CN118294648A

CN118294648A - Alzheimer disease marker detection kit and method thereof

Info

Publication number: CN118294648A
Application number: CN202410723316.7A
Authority: CN
Inventors: 李振韬; 赵海涛; 陈卓; 张欢欢; 刘云飞
Original assignee: Yihang Suzhou Biomedical Co ltd
Current assignee: Yihang Suzhou Biomedical Co ltd
Priority date: 2024-06-05
Filing date: 2024-06-05
Publication date: 2024-07-05
Anticipated expiration: 2044-06-05
Also published as: CN118294648B

Abstract

The invention relates to a kit for detecting Alzheimer disease markers and a method thereof, wherein the kit comprises a first reagent, a second reagent and streptavidin-phycoerythrin; the first reagent comprises microspheres coupled with Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL capture antibodies and a first buffer solution, and the second reagent comprises a biotin-labeled detection antibody and a second buffer solution; the detection antibodies respectively resist a corresponding Alzheimer disease marker and correspond to the capture antibodies; the second buffer solution contains 0.1-1 wt% of casein and 5-vol-15-vol% of animal serum. The kit has the advantages of high sensitivity, good stability, good repeatability, high flux, rapid and accurate detection and the like, and can simultaneously perform qualitative and quantitative detection on various Alzheimer disease related protein markers.

Description

Alzheimer disease marker detection kit and method thereof

Technical Field

The invention relates to the technical field of in-vitro diagnosis and detection, in particular to a kit and a method for detecting Alzheimer disease markers.

Background

Alzheimer's disease (Alzheimer disease, AD) is a degenerative change of the central nervous system. The diagnosis of which requires a large number of tests to be performed at present includes: medical history, mental state assessment, neuropsychological assessment, brain nuclear magnetic resonance, cerebrospinal fluid examination. Treatment history, mental state assessment, neuropsychological assessment are greatly affected by subjective factors of patients, are difficult to identify in early stages, and are easily misdiagnosed. In the related clinical research, mainly rely on PET scanning or cerebrospinal fluid detection to confirm the existence of Abeta pathology, but brain nuclear magnetic resonance and cerebrospinal fluid detection cost are higher, and brain nuclear magnetic resonance cannot be used as a conventional detection means due to certain radiation hazard, and cerebrospinal fluid detection needs invasive sampling means, so that the cost is high, the detection is highly dependent on professionals, even has certain invasiveness, and the detection is difficult to popularize in a large scale in clinical practice. The blood sample collection is micro-invasive, quick and cost-effective, and part of blood biomarkers are significantly changed before the clinical symptoms of AD appear, so that the detection of the blood biomarkers can be used as an ideal means for screening patients in early stages of AD.

At present, the number of Alzheimer's diagnosis kits on the market in China is small, and most of the kits are methods based on the traditional ELISA principle. However, the diagnosis kit product based on the traditional immunodetection method-enzyme-linked immunosorbent assay generally has the problems of poor detection sensitivity and poor repeatability, and is difficult to realize stable detection.

The liquid phase chip technology (xMAP) is a biochip technology platform which can be widely applied to various biological reactions such as proteins, genes, receptors/ligands and the like, and mainly comprises four components of microspheres, probe molecules, detected objects and reporter molecules. However, the detection sensitivity, specificity, stability and repeatability of the detection AD kit based on the liquid-phase chip technology still need to be improved.

Disclosure of Invention

Problems to be solved by the invention

The invention aims to provide a diagnostic kit for detecting Alzheimer disease peripheral blood protein markers and a detection method thereof, wherein the detection method and the kit comprise the combined parallel detection of six AD markers such as beta amyloid 1-42 (Abeta 1-42), beta amyloid 1-40 (Abeta 1-40), microtubule-related Tau protein (T-Tau), phosphorylated Tau181 (p-Tau-181), glial Fiber Acid Protein (GFAP) and neurofilament light chain protein (NfL), and the like, and have the advantages of high sensitivity, high specificity, good stability, rapid detection, good repeatability, wide linear range and the like.

Solution for solving the problem

The first object of the invention is to provide a kit for detecting Alzheimer disease markers, which comprises a first reagent, a second reagent and streptavidin-phycoerythrin;

The first reagent comprises microspheres coupled with different capture antibodies and a first buffer; the microspheres coupled with different capture antibodies comprise microspheres coupled with beta-amyloid 1-42 capture antibodies, microspheres coupled with beta-amyloid 1-40 capture antibodies, microspheres coupled with microtubule-associated Tau protein capture antibodies, microspheres coupled with phosphorylated Tau181 capture antibodies, microspheres coupled with glial fibrillary acidic protein capture antibodies and microspheres coupled with neurofilament light chain protein capture antibodies;

The second reagent comprises a biotin-labeled detection antibody and a second buffer; the detection antibodies respectively resist a corresponding Alzheimer disease marker and correspond to the capture antibodies; the second buffer solution contains 0.1-1 wt% of casein and 5-vol-15-vol% of animal serum.

Preferably, the concentration of casein is 0.1 wt% -0.5 wt%, preferably 0.1 wt% -0.3 wt%, more preferably 0.2 wt%.

Preferably, the concentration of the animal serum is 8 vol% -12 vol%, preferably 10 vol%.

Preferably, the animal serum is sheep serum and/or bovine serum, preferably sheep serum.

Preferably, the second buffer solution further contains 0.1wt% -10 wt% sodium chloride and 0.05 vol% -1 vol% pra Luo Kelin 300 (Proclin 300).

Preferably, the first buffer solution contains 0.1-10wt% of sodium chloride, 0.1-10wt% of bovine serum albumin and 0.05 vol-vol% of Pr Luo Kelin (Proclin 300).

Preferably, the first buffer solution and the second buffer solution are phosphate buffer solutions.

Preferably, the working concentrations of the biotin-labeled amyloid beta 1-42 detection antibody and the phosphorylated tau181 detection antibody in the second reagent are respectively and independently 0.1-1 μg/ml, preferably 0.3-0.8 μg/ml, and more preferably 0.5 μg/ml.

Preferably, the working concentrations of the biotin-labeled beta amyloid 1-40 detection antibody, the microtubule-associated Tau protein detection antibody and the glial fibrillary acidic protein detection antibody in the second reagent are respectively and independently 0.1-0.5 mug/ml; preferably 0.1 to 0.3. Mu.g/ml, more preferably 0.25. Mu.g/ml.

Preferably, the working concentration of the biotin-labeled neurofilament light chain protein detection antibody in the second reagent is 0.5-2 μg/ml, preferably 0.5-1.5 μg/ml, and more preferably 1 μg/ml.

Preferably, the working concentrations of the amyloid beta 1-42 capture antibody, the amyloid beta 1-40 capture antibody, the microtubule-associated Tau protein capture antibody, the glial acidic protein capture antibody and the neurofilament light chain protein capture antibody in the first reagent are respectively and independently 0.5-2 μg/ml, preferably 0.5-1.5 μg/ml, and more preferably 1 μg/ml.

Preferably, the working concentration of phosphorylated tau181 capture antibody in the first reagent is 1-5 μg/ml, preferably 1-3 μg/ml, more preferably 2 μg/ml.

Preferably, the kit further comprises a dilution buffer, wherein the dilution buffer is a phosphate buffer containing 0.1-10wt% of sodium chloride, 0.1-10wt% of bovine serum albumin, 0.1-0.5wt% of casein, 0.05 vol-0.2-vol% of tween-20 and 0.05 vol-10 vol% of Pr Luo Kelin 300 (Proclin 300).

Preferably, the concentration of casein in the dilution buffer is 0.1 wt% -0.3 wt%, preferably 0.2 wt%.

Preferably, the kit further comprises a reaction buffer and a wash buffer.

Preferably, the reaction buffer is a phosphate buffer containing 0.1-10wt% sodium chloride, 0.05 vol-vol% pra Luo Kelin 300 (Proclin 300).

Preferably, the cleaning buffer is phosphate buffer containing 0.05 vol% -0.2. 0.2 vol% tween-20.

A second object of the present invention is the use of a kit according to any one of the above in the preparation of a detection product for markers of alzheimer's disease.

A third object of the present invention is to provide a method of using the kit of any one of the above, the method comprising:

(1) Adding a reaction buffer solution, a first reagent and a sample to be tested into a detection tube;

(2) Adding a second reagent into the system in the step (1), and incubating;

(3) Adding streptavidin-phycoerythrin into the system in the step (2), and incubating;

(4) Adding a cleaning buffer solution into the system in the step (3), centrifuging, taking the precipitate, re-suspending, and detecting the fluorescence intensity.

Preferably, in the step (1), the volume ratio of the reaction buffer, the first reagent and the sample to be tested is 1: (0.1 to 1): (0.5-2).

Preferably, the incubation time in the step (2) is 12-24 hours.

Preferably, the incubation time in the step (3) is 0.2-1 hour.

Preferably, the incubation in step (2) and step (3) is performed under light-shielding conditions.

ADVANTAGEOUS EFFECTS OF INVENTION

The invention solves the problems of low multi-marker detection sensitivity, poor repeatability and poor stability in the traditional flow type fluorescence detection by optimizing an antibody coupling process, a microsphere coating process and a buffer liquid system. According to the invention, through utilizing the coupling modeling of the abundance of the 6 protein markers, the purposes of clinical sample typing, early diagnosis of Alzheimer's disease and timely treatment are realized so as to improve the life quality of a patient, and simultaneously, the cognitive impairment of the patient is avoided. The method has the advantages of rapidness, accuracy, high sensitivity, good repeatability, good stability and the like.

Drawings

FIG. 1 is a graph of ROC curve analysis of the single target and combined detection model of 6 proteins of the present invention.

Detailed Description

In order to make the technical scheme and the beneficial effects of the application more obvious and understandable, the following detailed description is given by way of example. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Aβ1-42, Aβ1-40 and P-Tau are currently widely accepted AD biomarkers at home and abroad. The ratio of plasma Aβ1-42 to Aβ1-42/Aβ1-40 reflects Aβpathology in the brain and can be used to assess early risk of AD dementia and mild cognitive impairment. Tau protein is a microtubule-associated protein, which is present in high levels in neurons of the central nervous system and has the main function of regulating the stability of axonal microtubules. Plasma T-Tau is an important predictor of Creutzfeldt-Jacob disease and frontotemporal dementia. Plasma p-Tau-181 levels can reflect the pathological conditions of aβ and Tau in AD, distinguish between alzheimer's disease and other neurodegenerative diseases (e.g., parkinson's disease and vascular dementia, etc.), and monitor the progression of AD patients throughout the clinical course. Glial Fibrillary Acidic Protein (GFAP) and neurofilament light chain protein (NfL) are considered to be very important non-specific markers for AD pathology. Wherein GFAP is a marker of astrocyte activation, which marker abnormality is associated with a higher risk of dementia onset and a faster rate of cognitive decline. NfL is a marker of axonal injury, and is used clinically in a variety of diseases, including multiple sclerosis, ALS, and traumatic brain injury.

The flow type fluorescent technology is an innovative high-flux fluorescent diagnosis technology after the chemiluminescence technology, the sensitivity of the flow type fluorescent diagnosis technology is far higher than that of the traditional chemiluminescence technology, and the flow type fluorescent diagnosis technology is commonly called as a liquid chip technology, and can be widely applied to a plurality of fields such as tumor immunity, autoimmune screening, HPV gene full typing screening and the like. Different from the traditional immunodetection flow type fluorescence technology, the detection sensitivity can be greatly improved by utilizing the fluorescence encoding microsphere technology and the sheath flow technology, the encoding microsphere marked with PE sequentially passes through the detection tube under the action of sheath flow, the encoding microsphere and PE are simultaneously excited by light with different wavelengths, and signals are counted for different encoding microspheres respectively by respectively detecting the excitation light encoded by the microsphere and the signal value represented by the PE, so that multi-item joint inspection is realized. Its low sample size, high sensitivity, high selectivity and high throughput make it a desirable diagnostic tool for alzheimer's pathology.

Based on the above, the kit contains microspheres with different fluorescence intensities (different numbers), namely 6 microspheres coupled with capture antibodies, microspheres coated with beta amyloid 1-42 (Abeta 1-42) capture antibodies, microspheres coated with beta amyloid 1-40 (Abeta 1-40) capture antibodies, microspheres coated with microtubule-associated Tau protein (T-Tau) capture antibodies, microspheres coated with phosphorylated Tau181 (p-Tau-181) capture antibodies, microspheres coated with Glial Fibrillary Acidic Protein (GFAP) capture antibodies and microspheres coated with neurofilament light chain protein (NfL) capture antibodies. Specifically binding Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL in a sample to be tested with capture microspheres and biotin-marked detection antibodies to form a double-antibody sandwich compound (capture microspheres-sample to be tested-detection antibodies), reacting with added phycoerythrin-marked streptavidin (SA-PE), and analyzing the fluorescence intensity of the compound and matching a standard curve to obtain the content of Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL in the sample to be tested.

In the application, the microsphere surface with the same fluorescence intensity is coated with the same specific antibody.

The biotin-labeled detection antibody comprises a biotin-labeled beta amyloid 1-42 detection antibody, a biotin-labeled beta amyloid 1-40 detection antibody, a biotin-labeled microtubule-associated Tau protein detection antibody, a biotin-labeled phosphorylated Tau181 detection antibody, a biotin-labeled glial fibrillary acidic protein detection antibody and a biotin-labeled neurofilament light chain protein detection antibody.

In the present invention, the capture antibody and the corresponding detection antibody bind to different epitopes of the same antigen, respectively. Wherein the capture antibody and the detection antibody are directed against the following 6 AD markers: aβ1-42, Aβ1-40, T-Tau, p-Tau-181, GFAP and NfL.

The SA-PE is not limited in the present application, and a commercial product such as SA-PE of the present application purchased from Sieimer, cat No. S866 may be used.

In one embodiment, the casein concentration is 0.1 wt% -0.5 wt%, for example 0.1 wt%, 0.15 wt%, 0.2 wt%, 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt%, 0.5 wt%, etc.

In one embodiment, the casein is present at a concentration of 0.1wt% to 0.3wt%.

In a certain embodiment, the casein is at a concentration of 0.2 wt%.

In certain embodiments, the concentration of the animal serum is 8 vol% -12% vol%, e.g., 8.5 vol%, 9 vol%, 9.5 vol%, 10 vol%, 10.5 vol%, 11 vol%, 11.5 vol%, etc.

In certain embodiments, the concentration of the animal serum is 10 vol%.

In certain embodiments, the animal serum is sheep serum and/or bovine serum.

In certain embodiments, the animal serum is sheep serum.

In some embodiments, the second buffer further comprises 0.1wt% -10 wt% sodium chloride (e.g., 0.2 wt%、0.3 wt%、0.4 wt%、0.5 wt%、0.6 wt%、0.7 wt%、0.8 wt%、0.9 wt%、1 wt%、1.5 wt%、2 wt%、2.5 wt%、3 wt%、3.5 wt%, etc.) and 0.05 vol% -1 vol% pra Luo Kelin 300 (e.g., 0.05 vol %、0.06 vol %、0.07 vol %、0.08 vol %、0.09 vol %、0.1 vol %、0.15 vol %、0.2 vol %、0.25 vol %、0.3 vol %、0.4vol %, etc.).

In certain embodiments, the second buffer further comprises 0.5wt% to 1wt% sodium chloride and 0.05 vol wt% to 0.5 vol wt% pra Luo Kelin 300 (Proclin 300).

In certain embodiments, the second buffer further comprises 0.9wt% sodium chloride and 0.1vol% Proclin Luo Kelin (Proclin 300).

In certain embodiments, the first buffer comprises 0.1wt% to 10wt% sodium chloride (e.g., 0.2 wt%、0.3 wt%、0.4 wt%、0.5 wt%、0.6 wt%、0.7 wt%、0.8 wt%、0.9 wt%、1 wt%、1.5 wt%、2 wt%、2.5 wt%、3 wt%、3.5 wt%, etc.), 0.1wt% to 10wt% bovine serum albumin (e.g., 0.2 wt%、0.3 wt%、0.4 wt%、0.5 wt%、0.6 wt%、0.7 wt%、0.8 wt%、0.9 wt%、1 wt%、1.5 wt%、2 wt%、2.5 wt%、3 wt%、3.5 wt%, etc.), 0.05vol% to 10 vol% pra Luo Kelin 300 (e.g., 0.05 vol %、0.06 vol %、0.07 vol %、0.08 vol %、0.09 vol %、0.1 vol %、0.15 vol %、0.2 vol %、0.25 vol %、0.3 vol %、0.4vol %、1 vol %、2 vol %、3 vol %、4 vol %、5 vol %, etc.).

In some embodiments, the first buffer contains 0.5wt% to 1wt% sodium chloride, 0.1wt% to 5wt% bovine serum albumin, 0.05 vol wt% to 0.5vol% Pr Luo Kelin 300 (Proclin 300).

In certain embodiments, the first buffer comprises 0.9wt% sodium chloride, 1wt% bovine serum albumin, 0.1vol% prantin Luo Kelin (Proclin 300).

In certain embodiments, the first buffer and the second buffer are both phosphate buffers.

In certain embodiments, the working concentrations of the biotin-labeled amyloid beta 1-42 detection antibody and phosphorylated tau181 detection antibody in the second reagent are, separately and independently, 0.1-1 μg/ml, e.g., 0.2 μg/ml, 0.3 μg/ml, 0.4 μg/ml, 0.5 μg/ml, 0.6 μg/ml, 0.7 μg/ml, 0.8 μg/ml, 0.9 μg/ml, etc.

In some embodiments, the working concentrations of the biotin-labeled amyloid beta 1-42 detection antibody and phosphorylated tau181 detection antibody in the second reagent are each and independently 0.3-0.8 μg/ml.

In certain embodiments, the working concentration of biotin-labeled amyloid beta 1-42 detection antibody and phosphorylated tau181 detection antibody in the second agent is 0.5 μg/ml separately and independently.

In certain embodiments, the working concentration of the biotin-labeled amyloid-beta 1-40 detection antibody, microtubule-associated Tau protein detection antibody, glial fibrillary acidic protein detection antibody in the second reagent is, respectively and independently, 0.1-0.5 μg/ml, e.g., 0.15 μg/ml, 0.2 μg/ml, 0.25 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4 μg/ml, 0.45 μg/ml, etc.

In some embodiments, the working concentrations of the biotin-labeled amyloid beta 1-40 detection antibody, the microtubule-associated Tau protein detection antibody, and the glial fibrillary acidic protein detection antibody in the second reagent are respectively and independently 0.1-0.3 μg/ml.

In certain embodiments, the working concentration of the biotin-labeled amyloid-beta 1-40 detection antibody, microtubule-associated Tau protein detection antibody, glial fibrillary acidic protein detection antibody in the second agent is 0.25 μg/ml, respectively and independently.

In some embodiments, the working concentration of the biotin-labeled neurofilament light chain protein detection antibody in the second reagent is 0.5-2 μg/ml, e.g., 0.6μg/ml、0.7μg/ml、0.8μg/ml、0.9μg/ml、1.0μg/ml、1.1μg/ml、1.2μg/ml、1.3μg/ml、1.4μg/ml、1.5μg/ml、1.6μg/ml、1.7μg/ml、1.8μg/ml、1.9μg/ml, etc.

In some embodiments, the working concentration of the biotin-labeled neurofilament light chain protein detection antibody in the second reagent is 0.5-1.5 μg/ml.

In certain embodiments, the working concentration of biotin-labeled neurofilament light chain protein detection antibody in the second agent is 1 μg/ml.

In some embodiments, the working concentrations of the amyloid beta 1-42 capture antibody, the amyloid beta 1-40 capture antibody, the microtubule-associated Tau protein capture antibody, the glial acidic protein capture antibody and the neurofilament light chain protein capture antibody in the first reagent are respectively and independently 0.5-2 μg/ml, such as 0.6μg/ml、0.7μg/ml、0.8μg/ml、0.9μg/ml、1.0μg/ml、1.1μg/ml、1.2μg/ml、1.3μg/ml、1.4μg/ml、1.5μg/ml、1.6μg/ml、1.7μg/ml、1.8μg/ml、1.9μg/ml and the like.

In some embodiments, the working concentrations of the amyloid beta 1-42 capture antibody, the amyloid beta 1-40 capture antibody, the microtubule-associated Tau protein capture antibody, the glial acidic protein capture antibody and the neurofilament light chain protein capture antibody in the first reagent are respectively and independently 0.5-1.5 μg/ml.

In certain embodiments, the working concentration of amyloid-beta 1-42 capture antibody, amyloid-beta 1-40 capture antibody, microtubule-associated Tau protein capture antibody, glial fibrillary acidic protein capture antibody, and neurofilament light chain protein capture antibody in the first agent is 1 μg/ml, respectively and independently.

In certain embodiments, the working concentration of phosphorylated tau181 capture antibody in the first reagent is 1-5 μg/ml, e.g., 1.1μg/ml、1.2μg/ml、1.3μg/ml、1.4μg/ml、1.5μg/ml、1.6μg/ml、1.7μg/ml、1.8μg/ml、2μg/ml、2.2μg/ml、2.4μg/ml、2.6μg/ml、2.8μg/ml、3μg/ml、3.2μg/ml、3.4μg/ml、3.6μg/ml、3.8μg/ml、4μg/ml、4.2μg/ml、4.4μg/ml、4.6μg/ml、4.8μg/ml, etc.

In certain embodiments, the working concentration of phosphorylated tau181 capture antibody in the first reagent is 1-3 μg/ml.

In certain embodiments, the working concentration of phosphorylated tau181 capture antibody in the first reagent is 2 μg/ml.

In a preferred example, the method for preparing the first reagent includes:

(1) Respectively activating microspheres with different numbers, mixing the activated microspheres with corresponding capture antibodies, and incubating at 20-40 ℃ for 1-3 hours;

(2) Washing, then placing the washed product into 0.5-5wt% of BSA, incubating the washed product at 20-40 ℃ for 1-3 hours, and storing the washed product into 0.5-5wt% of BSA after the washing;

(3) And (3) mixing the different capture antibodies coupled and prepared in the step (2) and diluting the different capture antibodies with a first buffer solution to obtain the first reagent.

In certain embodiments, the ratio of Abeta 1-42 capture antibody, abeta 1-40 capture antibody, T-Tau capture antibody, p-Tau-181 capture antibody, GFAP capture antibody and NfL capture antibody to microspheres is respectively and independently coated with 0.01-0.5 mg Abeta 1-42 capture antibody, 0.01-0.5 mg Abeta 1-40 capture antibody, 0.01-0.5 mg T-Tau capture antibody, 0.01-0.5 mg p-Tau-181 capture antibody, 0.01-0.5 mg GFAP capture antibody and 0.01-0.5 mg NfL capture antibody per 10- ⁹ microspheres.

In certain embodiments, the ratio of Abeta 1-42 capture antibody, abeta 1-40 capture antibody, T-Tau capture antibody, p-Tau-181 capture antibody, GFAP capture antibody and NfL capture antibody to microsphere is 0.05mg Abeta 1-42 capture antibody, 0.1mg Abeta 1-40 capture antibody, 0.1mg T-Tau capture antibody, 0.1mg p-Tau-181 capture antibody, 0.1mg GFAP capture antibody and 0.05mg NfL capture antibody, respectively, per 10 ⁹ microspheres.

In a preferred example, the method for preparing the first reagent includes:

(1) Mixing the microspheres with different numbers with corresponding capture antibodies after activation, and incubating for 2 hours at 37 ℃;

(2) Washing, placing in 1wt% BSA, incubating at 37 ℃ for 2 hours, and preserving in 1wt% BSA after the washing is finished;

In the invention, streptavidin-phycoerythrin (SA-PE) can be specifically combined with biotin to form a detection antibody marked by Phycoerythrin (PE) fluorescein, and the phycoerythrin is excited by green laser of a liquid phase chip instrument to carry out fluorescence detection.

In a preferred example, the method for preparing the second reagent includes:

(1) Mixing different detection antibodies with activated biotin, and incubating for 20-60 min at 20-40 ℃;

(2) Purifying the conjugate of the biotin-detection antibody prepared in the step (1), mixing and diluting with a second buffer solution to obtain the second reagent.

In certain embodiments, the molar ratio of the detection antibody to aβ1-42, the detection antibody to aβ1-40, the detection antibody to T-Tau, the detection antibody to p-Tau-181, the detection antibody to GFAP, and the detection antibody to NfL to activated biotin is 1, respectively and independently: 10-40, 1: 10-40, 1: 10-40, 1: 10-40, 1: 10-40, 1: 10-40.

In certain embodiments, the molar ratio of the detection antibody to aβ1-42, the detection antibody to aβ1-40, the detection antibody to T-Tau, the detection antibody to p-Tau-181, the detection antibody to GFAP, and the detection antibody to NfL to activated biotin is 1, respectively and independently: 10-30, 1: 10-30, 1: 15-35, 1: 10-30, 1: 10-30, 1: 10-30.

In certain embodiments, the molar ratio of the detection antibody to aβ1-42, the detection antibody to aβ1-40, the detection antibody to T-Tau, the detection antibody to p-Tau-181, the detection antibody to GFAP, and the detection antibody to NfL to activated biotin is 1, respectively and independently: 20. 1:20. 1: 30. 1:20. 1:20. 1:20.

In a preferred example, the method for preparing the second reagent includes:

(1) Mixing different detection antibodies with activated biotin, and incubating at 37 ℃ for 40min;

In certain embodiments, the kit further comprises a dilution buffer that is a phosphate buffer containing 0.1wt% to 10wt% sodium chloride (e.g., 0.2 wt%、0.3 wt%、0.4 wt%、0.5 wt%、0.6 wt%、0.7 wt%、0.8 wt%、0.9 wt%、1 wt%、1.5 wt%、2 wt%、2.5 wt%、3 wt%、3.5 wt%, etc.), 0.1wt% to 10wt% bovine serum albumin (e.g., 0.2 wt%、0.3 wt%、0.4 wt%、0.5 wt%、0.6 wt%、0.7 wt%、0.8 wt%、0.9 wt%、1 wt%、1.5 wt%、2 wt%、2.5 wt%、3 wt%、3.5 wt%, etc.), 0.1wt wt% to 0.5 wt% casein (e.g., 0.1wt%, 0.15 wt%, 0.2 wt%, 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt%, 0.5 wt%, etc.), 0.05 vol% -0.2 vol% tween-20 (0.06 vol%, 0.07 vol%, 0.08 vol%, 0.09 vol%, 0.1 vol%, 0.12 vol%, 0.14 vol%, 0.16 vol%, 0.18 vol%, etc.), 0.05 vol% -10 vol% p Luo Kelin (e.g., 0.05 vol %、0.06 vol %、0.07 vol %、0.08 vol %、0.09 vol %、0.1 vol %、0.15 vol %、0.2 vol %、0.25 vol %、0.3 vol %、0.4 vol %, etc.).

In certain embodiments, the dilution buffer is a phosphate buffer containing 0.1wt% to 5wt% sodium chloride, 0.1wt% to 5wt% bovine serum albumin, 0.1wt wt% to 0.3 wt% casein, 0.05 vol% to 0.15 vol% tween-20, 0.05 vol% to 5vol% common Luo Kelin.

In some embodiments, the dilution buffer is a phosphate buffer containing 0.1wt% to 1wt% sodium chloride, 0.1wt% to 2wt% bovine serum albumin, 0.1wt wt% to 0.3 wt% casein, 0.05 to 0.15 vol% tween-20, 0.05vol% to 0.5vol% p Luo Kelin 300.

In certain embodiments, the dilution buffer is a phosphate buffer containing 0.9wt% sodium chloride, 2wt% bovine serum albumin, 0.2 wt% casein, 0.1vol% tween-20, 0.1vol% p Luo Kelin 300.

In certain embodiments, the concentration of casein in the dilution buffer is 0.1 wt% > -0.5 wt%, e.g., 0.15 wt%, 0.2 wt%, 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt%, etc.

In certain embodiments, the concentration of casein in the dilution buffer is 0.1 wt% -0.3 wt%.

In certain embodiments, the concentration of casein in the dilution buffer is 0.2 wt%.

In certain embodiments, the kit further comprises a calibrator, wherein the mixed 6 markers of alzheimer's disease are diluted to different concentrations of the calibrator, respectively, using the dilution buffer.

In the present invention, various marker calibrators and reagents are commercially available or synthesized according to known methods, and commercial manufacturers include, but are not limited to, the company Simerfei, abcam and R & D Systems, unless otherwise indicated.

In certain embodiments, the kit further comprises a reaction buffer and a wash buffer.

In some embodiments, the reaction buffer is a phosphate buffer containing 0.1-10 wt% sodium chloride, 0.05-10 vol% Pr Luo Kelin 300.

In certain embodiments, the reaction buffer is a phosphate buffer containing 0.9wt% sodium chloride, 0.1 vol% p Luo Kelin 300.

In certain embodiments, the wash buffer is a phosphate buffer containing 0.05 vol% -0.2. 0.2 vol% tween-20.

In certain embodiments, the wash buffer is a phosphate buffer containing 0.1 vol% tween-20.

In the present application, the phosphate buffer is PBS.

(2) Adding a second reagent into the system in the step (1), and incubating;

In certain embodiments, the volume ratio of the reaction buffer, the first reagent, and the sample to be tested in step (1) is 1: (0.1 to 1): (0.5-2), such as 1:0.2:1, 1:0.3:1, 1:0.4:1, 1:0.5:1, 1:0.6:1, 1:0.8:1, etc.

In certain embodiments, the incubation time in step (2) is 12-24 hours, e.g., 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, etc.

In certain embodiments, the incubation time in step (3) is 0.2-1 hour, e.g., 0.4 hours, 0.6 hours, 0.8 hours, 1 hour, etc.

In certain embodiments, the incubation in step (2) and step (3) is performed under light-protected conditions. Compared with the PE direct labeling detection antibody reagent biotin, the method provided by the application has the advantages of higher labeling noise ratio, simplicity, easiness in operation and better control of batch-to-batch stability.

The application also provides a prediction model for predicting the Alzheimer's disease by combining the six peripheral blood markers of Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL, and whether the Alzheimer's disease exists or not is judged according to the constructed logistic regression equation. The logistic regression equation is:

Y= -0.208 x Abeta 42-0.026 x Abeta 40+0.566 x T-tau+1.598 x p-Tau181+0.03 x GFAP+0.054 x nfl-2.214, the cut-off value is 0.84893, the content of each marker in the sample to be tested is brought into the equation, and if the result is higher than the cut-off value, the patient is predicted to be the Alzheimer disease, otherwise, the patient is healthy.

The present invention will be further illustrated by the following examples, wherein "%" represents mass percent unless otherwise specified. The materials and reagents used in the examples which follow, unless otherwise indicated, are those commonly used in the art and may be obtained commercially or synthesized by known methods. The experimental methods, which are not specified in the following examples, are generally carried out according to conventional experimental conditions or conditions recommended by the manufacturer of the relevant reagent (kit).

Examples

The invention provides an Alzheimer disease marker detection kit which comprises a first reagent, a second reagent, streptavidin-phycoerythrin, a dilution buffer solution, a reaction buffer solution and a cleaning buffer solution. Alzheimer's disease markers are Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL.

The preparation method of the first reagent comprises the following steps:

the first reagent comprises microspheres coupled with different capture antibodies and a first buffer.

(1) Purifying the capture antibodies of Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL respectively by commercial purification columns (Sieimer, cat# 89882) to remove sodium azide and related compounds, and collecting the purified 6 capture antibodies;

(2) Mixing the capture antibody of Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL with microspheres activated by EDC according to the corresponding proportion, and incubating for 2 hours at 37 ℃;

(3) Washing, incubating for 2 hours at 37 ℃ by using a blocking solution containing 1% BSA, and preserving in the blocking solution containing 1% BSA after finishing to obtain capture antibody-coated microspheres;

(4) Preparing phosphate buffer solution containing 0.9wt% sodium chloride, 1wt% bovine serum albumin and 0.1vol% common Luo Kelin;

(5) Mixing the different capture antibodies coupled prepared in the step (3) and diluting the different capture antibodies with the buffer solution prepared in the step (4) to obtain the first reagent.

The preparation method of the second reagent comprises the following steps:

The second reagent comprises a biotin-labeled detection antibody and a second buffer.

(1) Purifying the detection antibody of Abeta 1-42, the detection antibody of Abeta 1-40, the detection antibody of T-Tau, the detection antibody of p-Tau-181, the detection antibody of GFAP and the detection antibody of NfL by a commercial purification column (Sieimer, product number: 89882) to remove sodium azide and related compounds, and collecting 6 detection antibodies after purification;

(2) Mixing 6 different detection antibodies prepared in the step (1) with commercial biotin NHS (Siemens, product number: 21336) according to a molar ratio, incubating for 45min at 25 ℃, and purifying biotin-detection antibody conjugate through a purification column after incubation to obtain a biotinylated detection antibody;

(3) Preparing phosphate buffer solution containing 0.9wt% of sodium chloride, 0.2wt% of casein, 10vol% of sheep serum and 0.1 vol% of Pr Luo Kelin;

(4) Mixing and purifying the biotin-detection antibody conjugate prepared in the step (2) and diluting with the buffer solution prepared in the step (3) to obtain the second reagent.

Preparation of a calibrator:

(1) Preparing a dilution buffer solution: phosphate buffer containing 0.9 wt% sodium chloride, 2% bovine serum albumin, 0.2% casein, 0.1 vol% tween-20, 0.1 vol% pra Luo Kelin;

(2) And (3) 6 corresponding AD markers are taken, 6 standard substances are prepared by diluting the buffer solution prepared in the step (1) according to a certain proportion, wherein the standard substances are respectively standard substance S1, standard substance S2, standard substance S3, standard substance S4, standard substance S5 and standard substance S6, and the concentrations of Abeta 1-42, abeta 1-40, T-Tau, p-Tau181, GFAP and NfL antigens in the standard substances are shown in the following table, so that the standard substances are the calibrator, and the calibrator is shown in the table 1.

TABLE 1

Streptavidin-phycoerythrin was purchased from zemoeid, cat No. S866.

Preparing a reaction buffer solution: contains 0.9 wt% sodium chloride and 0.1 vol% pra Luo Kelin 300 phosphate buffer.

Preparing 10 Xcleaning buffer: contains 0.1vol% Tween-20 phosphate buffer solution.

The invention also provides a using method of the Alzheimer disease marker detection kit, which comprises the following steps:

(1) Add 20. Mu.L of reaction buffer to the tube;

(2) Add 10 μl of capture microsphere cocktail (first reagent) to all tubes;

(3) Adding 20 μl of sample to the test tube;

(4) Adding 10 mu L of detection antibody reagent (second reagent) into all the tubes, and vibrating at room temperature and keeping away from light for incubation for 18+/-1 h;

(5) Add 20. Mu.L SA-PE (working concentration 200. Mu.g/mL) to all tubes and incubate at room temperature with shaking in the dark for 0.5h;

(6) To each tube, 300 μl of 1 x wash buffer was added, the microspheres were resuspended by vortexing, and the supernatant was removed 2 times by centrifugation at 5min 500g, taking care to remove the supernatant;

(7) According to the loading requirements, 150-300 mu L of 1 Xwashing buffer is added into each tube, and the microspheres are resuspended by vortexing;

(8) And (3) detecting by a machine to obtain the fluorescence intensity of the compound, and calculating according to the standard curve of each marker to obtain the protein content of Abeta 1-42, abeta 1-40, T-Tau, p-Tau181, GFAP and NfL in the sample.

The method for establishing standard curves and linear regression correlation coefficients (r) for each marker is described in example 6.

Example 1

According to the use method of the detection kit, the proportions of the antibody-coupled microsphere and the antibody-labeled biotin are respectively optimized, and Abeta 1-42, abeta 1-40, T-Tau, p-Tau181, GFAP and NfL antigen calibrators S1-S6 are respectively detected, and the comparison linearity r is shown in Table 2.

TABLE 2

The test results showed that the optimal labeling ratios of Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL to the capture microspheres were 0.05mg/1x10⁹、0.1mg/1x10⁹、0.1mg/1x10⁹、0.1mg/1x10⁹、0.1mg/1x10⁹ and 0.05mg/1X10 ⁹, respectively, based on the linear r values; the optimal ratios (moles) of Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL detection antibodies to biotin are 1:20. 1:20. 1:20. 1: 30. 1:20 and 1:20.

Example 2

In order to improve the signal-to-noise ratio of the reagent, according to the use method of the detection kit, the working concentrations of the capture reagent and the detection reagent are respectively optimized, the calibrators S1-S6 are detected, and the signal-to-noise ratio S5/S1 is compared, and the result is shown in Table 3.

TABLE 3 Table 3

The test results showed that the working concentrations of Aβ1-42, Aβ1-40, T-Tau, p-Tau-181, GFAP and NfL capture antibodies in the capture reagent (first reagent) were 1. Mu.g/ml, 2. Mu.g/ml, 1. Mu.g/ml and 1. Mu.g/ml, respectively, according to the signal to noise ratio S5/S1; the working concentrations of the Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL detection antibodies in the detection reagent (second reagent) were 0.5. Mu.g/ml, 0.25. Mu.g/ml, 0.5. Mu.g/ml, 0.25. Mu.g/ml and 1. Mu.g/ml, respectively.

Example 3

According to the use method of the detection kit, the second reagent is prepared by optimizing the components in the second buffer solution, other reagents and the condition-unchanged detection calibrators S1-S6 are used, and the background signal luminescence value and the signal to noise ratio are compared, and the result is shown in Table 4.

TABLE 4 Table 4

The test result shows that the casein addition amount of the second buffer solution containing the antibody second reagent for Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL is 0.2 percent according to the average fluorescence intensity of background S1 and the comparison of signal to noise ratio S5/S1 data, and has better detection effect.

Example 4

In this example, according to the method of using the above-described test kit, a second reagent, other reagents and a condition-unchanged test standard S1 and a standard S4 were prepared, 10 wells were repeated for each calibrator, and CV values were compared, and the results are shown in table 5, wherein% is the volume ratio.

TABLE 5

Experimental results show that the second buffer solution prepared from 10% sheep serum has a better luminescence value and good repeatability.

Example 5

The components of the dilution buffer are optimized according to the use method of the detection kit, specifically, the dilution buffer added with different proteins, saccharides and alcohols is screened, and the deviation of the dilution buffer after being placed at 37 ℃ for 3 days is tested, and the results are shown in table 6, wherein the control group is phosphate buffer which does not contain glycerol, trehalose or casein and only contains sodium chloride, bovine serum albumin and common Luo Kelin.

TABLE 6

Experimental results show that after 0.2% casein is added into a diluent buffer system and the temperature is increased by 3 days at 37 ℃, the fluorescence intensity deviation of the standard substance is within +/-15%, and the standard substance still has better detection performance.

Example 6

The standard curve is established according to the using method of the detection kit.

Standard curve establishment:

1. Preparing the 6 calibration materials S1-S6;

2. remove and add 20 μl reaction buffer to the tube;

3. Add 10. Mu.L of capture microsphere mix to all tubes;

4. add 10. Mu.L of calibrator to calibrator tube;

5. add 10 μl of detection antibody reagent to all tubes;

6. shake at room temperature and incubate for 18+ -1 h under dark condition;

7. Add 20. Mu.L SA-PE (working concentration 200. Mu.g/mL) to all tubes;

8. Shake at room temperature and incubate for 0.5h in dark place;

9. to each tube, 300 μl of 1 x wash buffer was added, the microspheres were resuspended by vortexing, and the supernatant was removed 2 times by centrifugation at 5min 500g, taking care to remove the supernatant;

10. According to the loading requirements, 150-300 mu L of 1 Xwashing buffer is added into each tube, and the microspheres are resuspended by vortexing;

11. And (5) detecting by a machine to obtain the fluorescence intensity of the compound, and calculating to obtain a standard curve. The method comprises the following steps: a linear regression equation was obtained using the dilution concentration (xi) as an independent variable and the measurement result mean value (yi) as a dependent variable. The correlation coefficient (r) of the linear regression is calculated as follows:

The standard curves of the 6 markers of the kit are as follows:

Aβ42 standard curve (table 7): in the measurement range of (0-8000) pg/mL, the correlation coefficient (r) of the kit should be more than or equal to 0.9900.

TABLE 7

Aβ40 standard curve (table 8): in the measurement range of (0-3000) pg/mL, the correlation coefficient (r) of the kit should be more than or equal to 0.9900.

TABLE 8

TTau standard curve (table 9): in the measurement range of (0-1000) pg/mL, the correlation coefficient (r) of the kit should be larger than or equal to 0.97800.

TABLE 9

PTau181 standard curve (table 10): in the measurement range of (0-160) pg/mL, the correlation coefficient (r) of the kit should be more than or equal to 0.9900.

Table 10

GFAP standard curve (table 11): in the measurement range of (0-10000) pg/mL, the correlation coefficient (r) of the kit should be more than or equal to 0.9900.

TABLE 11

NfL standard curve (table 12): nfL in the measurement range of (0-8000) pg/mL, the correlation coefficient (r) of the kit should be more than or equal to 0.9900.

Table 12

(3) Sample detection: 20 clinical plasma samples were collected, including 10 healthy physical examination populations and 10 AD patients, with a simple intelligent mental state examination scale score of <10 points, and were confirmed by diagnosis according to AD diagnostic criteria recommended by national institute of neurological and linguistic disorders and stroke, alzheimer's disease and related disease association.

1. Add 20. Mu.L of reaction buffer to the tube;

2. Add 10 μl of capture microsphere cocktail (first reagent) to all tubes;

3. Add 20 μl sample to the tube;

4. Add 10 μl of detection antibody reagent (second reagent) to all tubes;

5. shake at room temperature and incubate for 18+ -1 h under dark condition;

6. Add 20. Mu. LSA-PE (working concentration 200. Mu.g/mL) to all tubes;

7. shake at room temperature and incubate for 0.5h in dark place;

8. To each tube, 300 μl of 1x wash buffer was added, the microspheres were resuspended by vortexing, and the supernatant was removed 2 times by centrifugation at 5min 500g, taking care to remove the supernatant;

9. according to the loading requirements, 150-300 mu L of 1 Xwashing buffer is added into each tube, and the microspheres are resuspended by vortexing;

10. And (3) detecting by a machine to obtain the fluorescence intensity of the compound, and calculating according to standard curves of all the markers to obtain the protein contents of Abeta 1-42, abeta 1-40, T-Tau, p-Tau181, GFAP and NfL in the sample, wherein the detection results of 6 protein contents and the comparison among groups are shown in Table 13, and the protein content unit is pg/ml.

TABLE 13

The results of ROC curve analysis of the 6 proteins to identify AD dementia and normal elderly are shown in Table 14 and FIG. 1.

The single protein index can distinguish samples, but the single protein index has poor specificity and misjudgment, so the measured six protein marker contents are subjected to modeling analysis, and the detection results are shown in table 14 and fig. 1.

TABLE 14

Analysis of biomarker and clinical sample outcome consistency: the kit is used for detecting clinical AD samples and normal human samples, SPSS software is used for respectively counting AUC values under ROC curves of each marker, the triple detection combination (Abeta 1-42/Abeta 1-40/p-Tau 181) and the six-combined detection combination for detecting the clinical samples, and consistency of different combination results and the clinical samples is compared.

Compared with triple detection, the kit increases the variety of Alzheimer disease detection markers. AUC values under ROC curve for six markers when differentiating clinical AD samples were respectively: aβ1-42 was 0.755, Aβ1-40 was 0.710, T-Tau was 0.820, p-Tau181 was 0.885, GFAP was 0.840, nfL was 0.8400. The AUC value of the combined three-joint detection Aβ1-42/Aβ1-40/p-Tau181 is 0.920, the AUC value of the combined six-joint detection is 0.940, and the six-joint detection can improve the diagnosis sensitivity and specificity of the Alzheimer disease.

The following discriminant function is established through discriminant analysis, and discriminant function scores are calculated, and a function score interpretation formula is as follows:

Y= -0.208 x aβ42-0.026 x aβ40+0.566 x t_tau+1.598 x p_tau181+0.03 x gfap+0.054 x nfl-2.214, cut-off 0.84893, specificity 0.9, sensitivity 0.8.

Clinical sample verification:

Example 7

And detecting by using the zero-concentration calibrator as a sample, repeatedly measuring for 20 times to obtain MFI values (relative luminescence values) of 20 measurement results, calculating an average value (M) and a Standard Deviation (SD) of the MFI values to obtain M+2SD, performing two-point regression fitting according to concentration-RLU value results between the zero-concentration calibrator and adjacent calibrators to obtain a primary equation, introducing the RLU value corresponding to the M+2SD into the primary equation, and obtaining a corresponding concentration value, namely a blank limit.

LOB is a margin;

M is the average value of the detection results of the blank samples;

SD _k is the standard deviation of the blank sample detection result;

n is the detection times of the blank sample;

N is the number of blank samples;

N-N is the degree of freedom of the estimated SDk.

The calculated limits were 2.5pg/mL for Abeta 1-42, 2.5pg/mL for Abeta 1-40, 1.5pg/mL for T-Tau, 1.0pg/mL for p-Tau181, 2.5pg/mL for GFAP, and 2.5pg/mL for NfL.

Example 8

Pure products of antigens Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL are respectively taken, and standard solutions with the concentration of 500pg/mL, 1000pg/mL, 150pg/mL, 1000pg/mL and 500pg/mL are prepared by using a dilution buffer solution. Adding the mixture into a dilution buffer according to the volume ratio of 1:9, repeatedly measuring for 3 times, and respectively calculating the recovery rate according to the formula below.

Wherein: r-recovery rate;

v-adding the volume of the standard solution;

V ₀ -the volume of the sample;

C-measuring the concentration of the sample after the standard solution is added;

C ₀ -the measured concentration of the sample;

Concentration of Cs-standard solution.

The results showed that the recovery rate of Abeta 1-42 was 105.2%, the recovery rate of Abeta 1-40 was 99.2%, the recovery rate of T-Tau was 98.9%, the recovery rate of p-Tau181 was 88.7%, the recovery rate of GFAP was 110.2% and the recovery rate of NfL was 103.5%.

Example 9

The pure products of the antigens Abeta 1-38, abeta 1-43, TNF alpha and IP-10 are respectively taken and respectively diluted to 1000pg/ml by sample diluent, the content of Abeta 1-42, abeta 1-40, T-Tau, p-Tau-181, GFAP and NfL is detected, and the ratio analysis is carried out on the products with theoretical concentration, namely the cross reaction rate (see Table 15).

TABLE 15

The cross-reactivity of Abeta 1-38, abeta 1-43, TNF alpha and IP-10 with the kit was less than 1%.

The kit provided by the invention has the advantages of high signal-to-noise ratio, good stability, good repeatability, strong specificity, high accuracy, high flux, wide linear range and the like. Meanwhile, the qualitative and quantitative detection can be simultaneously carried out on 6 protein markers related to the Alzheimer's disease, the comprehensive diagnosis specificity of the combined detection of the 6 protein markers is 90%, and the diagnosis accuracy is obviously higher than that of a single protein marker. The marker joint inspection has higher specificity or sensitivity; compared with the traditional imaging detection means, the diagnosis sensitivity can reach 80% under the condition of 90% of specificity, which is far higher than that of similar products in the market.

It should be understood that the above examples are illustrative and are not intended to encompass all possible implementations encompassed by the claims. Various modifications and changes may be made in the above embodiments without departing from the scope of the disclosure. Likewise, the individual features of the above embodiments can also be combined arbitrarily to form further embodiments of the invention which may not be explicitly described. Therefore, the above examples merely represent several embodiments of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. The Alzheimer disease marker detection kit is characterized by comprising a first reagent, a second reagent and streptavidin-phycoerythrin;

2. The kit of claim 1, wherein the casein concentration is 0.1 wt% -0.5 wt%;

and/or the concentration of the animal serum is 8 vol-12 vol%;

And/or, the animal serum is sheep serum and/or bovine serum.

3. The kit of claim 1, wherein the second buffer solution further comprises 0.1 wt% -10 wt% sodium chloride and 0.05 vol% -1 vol% common Luo Kelin 300;

And/or the first buffer solution contains 0.1-10wt% of sodium chloride, 0.1-10wt% of bovine serum albumin and 0.05-10vol% of common Luo Kelin & lt 300 & gt;

And/or, the first buffer solution and the second buffer solution are phosphate buffer solutions.

4. The kit according to claim 1, wherein the working concentrations of the biotin-labeled amyloid beta 1-42 detection antibody and the phosphorylated tau181 detection antibody in the second reagent are respectively and independently 0.1-1 μg/ml;

And/or the working concentration of the biotin-labeled beta amyloid 1-40 detection antibody, the microtubule-associated Tau protein detection antibody and the glial fibrillary acidic protein detection antibody in the second reagent is respectively and independently 0.1-0.5 mug/ml;

and/or the working concentration of the biotin-labeled neurofilament light chain protein detection antibody in the second reagent is 0.5-2 mug/ml.

5. The kit according to claim 1, wherein the working concentrations of the amyloid beta 1-42 capture antibody, the amyloid beta 1-40 capture antibody, the microtubule-associated Tau protein capture antibody, the glial fibrillary acidic protein capture antibody and the neurofilament light chain protein capture antibody in the first reagent are respectively and independently 0.5-2 μg/ml;

and/or the working concentration of the phosphorylated tau181 capture antibody in the first reagent is 1-5 mug/ml.

6. The kit of claim 1, further comprising a dilution buffer comprising 0.1wt% to 10wt% sodium chloride, 0.1wt% to 10wt% bovine serum albumin, 0.1wt wt% to 0.5 wt% casein, 0.05 vol% to 0.2 vol% tween-20, 0.05 vol% to 10 vol% p Luo Kelin.

7. The kit according to any one of claims 1 to 6, further comprising a reaction buffer and a wash buffer;

Wherein the reaction buffer solution is phosphate buffer solution containing 0.1-10wt% of sodium chloride, 0.05 vol-vol% of Pr Luo Kelin 300;

the cleaning buffer solution is phosphate buffer solution containing 0.05 vol% -0.2 vol% Tween-20.

8. Use of the kit according to any one of claims 1 to 7 in the preparation of a detection product for a marker of alzheimer's disease.

9. The method of using the kit of any one of claims 1 to 7, comprising:

(2) Adding a second reagent into the system in the step (1), and incubating;

10. The method of claim 9, wherein the volume ratio of the reaction buffer, the first reagent and the sample to be tested in step (1) is 1: (0.1 to 1): (0.5-2);

And/or, the incubation time in the step (2) is 12-24 hours;

and/or, the incubation time in the step (3) is 0.2-1 hour;

And/or, the incubation in the step (2) and the incubation in the step (3) are carried out under the light-shielding condition.