Kit for detecting Alzheimer's disease marker and preparation method thereof

Abstract

The invention discloses a kit for detecting Alzheimer's disease markers and a preparation method thereof, and relates to the technical field of immunodetection. The kit for detecting the Alzheimer's disease marker comprises a shell and a test strip, wherein the test strip comprises a bottom plate extending along a first direction as well as a sample pad, a combination pad, a coating pad and absorbent paper which are arranged on the bottom plate at intervals along the first direction; the combination pad is coated with an A beta 1-40 labeled primary antibody, an A beta 1-42 labeled primary antibody and a quality control labeled primary antibody; the coating pad is sequentially provided with a first detection piece, a second detection piece and a quality control piece along a first direction, the first detection piece is coated with one of an A beta 1-40 secondary antibody and an A beta 1-42 secondary antibody, the second detection piece is coated with the other of the A beta 1-40 secondary antibody and the A beta 1-42 secondary antibody, and the quality control piece is coated with a quality control secondary antibody. According to the technical scheme, A beta 1-40 and A beta 1-42 can be detected at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a kit for detecting Alzheimer's disease markers and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) has a huge impact on the lives of patients and their families due to impaired thinking, memory and independence, which affects the quality of life. Abnormal levels of amyloid-β (Aβ) form plaques between neurons in the brain that are neurotoxic and lead to neuronal degeneration. Currently, quantitative detection of amyloid-β 1-40 (Aβ1-40) and amyloid-β 1-42 (Aβ1-42) in vitro is one of the clinically recognized tools for auxiliary diagnosis of Alzheimer's disease (AD). By detecting the Aβ1-42 content in plasma and the ratio of Aβ1-42 / Aβ1-40, which reflects the Aβ pathology in the brain, it can be used to assess the risk of AD and mild cognitive impairment at an early stage.

[0003] Common methods for detecting Aβ1-40 and Aβ1-42 include chemiluminescence and enzyme-linked immunosorbent assay. Among them, the enzyme-linked immunosorbent assay has complicated operation steps, is time-consuming, and has poor precision; the chemiluminescence assay is costly. Although some immunofluorescence rapid detection kits have been developed 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 first, and then the ratio between the two is calculated. The use of a kit with a single detection mode not only causes a waste of test samples but also has a high cost of use.

[0004] Therefore, the existing kits for detecting Alzheimer's disease markers have the defect of not being able to detect Aβ1-40 and Aβ1-42 at the same time. Summary of the invention

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

[0006] To achieve the above object, the present invention provides a kit for detecting Alzheimer's disease markers, comprising a housing and a test strip, wherein the test strip comprises a bottom plate extending along a first direction, and a sample pad, a binding pad, a coating pad and absorbent paper arranged on the bottom plate at intervals along 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 along a first direction, the first detection element is coated with one of the Aβ1-40 secondary antibody and the 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 one embodiment, the markers 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 one embodiment, the fluorescent microspheres are time-resolved fluorescent microspheres, and the particle size of the fluorescent microspheres is 150 to 300 nm; and / or,

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

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

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

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

[0014] Providing a substrate, placing an Aβ1-40 labeled primary antibody, an Aβ1-42 labeled primary antibody and a quality control labeled primary antibody on the substrate, and drying to obtain a conjugate pad;

[0015] Providing a base film, sequentially arranging a first detection element, a second detection element and a quality control element on the base film along a first direction, and drying to obtain a coated pad;

[0016] Providing a base plate and absorbent paper, assembling the base plate, sample pad, absorbent paper, conjugate pad and coating pad to obtain a test strip;

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

[0018] In one embodiment, the steps of providing a substrate, placing an Aβ1-40 labeled primary antibody, an Aβ1-42 labeled primary antibody and a quality control labeled primary antibody on the substrate, and drying to obtain a conjugate pad include:

[0019] Mixing the fluorescent microspheres and the activator to activate them, thereby obtaining activated fluorescent microspheres;

[0020] The activated fluorescent microspheres are mixed with Aβ1-40 primary antibody, Aβ1-42 primary antibody and chicken IgY antibody respectively for coupling 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;

[0021] Fluorescent microspheres coupled to Aβ1-40 primary antibody, fluorescent microspheres coupled to Aβ1-42 primary antibody and fluorescent microspheres coupled to chicken IgY antibody were mixed with blocking agents respectively, and blocked to obtain Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody and quality control labeled primary antibody.

[0022] In one embodiment, the steps of providing a substrate, placing an Aβ1-40 labeled primary antibody, an Aβ1-42 labeled primary antibody and a quality control labeled primary antibody on the substrate, and drying to obtain a conjugate pad include:

[0023] A substrate is provided, and Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody, quality control labeled primary antibody and marker diluent are mixed to obtain a conjugate pad coating solution, and the conjugate pad coating solution is sprayed on the substrate and dried to obtain a conjugate pad.

[0024] In one 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 to 9.0; and / or,

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

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

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

[0029] The substrate is made of glass cellulose membrane, has a width of 6-7 mm and a thickness of 335-405 μm, and when the conjugate pad coating liquid is sprayed on the substrate, the spraying amount of the conjugate pad coating liquid is 8-10 μL / cm.

[0030] In one embodiment, the steps of providing a base film, sequentially arranging a first detection element, a second detection element and a quality control element on the base film along a first direction, and drying to obtain a coated pad include:

[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 basement membrane is provided, and Aβ1-40 secondary antibody coating solution, Aβ1-42 secondary antibody coating solution and goat anti-chicken IgY antibody coating solution are respectively streaked onto the basement membrane, and dried to obtain a coating pad.

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

[0034] The 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 to 2.5 mg / mL; and / or,

[0035] The basement membrane is a nitrocellulose membrane, the pore size of the basement 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, Aβ1-42 secondary antibody coating solution and goat anti-chicken IgY antibody coating solution used in the membrane drawing is 1-1.5 μL / cm.

[0036] The technical solution of the present invention adopts the principle of immunofluorescence chromatography and double antibody sandwich method to detect Aβ1-40 and Aβ1-42. Aβ1-40 labeled primary antibody and Aβ1-42 labeled primary antibody are coated on the binding pad respectively, and Aβ1-40 secondary antibody and Aβ1-42 secondary antibody are coated on the coating pad respectively. When the sample to be tested is added, Aβ1-40 and Aβ2-40 in the sample are combined with Aβ1-40 labeled primary antibody and Aβ1-42 labeled primary antibody in the binding pad area to form a complex of Aβ1-40 and Aβ1-40 labeled primary antibody and a complex of Aβ1-42 and Aβ1-42 labeled primary antibody. Due to the effect of chromatography, 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 and bind to the Aβ1-40 secondary antibody or Aβ1-42 secondary antibody in the first detection element and the second detection element to form a corresponding complex of Aβ1-40, Aβ1-40 labeled primary antibody, Aβ1-40 secondary antibody and a complex of Aβ1-42, Aβ1-42 labeled primary antibody, Aβ1-42 secondary antibody. The higher the concentration of Aβ1-40 and Aβ2-40 in the sample to be tested, the more the corresponding labeled primary antibody-antigen-secondary antibody will gather 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 achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0038] Figure 1 A schematic plan view of an embodiment of a kit for detecting Alzheimer's disease markers provided by the present invention;

[0039] Figure 2 for Figure 1 A plan view of a test strip in FIG.

[0040] Figure 3 This is a result diagram of the linear range of Aβ1-40 and Aβ1-42 detected by the kit for detecting Alzheimer's disease markers in Example 1 provided by the present invention;

[0041] Figure 4 This is a result diagram of the linear range of Aβ1-40 and Aβ1-42 detected by the kit for detecting Alzheimer's disease markers in Example 2 provided by the present invention;

[0042] Figure 5 This is a result diagram of the linear range of Aβ1-40 and Aβ1-42 detected by the kit for detecting Alzheimer's disease markers in Example 3 provided by the present invention;

[0043] Figure 6 This is a graph showing the linear range results of the kit for detecting Alzheimer's disease markers in Example 4 provided by the present invention for detecting Aβ1-40 and Aβ1-42.

[0044] Description of Figure Numbers:

[0045] 100. A test kit for detecting markers of Alzheimer's disease; 1. outer shell; 11. sample addition hole 2. test strip; 21. bottom plate; 22. sample pad; 23. binding pad; 24. coating pad; 241. first detection element; 242. second detection element; 243. quality control element; 25. absorbent paper.

[0046] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] It should be noted that if the embodiments of the present invention 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 status, etc. between the components in a certain specific posture. If the specific posture changes, the directional indication will also change accordingly.

[0049] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features limited to "first" and "second" may explicitly or implicitly include at least one of the features. In addition, if "and / or" or "and / or" appears in the full text, its meaning includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or solutions that satisfy both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0050] Alzheimer's disease (AD) is the most common type of dementia in the elderly. It affects the quality of life of patients and their families due to impaired thinking, memory and independence. At present, quantitative detection of β-amyloid 1-40 (Aβ1-40) and β-amyloid 1-42 (Aβ1-42) in vitro is one of the clinically recognized tools for auxiliary diagnosis of Alzheimer's disease (AD). By detecting the Aβ1-42 content in plasma and the ratio of Aβ1-42 / Aβ1-40, which reflects the Aβ pathology in the brain, it can be used to assess the risk of AD dementia and mild cognitive impairment at an early stage.

[0051] Common methods for detecting Aβ1-40 and Aβ1-42 include chemiluminescence and enzyme-linked immunosorbent assay, among which the enzyme-linked immunosorbent assay has complicated operation steps, is time-consuming, and has poor precision; the chemiluminescence method is costly. 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 first, and then the ratio between the two is calculated. The use of a kit with a single detection mode not only causes a waste of test samples but also has a high cost of use. Therefore, the existing kits for detecting Alzheimer's disease markers have the defect that they cannot detect Aβ1-40 and Aβ1-42 at the same time.

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

[0053] See also Figure 1 and Figure 2In one embodiment of the present invention, the kit 100 for detecting Alzheimer's disease markers comprises a housing 1 and a test strip 2, wherein the test strip 2 comprises a bottom plate 21 extending along a first direction and a sample pad 22, a binding pad 23, a coating pad 24 and a blotting paper 25 arranged on the bottom plate 21 at intervals along the first direction;

[0054] The binding pad 23 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 24 is provided with a first detection element 241, a second detection element 242 and a quality control element 243 in sequence along the first direction, the first detection element 241 is coated with one of the Aβ1-40 secondary antibody and the Aβ1-42 secondary antibody, the second detection element 242 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 the quality control secondary antibody.

[0055] In the technical solution of the present invention, the Aβ1-40 labeled 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 sites where the Aβ1-40 labeled primary antibody and the Aβ1-40 secondary antibody recognize Aβ1-40 do not overlap; the Aβ1-42 labeled 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 sites where the Aβ1-42 labeled primary antibody and the Aβ1-42 secondary antibody recognize Aβ1-42 do not overlap; the quality control secondary antibody is used to capture the quality control labeled primary antibody.

[0056] The technical solution of the present invention adopts the principle of immunofluorescence chromatography and the double antibody sandwich method to detect the Alzheimer's disease markers Aβ1-40 and Aβ1-42. The kit provided by the present invention coats the Aβ1-40 labeled primary antibody and the Aβ1-42 labeled primary antibody on the binding pad 23, respectively, and coats the Aβ1-40 secondary antibody and the Aβ1-42 secondary antibody on the coating pad 24, respectively. After the sample to be tested is added through the sample addition hole 11, the Aβ1-40 labeled primary antibody in the binding pad 23 area specifically recognizes the Aβ1-40 in the sample to be tested, forming a complex of Aβ1-40 and the Aβ1-40 labeled primary antibody, and the Aβ1-42 labeled primary antibody in the binding pad 23 area specifically recognizes the Aβ1-42 in the sample to be tested, forming a complex of Aβ1-42 and the Aβ1-42 labeled primary antibody. Due to the effect of chromatography, 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 the Aβ1-40 secondary antibody or Aβ1-42 secondary antibody coated on the first detection element 241 and the second detection element 242 to form a complex of Aβ1-40, Aβ1-40 labeled primary antibody, Aβ1-40 secondary antibody, and a complex of Aβ1-42, Aβ1-42 labeled primary antibody, Aβ1-42 secondary antibody. The higher the concentration of Aβ1-40 and Aβ2-40 in the sample to be tested, the more the corresponding labeled primary antibody-antigen-secondary antibody complexes will gather 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, thereby realizing the simultaneous detection of Aβ1-40 and Aβ1-42.

[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 solution of the present invention can simultaneously obtain the content of Aβ1-40 and Aβ1-42 using the same sample to be tested, without changing the sample for two tests, which can reduce the sample amount and improve the credibility of the test results. Moreover, the kit provided by the present invention has been proven through interference tests that the relevant interference when simultaneously detecting Aβ1-40 and Aβ1-42 is small.

[0058] It should be noted that the first detection element 241 is coated with one of the Aβ1-40 secondary antibody and the Aβ1-42 secondary antibody, and the second detection element 242 is coated with the other of the Aβ1-40 secondary antibody and the Aβ1-42 secondary antibody, which means that the positions of the Aβ1-40 secondary antibody and the Aβ1-42 secondary antibody are not limited. The first detection element 241 can be coated with the Aβ1-40 secondary antibody and the second detection element 242 can be coated with the Aβ1-42 secondary antibody, or the first detection element 241 can be coated with the Aβ1-42 secondary antibody and the second detection element 242 can be coated with the Aβ1-40 secondary antibody. The above two settings both fall within the protection scope of the present invention, can achieve rapid detection, and have high detection sensitivity.

[0059] In an embodiment of the present invention, the markers 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] Fluorescent microspheres have the advantages of good stability, dispersibility and high sensitivity. The technical solution of the present invention uses fluorescent microspheres as markers, which helps to improve the stability and sensitivity of the kit.

[0061] In an embodiment of the present invention, the fluorescent microspheres are time-resolved fluorescent microspheres, and the particle size of the fluorescent microspheres is 150 to 300 nm.

[0062] Time-resolved fluorescent microspheres have higher sensitivity and specificity, good stability and repeatability, and are not easily quenched. The technical solution of the present invention selects time-resolved fluorescent microspheres to label antibodies, which can obtain higher detection sensitivity and good batch-to-batch consistency, making the prepared kit suitable for long-term storage and application.

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

[0064] In an embodiment of the present invention, the Aβ1-40 labeled primary antibody is obtained by coupling fluorescent microspheres and Aβ1-40 primary antibody by adding 0.2 to 0.4 mg / mL Aβ1-40 primary antibody per 1 mL of fluorescent microspheres. The dosage of fluorescent microspheres and Aβ1-40 primary antibody is set within the above range, and the coupling efficiency of fluorescent microspheres and Aβ1-40 primary antibody is high, which can ensure the reliability and sensitivity of the test results while controlling the production cost. Within this range, the added concentration of the Aβ1-40 primary antibody can be 0.2 mg / mL, 0.3 mg / mL and 0.4 mg / mL. Preferably, the added concentration of the Aβ1-40 primary antibody is set to 0.3 mg / mL.

[0065] In an embodiment of the present invention, the Aβ1-42 labeled primary antibody is obtained by coupling fluorescent microspheres and Aβ1-42 primary antibody by adding 0.2 to 0.4 mg / mL Aβ1-42 primary antibody per 1 mL of fluorescent microspheres. The dosage of fluorescent microspheres and Aβ1-42 primary antibody is set within the above range, and the coupling efficiency of fluorescent microspheres and Aβ1-42 primary antibody is high, which can ensure the reliability and sensitivity of the test results while controlling the production cost. Within this range, the added concentration of the Aβ1-42 primary antibody can be 0.2 mg / mL, 0.3 mg / mL and 0.4 mg / mL. Preferably, the added concentration of the Aβ1-42 primary antibody is set to 0.3 mg / mL.

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

[0067] In an embodiment of the present invention, the IgY-labeled primary antibody is obtained by coupling fluorescent microspheres and chicken IgY antibodies by adding 0.2-0.4 mg / mL chicken IgY antibodies per 1 mL of fluorescent microspheres. The IgY-labeled primary antibody is prepared by coupling fluorescent microspheres and chicken IgY antibodies. Setting the dosage of fluorescent microspheres and chicken IgY antibodies in the above range can meet the effectiveness requirements of the kit and is conducive to controlling production costs. Within this range, the added concentration of the chicken IgY antibody can be 0.2 mg / mL, 0.3 mg / mL and 0.4 mg / mL.

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

[0069] Providing a substrate, placing an Aβ1-40 labeled primary antibody, an Aβ1-42 labeled primary antibody and a quality control labeled primary antibody on the substrate, and drying to obtain a conjugate pad 23;

[0070] Providing a base film, sequentially arranging a first detection component 241, a second detection component 242 and a quality control component 243 on the base film along a first direction, and drying to obtain a coating pad 24;

[0071] Provide a base plate 21 and absorbent paper 25, assemble the base plate 21, sample pad 22, absorbent paper 25, conjugate pad 23 and coating pad 24 to obtain a test strip 2;

[0072] A housing 1 is provided, and the housing 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 present invention arranges the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody on the binding pad 23 respectively, and arranges the first detection element 241, the second detection element 242 and the quality control element 243 in sequence along the first direction on the coating pad 24, wherein the first detection element 241 is coated with one of the Aβ1-40 secondary antibody and the Aβ1-42 secondary antibody, and the second detection element 242 is coated with the other 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, and the bottom plate 21, the sample pad 22, the above-treated binding pad 23, the above-treated coating pad 24 and the absorbent 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 Alzheimer's disease markers. The kit 100 for detecting Alzheimer's disease markers prepared by the above preparation method has a simple structure, is easy to operate, and can realize the simultaneous detection of Aβ1-40 and Aβ1-42.

[0074] In an embodiment of the present invention, the sample pad 22 is a sample pad that has been soaked in a sample pad treatment solution. The method for preparing the sample pad that has been soaked in a sample pad treatment solution comprises the following steps:

[0075] Soak the sample pad in the sample pad treatment solution and dry it at 40-50℃;

[0076] Wherein, the material of the sample pad 22 is glass cellulose membrane;

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

[0078] In an embodiment of the present invention, the binding pad 23 is a binding pad that has been soaked in a binding pad treatment solution. The method for preparing the binding pad that has been soaked in a binding pad treatment solution comprises the following steps:

[0079] Soak the conjugate pad in the conjugate pad treatment solution and dry it at 40-50°C;

[0080] The conjugate pad treatment solution is a mixed solution of phosphate buffer with a molar concentration of 0.01M, trehalose with a mass concentration of 5%, and casein with a mass concentration of 0.5%. The use of the conjugate pad treatment solution with the above components is conducive to ensuring the uniform release of microspheres and protecting the labeled antibodies to extend the shelf life of the kit.

[0081] In an embodiment of the present invention, the steps of providing a substrate, placing an Aβ1-40 labeled primary antibody, an Aβ1-42 labeled primary antibody and a quality control labeled primary antibody on the substrate, and drying to obtain a conjugate pad 23 include:

[0082] Mixing the fluorescent microspheres and the activator to activate them, thereby obtaining activated fluorescent microspheres;

[0083] The activated fluorescent microspheres are mixed with Aβ1-40 primary antibody, Aβ1-42 primary antibody and chicken IgY antibody respectively for coupling 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;

[0084] Fluorescent microspheres coupled to Aβ1-40 primary antibody, fluorescent microspheres coupled to Aβ1-42 primary antibody and fluorescent microspheres coupled to chicken IgY antibody were mixed with blocking agents respectively, and blocked to obtain Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody and quality control labeled primary antibody.

[0085] In the implementation of the present invention, the step of mixing the fluorescent microspheres and the activator to activate the activated fluorescent microspheres comprises:

[0086] The fluorescent microspheres were mixed with a first boric acid-borax buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), shaken on a shaker in the dark at room temperature for 30 minutes, centrifuged at 14000 r / m for 25 minutes, the supernatant was removed, and the mixture was re-dissolved with a second boric acid-borax buffer, washed by centrifugation at 14000 r / m for 25 minutes, and the supernatant was removed to obtain activated fluorescent microspheres;

[0087] Wherein, 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 molar concentration of the first boric acid-borax buffer is 0.05 M and the pH is 6.0;

[0089] The molar concentration of the second boric acid-borax buffer is 0.05 M, and the pH value is 7.5.

[0090] In an embodiment of the present invention, the step of performing a blocking treatment comprises: adding bovine serum albumin (BSA) with a mass concentration of 10%, blocking on a shaker at room temperature for 30 minutes, and after blocking, centrifuging at 14000r / m for 25 minutes, removing the supernatant, re-dissolving with a second boric acid-borax buffer, washing by centrifugation at 14000r / m for 25 minutes, and removing the supernatant.

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

[0092] In an embodiment of the present invention, the steps of providing a substrate, placing an Aβ1-40 labeled primary antibody, an Aβ1-42 labeled primary antibody and a quality control labeled primary antibody on the substrate, and drying to obtain a conjugate pad 23 include:

[0093] A substrate is provided, and Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody, quality control labeled primary antibody and marker diluent are mixed to obtain a conjugate pad coating solution, and the conjugate pad coating solution is sprayed on the substrate and dried to obtain a conjugate pad 23.

[0094] In an embodiment of the present invention, the marker diluent is a mixed solution of bovine serum albumin with a mass concentration of 0.5-1%, trehalose with a mass concentration of 5-10%, and Tris-HCl buffer with a molar concentration of 0.05M. The use of high concentration of bovine serum albumin is beneficial to protect the labeled protein, while blocking the sites on the conjugate pad that are easy to bind to the protein, and flowing to block the base membrane (such as nitrocellulose membrane) to prevent the problem of non-specific binding; trehalose is used as a drying protective agent to prevent the protein from deteriorating during drying. Tris-HCl provides a high ion concentration and increases the fluidity of the sample.

[0095] In an embodiment of the present invention, the pH value of the marker diluent is 8.0-9.0.

[0096] In an embodiment of the present invention, the mass ratio of the Aβ1-40 labeled primary antibody to the marker diluent is 1:(5-10). Within this range, the mass ratio of the Aβ1-40 labeled primary antibody to the marker 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 invention.

[0097] In an embodiment of the present invention, the mass ratio of the Aβ1-42 labeled primary antibody to the marker diluent is 1:(5-10). Within this range, the mass ratio of the Aβ1-42 labeled primary antibody to the marker 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 invention.

[0098] In an embodiment of the present invention, 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 invention.

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

[0100] Controlling the spraying amount of the conjugate pad coating liquid within the above range helps to ensure the uniform distribution and effective fixation of the labeled antibody on the conjugate pad 23, so that the final concentration of the antibody fixed on the conjugate 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 conjugate pad coating liquid is lower than 8 μL / cm, the high-concentration sample will produce a HOOK effect (Hook Effect), which reduces the high-value sensitivity of the entire detection system; when the spraying amount of the conjugate pad coating liquid is higher than 10 μL / cm, there is too much conjugate pad coating liquid on the conjugate pad 23, which makes the release of the fluorescent microspheres uneven, and reduces the specificity of the entire detection system. Taking the above factors into consideration, the spraying amount of the conjugate pad coating liquid is set to 8-10 μL / cm.

[0101] In an embodiment of the present invention, the steps of providing a base film, sequentially arranging a first detection member 241, a second detection member 242 and a quality control member 243 on the base film along a first direction, and drying to obtain a coating pad 24 include:

[0102] 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;

[0103] A basement membrane is provided, and Aβ1-40 secondary antibody coating solution, Aβ1-42 secondary antibody coating solution and goat anti-chicken IgY antibody coating solution are respectively streaked onto the basement membrane and dried to obtain a coating pad 24 .

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

[0105] In an embodiment of the present invention, 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 to 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 an embodiment of the present invention, the base membrane is a nitrocellulose membrane, the base membrane has a pore size of 8 to 15 μm, a width of 24.5 to 25.5 mm, and a thickness of 225 to 255 μm.

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

[0108] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0109] In the following examples, 150-3200 nm fluorescent microspheres were purchased from Changsha Meiniu Biotechnology Co., Ltd., model MF02;

[0110] 200-300 nm fluorescent microspheres were purchased from Changsha Meiniu Biotechnology Co., Ltd., model MF03;

[0111] The primary antibody for Aβ1-40 was Anti-Amyloid-β1-40McAb, catalog number AB1-40101, purchased from Hangzhou Boyue Biotechnology Co., Ltd.;

[0112] The primary antibody for Aβ1-42 was Anti-beta-amyloid, catalog number 4BA3, purchased from HyTest (HyTest Biotechnology Shanghai Co., Ltd.);

[0113] The Aβ1-40 secondary antibody was Anti-Amyloid-β1-40McAb, catalog number AB1-40102, purchased from Hangzhou Boyue Biotechnology Co., Ltd.;

[0114] The secondary antibody for Aβ1-42 was Anti-beta-amyloid, catalog number 4BA3, purchased from HyTest;

[0115] Chicken IgY antibody, catalog number C-IgY303, was purchased from Hangzhou Boyue Biotechnology Co., Ltd.;

[0116] Sheep anti-chicken IgY antibody, catalog number C-IgY202, was purchased from Hangzhou Boyue Biotechnology Co., Ltd.

[0117] Example 1

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

[0119] 1. Preparation of conjugate pad:

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

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

[0122] b. Coupling: After the fluorescent microspheres are redissolved, Aβ1-40 primary antibody, Aβ1-42 primary antibody and chicken IgY antibody are added respectively, and the mixture is shaken on a shaker at room temperature in a dark place for 2 hours 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: Add 50 μL of coupling blocking solution to the fluorescent microspheres coupled to Aβ1-40 primary antibody, fluorescent microspheres coupled to Aβ1-42 primary antibody and fluorescent microspheres coupled to chicken IgY antibody respectively, and block for 30 minutes on a shaker at room temperature. After blocking, centrifuge at 14000r / m for 25 minutes and wash once to obtain Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody and chicken IgY labeled antibody;

[0124] d. Storage: After washing, re-dissolve with coupling storage solution, and store at 2-8°C after ultrasonic dispersion; the coupling storage solution is a mixed solution of 0.5% bovine serum albumin, 10% trehalose, 0.05M Tris-HCl buffer, 0.05% casein and 0.05% Tween 20.

[0125] 2) Coating with Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody and chicken IgY labeled antibody:

[0126] A glass cellulose membrane with a width of 6.8 mm and a thickness of 360 μm was selected as a substrate; Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody, chicken IgY antibody and a marker diluent were mixed to obtain a conjugate pad coating solution, and the conjugate pad coating solution was evenly sprayed onto the glass cellulose membrane using a film sprayer (the spraying volume was 10 μL / cm), and placed in a 45°C oven for overnight drying to obtain a conjugate pad; wherein the marker diluent included: 0.5% BSA, 5% trehalose, and 0.05M Tris-HCl (pH=8.2). The concentrations of the diluted Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody, and chicken IgY antibody were all 1.0 mg / mL.

[0127] 2. Preparation of coated 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 membrane; Aβ1-40 secondary antibody, Aβ1-42 secondary antibody, and goat anti-chicken IgY antibody were diluted with NC coating solution (0.01 M phosphate buffer, 1-3% trehalose), and the concentration after dilution was 2.0 mg / mL;

[0129] The first detection piece, the second detection piece and the quality control piece were set up respectively. The first detection piece was coated with Aβ1-40 secondary antibody, the second detection piece was coated with Aβ1-42 secondary antibody, and the quality control piece was coated with sheep anti-chicken IgY antibody. The film-stripping volume was 1.0 μL / cm. The samples were placed in a 60°C oven for 1-3 days to dry to obtain a coated pad.

[0130] 3. Sample pad preparation:

[0131] A glass cellulose membrane with a width of 20 mm was selected, and the glass cellulose membrane was soaked in a sample pad treatment solution, and then placed in a 45°C oven for overnight drying to obtain a sample pad; the sample pad treatment solution was a mixed solution of phosphate buffer with a molar concentration of 0.01M, bovine serum albumin with a mass concentration of 1%, trehalose with a mass concentration of 4%, Tween 20 with a mass concentration of 1.0%, and EDTA-2Na with a mass concentration of 1%.

[0132] 4. Assemble the test card:

[0133] Provide absorbent paper with a width of 17 mm and a PVC base plate with a width of 60 mm, and sequentially paste the sample pad, conjugate pad, coating pad and absorbent paper obtained after the above treatment on the PVC base plate to obtain a test strip, as shown in the schematic diagram Figure 2 As shown;

[0134] Install the reagent strip into the housing to complete the test card assembly.

[0135] Example 2

[0136] Compared with Example 1, the difference is that time-resolved fluorescent microspheres with a particle size of 150 to 200 nm are used; the fluorescent microspheres coupled to the Aβ1-40 primary antibody are obtained by coupling the fluorescent microspheres and the Aβ1-40 primary antibody at a rate of 0.3 mg / mL Aβ1-40 primary antibody per 1 mL of fluorescent microspheres; the fluorescent microspheres coupled to the Aβ1-42 primary antibody are obtained by coupling the fluorescent microspheres and the Aβ1-42 primary antibody at a rate of 0.3 mg / mL Aβ1-42 primary antibody per 1 mL of fluorescent microspheres; the fluorescent microspheres coupled to the chicken IgY antibody are obtained by coupling the fluorescent microspheres and the chicken IgY antibody at a rate of 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 to 300 nm are used; the fluorescent microspheres coupled to the Aβ1-40 primary antibody are obtained by coupling the fluorescent microspheres and the Aβ1-40 primary antibody at a rate of 0.2 mg / mL Aβ1-40 primary antibody per 1 mL of fluorescent microspheres; the fluorescent microspheres coupled to the Aβ1-42 primary antibody are obtained by coupling the fluorescent microspheres and the Aβ1-42 primary antibody at a rate of 0.2 mg / mL Aβ1-42 primary antibody per 1 mL of fluorescent microspheres; the fluorescent microspheres coupled to the chicken IgY antibody are obtained by coupling the fluorescent microspheres and the chicken IgY antibody at a rate of 0.2 mg / mL chicken IgY antibody per 1 mL of fluorescent microspheres.

[0139] Example 4

[0140] Compared with Example 1, the difference is that the first detection element is coated with Aβ1-42 secondary antibody, and the second detection element is coated with Aβ1-40 secondary antibody.

[0141] Performance Testing

[0142] Aβ1-40 and Aβ1-42 samples with a concentration of 0 to 8000 pg / mL were selected respectively, and the kits provided in Examples 1-4 and Comparative Example 1 were used for detection respectively, and the T1 line signal value detected by the first detection element, the T1 line signal value detected by the second detection element, and the C line signal value detected by the quality control were recorded, and the T / C ratio was calculated. The specific experimental process includes:

[0143] 1) The experiment was completed at a temperature of 15-30°C and a humidity of 35%-85%. When stored at low temperatures, the kit should 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 reagent kit batch number. Do not touch the insertion end of the ID chip when inserting it.

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

[0147] 5) Sampling method: Hold the pipette vertically, draw 70 μL of evenly mixed serum (or plasma, reconstituted quality control product) and add it dropwise into the sample well of the test card. Avoid inhaling bubbles when sampling.

[0148] 6) After standing for 15 minutes at 15-30℃, place the test card into the card slot of the analyzer and read the card according to the requirements of the instrument manual. The test results will be automatically displayed on the instrument screen and can be saved and printed.

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

[0150] Interference test: The kit of Example 1 was used to test whether relevant interference was generated when Aβ1-40 alone, Aβ1-42 alone, and Aβ1-40 and Aβ1-42 were tested simultaneously. 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 Table 1 and Figure 3 It is not difficult to find from the test results that when the concentration of Aβ1-40 or Aβ1-42 of the kit of Example 1 is 0-8000 pg / mL, R 2 All of them were greater than 0.99, and there was no HOOK effect, indicating that when the kit provided in Example 1 was used, the specific concentration of Aβ1-40 or Aβ1-42 in the concentration range of 0-8000 pg / mL could be measured.

[0161] According to Table 2-3, Figure 4-5 The test results show that when the concentration of Aβ1-40 or Aβ1-42 is 0-8000 pg / mL, R 2 All of them are less than 0.95, and there is a certain HOOK effect, and the specific concentration of Aβ1-40 or Aβ1-42 in the concentration range of 0-8000pg / mL cannot be measured. It can be seen that compared with the kits of Examples 2-3, the kit provided in Example 1 can better measure the specific concentration of Aβ1-40 or Aβ1-42 in the concentration range of 0-8000pg / mL, which can explain that the kit provided in Example 1 using the fluorescent microsphere activation method of the present invention has a lower minimum detection limit and thus has a higher sensitivity.

[0162] According to Table 1 and Table 4, Figure 3 and Figure 6 It can be seen from the test results that there is no obvious difference between the test results when the first detection piece is coated with Aβ1-40 secondary antibody and the second detection piece is coated with Aβ1-42 secondary antibody and the test results when the first detection piece is coated with Aβ1-42 secondary antibody and the second detection piece is coated with Aβ1-40 secondary antibody, that is, exchanging the positions of the first detection piece and the second detection piece does not affect the test results.

[0163] Table 5 Interference test results when Aβ1-40 or Aβ1-42 is detected alone and when it is mixed

[0164]

[0165]

[0166] According to the test results in Table 5, there is no significant difference between the results of testing Aβ1-40 or Aβ1-42 alone and the results of measuring Aβ1-40 and Aβ1-42 simultaneously.

[0167] The above description is only an exemplary embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A kit for detecting Alzheimer's disease markers, characterized in that: The test strip comprises a housing and a test strip, wherein the test strip comprises a bottom plate extending along a first direction, and a sample pad, a binding pad, a coating pad and absorbent paper arranged on the bottom plate at intervals along the first direction; 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 along a first direction, the first detection element is coated with one of the Aβ1-40 secondary antibody and the 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.

2. The kit for detecting Alzheimer's disease markers according to claim 1, characterized in that: The markers in the Aβ1-40 labeled primary antibody, the Aβ1-42 labeled primary antibody and the quality control labeled primary antibody are all fluorescent microspheres.

3. The kit for detecting Alzheimer's disease markers according to claim 2, characterized in that: The fluorescent microspheres are time-resolved fluorescent microspheres, and the particle size of the fluorescent microspheres is 150 to 300 nm; and / or, The Aβ1-40 labeled primary antibody is obtained by coupling fluorescent microspheres and Aβ1-40 primary antibody by adding 0.2 to 0.4 mg / mL Aβ1-40 primary antibody per 1 mL of fluorescent microspheres; and / or, The Aβ1-42 labeled primary antibody is obtained by coupling fluorescent microspheres and Aβ1-42 primary antibody by adding 0.2 to 0.4 mg / mL Aβ1-42 primary antibody per 1 mL of fluorescent microspheres.

4. The kit for detecting Alzheimer's disease markers according to claim 1, characterized in that: The antibody in the quality control labeled primary antibody is chicken IgY antibody, and the quality control secondary antibody is goat anti-chicken IgY antibody.

5. A method for preparing a kit for detecting Alzheimer's disease markers according to any one of claims 1 to 4, characterized in that: The following steps are involved: Providing a substrate, placing an Aβ1-40 labeled primary antibody, an Aβ1-42 labeled primary antibody and a quality control labeled primary antibody on the substrate, and drying to obtain a conjugate pad; Providing a base film, sequentially arranging a first detection element, a second detection element and a quality control element on the base film along a first direction, and drying to obtain a coated pad; Providing a base plate and absorbent paper, assembling the base plate, sample pad, absorbent paper, conjugate pad and coating pad to obtain a test strip; A shell is provided, and the shell and the test strip are assembled to obtain the kit for detecting Alzheimer's disease markers.

6. The method for preparing a kit for detecting Alzheimer's disease markers according to claim 5, characterized in that: The steps of providing a substrate, placing an Aβ1-40 labeled primary antibody, an Aβ1-42 labeled primary antibody and a quality control labeled primary antibody on the substrate, and drying to obtain a conjugate pad include: Mixing the fluorescent microspheres and the activator to activate them, thereby obtaining activated fluorescent microspheres; The activated fluorescent microspheres are mixed with Aβ1-40 primary antibody, Aβ1-42 primary antibody and chicken IgY antibody respectively for coupling 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; Fluorescent microspheres coupled to Aβ1-40 primary antibody, fluorescent microspheres coupled to Aβ1-42 primary antibody and fluorescent microspheres coupled to chicken IgY antibody were mixed with blocking agents respectively, and blocked to obtain Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody and quality control labeled primary antibody.

7. The method for preparing a kit for detecting Alzheimer's disease markers according to claim 5, characterized in that: The steps of providing a substrate, placing an Aβ1-40 labeled primary antibody, an Aβ1-42 labeled primary antibody and a quality control labeled primary antibody on the substrate, and drying to obtain a conjugate pad include: A substrate is provided, and Aβ1-40 labeled primary antibody, Aβ1-42 labeled primary antibody, quality control labeled primary antibody and marker diluent are mixed to obtain a conjugate pad coating solution, and the conjugate pad coating solution is sprayed on the substrate and dried to obtain a conjugate pad.

8. The method for preparing a kit for detecting Alzheimer's disease markers according to claim 7, characterized in that: 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, The pH value of the marker diluent is 8.0 to 9.0; and / or, The mass ratio of the Aβ1-40 labeled primary antibody to the labeling substance diluent is 1:(5-10); and / or, The mass ratio of the Aβ1-42 labeled primary antibody to the labeling substance diluent is 1:(5-10); and / or, The mass ratio of the quality control labeled primary antibody to the label diluent is 1:(30-40); and / or, The substrate is made of glass cellulose membrane, has a width of 6-8 mm and a thickness of 335-405 μm, and when the conjugate pad coating liquid is sprayed on the substrate, the spraying amount of the conjugate pad coating liquid is 8-10 μL / cm.

9. The method for preparing a kit for detecting Alzheimer's disease markers according to claim 5, characterized in that: The steps of providing a base film, sequentially arranging a first detection element, a second detection element and a quality control element on the base film along a first direction, and drying to obtain a coated pad include: 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; A basement membrane is provided, and Aβ1-40 secondary antibody coating solution, Aβ1-42 secondary antibody coating solution and goat anti-chicken IgY antibody coating solution are respectively streaked onto the basement membrane, and dried to obtain a coating pad.

10. The method for preparing a kit for detecting Alzheimer's disease markers according to claim 9, characterized in that: The coating solution is a mixed solution of 0.01M phosphate buffer and 1-3% trehalose; and / or, The 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 to 2.5 mg / mL; and / or, The basement membrane is a nitrocellulose membrane, the pore size of the basement membrane is 8-15 μm, the width is 24.5-25 mm, and the thickness is 225-255 μm; the amount of the Aβ1-40 secondary antibody coating solution, the Aβ1-42 secondary antibody coating solution and the sheep anti-chicken IgY antibody coating solution used in the membrane drawing is 1-1.5 μL / cm.

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