant.

[0091] In the embodiment of the present application, after the preparation of the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody, the labeled antibodies are reconstituted with a conjugate storage solution and stored in an environment of 2-8°C. The conjugate storage solution is a mixed solution of 0.5-1% bovine serum albumin, 5-10% trehalose, 0.05M Tris-HCl buffer, 0.05-0.1% casein and 0.05-0.1% Tween 20.

[0092] In the embodiment of the present application, the step of providing a substrate, placing the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody on the substrate, drying and obtaining the binding pad 23 comprises:

[0093] providing a substrate, mixing the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody, the quality control labeled primary antibody and the label diluent to obtain a binding pad coating solution, spraying the binding pad coating solution on the substrate and drying to obtain the binding pad 23.

[0094] In the embodiment of the present application, the label diluent is a mixed solution of 0.5-1% bovine serum albumin, 5-10% trehalose and 0.05M Tris-HCl buffer. The use of high concentration of bovine serum albumin is beneficial to protect the labeled protein, block the sites on the binding pad that are easy to bind with the protein and flow block the base film (such as nitrocellulose membrane) to prevent the problem of non-specific binding; trehalose as a drying protective agent prevents the deterioration of the protein during drying. Tris-HCl provides high ionic strength to increase the flowability of the sample.

[0095] In the embodiment of the present application, the pH value of the label diluent is 8.0-9.0.

[0096] In the embodiment of the present application, the mass ratio of the Aβ1-40 labeled primary antibody to the label diluent is 1:(5-10). Within this range, the mass ratio of the Aβ1-40 labeled primary antibody to the label diluent can be 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10, all of which belong to the protection scope of the present application.

[0097] In the embodiment of the present application, the mass ratio of the Aβ1-42 labeled primary antibody to the label diluent is 1:(5-10). Within this range, the mass ratio of the Aβ1-42 labeled primary antibody to the label diluent can be 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10, all of which belong to the protection scope of the present application.

[0098] In the embodiment of the present application, the mass ratio of the quality control labeled primary antibody to the marker diluent is 1: (30-40). Within this range, the mass ratio of the quality control labeled primary antibody to the marker diluent can be 1:30, 1:32, 1:34, 1:36, 1:38 and 1:40, all of which belong to the protection scope of the present application.

[0099] In the embodiment of the present application, the substrate is a glass cellulose membrane, the width of the substrate is 6-7 mm, and the thickness of the substrate is 225-255 μm. When the binding pad coating liquid is sprayed on the substrate, the spraying amount of the binding pad coating liquid is 8-10 μL / cm.

[0100] Controlling the spraying amount of the binding pad coating liquid in the above range helps to ensure the uniform distribution and effective fixation of the labeled antibody on the binding pad 23, so that the final concentration of the antibody fixed on the binding pad 23 is in a suitable range, and the sensitivity, specificity and stability of the entire detection system are better. When the spraying amount of the binding pad coating liquid is less than 8 μL / cm, a high concentration of sample will produce a hook effect, which reduces the high value sensitivity of the entire detection system; when the spraying amount of the binding pad coating liquid is more than 10 μL / cm, there is too much binding pad coating liquid on the binding pad 23, which causes uneven release of the fluorescent microspheres, and the specificity of the entire detection system decreases. Considering the above factors, the spraying amount of the binding pad coating liquid is set to 8-10 μL / cm.

[0101] In the embodiment of the present application, the step of providing a base film, sequentially arranging the first detection member 241, the second detection member 242 and the quality control member 243 on the base film along a first direction, and drying to obtain the coated pad 24 comprises:

[0102] The coating liquid is mixed with the Aβ1-40 secondary antibody, the Aβ1-42 secondary antibody and the goat anti-chicken IgY antibody respectively to obtain the Aβ1-40 secondary antibody coating liquid, the Aβ1-42 secondary antibody coating liquid and the goat anti-chicken IgY antibody coating liquid.

[0103] The base film is provided, the Aβ1-40 secondary antibody coating liquid, the Aβ1-42 secondary antibody coating liquid and the goat anti-chicken IgY antibody coating liquid are respectively drawn on the base film, and drying is performed to obtain the coated pad 24.

[0104] In the embodiment of the present application, the coating liquid is a mixed solution of a phosphate buffer with a molar concentration of 0.01 M and trehalose with a mass concentration of 1-3%.

[0105] In the embodiments of the present application, the volume concentrations of the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the goat anti-chicken IgY antibody coating solution are all 1.5-2.5 mg / mL. Within this range, the volume concentrations of the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the goat anti-chicken IgY antibody coating solution can all be set to 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, 2.2 mg / mL and 2.5 mg / mL.

[0106] In the embodiments of the present application, the base film is a nitrocellulose membrane, the pore size of the base film is 8-15 μm, the width is 24.5-25.5 mm, and the thickness is 225-255 μm.

[0107] In the embodiments of the present application, the amounts of the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the goat anti-chicken IgY antibody coating solution used when the membrane is drawn are all 1-1.5 μL / cm. When the amounts of the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the goat anti-chicken IgY antibody coating solution used when the membrane is drawn are less than 1 μL / cm, high-concentration samples will produce a HOOK effect, which reduces the high-value sensitivity of the entire detection system; when the amounts of the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the goat anti-chicken IgY antibody coating solution used when the membrane is drawn are greater than 1.5 μL / cm, the coating solution will spread on the membrane, which reduces the sensitivity, specificity and stability of the kit. Taking the above factors into consideration, the amounts of the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the goat anti-chicken IgY antibody coating solution used when the membrane is drawn are all set to 1-1.5 μL / cm. Within this range, the amounts of the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the goat anti-chicken IgY antibody coating solution used when the membrane is drawn can all be 1 μL / cm, 1.2 μL / cm and 1.5 μL / cm.

[0108] The technical solutions of the present application are further described in detail below in combination with specific embodiments. It should be understood that the following embodiments are only used to explain the present application and do not limit the present application.

[0109] In the following embodiments, the 150-3200 nm fluorescent microspheres are purchased from Changsha Meiniu Biological Technology Co., Ltd., and the model number is MF02;

[0110] The 200-300 nm fluorescent microspheres are purchased from Changsha Meiniu Biological Technology Co., Ltd., and the model number is MF03;

[0111] The Aβ1-40 primary antibody is Anti-Amyloid-β1-40 McAb, and the item number is AB1-40101, which is purchased from Hangzhou Boyue Biological Technology Co., Ltd.

[0112] Aβ1-40 primary antibody is Anti-Amyloid-β1-40 McAb, product number AB1-40102, purchased from Hangzhou Boyue Biotechnology Co., Ltd.

[0113] Aβ1-40 primary antibody is Anti-Amyloid-β1-40 McAb, product number AB1-40102, purchased from Hangzhou Boyue Biotechnology Co., Ltd.

[0114] Aβ1-40 primary antibody is Anti-Amyloid-β1-40 McAb, product number AB1-40102, purchased from Hangzhou Boyue Biotechnology Co., Ltd.

[0115] Aβ1-40 primary antibody is Anti-Amyloid-β1-40 McAb, product number AB1-40102, purchased from Hangzhou Boyue Biotechnology Co., Ltd.

[0116] Aβ1-40 primary antibody is Anti-Amyloid-β1-40 McAb, product number AB1-40102, purchased from Hangzhou Boyue Biotechnology Co., Ltd.

[0117] Example 1

[0118] A method for preparing a kit for detecting Alzheimer's disease markers, comprising the following steps:

[0119] 1. Preparation of the combination pad:

[0120] 1) Preparation of Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody and quality control labeled primary antibody:

[0121] a. Activation: Take 400 μL of 0.05M boric acid-borax buffer (pH 6.0) and 40 μL of 200-300 nm fluorescent microspheres in a centrifuge tube, mix well. Add 50 μL of EDC, 50 μL of NHS solution for activation, shake on a shaker at room temperature for 30 min. After activation, centrifuge at 14000 r / m for 25 min, remove the supernatant, and resuspend with 0.05M boric acid-borax buffer (pH 7.5), and centrifuge for washing once;

[0122] b. Coupling: After resuspension of the fluorescent microspheres, add Aβ1-40 primary antibody, Aβ1-42 primary antibody and chicken IgY antibody, respectively, and shake on a shaker at room temperature for 2 h to obtain fluorescent microspheres coupled with Aβ1-40 primary antibody, fluorescent microspheres coupled with Aβ1-42 primary antibody and fluorescent microspheres coupled with chicken IgY antibody; wherein, 0.3 mg / mL of Aβ1-40 primary antibody, 0.3 mg / mL of Aβ1-42 primary antibody and 0.3 mg / mL of chicken IgY antibody are added to each 1 mL of fluorescent microspheres for coupling;

[0123] c. Blocking: 50 μL of conjugate blocking solution was added to the fluorescent microsphere-coupled Abeta1-40 primary antibody, fluorescent microsphere-coupled Abeta1-42 primary antibody and fluorescent microsphere-coupled chicken IgY antibody, respectively, and the mixture was blocked on a shaker at room temperature for 30 min. After blocking, the mixture was centrifuged at 14,000 r / m for 25 min to obtain Abeta1-40 labeled primary antibody, Abeta1-42 labeled primary antibody and chicken IgY labeled antibody;

[0124] d. Preservation: After washing, the mixture was reconstituted with a conjugate storage solution, ultrasonically dispersed and stored at 2-8°C; the conjugate storage solution was a mixture of 0.5% bovine serum albumin, 10% trehalose, 0.05M Tris-HCl buffer, 0.05% casein and 0.05% Tween 20.

[0125] 2) Coating Abeta1-40 labeled primary antibody, Abeta1-42 labeled primary antibody and chicken IgY labeled antibody:

[0126] A glass fiber cellulose membrane with a width of 6.8 mm and a thickness of 360 μm was selected as the substrate; Abeta1-40 labeled primary antibody, Abeta1-42 labeled primary antibody, chicken IgY antibody and marker diluent were mixed to obtain a conjugate pad coating solution, which was uniformly sprayed onto the glass fiber cellulose membrane using a film sprayer (10 μL / cm of film spraying amount), and the mixture was dried in an oven at 45°C overnight to obtain a conjugate pad; the marker diluent included 0.5% BSA, 5% trehalose and 0.05M Tris-HCl (pH=8.2). The concentration of Abeta1-40 labeled primary antibody, Abeta1-42 labeled primary antibody and chicken IgY antibody after dilution was 1.0 mg / mL.

[0127] 2. Preparation of the coating pad:

[0128] A nitrocellulose membrane (NC membrane) with a pore size of 10 μm, a width of 25 mm and a thickness of 240 μm was selected as the base film; Abeta1-40 secondary antibody, Abeta1-42 secondary antibody and goat anti-chicken IgY antibody were diluted with an NC coating solution (0.01M phosphate buffer, 1-3% trehalose) to obtain a concentration of 2.0 mg / mL;

[0129] A first detection member, a second detection member and a quality control member were set up, respectively; the first detection member was coated with Abeta1-40 secondary antibody, the second detection member was coated with Abeta1-42 secondary antibody, and the quality control member was coated with goat anti-chicken IgY antibody; the film amount was 1.0 μL / cm, and the mixture was dried in an oven at 60°C for 1-3 days to obtain a coating pad.

[0130] 3. Preparation of the sample pad:

[0131] Select a glass cellulose membrane with a width of 20 mm, immerse the glass cellulose membrane in a sample pad treatment solution, and place it in a 45°C oven to dry overnight to obtain a sample pad; the sample pad treatment solution is a mixed solution of a 0.01M molar concentration phosphate buffer, 1% bovine serum albumin, 4% trehalose, 1.0% Tween 20, and 1% EDTA-2Na.

[0132] 4. Assemble the test card:

[0133] Provide a water-absorbing paper with a width of 17 mm and a PVC base plate with a width of 60 mm, and sequentially paste the sample pad, the conjugate pad, the coated pad, and the water-absorbing paper obtained after the above treatment on the PVC base plate to obtain a test strip, as shown in Figure 2

[0134] Assemble the reagent strip into a shell to complete the assembly of the test card.

[0135] Example 2

[0136] Compared with Example 1, the difference is that time-resolved fluorescent microspheres with a particle size of 150-200 nm are used; the fluorescent microsphere-coupled Aβ1-40 primary antibody is obtained by coupling 0.3 mg / mL Aβ1-40 primary antibody per 1 mL of fluorescent microspheres; the fluorescent microsphere-coupled Aβ1-42 primary antibody is obtained by coupling 0.3 mg / mL Aβ1-42 primary antibody per 1 mL of fluorescent microspheres; and the fluorescent microsphere-coupled chicken IgY antibody is obtained by coupling 0.3 mg / mL chicken IgY antibody per 1 mL of fluorescent microspheres.

[0137] Example 3

[0138] Compared with Example 1, the difference is that time-resolved fluorescent microspheres with a particle size of 200-300 nm are used; the fluorescent microsphere-coupled Aβ1-40 primary antibody is obtained by coupling 0.2 mg / mL Aβ1-40 primary antibody per 1 mL of fluorescent microspheres; the fluorescent microsphere-coupled Aβ1-42 primary antibody is obtained by coupling 0.2 mg / mL Aβ1-42 primary antibody per 1 mL of fluorescent microspheres; and the fluorescent microsphere-coupled chicken IgY antibody is obtained by coupling 0.2 mg / mL chicken IgY antibody per 1 mL of fluorescent microspheres.

[0139] Example 4

[0140] ​The difference compared with Example 1 is that the first detection member is coated with Aβ1-42 secondary antibody and the second detection member is coated with Aβ1-40 secondary antibody.

[0141] Performance test

[0142] Select Aβ1-40 and Aβ1-42 samples with concentrations of 0-8000 pg / mL respectively, and use the kits provided by Examples 1-4 and Comparative Example 1 to perform detection, record the T1 line signal value detected by the first detection member, the T1 line signal value detected by the second detection member, and the C line signal value detected by the quality control member, and calculate the T / C ratio. The specific experimental process includes:

[0143] 1) The experiment is completed at a temperature of 15-30°C and a humidity of 35%-85%. When stored at low temperature, the kit must be restored to 15-30°C before use.

[0144] 2) Turn on the instrument according to the instrument instruction manual.

[0145] 3) Check whether the ID chip is consistent with the kit batch number, and do not touch the insertion end of the ID chip when inserting it.

[0146] 4) Tear open the outer package, take out the detection card, and place it on a horizontal operation table. The detection card taken out should be used within 1 hour.

[0147] 5) Sample addition method: vertically hold the pipette, and suck 70 μL of uniformly mixed serum (or plasma, or reconstituted quality control) and drop it into the sample addition hole of the detection card. Avoid sucking air bubbles when taking samples.

[0148] 6) After standing at 15-30°C for 15 minutes, place the detection card into the analyzer card slot, and perform card reading according to the requirements of the instrument instruction manual. The detection result will be automatically displayed on the instrument screen, and the result can be saved and printed.

[0149] The sensitivity test results are shown in Tables 1-4 and Figures 1 to 4 .

[0150] Interference test: use the kit of Example 1 to perform testing, compare whether Aβ1-40 is tested alone, Aβ1-42 is tested alone, and Aβ1-40 and Aβ1-42 are tested simultaneously, and whether relevant interference is generated. The test results are shown in Table 5.

[0151] Table 1 Sensitivity test results of Example 1

[0152]

[0153]

[0154] Table 2 Sensitivity test results of Example 2

[0155]

[0156] Table 3 Sensitivity test results of Example 3

[0157]

[0158] Table 4 Sensitivity test results of Example 4

[0159]

[0160] According to the test results in Table 1 and Figure 3 It is not difficult to find that the kit of Example 1 has R 2 all greater than 0.99, and all have no HOOK effect, which indicates that when the kit provided by Example 1 is applied, the specific concentration of Aβ1-40 or Aβ1-42 in the concentration range of 0-8000 pg / mL can be measured.

[0161] According to the test results in Table 2-3, Figures 4-5 It can be found that the kits of Examples 2-3 have R 2 all less than 0.95, and have certain HOOK effect, which cannot measure the specific concentration of Aβ1-40 or Aβ1-42 in the concentration range of 0-8000 pg / mL. It can be seen that compared with the kits of Examples 2-3, the kit provided by Example 1 can better measure the specific concentration of Aβ1-40 or Aβ1-42 in the concentration range of 0-8000 pg / mL, which can indicate that the kit provided by Example 1 using the fluorescence microsphere activation method of the application has lower minimum detection limit, and thus has higher sensitivity.

[0162] According to the test results in Table 1 and Table 4, Figure 3 and Figure 6 It can be found that there is no obvious difference between the test results of coating Aβ1-40 secondary antibody on the first detection member and coating Aβ1-42 secondary antibody on the second detection member, and coating Aβ1-42 secondary antibody on the first detection member and coating Aβ1-40 secondary antibody on the second detection member, that is, the interchanging of the first detection member and the second detection member does not affect the test results.

[0163] Table 5 Interference test results of separate detection of Aβ1-40 or Aβ1-42 and mixed detection

[0164]

[0165]

[0166] From the test results according to Table 5, it can be seen that there is no significant difference between the results of testing Aβ1-40 or Aβ1-42 alone and testing Aβ1-40 and Aβ1-42 simultaneously.

[0167] The above merely describes exemplary embodiments of the present application, and is not intended to limit the patent scope of the present application. Any equivalent structural transformation made according to the technical concept of the present application, or direct / indirect application in other related technical fields, is included in the patent protection scope of the present application.

Claims

1. A kit for detecting a marker of Alzheimer's disease, characterized by, The kit comprises a housing and a test strip, the test strip comprises a base plate extending along a first direction, and a sample pad, a conjugate pad, a coated pad and an absorbent paper arranged on the base plate along the first direction; The conjugate pad is coated with an Aβ1-40 labeled primary antibody, an Aβ1-42 labeled primary antibody and a quality control labeled primary antibody; wherein the labels in the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody are all fluorescent microspheres, the fluorescent microspheres are time-resolved fluorescent microspheres, the particle size of the fluorescent microspheres is 200-300 nm; the Aβ1-40 labeled primary antibody is obtained by coupling 0.3 mg / mL Aβ1-40 primary antibody with fluorescent microspheres per 1 mL of fluorescent microspheres; the Aβ1-42 labeled primary antibody is obtained by coupling 0.3 mg / mL Aβ1-42 primary antibody with fluorescent microspheres per 1 mL of fluorescent microspheres; the antibody in the quality control labeled primary antibody is chicken IgY antibody; the conjugate pad is obtained by spraying the conjugate pad coating solution on the substrate and drying, the conjugate pad coating solution is a mixed solution of the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody, the quality control labeled primary antibody and a label diluent, the label diluent is a mixed solution of 0.5% bovine serum albumin, 5% trehalose and 0.05M Tris-HCl buffer, the pH value of the label diluent is 8.2; the concentration of the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody in the conjugate pad coating solution is 1.0 mg / mL; The coated pad sequentially comprises a first detection element, a second detection element and a quality control element along the first direction, one of the Aβ1-40 secondary antibody and the Aβ1-42 secondary antibody is coated on the first detection element, the other of the Aβ1-40 secondary antibody and the Aβ1-42 secondary antibody is coated on the second detection element, and the quality control secondary antibody is coated on the quality control element; wherein the quality control secondary antibody is a goat anti-chicken IgY antibody; the coated pad is obtained by separately coating the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the quality control secondary antibody coating solution on the substrate and drying, the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the quality control secondary antibody coating solution are obtained by mixing the Aβ1-40 secondary antibody, the Aβ1-42 secondary antibody and the quality control secondary antibody with a coating solution respectively, the coating solution is a mixed solution of 0.01M phosphate buffer and 1-3% trehalose; the concentration of the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the quality control secondary antibody coating solution is 2.0 mg / mL; The kit for detecting Alzheimer's disease markers is used for simultaneously detecting the contents of Aβ1-40 and Aβ1-42, and the range is 0-8000 pg / mL.

2. A method for preparing a kit for detecting a marker of Alzheimer's disease according to claim 1, characterized in that, The kit comprises the following steps: providing a substrate, arranging the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody on the substrate, and drying to obtain a conjugate pad; Providing a base film, sequentially arranging a first detection member, a second detection member and a quality control member on the base film along a first direction, drying to obtain a coated pad; Providing a base plate and an absorbent paper, assembling the base plate, a sample pad, the absorbent paper, a combination pad and the coated pad to obtain a test strip; Providing a shell, assembling the shell and the test strip to obtain the kit for detecting the Alzheimer's disease marker.

3. The method for preparing a kit for detecting a marker for Alzheimer's disease according to claim 2, wherein Before the step of providing a base, arranging Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody and quality control labeled primary antibody on the base, and drying to obtain a combination pad, the step comprises: Mixing fluorescent microspheres and an activating agent, activating to obtain activated fluorescent microspheres; Mixing the activated fluorescent microspheres with Aβ1-40 primary antibody, Aβ1-42 primary antibody and chicken IgY antibody respectively, coupling to obtain fluorescent microsphere-coupled Aβ1-40 primary antibody, fluorescent microsphere-coupled Aβ1-42 primary antibody and fluorescent microsphere-coupled chicken IgY antibody; Mixing the fluorescent microsphere-coupled Aβ1-40 primary antibody, the fluorescent microsphere-coupled Aβ1-42 primary antibody and the fluorescent microsphere-coupled chicken IgY antibody with a blocking agent respectively, and performing blocking treatment to obtain Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody and quality control labeled primary antibody.

4. The method for preparing a kit for detecting Alzheimer's disease markers according to claim 2, wherein Before the step of providing a base, arranging Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody and quality control labeled primary antibody on the base, and drying to obtain a combination pad, the step comprises: Providing a base, mixing Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody, quality control labeled primary antibody and a marker diluent to obtain a combination pad coating liquid, spraying the combination pad coating liquid on the base, and drying to obtain a combination pad.

5. The method for preparing a kit for detecting a marker for Alzheimer's disease according to claim 4, wherein The marker diluent is a mixed solution of 0.5% bovine serum albumin, 5% trehalose and 0.05M Tris-HCl buffer; and / or, The pH value of the marker diluent is 8.2; and / or, The base is made of glass cellulose membrane, the width of the base is 6-8 mm, and the thickness of the base is 335-405 μm, and when the combination pad coating liquid is sprayed on the base, the spraying amount of the combination pad coating liquid is 8-10 μL / cm.

6. The method for preparing a kit for detecting Alzheimer's disease markers according to claim 2, wherein the antibody is an antibody recognizing the amyloid β protein. Before the step of providing a base film, sequentially arranging a first detection member, a second detection member and a quality control member on the base film along a first direction, and drying to obtain a coated pad, the step comprises: Mixing the coating liquid with Aβ1-40 secondary antibody, Aβ1-42 secondary antibody and goat anti-chicken IgY antibody respectively to obtain Aβ1-40 secondary antibody coating liquid, Aβ1-42 secondary antibody coating liquid and goat anti-chicken IgY antibody coating liquid; Providing a base film, drawing Aβ1-40 secondary antibody coating liquid, Aβ1-42 secondary antibody coating liquid and goat anti-chicken IgY antibody coating liquid on the base film respectively, and drying to obtain a coated pad.

7. The method for preparing a kit for detecting Alzheimer's disease markers according to claim 6, wherein The coating liquid is a mixed solution of 0.01 M phosphate buffer and 1-3% trehalose; and / or, The concentrations of the Aβ1-40 secondary antibody coating liquid, the Aβ1-42 secondary antibody coating liquid and the goat anti-chicken IgY antibody coating liquid are all 2 mg / mL; and / or, The base film is a nitrocellulose film, the pore size of the base film is 8-15 μm, the width is 24.5-25 mm, and the thickness is 225-255 μm; the use amount of the Aβ1-40 secondary antibody coating liquid, the Aβ1-42 secondary antibody coating liquid and the goat anti-chicken IgY antibody coating liquid when marking the film is 1-1.5 μL / cm.

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