Chemiluminescence detection kit for beta amyloid protein 1-40

Abstract

The beta amyloid protein 1-40 (A beta 1-40) detection kit is developed by adopting a sandwich method in a chemiluminescence method and is matched with a full-automatic chemiluminescence analyzer for use, and human intervention is basically not needed. Compared with an existing beta amyloid 1-40 (A beta 1-40) detection method (CN114966047A linear range is 500 pg / mL, the reaction time is 50 minutes, CN11919099A linear range is 800 pg / mL, and the lowest detection limit is 5 pg / mL), the linear range is wider and reaches 1500 pg / mL, the sensitivity is higher and reaches 0.075 pg / mL, the detection time is shortened to 35 minutes, and the detection efficiency is improved. The lowest detection limit of the kit is 0.075 pg / mL, the linear R is equal to 0.9999, the anti-interference performance is good, the stability lasts for 12 months, the correlation R2 with a kit of beta amyloid protein 1-40 (A beta 1-40) sold on the market is equal to 0.9503, and the kit is superior to the detection kit on the market.

Description

Technical Field

[0001] The present application belongs to the field of clinical in vitro diagnosis and medical immunology, and relates to an immunoassay reagent, specifically to a chemiluminescent detection kit for beta-amyloid protein 1-40 (Aβ1-40) and its preparation, which is suitable for the quantitative detection of beta-amyloid protein 1-40 in human plasma. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease that mainly manifests as impairment of memory, thinking ability, emotion and behavior. More than 50 million people suffer from the disease worldwide. By 2050, the number of patients will reach 150 million. At present, the treatment of AD remains a global challenge, and the research on its therapeutic drugs has progressed slowly, and many patients have not received effective treatment. The most prominent pathological features of AD are the deposition of β-amyloid (Aβ) in the brain, hyperphosphorylation of tau protein, and the dysfunction and even loss of neurons and their synapses. AD often has a slow onset and is insidious. There is currently a lack of effective treatment and prevention methods. Early diagnosis and early treatment are the key, but the early diagnosis of AD is still very challenging. The main reason is the lack of ideal diagnostic markers, especially those that are well correlated with cognitive function changes and outcomes. Therefore, we urgently need a diagnostic technology that can detect changes in brain-related indicators in the body at the early stage of AD formation.

[0003] At present, neuromolecular imaging is the frontier and hotspot in the field of nuclear medicine. In particular, nuclear medicine molecular imaging technology represented by positron emission tomography (PET) provides a new and powerful high-tech platform for the rapid development of drugs, and provides great help for the exploration of the mechanism and early diagnosis of AD. As one of the molecular imaging technologies, PET can reflect pathological changes at the molecular level, and can non-invasively detect the distribution of radionuclides in the body. Through this distribution, it reflects abnormal changes in physiology, biochemistry, metabolism, receptors, gene expression, etc. For example, Aβ-PET and tau-PET scans have been used as the gold standard for Aβ and tau pathology in clinical practice, and can be used for early clinical diagnosis of AD, differential diagnosis with other cognitive disorders, and selection of subjects for Aβ targeted therapeutic drugs and efficacy evaluation, but it is also a non-specific injury indicator for many other causes, such as cerebrovascular injury.

[0004] Aβ protein is the main pathological marker of AD. It is a short peptide formed by the cleavage of amyloid precursor protein (APP) by β-secretase and v-secretase in sequence. Due to different cleavage sites, different types of Aβ are produced, mainly including: A338, A340 and Aβ42: Aβ40 and Aβ42 are considered to be neurotoxic, can aggregate in the brain to form senile plaques, and damage neurons and other nerve cells; CSF / plasma Aβ42 / 40 is included in the core biomarkers of AD in the 2023 "NIA-AA Revised Clinical Criteria for AD Diagnosis (Draft)".

[0005] Aβ1-40 and Aβ1-42, as the main components of amyloid plaques, are often considered to be related to cognitive impairment caused by Alzheimer's disease. However, since Aβ1-40 does not change significantly during the progression of the disease, it is often used in combination with Aβ1-42 in the form of the Aβ1-42 / Aβ1-40 ratio to evaluate cognitive impairment caused by Alzheimer's disease (AD) and cognitive impairment caused by other reasons. The Aβ1-42 / Aβ1-40 ratio can help identify subjects with lower and higher risks of cognitive decline identified by changes in clinical scores within 3 years.

[0006] At present, AD diagnosis mainly includes three detection methods: PET imaging diagnosis, cerebrospinal fluid puncture examination, and blood marker screening. Among them, PET biomarker tracing is a relatively mature method in clinical application, with high diagnostic sensitivity, but due to the high cost of examination, the scarcity of tracers and the incomplete medical infrastructure in China, the overall application is still relatively limited. Cerebrospinal fluid examination has good accuracy and sensitivity for patients in the MCI stage, and the technology is relatively mature. The consistency of Roche Elecsys AD cerebrospinal fluid (CSF) detection and amyloid protein PET scanning imaging reaches 90%, which may provide a more affordable and more accessible routine option for confirming the presence of amyloid protein in the brain. They can also detect multiple biomarkers from a single image, without radiation, and may detect Alzheimer's pathology in the early stages of the disease, but their clinical application is also limited due to their invasiveness.

[0007] In comparison, the cost of detecting amyloid beta 1-40 (Aβ1-40) in patient serum is lower than that of PET and CSF. It is easier to carry out, less invasive than cerebrospinal fluid testing, and can be easily repeated multiple times. At present, the detection methods disclosed in invention patents such as CN114966047A and CN119199099A have disadvantages such as low sensitivity and difficulty in meeting clinical needs in linear range. Chemiluminescent immunoassay is an immunoassay technology developed on the basis of enzyme-linked immunosorbent assay. It has the advantages of high sensitivity, wide linear range of detection, simple operation, and high degree of automation, and has been widely used. However, there are currently few chemiluminescent detection kits for detecting the content of amyloid beta 1-40 (Aβ1-40) at home and abroad, and the technology in related fields needs further development. Therefore, it is very necessary to develop a more sensitive, specific and safe chemiluminescent detection kit for amyloid beta 1-40 (Aβ1-40). Summary of the invention

[0008] The present invention aims to develop a chemiluminescent detection kit for determining the content of human amyloid beta protein 1-40 (Aβ1-40) which has the advantages of simple operation, wide linear range, fast detection speed, high sensitivity, no pollution, good specificity and low cost.

[0009] Detection principle: The beta amyloid protein 1-40 (Aβ1-40) detection kit of the present invention is based on the principle of chemiluminescence sandwich method and uses magnetic beads as solid phase carriers. In the detection system, the magnetic microparticle suspension is coated with Aβ1-40 antibody, and the Aβ1-40 antibody is labeled with acridinium ester to prepare an acridinium ester marker. An antibody-antigen-antibody complex is formed through immune reaction, and the complex emits light under the action of the excitation liquid and the pre-excitation liquid, so that the relative luminescence unit (RLUs) is detected by the fully automatic chemiluminescence immunoassay analyzer, and the relative luminescence unit is proportional to the content of the analyte Aβ1-40.

[0010] Based on the above purpose, the present invention provides a chemiluminescent detection kit for detecting the content of amyloid β 1-40 (Aβ1-40) in human plasma. The invention includes: Magnetic bead reagent: Contains magnetic beads coated with streptavidin. The average particle size of the magnetic beads is 2.8 μm and the concentration is 10 mg / mL. The magnetic beads are suspended in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer (pH 7.4) to maintain their stability and activity.

[0011] Calibrators: A set of solutions of known concentrations of amyloid beta 1-40 (Aβ1-40) for drawing the standard curve. The concentrations of the calibrators are 0 pg / mL, 20 pg / mL, 100 pg / mL, 500 pg / mL, 1000 pg / mL, and 1500 pg / mL.

[0012] Quality control: Dilute amyloid β-1-40 (Aβ1-40) to a concentration of 50 pg / mL and 500 pg / mL in phosphate buffered saline.

[0013] Marker solution: Contains amyloid β-1-40 monoclonal antibody labeled with acridinium ester. The monoclonal antibody in the marker solution is 1 μg / ml, and the labeling efficiency of acridinium ester reaches more than 90%, ensuring that the marker has high chemiluminescent activity.

[0014] Biotin-antibody solution: Contains a monoclonal antibody that specifically binds to amyloid β-1-40, and the antibody is labeled with biotin. The antibody concentration is 2 μg / mL, and the molar ratio of biotin to antibody is 1:5. The solution is prepared with a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer (pH 7.4).

[0015] Substrate solution: A specific substrate solution used to excite the marker to produce a chemiluminescent signal. The main components of the substrate solution are hydrogen peroxide and sodium hydroxide, with concentrations of 0.1 mol / L and 0.25 mol / L, respectively. When the substrate comes into contact with the marker, a strong chemiluminescent reaction is triggered.

[0016] Washing solution: used to wash the magnetic beads and remove unbound substances to reduce the impact of nonspecific binding on the test results. The main components of the washing solution are 0.05 M Tris-HCl buffer (pH 7.4) and 0.1% Tween-20 surfactant.

[0017] The present invention uses the sandwich method in the chemiluminescence method to develop a β-amyloid protein 1-40 (Aβ1-40) detection kit, which is used in conjunction with a fully automatic chemiluminescence analyzer and basically does not require human intervention. Compared with the existing β-amyloid protein 1-40 (Aβ1-40) detection method (CN114966047A linear range 500 pg / mL, reaction time 50 minutes, CN119199099A linear range 800 pg / mL, minimum detection limit 5 pg / mL), the linear range is wider to 1500 pg / mL, the sensitivity is higher to 0.075pg / mL, the detection time is shortened to 35 minutes, and the detection efficiency is improved. The kit of the present invention has a minimum detection limit of 0.075 pg / mL, a linear R=0.9999, good anti-interference, and stability of 12 months. The correlation with the β-amyloid protein 1-40 (Aβ1-40) kit on the market is R 2 =0.9503, which is better than the detection kits on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the detection principle of the beta amyloid protein 1-40 (Aβ1-40) detection kit of the present invention; Figure 2 This is a schematic diagram of the detection kit for amyloid beta 1-40 (Aβ1-40) of the present invention; Figure 3 Schematic diagram of the linearity results of the beta amyloid 1-40 (Aβ1-40) detection kit developed for the present invention; Figure 4 A schematic diagram of the correlation results between the kit developed for the present invention and the amyloid β-protein 1-40 (Aβ1-40) kit samples sold on the market. DETAILED DESCRIPTION

[0019] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0020] The following are the main raw materials used in the implementation: β-amyloid 1-40 antigen (Cat. No.: MA2341), β-amyloid 1-40 antibody (Cat. No.: AB3221, AC3223), provided by Zhenjiang Arenac Biotechnology Co., Ltd. (Zhenjiang, China); magnetic bead reagent was purchased from Thermo Fisher Scientific (China), Cat. No. 88817.

[0021] Example 1: Kit preparation Preparation of magnetic bead reagent: First, select magnetic beads with a particle size of 2.8 μm, activate the surface of the magnetic beads with 0.1 M hydrochloric acid solution, and then rinse with deionized water until neutral. Next, mix the streptavidin solution (concentration of 10 μg / mL) with the magnetic beads and incubate at 37 °C for 2 h to coat the streptavidin on the surface of the magnetic beads. Finally, suspend the magnetic beads in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer (pH 7.4) to adjust the concentration of the magnetic beads to 10 mg / mL.

[0022] Preparation of calibrators: Phosphate buffer was used as the calibrator matrix. Then, accurate amounts of amyloid β 1-40 standards were added according to the predetermined concentrations to prepare calibrators with concentrations of 0 pg / mL, 20 pg / mL, 100 pg / mL, 500 pg / mL, 1000 pg / mL, and 1500 pg / mL. During the preparation process, high-precision pipettes and balances were used to ensure the accuracy and repeatability of the calibrator concentrations.

[0023] Calibrator diluent: 1000 mL pure water, NaN 3 2.0 g, tetracycline 43 mg (tetracycline was dissolved in 0.1 M Tris, pH = 7.5 and then added (50 mg / 10 ml)), 0.1% ProClin 300. After stirring, the pH value was tested to be 7.0 ± 0.1, filtered with 0.22 μm filter paper, and stored at 4 °C.

[0024] Preparation of marker solution: Select amyloid β 1-40 monoclonal antibody (AB3221), label the antibody with acridinium ester, and dilute the bound marker with diluent. During the labeling process, optimize the reaction conditions such as temperature, time, and reagent concentration to ensure that the labeling efficiency reaches more than 90%. Dissolve the labeled antibody in the diluent and adjust the antibody concentration to 1 μg / mL.

[0025] Preparation of biotin-antibody solution: First, adjust the β-amyloid 1-40 monoclonal antibody (AC3223) to 1 mg / mL with PBS buffer, and take 250 μL of the antibody solution with a concentration of 1 mg / mL and add it to a centrifuge tube. Dissolve the Sulfo-NHS-LC-Biotin solid with PBS buffer and adjust the concentration to 1 mg / mL. Take 4 μL of the dissolved biotin solution to a centrifuge tube. Gently vortex and shake, then seal with sealing film, place on a mixer in a constant temperature incubator, and incubate at 37°C for 1 hour. After incubation, dialyze in 500mL PBS buffer with a 14000 Da dialysis bag for 4 hours, then replace the PBS buffer and continue dialysis overnight (16 to 18 hours). After dialysis, measure the antibody concentration with UV OD280nm / 1.4, and then add 0.1% BSA and 0.1% Proclin 300 for storage. Dissolve the labeled antibody in the diluent and adjust the antibody concentration to 2 μg / mL.

[0026] Preparation of labeling substance and biotin-antibody diluent: prepare 1 L of pure water, weigh 12.05 g of Tris, 5.82 g of NaCl, and 10 g of NaN 3 2.0 g was stirred at room temperature for 1 h. 1 M MgCl was added 2 6H 2 O 0.998 mL, 0.1 M ZnCl 2 1.027 mL, 2 mL of sheep serum, adjust the pH to 8.0±0.05, filter, and store at 4 ℃.

[0027] Example 2: Detection steps Preparation of magnetic beads: Take out the magnetic bead reagent from the kit and gently invert to mix so that the magnetic beads are evenly suspended.

[0028] Sample addition: Add sample, marker solution and biotin-antibody solution to the reaction tube in sequence, with the added amount of 30 μL each.

[0029] Incubation: Place the reaction tube in an incubator and incubate at 37°C for 30 min. During the incubation, the amyloid β-protein 1-40 (Aβ1-40) in the sample binds to the two specific monoclonal antibodies.

[0030] Washing: After the incubation, add 100 μL of magnetic bead solution, incubate for 3 min, separate the magnetic beads from the reaction solution using a magnetic separator, and then wash the magnetic beads with washing solution. The amount of washing solution used is 300 μL per tube, and the number of washes is 3 times.

[0031] Add luminescent substrate: Add substrate solution to the washed magnetic bead reaction tube, the amount of substrate solution added is 100 μL. After adding the substrate, the reaction tube should be immediately placed in the chemiluminescence detector to start the chemiluminescence reaction.

[0032] Detection: In the chemiluminescence detector, the reaction tubes are detected according to the instrument's operating procedures. The instrument measures the chemiluminescence signal intensity of each reaction tube and transmits the data to the computer for analysis.

[0033] Result calculation: A standard curve is drawn based on the concentration of the calibrator and the corresponding chemiluminescent signal intensity. Using the standard curve, the content of amyloid β 1-40 (Aβ1-40) in the sample is calculated by the chemiluminescent signal intensity of the sample, which is automatically completed by computer software.

[0034] Example 3: Performance Evaluation 1. Determine the antibody concentration and standard curve: The biotin-labeled antibody solution was prepared in diluents with concentrations of 1 μg / mL, 2 μg / mL, and 3 μg / mL. The acridinium ester-labeled antibody solution was prepared in diluents with concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, and the calibration standards were prepared at concentrations of 30 pg / mL (point A), 400 pg / mL (point B), and 900 pg / mL (point C). The test was performed using the chessboard method. According to the test results, the concentration of biotinylated antibody was 2 μg / mL, and the concentration of acridinium ester-labeled antibody was 1 μg / mL (Table 1).

[0035] Table 1 Beta-amyloid protein 1-40 (Aβ1-40) chessboard method test data

[0036] The amyloid β-protein 1-40 (Aβ1-40) complex described in Example 1 was diluted with a standard diluent to prepare calibration solutions S1-S6 of different concentrations, with concentrations of 0 pg / mL, 20 pg / mL, 100 pg / mL, 500 pg / mL, 1000 pg / mL, and 1500 pg / mL, respectively, and stored at 4°C for later use. The calibrators were then tested using the kit provided in Example 1 of the present invention, and the luminescence intensity values ​​corresponding to each calibrator were read respectively. The results are shown in Table 2 below. The standard curve was obtained by fitting with concentration as the horizontal axis and luminescence intensity as the vertical axis, as shown in Table 2 below. Figure 2 .

[0037] Table 2 Detection range of amyloid β 1-40 (Aβ1-40) test kit Calibrator concentration (pg / mL) Luminescence value (RLU) 0 3148 20 30182 100 150710 500 701684 1000 1426354 1500 2082766 2. Linear The calibrator with added antigen is used as the high concentration sample, and the calibrator diluent is used as the low concentration sample. The high-value sample close to the upper limit of the linear range is diluted to several concentrations in a certain proportion (for example: linear sample preparation table), and the low-value concentration sample must be close to the lower limit of the linear range. The sample of each concentration is tested 3 times and the average value is calculated. The concentration value of the analyte (as the horizontal axis) and the average value of the test result (as the vertical axis) are fitted with a straight line using the least squares method. The correlation coefficient r is calculated within the linear range, and the acceptance standard is the correlation coefficient r ≥ 0.99. The linear range of this kit is better than 800 pg / mL in (CN119199099A).

[0038] Figure 3 This is the linear analysis of the chemiluminescent assay kit for amyloid beta 1-40 (Aβ1-40) in the present invention. The results show that the linear relationship between the theoretical concentration and the actual concentration of the sample is good, R>0.99, and the linear regression equation after fitting is Y = 0.9923x - 1.8276.

[0039] 3. Minimum detection limit Use the zero-concentration calibrator dilution as the sample for detection, repeat the measurement 20 times, and obtain the light signal values ​​of the 20 measurement results. Calculate the mean (M) and standard deviation (SD) to obtain M+2SD. Perform two-point regression fitting on the concentration and light signal value results between the zero-concentration enterprise linear reference and the adjacent concentration calibrator to obtain a linear equation. Substitute the light signal value of M+2SD into the equation to find the corresponding concentration value, which is the minimum detection limit.

[0040] The results are shown in Table 3. The concentration values ​​of amyloid β 1-40 (Aβ1-40) were 0.054 pg / mL, 0.075 pg / mL, and 0.059 pg / mL, respectively, and the minimum detection limit of amyloid β 1-40 (Aβ1-40) was 0.075 pg / mL, which was significantly better than (CN119199099A) 5 pg / mL.

[0041] Table 3 Validation results of detection limit of amyloid β 1-40 (Aβ1-40) Test 1 Test 2 Test 3 M 3220.967783 3249.012224 3228.18012 SD 62.01 71.36 57.04 M+2SD 3220.967783 3249.012224 3228.18012 LOD(pg / mL) 0.054 0.075 0.059 4. Accuracy Prepare samples of high and low concentrations of amyloid β-protein 1-40 (Aβ1-40) complex, repeat the measurement three times, calculate the relative deviation between the average value (denoted as M) and the labeled value, and calculate the relative deviation B of the measured concentration according to formula (1). Alternatively, use the reagent to be evaluated to test samples of high and low concentrations that have been determined by the reference method, repeat the measurement three times for each concentration sample, take the average of the test results, and calculate its relative deviation from the labeled value.

[0042] B=(MT) / T×100%---------(1) Where: B—relative deviation; M—average value of measured concentration; T—indicative value; The above experimental results are shown in Table 4. The relative deviation of the β-amyloid protein 1-40 (Aβ1-40) kit of the present invention is within 5%, which has good accuracy.

[0043] Table 4 Accuracy test of amyloid β 1-40 (Aβ1-40) kit Concentration (pg / mL) Test 1 Test 2 Test 3 Average value (pg / mL) Relative deviation (%) 50 51.16 50.48 50.35 50.66 1.3% 500 495.61 514.34 500.39 503.45 0.7% 5. Precision The test reagent is used to test the same batch of samples multiple times to see if the results are consistent, and this indicator is used as precision. Usually, the CV value within the batch is within 8%, and the CV value between batches is within 10%. According to the NCCLS EP5-A2 standard, this technology is used to measure two plasma samples of different concentrations. β-amyloid protein 1-40 (Aβ1-40) sample 1: a low-concentration sample with a concentration of 50pg / mL, sample 2: a high-concentration sample with a concentration of 500 pg / mL.

[0044] (1) Intra-batch precision: The two samples were measured 20 times using three batches of reagents respectively, and the average M and standard deviation SD of the 20 measured concentrations were calculated, and the CV was calculated according to Formula 2.

[0045] CV=M / SD×100%---------(2) Where CV is the coefficient of variation and M is the mean; SD is standard deviation.

[0046] (2) Inter-batch precision: Two samples were measured using three batches of reagents, 20 times each. The CV value was calculated according to Formula 2.

[0047] The precision of this kit is within 5%, which meets clinical requirements. See Table 5.

[0048] Table 5 Comparison of precision measurement results of β-amyloid 1-40 (Aβ1-40) detection kits Low value quality control High value quality control 1 52.60 519.13 2 49.58 498.24 3 50.43 499.18 4 51.18 503.73 5 50.30 499.84 6 51.38 499.27 7 50.81 498.01 8 50.08 494.78 9 49.14 491.38 10 49.16 506.23 Fitting concentration M 1.08 7.59 Fitted concentration SD 50.47 500.98 Fitting concentration CV 2.13% 1.52% 6. Thermal stability test The reagent must meet the quality standards of the product within the validity period, and the various performance indexes of the product must be stable. The stability test includes: thermal stability when stored at 2-8 ℃ for 15 months and 37 ℃ for 10 days, and the stability of each index is determined by a three-day simulation experiment. At the same time, the stability of the standard sample and the quality control material was evaluated, and the stability of the sample stored at 2-8 ℃ for 14-15 months was determined, the stability of the sample after 20 days of opening the bottle was determined, and the stability of the simulated transport sample for 3 days was evaluated. In addition, the stability of clinical samples placed under different conditions also needs to be considered. To verify the stability of the reagent, a standard experimental process needs to be carried out in a storage environment of 37 ℃, so that the experimental determination can be completed quickly. The detailed operation process requires three different batches of plasma reagents to be stored in an incubator, and the stability of the samples is determined at different time points. The sample can be tested on the 1st, 3rd, 6th and 10th days as 4 different measurement time points, and then the final test results are compared and analyzed with the reagents stored at 4 ℃, and each performance index is evaluated.

[0049] The calibrators of the prepared amyloid beta protein 1-40 (Aβ1-40) kit were placed at 4°C and 37°C for 1 day, 3 days, 6 days, 10 days, 20 days, 25 days, and 30 days, respectively, and the luminescence value was tested. The relative error (B%) was within ±20%. The accelerated stability of the amyloid beta protein 1-40 (Aβ1-40) kit of the present invention was comprehensively analyzed to meet the test requirements, and the accelerated stability results were better than similar amyloid beta protein 1-40 (Aβ1-40) chemiluminescence detection kits on the market. The results are shown in Table 6.

[0050] Table 6 Results of accelerated stability test of amyloid β 1-40 (Aβ1-40) kit

[0051] The beta amyloid protein 1-40 (Aβ1-40) test kit was taken out at the 3rd, 6th, 9th and 12th month, and the CV values ​​were within 10% by measuring the quality control products, which all met the specified standards. Therefore, the beta amyloid protein 1-40 (Aβ1-40) test kit can be effectively stored at room temperature for 12 months, and the results are shown in Table 7: Table 7 Real-time stability evaluation results of the β-amyloid protein 1-40 (Aβ1-40) kit

[0052] 7. Kit specificity test The specific substances were configured to corresponding concentrations, and one batch of the beta amyloid protein 1-40 (Aβ1-40) detection kit of the present invention was used to determine the specific substances and calculate the cross-reaction rate. Cross-reaction rate % = (beta amyloid protein 1-40 (Aβ1-40) determination concentration / specific substance experimental concentration) × 100%. The specificity was <0.1%, which met the requirements, and the results are shown in Table 8 below.

[0053] Table 8 Specificity test results of the beta amyloid protein 1-40 (Aβ1-40) kit Cross-reactants Experimental concentration (pg / mL) Measured concentration (pg / mL) Cross-reaction rate Aβ1-42 1000 0.03 0.0003% Aβ1-38 1000 0.09 0.009% Example 4: Clinical relevance assessment In order to better verify the detection effect of the kit developed in this study, clinical performance evaluation is required. 124 samples of patient plasma (provided by the Affiliated Hospital of Jiangnan University) were collected and tested using a commercially available amyloid β-1-40 (Aβ1-40) detection kit, and the data were exported. The concentration in plasma was measured using a self-developed amyloid β-1-40 (Aβ1-40) chemiluminescence kit and compared with the measured values ​​of the commercially available kit. The values ​​measured by the homemade kit were used as the horizontal axis, and the values ​​measured by the commercially available kit were used as the vertical axis. The corresponding method was fitted using this method. The linear regression results are shown in Figure 4 The linear correlation coefficient of amyloid β 1-40 (Aβ1-40) is R 2 =0.9503. Its detection effect is well linearly correlated with the results of existing kits on the market, indicating that the developed kit can meet the needs of clinical testing.

Claims

1. A chemiluminescent detection kit for detecting the content of amyloid β-protein 1-40 in human plasma, characterized in that: The components include: Magnetic bead reagent: containing magnetic beads, the surface of which is coated with streptavidin; Calibrators; Labeling solution: containing amyloid-β 1-40 monoclonal antibody labeled with acridinium ester; Biotin-antibody solution: contains a monoclonal antibody specifically binding to amyloid β-protein 1-40, wherein the antibody is labeled with biotin; Substrate solution: used to excite the marker to produce chemiluminescent signal.

2. The chemiluminescent detection kit for detecting the content of amyloid β-protein 1-40 in human plasma according to claim 1, characterized in that: Also included are controls, wash solutions and calibrators.

3. The chemiluminescent detection kit for detecting the content of amyloid β-protein 1-40 in human plasma according to claim 1 or 2, characterized in that: The average particle size of the magnetic beads is 2.8 μm, and the concentration is 10 mg / mL. The magnetic beads are suspended in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer to maintain their stability and activity.

4. The chemiluminescent detection kit for detecting the content of amyloid β-protein 1-40 in human plasma according to any one of claims 1 to 3, characterized in that: The concentrations of the calibrators are 0 pg / mL, 20 pg / mL, 100 pg / mL, 500 pg / mL, 1000 pg / mL, and 1500 pg / mL, respectively.

5. The chemiluminescent detection kit for detecting the content of amyloid β-protein 1-40 in human plasma according to any one of claims 1 to 3, characterized in that: The concentration of the amyloid beta 1-40 monoclonal antibody in the marker solution is 1 μmol / L, and the labeling efficiency of the acridinium ester reaches more than 90%, ensuring that the marker has a high chemiluminescent activity.

6. The chemiluminescent detection kit for detecting the content of amyloid β-protein 1-40 in human plasma according to any one of claims 1 to 3, characterized in that: The concentration of the monoclonal antibody in the labeling solution in the biotin-antibody solution is 2 μg / ml.

7. A method for preparing a chemiluminescent detection kit for detecting the content of amyloid β-protein 1-40 in human plasma according to any one of claims 1 to 6, characterized in that: Here are the steps: (1) Preparation of calibrators: Phosphate buffer was used as the calibrator matrix to prepare calibrators with concentrations of 0 pg / mL, 20 pg / mL, 100 pg / mL, 500 pg / mL, 1000 pg / mL, and 1500 pg / mL. (2) Preparation of magnetic bead reagent: Select magnetic beads with a particle size of 2.8 μm, activate the surface of the magnetic beads with 0.1 M hydrochloric acid solution, then rinse with deionized water until neutral, mix the streptavidin solution with the magnetic beads, incubate at 37 °C for 2 h to coat the surface of the magnetic beads with streptavidin, suspend the magnetic beads in a solution containing 0.1% bovine serum albumin and 0.05 M Tris-HCl buffer, and adjust the magnetic bead concentration to 10 mg / mL; (3) Preparation of substrate solution: prepare hydrogen peroxide and sodium hydroxide stock solutions, and further prepare them into solutions with final concentrations of 0.1 mol / L and 0.25 mol / L, respectively; (4) Preparation of marker solution: Select β-amyloid protein 1-40 monoclonal antibody, label the β-amyloid protein 1-40 monoclonal antibody with acridinium ester by chemical synthesis method, and dilute the bound marker with diluent; (5) Preparation of biotin-antibody solution: Label the antibody that specifically binds to amyloid β-protein 1-40 with biotin and adjust the antibody concentration to 2 μg / mL.

8. The method for preparing the chemiluminescent detection kit for detecting the content of amyloid β-protein 1-40 in human plasma according to claim 7, characterized in that: Preparation of labeling substance and biotin-antibody diluent: add 0.998 mL of 1 M MgCl2·6H2O, 1.027 mL of 0.1 M ZnCl2, and 2 mL of sheep serum, adjust the pH to 8.0±0.05, filter, and store at 4 ℃.

9. Use of the chemiluminescent detection kit for detecting the content of amyloid β-protein 1-40 in human plasma as claimed in any one of claims 1 to 6 in detecting the content of amyloid β-protein 1-40 in human plasma.

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