Novel magnetic particle chemiluminiscence detection kit for A beta 1-42 and preparation method of novel magnetic particle chemiluminiscence detection kit

Abstract

The invention relates to the technical field of A beta 1-42 detection, and discloses a novel magnetic particle chemiluminescence detection kit for A beta 1-42 and a preparation method of the novel magnetic particle chemiluminescence detection kit for A beta 1-42. A double-antibody sandwich method is adopted, A beta 1-42 and reagents form a sandwich structure compound when a sample is incubated with a reagent R1 and a reagent R2, and the sandwich structure compound is used for detecting A beta 1-42. The acridinium ester in the reagent can emit light with specific wavelength under the action of excitation liquid, a full-automatic chemiluminescence immunoassay analyzer is used for detecting a luminescence value, and the content of Abeta1-42 is measured according to a standard curve of concentration-luminescence value of Abeta1-42. According to the present invention, the damage of the antibody and biotin coupling process and the labeled antibody and marker coupling process to the immunocompetence of the Abeta1-42 mouse anti-human IgG antibody is avoided, the reactivity and the sensitivity of the detection are significantly improved, the reaction time is shortened, particularly, the sensitivity is significantly higher than that of other methodologies, and the detection rate of the Abeta1-42 in the human serum is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of Aβ1-42 detection, in particular to a novel magnetic particle chemiluminescence detection kit for Aβ1-42 and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD), also known as senile dementia, is a chronic, persistent, degenerative brain disease that occurs in the elderly or pre-elderly. The pathological characteristics of AD mainly include the appearance of senile plaques with β-amyloid protein (Aβ) as the core outside the cells of the cerebral cortex and hippocampus, and intracellular neurofibrillary tangles.

[0003] Aβ is a polypeptide containing 39 to 43 amino acids produced by the proteolysis of amyloid precursor protein by β- and γ-secretase. The large amount of Aβ deposition is the main cause of neuronal degeneration and death around senile plaques in the brains of AD patients. Aβ is divided into Aβ1-39, Aβ1-40, Aβ1-41, Aβ1-42 and Aβ1-43. Among them, the neurotoxic effect of Aβ1-42 plays a major role in the progression of Alzheimer's disease AD. AD patients produce a large amount of Aβ1-42, and the complete C-terminus of Aβ1-42 can induce cell toxicity and destroy the blood-brain barrier, allowing macromolecules in brain tissue to enter blood vessels. In the analysis of etiology research, the neurotoxicity of Aβ has been recognized, so the detection of Aβ levels in serum can be used for auxiliary diagnosis of mild AD.

[0004] At present, the commonly used Aβ1-42 detection methods mainly include enzyme-linked immunosorbent assay and magnetic microparticle chemiluminescence immunoassay. Among them, the detection sensitivity of Aβ1-42 content determination by enzyme-linked immunosorbent assay is low, the detection range is narrow, it takes a long time, and it is greatly affected by the operator. When using magnetic microparticle chemiluminescence immunoassay to determine the Aβ1-42 content, most of them use the biotin-avidin system to capture the antibody and coat it with biotin, and then combine it with streptavidin magnetic beads; because the Aβ1-42 content in human serum is extremely low, the reagent sensitivity requirements are extremely high, and the "antibody and biotin coupling process" and "labeled antibody and marker coupling process" in the existing magnetic microparticle chemiluminescence immunoassay technology both destroy the immune activity of the antibody, resulting in low sensitivity of the detection reagent of this method.

[0005] Therefore, there is an urgent need for a novel magnetic microparticle chemiluminescence detection kit for Aβ1-42 and a preparation method thereof that can maintain the immunological activity of the antibody to the greatest extent and greatly improve the detection sensitivity. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a new magnetic particle chemiluminescence detection kit for Aβ1-42 and a preparation method thereof, which can avoid the technical problem of low detection sensitivity caused by the destruction of antibody immunoactivity in the "coupling process of antibody and biotin" and the "coupling process of labeled antibody and marker" when using the existing magnetic particle chemiluminescence immunoassay method.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A novel magnetic microparticle chemiluminescent detection kit for Aβ1-42, the kit comprising an R1 reagent and an R2 reagent which are independent of each other;

[0009] The R1 reagent comprises: an R1 complex formed by a chicken anti-mouse IgY antibody coated with magnetic microparticles and an Aβ1-42 mouse anti-human IgG capture antibody, and an R1 buffer;

[0010] The R2 reagent comprises: an R2 complex formed by a biotin-avidin complex and an Aβ1-42 mouse anti-human IgG detection antibody, and an R2 buffer; wherein the biotin-avidin complex is composed of a streptavidin-ylated protein A and a biotinylated acridinium ester;

[0011] The kit adopts a double antibody sandwich method. When the clinical sample to be tested is incubated with R1 reagent and R2 reagent, the analyte Aβ1-42 in the sample forms a sandwich structure complex with the R1 complex and the R2 complex. The acridinium ester emits light of a specific wavelength under the action of an excitation solution. The luminescence value is detected using a fully automatic chemiluminescence immunoassay instrument. The content of the analyte Aβ1-42 in the clinical sample to be tested is calculated based on a standard curve of Aβ1-42 concentration-luminescence value.

[0012] Preferably, in the R1 reagent, the surface of the magnetic particles is modified with -COOH or -NH2 or -SH functional groups, the particle size of the magnetic particles is 0.5 μm to 20 μm, the mass ratio of the magnetic particles to the chicken anti-mouse IgY antibody is 1:(0.01-0.1), and the mass ratio of the chicken anti-mouse IgY antibody coated with the magnetic particles to the Aβ1-42 mouse anti-human IgG capture antibody is 1:(0.02-0.1);

[0013] Preferably, the mass ratio of the chicken anti-mouse IgY antibody coated with magnetic particles to the Aβ1-42 mouse anti-human IgG capture antibody is 1:0.04.

[0014] Preferably, in the R1 reagent, the R1 buffer is a PBS buffer, and (1-5)%-BSA, (0.5-2)% Proclin300 and (0.1-1)% TWEEN-20 are added to the PBS buffer.

[0015] Preferably, in the R2 reagent, the mass ratio of the biotin-avidin complex to the Aβ1-42 mouse anti-human IgG detection antibody is 1:(0.2-0.8).

[0016] Preferably, in the R2 reagent, the R2 buffer is a PBS buffer, and the PBS buffer is supplemented with (0.5-0.8)% BSA, (0.5-1.5)% sucrose, (0.05-1)% Proclin300 and (0.01-0.1)% TWEEN-20.

[0017] Preferably, in the R1 reagent, the final concentration of the Aβ1-42 mouse anti-human IgG capture antibody is (0.5-1.2) μg / mL;

[0018] In the R2 reagent, the final concentration of the Aβ1-42 mouse anti-human IgG detection antibody is (0.5-1.2) μg / mL.

[0019] Preferably, the kit further comprises an Aβ1-42 calibrator;

[0020] The Aβ1-42 calibrators are Aβ1-42 standard solutions of different concentrations prepared by calibrator buffer, and the final concentrations of Aβ1-42 antigen are 1500pg / mL, 750pg / mL, 150pg / mL, 50pg / mL, 10pg / mL, and 0pg / mL, respectively.

[0021] Preferably, the calibration buffer is a calf serum solution, and (0.05-0.2)% Proclin 300 is added to the calf serum solution.

[0022] A method for preparing a novel magnetic particle chemiluminescent detection kit for Aβ1-42 comprises the following steps:

[0023] Step 1, combining the magnetic particles with chicken anti-mouse IgY antibodies to prepare a semi-finished magnetic particle reagent;

[0024] Step 2, combining the semi-finished magnetic particle reagent with the Aβ1-42 mouse anti-human IgG capture antibody, mixing at a temperature of 37° C. to prepare an R1 complex, and adding the R1 complex into an R1 buffer to prepare an R1 reagent;

[0025] Step 3, prepare biotin-avidin complex: add biotinylated acridinium ester to streptavidin-protein A, mix well at 37°C in the dark, react fully, purify by ultrafiltration, take the purified solution and dilute with 1× PBS;

[0026] Step 4, combining the biotin-avidin complex with the Aβ1-42 mouse anti-human IgG detection antibody, slowly mixing at a temperature of 37° C. to prepare an R2 complex, and adding the R2 complex into an R2 buffer to prepare an R2 reagent;

[0027] Step 5, preparation of calibrators: Aβ1-42 antigen standards were prepared using calibrator buffer to prepare Aβ1-42 standard solutions of different concentrations.

[0028] A method for using a novel magnetic particle chemiluminescence detection kit for Aβ1-42 comprises the following steps:

[0029] Drawing of standard curve: Add R1 reagent, R2 reagent and Aβ1-42 calibrator into the luminescent reaction cup at the same time, react fully at 37°C, separate solid and liquid in a magnetic field and wash, add luminescent substrate solution, and after sufficient reaction, use a fully automatic chemiluminescence immunoassay to detect the luminescence value, and draw a standard curve to obtain the standard curve of Aβ1-42 concentration-luminescence value;

[0030] Sample detection: Add R1 reagent, R2 reagent and sample to the luminescent reaction cup at the same time, react fully at 37°C, separate the solid and liquid in a magnetic field and wash, then add the luminescent substrate solution. After sufficient reaction, use a fully automatic chemiluminescence immunoassay to detect the luminescence value, and calculate the Aβ1-42 content in the sample based on the standard curve of Aβ1-42 concentration-luminescence value.

[0031] Compared with the prior art, the present invention has the following beneficial technical effects:

[0032] First: The present invention avoids the destruction of the immune activity of Aβ1-42 mouse anti-human IgG antibody by the "antibody and biotin coupling process" and the "labeled antibody and marker coupling process" in the traditional method, so that the Aβ1-42 mouse anti-human IgG antibody directly participates in the immune response, and relies on the non-covalent binding force between the antibody and the antigen to maintain the immune activity of the antibody to the greatest extent, significantly improves the reactivity and sensitivity of the detection, shortens the reaction time, and especially its sensitivity is significantly higher than other methodologies, thereby improving the detection rate of Aβ1-42 in human serum.

[0033] Second: The present invention couples chicken anti-mouse IgY antibodies with magnetic particles, with high coupling efficiency, firm binding, and stable process;

[0034] The streptavidin-ylated protein A is combined with the biotinylated acridinium ester to introduce the biotin-avidin amplification system. The biotin labeling efficiency is high without affecting other activities. The binding of biotin-avidin is also extremely stable and will not fall off during incubation and washing.

[0035] Third: The methodology of the present invention can become a model system, which is applicable to any project. The labeling process of chicken anti-mouse IgY antibody and magnetic microparticle conjugate is stable, and the labeling process of streptavidin-ylated protein A and biotinylated acridinium ester conjugate is stable;

[0036] Moreover, in the methodological system of the present invention, both conjugates are applicable to other projects. Different projects only need to change the type of mouse anti-human antibodies and the analyte, which simplifies the process of labeling process optimization and system optimization, ensures process stability and production stability to the greatest extent, and saves time, manpower and financial costs for project development.

[0037] Fourth: The present invention selects a direct chemiluminescence system and uses acridinium ester as a marker. Compared with traditional enzymatic chemiluminescence, the reaction does not require the participation of enzymes, which greatly saves costs;

[0038] In addition, the acridinium ester chemiluminescent immunoassay system has high repeatability, with an intra-batch precision of less than 3%, which is difficult to achieve with other chemiluminescent immunoassay systems;

[0039] The chemiluminescent immunoassay system has achieved sample quantification. By building a standard curve into the test software, the concentration value of the sample can be directly obtained by simply testing the sample.

[0040] The chemiluminescent immunoassay system can be fully automated. The addition of reagents and samples is all completed by the instrument, which makes the operation easier, the results are more accurate, the repeatability is good, and human errors are reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a standard curve diagram of Aβ1-42 concentration-luminescence value of the present invention;

[0042] Figure 2 It is a two-point regression fitting diagram of the blank limit equation of the present invention;

[0043] Figure 3 It is a correlation diagram of the Aβ1-42 detection results of the method of the present invention and the commercially available kit. DETAILED DESCRIPTION

[0044] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0045] The present invention provides a novel magnetic particle chemiluminescence detection kit for Aβ1-42. The detection principle of the kit is a double antibody sandwich method. The specific detection process is as follows:

[0046] The chicken anti-mouse IgY antibody coated with magnetic beads was combined with the Aβ1-42 mouse anti-human IgG antibody to prepare the R1 complex;

[0047] The streptavidin-ylated protein A is combined with the biotinylated acridinium ester to form a biotin-avidin complex, and the complex is combined with the Aβ1-42 mouse anti-human IgG antibody to form an R2 complex;

[0048] When the clinical sample to be tested is incubated with the R1 reagent and the R2 reagent, the analyte in the sample forms a sandwich structure complex with the R1 complex and the R2 complex;

[0049] After washing, other unbound components in the sample are removed;

[0050] Subsequently, pre-excitation solution and excitation solution are added. Acridinium ester (chemiluminescent marker) will emit light of a specific wavelength under the action of the excitation solution. The luminescence intensity is proportional to the concentration of the analyte in the sample. The content of the analyte in the sample can be obtained by processing the calibration curve, thereby quantitatively detecting the analyte in the clinical sample to be tested.

[0051] It should be noted that, unless otherwise specified, the test materials used in the present invention are all common commercial products and can be directly purchased from the market. The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0052] Embodiment 1:

[0053] A novel magnetic microparticle chemiluminescent detection kit for Aβ1-42 of this embodiment comprises: R1 reagent, R2 reagent and Aβ1-42 calibrator which are independent of each other;

[0054] The R1 reagent comprises: an R1 complex formed by a chicken anti-mouse IgY antibody coated with magnetic microparticles and an Aβ1-42 mouse anti-human IgG capture antibody, and an R1 buffer;

[0055] The R2 reagent includes: an R2 complex formed by a biotin-avidin complex and an Aβ1-42 mouse anti-human IgG detection antibody, and an R2 buffer;

[0056] Wherein, the biotin-avidin complex is composed of streptavidin-ylated protein A and biotinylated acridinium ester;

[0057] The Aβ1-42 calibrator comprises: Aβ1-42 standard solutions of different concentrations prepared by calibrator buffer; wherein the final concentrations of Aβ1-42 antigen are 1500pg / mL, 750pg / mL, 150pg / mL, 50pg / mL, 10pg / mL, and 0pg / mL respectively;

[0058] Specifically, in the R1 reagent, the magnetic particles are surface-modified magnetic particles, preferably modified magnetic beads with -COOH, -NH2 or -SH groups on the surface, more preferably MS160 carboxyl magnetic beads of model JSR, and the particle size of the magnetic particles is 0.5 μm to 20 μm;

[0059] The mass ratio of magnetic microparticles to chicken anti-mouse IgY antibody was 1:0.01; the mass ratio of magnetic microparticle-coated chicken anti-mouse IgY antibody to Aβ1-42 mouse anti-human IgG capture antibody was 1:0.04;

[0060] R1 buffer is PBS buffer, and the PBS buffer is supplemented with 2% BSA, 1% Proclin300 and 0.5% TWEEN-20;

[0061] In the final system after the R1 complex is mixed with the R1 buffer, the final concentration of the Aβ1-42 mouse anti-human IgG capture antibody is 0.5 μg / mL;

[0062] Specifically, in the R2 reagent, the mass ratio of the biotin-avidin complex to the Aβ1-42 mouse anti-human IgG detection antibody is 1:0.5;

[0063] The R2 buffer is a PBS buffer, and the PBS buffer is supplemented with 0.5% BSA, 1% sucrose, 0.1% Proclin 300 and 0.05% TWEEN-20.

[0064] In the final system after the R2 complex is mixed with the R2 buffer, the final concentration of the Aβ1-42 mouse anti-human IgG detection antibody is 0.5 μg / mL.

[0065] Specifically, in the Aβ1-42 calibration, the calibration buffer is a calf serum solution, and 0.1% Proclin300 is added to the calf serum solution.

[0066] Embodiment 2:

[0067] The preparation method of the novel magnetic microparticle chemiluminescent detection kit for Aβ1-42 in Example 1 comprises the following specific steps:

[0068] (1) Preparation of magnetic microparticle capture antibody: 5 mg of surface-modified magnetic microparticles (model: JSR MS160 carboxyl magnetic beads) and 0.05 mg of chicken anti-mouse IgY antibody were combined at a mass ratio of 1:0.01 to prepare a 10 mg / mL magnetic microparticle reagent semi-finished product with a volume of 500 μL;

[0069] (2) Preparation of R1 reagent: 500 μL of the semi-finished magnetic particle reagent was combined with 0.2 mg of Aβ1-42 mouse anti-human IgG capture antibody at a mass ratio of 1:0.04;

[0070] When the Aβ1-42 mouse anti-human IgG capture antibody was added to the semi-finished magnetic particle reagent, it was slowly mixed at 37°C for 30 minutes to prepare the R1 complex;

[0071] The R1 complex was added into the R1 buffer, and the final concentration of the Aβ1-42 mouse anti-human IgG capture antibody in the final system was made to be 0.5 μg / mL, thereby obtaining the R1 reagent;

[0072] (3) Preparation of biotin-avidin complex: 5 μL of biotinylated acridinium ester was added to 0.1 mg of streptavidin-protein A and mixed at 37°C in the dark for 1 h.

[0073] Afterwards, the product was purified by ultrafiltration tube, and the purified solution was diluted to 0.357 mg / mL with 1× PBS, with a volume of 280 μL;

[0074] Among them, the streptavidin-ylated protein A of this example was purchased from WuXi AppTec New Drug Development Co., Ltd., and the biotinylated acridinium ester was purchased from Shanghai Maxwell Chemical New Material Technology Co., Ltd.;

[0075] (4) Preparation of R2 reagent: 280 μL of the biotin-avidin complex prepared in step (3) was combined with 0.1785 mg of Aβ1-42 mouse anti-human IgG detection antibody at a mass ratio of 1:0.5;

[0076] When the Aβ1-42 mouse anti-human IgG detection antibody was added to the biotin-avidin complex, it was slowly mixed at 37°C for 30 min to prepare the R2 complex;

[0077] The R2 complex was added into the R2 buffer, and the final concentration of the Aβ1-42 mouse anti-human IgG detection antibody in the final system was made to be 0.5 μg / mL, thereby obtaining the R2 reagent;

[0078] (5) Preparation of Calibrators

[0079] Aβ1-42 antigen standards were prepared into Aβ1-42 standard solutions of different concentrations using a calibration buffer;

[0080] The final concentrations of Aβ1-42 antigen were 1500 pg / mL, 750 pg / mL, 150 pg / mL, 50 pg / mL, 10 pg / mL, and 0 pg / mL;

[0081] The calibration buffer was prepared by adding 0.1% Proclin 300 to calf serum, mixing thoroughly, and filtering through a 0.22 μm filter membrane.

[0082] Embodiment 3:

[0083] The method for using the novel magnetic microparticle chemiluminescent detection kit for Aβ1-42 in Example 1 comprises the following specific steps:

[0084] (1) The specific process of drawing the standard curve is as follows:

[0085] 50 μL R1 reagent, 50 μL R2 reagent and 100 μL Aβ1-42 calibrator (the final concentrations of Aβ1-42 antigen are 1500 pg / mL, 750 pg / mL, 150 pg / mL, 50 pg / mL, 10 pg / mL, and 0 pg / mL, respectively) were added to the luminescent reaction cup at the same time, and fully reacted at 37°C for 15 min; after solid-liquid separation and washing in a magnetic field, the luminescent substrate solution was added; after sufficient reaction, the luminescence value was detected using a fully automatic chemiluminescence immunoassay instrument, and a standard curve was drawn and built into the computer software to obtain a standard curve of Aβ1-42 concentration-luminescence value, such as Figure 1 As shown;

[0086] (2) Sample testing: The specific process is as follows:

[0087] 50 μL R1 reagent, 50 μL R2 reagent and 100 μL sample were added to the luminescent reaction cup at the same time, and fully reacted at 37°C for 15 minutes; after solid-liquid separation and washing in a magnetic field, the luminescent substrate solution was added; after sufficient reaction, the Aβ1-42 content was measured using the standard curve of Aβ1-42 concentration-luminescence value;

[0088] In this embodiment, the luminescent substrate solution includes a pre-excitation solution and an excitation solution, both of which are purchased from Diman Technology Co., Ltd. The pre-excitation solution is a H2O2 solution, and the excitation solution is a NaOH solution.

[0089] Embodiment 4:

[0090] The performance evaluation of the kit of the present invention is as follows:

[0091] (1) Sensitivity analysis, the specific method is as follows:

[0092] The blank calibrator buffer without Aβ1-42 antigen (0 pg / mL Aβ1-42 antigen concentration) was measured 20 times, and the signal mean and standard deviation were calculated;

[0093] Then, a two-point regression fitting is performed based on the concentration-signal value results between the zero-concentration calibrator and the adjacent calibrator (10 pg / mL Aβ1-42 antigen concentration) to obtain a linear equation, and the sum of the signal mean and two times the standard deviation is substituted into the above equation. The calculated concentration value is the analytical sensitivity of the kit of the present invention for determining Aβ1-42;

[0094] Among them, the data results of the blank calibrator buffer are shown in Table 1;

[0095] The concentration-signal value results between the zero-concentration calibrator and the adjacent calibrator are shown in Table 2. The blank limit equation is y = 541.7x + 2367.7, R 2 =1, such as Figure 2 As shown;

[0096] Finally, it was calculated that the analytical sensitivity of the kit of the present invention for determining Aβ1-42 was ≤5 pg / mL;

[0097] It can be seen that compared with the traditional Aβ1-42 antibody detection method, the sensitivity of the present invention is significantly improved;

[0098] Table 1 Blank calibration buffer test results

[0099]

[0100]

[0101] Table 2 Concentration-signal values ​​of zero-concentration calibrators and adjacent calibrators

[0102]

[0103] (2) Intra-batch precision analysis: the specific method is as follows:

[0104] Two Aβ1-42 samples with concentrations of 50 pg / mL and 750 pg / mL were taken, and 10 replicates were made for each concentration sample to calculate the intra-batch precision of the kit. The specific experimental results are shown in Table 3;

[0105] Table 3 Intra-batch precision analysis results of the kit of the present invention

[0106]

[0107]

[0108] As can be seen from Table 3, the intra-batch precision of the kit of the present invention is less than 3%.

[0109] (3) Correlation analysis between the kit of the present invention and commercially available kits, the specific method is as follows:

[0110] The correlation analysis of the Aβ1-42 detection results was performed using the kit of the present invention and the commercially available kit: 55 clinical value samples of Anqun reagent were detected using the method of the present invention, and the specific experimental results (clinical data correlation) are as follows: Figure 3 As shown;

[0111] Depend on Figure 3It can be seen that the clinical correlation between the test results of the test kit of the present invention and the commercially available test kit is >0.90, which indicates that the test results of the method of the present invention have sufficient credibility and accuracy.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A novel magnetic microparticle chemiluminescent detection kit for Aβ1-42, It is characterized in that The kit comprises an R1 reagent and an R2 reagent which are independent of each other; The R1 reagent comprises: an R1 complex formed by a chicken anti-mouse IgY antibody coated with magnetic microparticles and an Aβ1-42 mouse anti-human IgG capture antibody, and an R1 buffer; The R2 reagent comprises: an R2 complex formed by a biotin-avidin complex and an Aβ1-42 mouse anti-human IgG detection antibody, and an R2 buffer; wherein the biotin-avidin complex is composed of a streptavidin-ylated protein A and a biotinylated acridinium ester; The kit adopts a double antibody sandwich method. When the clinical sample to be tested is incubated with R1 reagent and R2 reagent, the analyte Aβ1-42 in the sample forms a sandwich structure complex with the R1 complex and the R2 complex. The acridinium ester emits light of a specific wavelength under the action of an excitation solution. The luminescence value is detected using a fully automatic chemiluminescence immunoassay instrument. The content of the analyte Aβ1-42 in the clinical sample to be tested is calculated based on a standard curve of Aβ1-42 concentration-luminescence value.

2. The novel magnetic microparticle chemiluminescence detection kit for Aβ1-42 according to claim 1, It is characterized in that In the R1 reagent, the surface of the magnetic particles is modified with -COOH or -NH 2 or -SH functional group, the particle size of the magnetic particles is 0.5μm to 20μm, the mass ratio of the magnetic particles to the chicken anti-mouse IgY antibody is 1:(0.01-0.1), and the mass ratio of the chicken anti-mouse IgY antibody coated with the magnetic particles to the Aβ1-42 mouse anti-human IgG capture antibody is 1:(0.02-0.1).

3. The novel magnetic microparticle chemiluminescence detection kit for Aβ1-42 according to claim 1, It is characterized in that In the R1 reagent, the R1 buffer is a PBS buffer, and (1-5)% BSA, (0.5-2)% Proclin300 and (0.1-1)% TWEEN-20 are added to the PBS buffer.

4. The novel magnetic microparticle chemiluminescent detection kit for Aβ1-42 according to claim 1, It is characterized in that In the R2 reagent, the mass ratio of the biotin-avidin complex to the Aβ1-42 mouse anti-human IgG detection antibody is 1:(0.2-0.8).

5. The novel magnetic microparticle chemiluminescence detection kit for Aβ1-42 according to claim 1, It is characterized in that In the R2 reagent, the R2 buffer is a PBS buffer, and the PBS buffer is added with (0.5-0.8)% BSA, (0.5-1.5)% sucrose, (0.05-1)% Proclin300 and (0.01-0.1)% TWEEN-20.

6. The novel magnetic microparticle chemiluminescent detection kit for Aβ1-42 according to claim 1, It is characterized in that In the R1 reagent, the final concentration of Aβ1-42 mouse anti-human IgG capture antibody is (0.5-1.2) μg / mL; In the R2 reagent, the final concentration of the Aβ1-42 mouse anti-human IgG detection antibody is (0.5-1.2) μg / mL.

7. The novel magnetic microparticle chemiluminescence detection kit for Aβ1-42 according to claim 1, It is characterized in that The kit also includes an Aβ1-42 calibrator; The Aβ1-42 calibrators are Aβ1-42 standard solutions of different concentrations prepared by calibrator buffer, and the final concentrations of Aβ1-42 antigen are 1500pg / mL, 750pg / mL, 150pg / mL, 50pg / mL, 10pg / mL, and 0pg / mL, respectively.

8. The novel magnetic microparticle chemiluminescence detection kit for Aβ1-42 according to claim 7, It is characterized in that The calibration buffer is a calf serum solution, and (0.05-0.2)% Proclin 300 is added to the calf serum solution.

9. A method for preparing a novel magnetic microparticle chemiluminescent detection kit for Aβ1-42 according to any one of claims 1 to 8, It is characterized in that The following steps are involved: Step 1, combining the magnetic particles with chicken anti-mouse IgY antibodies to prepare a semi-finished magnetic particle reagent; Step 2, combining the semi-finished magnetic particle reagent with the Aβ1-42 mouse anti-human IgG capture antibody, mixing at a temperature of 37° C. to prepare an R1 complex, and adding the R1 complex into an R1 buffer to prepare an R1 reagent; Step 3, prepare biotin-avidin complex: add biotinylated acridinium ester to streptavidin-protein A, mix well at 37°C in the dark, react fully, purify by ultrafiltration tube, take the purified solution and dilute with 1× PBS; Step 4, combining the biotin-avidin complex with the Aβ1-42 mouse anti-human IgG detection antibody, slowly mixing at a temperature of 37° C. to prepare an R2 complex, and adding the R2 complex into an R2 buffer to prepare an R2 reagent; Step 5, preparation of calibrators: Aβ1-42 antigen standards were prepared using calibrator buffer to prepare Aβ1-42 standard solutions of different concentrations.

10. A method for using a novel magnetic microparticle chemiluminescence detection kit for Aβ1-42 according to any one of claims 1 to 8, It is characterized in that The following steps are involved: Drawing of standard curve: Add R1 reagent, R2 reagent and Aβ1-42 calibrator into the luminescent reaction cup at the same time, react fully at 37°C, separate solid and liquid in a magnetic field and wash, add luminescent substrate solution, and after sufficient reaction, use a fully automatic chemiluminescence immunoassay to detect the luminescence value, and draw a standard curve to obtain the standard curve of Aβ1-42 concentration-luminescence value; Sample detection: Add R1 reagent, R2 reagent and sample to the luminescent reaction cup at the same time, react fully at 37°C, separate the solid and liquid in a magnetic field and wash, then add the luminescent substrate solution. After sufficient reaction, use a fully automatic chemiluminescence immunoassay to detect the luminescence value, and calculate the Aβ1-42 content in the sample based on the standard curve of Aβ1-42 concentration-luminescence value.

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