Combined detection kit for blood markers of Alzheimer's disease and manufacturing method of combined detection kit

Abstract

The invention belongs to the technical field of Alzheimer's disease marker detection, and particularly relates to an Alzheimer's disease blood marker joint detection kit and a manufacturing method thereof.The kit comprises a protein chip, the protein chip comprises a chip base and marker antibodies coating the chip base, the marker antibodies comprise anti-A beta (1-40) IgG, anti-A beta (1-42) IgG, anti-T-Tau IgG, anti-P-Tau181IgG, anti-P-Tau217 IgG and anti-S100 beta IgG, the chip base is coated with the marker antibodies, and the chip base is coated with the marker antibodies. The Alzheimer's disease blood marker joint detection kit is high in sensitivity, specificity and accuracy, stable in result, good in repeatability and convenient and fast to operate, saves manpower and material resources, is a reliable tool means for early diagnosis of Alzheimer's disease by medical workers, and improves the labor efficiency. The patient can be treated earlier, and the survival rate is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of Alzheimer's disease marker detection, and specifically relates to an Alzheimer's disease blood marker joint detection kit and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease that mainly affects the memory, thinking and behavior of the elderly. AD is the most common disease causing brain dementia. According to statistics, the global expenditure on this disease reached 604 billion US dollars in 2010. According to statistics from the Alzheimer's Disease International Association, there are currently about 44 million patients worldwide, and this number is expected to triple by 2050. The early diagnosis of AD has always attracted much attention and there are many difficulties. First, the disease has an insidious onset and slow development, and early symptoms are easily ignored; second, the existing AD early diagnosis markers are either invasive examinations, or expensive and have qualification requirements, and have not yet been widely used. In recent years, with the continuous advancement of science and technology, researchers have deepened their understanding of the disease and discovered some blood indicators that may become biomarkers of Alzheimer's syndrome. Blood β-amyloid protein (Amyloid-β, Aβ) and phosphorylated tau protein are the most potential AD-related blood biomarkers. At present, ultrasensitive detection technologies such as single molecule array detection, chemiluminescent enzyme immunoassay and immunoprecipitation combined with mass spectrometry are usually used to quantitatively detect low concentrations of Aβ and p-tau in the blood. AD biomarkers can not only accurately diagnose Alzheimer's disease, but also guide clinical treatment and efficacy monitoring, and determine the prognosis of Alzheimer's disease.

[0003] Currently, Aβ and Tau in cerebrospinal fluid are recognized as biomarkers of AD, but due to their invasive sampling methods, most patients find them difficult to accept and their clinical application is very limited. In addition, there are also a variety of detection methods for the determination of AD blood markers, including immunofluorescence analysis, enzyme-linked immunosorbent assay, magnetic separation enzyme-linked immunosorbent assay, chromatography, etc. However, these technologies can only detect one marker in the detection of AD markers, which cannot meet the needs of efficient and fast clinical diagnosis. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides an Alzheimer's disease blood marker joint detection kit and a preparation method thereof, so as to achieve the purpose of joint high-throughput detection of various AD blood markers, reduce the workload of testers, and improve the accuracy of test results.

[0005] The present invention solves the technical problem by adopting the following technical solutions:

[0006] The present invention aims to provide a combined detection kit for blood markers of Alzheimer's disease, comprising a protein chip, wherein the protein chip comprises a substrate and marker antibodies coated on the substrate, wherein the marker antibodies comprise anti-Aβ(1-40)IgG, anti-Aβ(1-42)IgG, anti-T-Tau IgG, anti-P-Tau181 IgG, anti-P-Tau217 IgG, and anti-S100βIgG.

[0007] Alzheimer's disease is a progressive degenerative disease of the central nervous system characterized by amyloid-β (Aβ) deposition and neurofibrillary tangles formed by Tau protein. Compared with CSF examination and PET examination, blood sample testing has significant advantages such as simple sampling, high safety, strong repeatability, and suitability for follow-up. Studies have found that many blood markers can be used for the early diagnosis of AD. Based on this, the present invention has developed a joint test kit that can simultaneously detect multiple blood markers of AD, providing more accurate prediction for the early diagnosis of AD.

[0008] Furthermore, at least one spotting module is provided on the substrate, and each spotting module is provided with a plurality of spotting grooves, and the marker antibodies are fixed in the spotting grooves.

[0009] Furthermore, there are sixteen spotting modules, and a plurality of spotting grooves are arranged in six rows and three columns on the spotting module, and a marker antibody is fixed in each row of spotting grooves.

[0010] Furthermore, the kit also includes a sample diluent, which is a 0.1 M PBS buffer solution containing 5% BSA and 0.5 g / L ProClin, with a pH of 7.4.

[0011] Furthermore, the kit also includes a washing buffer, which is a 0.1M PBS buffer containing 0.05% Tween 20, pH 7.4.

[0012] Furthermore, the kit also includes goat anti-human IgG labeled with a fluorescent group, the fluorescent group used for labeling is Cy5, and the final concentration used is 1 μg / mL.

[0013] Furthermore, the kit also includes calibrators and quality control materials. The calibrators are Aβ(1-40), Aβ(1-42), T-Tau, Tau181, P-Tau217, and S100β protein solutions with different concentrations, and the concentrations are 0pg / mL, 5-100pg / mL, and 100-200pg / mL, respectively; the quality control materials are Aβ(1-40), Aβ(1-42), T-Tau, Tau181, P-Tau217, and S100β protein solutions with different concentrations, and the concentrations are 5-100pg / mL and 100-200pg / mL, respectively.

[0014] A method for preparing a combined detection kit for blood markers of Alzheimer's disease comprises the following steps: preparation of a protein chip: preparing a solution of human-expressed anti-Aβ(1-40)IgG, anti-Aβ(1-42)IgG, anti-T-Tau IgG, anti-P-Tau181IgG, anti-P-Tau217 IgG, and anti-S100βIgG marker antibodies; and fixing the marker antibodies on a substrate of the protein chip.

[0015] Furthermore, after the marker antibody was fixed on the substrate of the protein chip, it was kept fixed at 35% RH for 2 hours at room temperature, and the protein chip was placed in a blocking buffer and blocked at room temperature for 1 hour on a shaker at 50 rpm.

[0016] Furthermore, after the sealing is completed, the shaker is 50 rpm, and the washing is performed for 5 min, which is repeated 3 times; after the treatment, the card is placed in a card box and sealed with tin foil.

[0017] Compared with the prior art, the beneficial technical effects of the present invention are:

[0018] 1. The Alzheimer's disease blood marker joint detection kit of the present invention has high sensitivity, strong specificity, high accuracy, stable results, good repeatability, convenient and fast operation, saving manpower and material resources, and is a reliable tool for medical workers to diagnose Alzheimer's disease in the early stage, which improves labor efficiency, enables patients to receive treatment earlier, and improves survival rate.

[0019] 2. The present invention highly integrates multiple markers, which can improve the utilization rate of the chip, the accuracy and sensitivity of the chip design itself. The establishment of a protein chip based on AD is a highly targeted serological test for a specific neurodegenerative disease, and can integrate multiple other neurodegenerative blood marker antibodies, providing a basis for the detection of multiple diseases. It is an effective means of clinically detecting elderly diseases, which has profound significance for the treatment and control of AD and effectively improves the survival rate of AD.

[0020] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention is a schematic diagram of a protein chip in a combined detection kit for Alzheimer's disease blood markers.

[0022] Figure 2 The standard curve diagram of Aβ(1-42) in the Alzheimer's disease blood marker joint detection kit of the present invention.

[0023] Figure 3 The standard curve diagram of Aβ(1-40) in the Alzheimer's disease blood marker joint detection kit of the present invention.

[0024] Figure 4 This is a standard curve diagram of T-Tau in an Alzheimer's disease blood marker joint detection kit of the present invention.

[0025] Figure 5 This is a standard curve diagram of P-Tau181 in an Alzheimer's disease blood marker joint detection kit of the present invention.

[0026] Figure 6 This is a standard curve diagram of P-Tau217 in an Alzheimer's disease blood marker joint detection kit of the present invention.

[0027] Figure 7 This is a standard curve diagram of S100β in an Alzheimer's disease blood marker joint detection kit of the present invention.

[0028] 1- substrate, 2- spotting module, 3- spotting groove. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0030] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0031] Example 1

[0032] A combined detection kit for Alzheimer's disease blood markers comprises a protein chip, a sample diluent, a cleaning buffer, a goat anti-human IgG labeled with a fluorescent group, a calibrator, a quality control product, a wet box and a washing box.

[0033] The protein chip for combined detection of AD blood markers comprises a substrate 1 and a marker antibody coated on the substrate 1. The substrate 1 is a glass sheet modified with active groups, and one or more spotting modules 2 are arranged on the protein chip substrate 1, on which the marker antibody is fixed.

[0034] The marker antibodies are anti-Aβ(1-40)IgG, anti-Aβ(1-42)IgG, anti-T-Tau IgG, anti-P-Tau181 IgG, anti-P-Tau217 IgG, and anti-S100βIgG.

[0035] Preferably, reference Figure 1 The protein chip substrate 1 is a rectangular parallelepiped structure, and 16 spotting modules 2 are arranged on its upper surface. The spotting modules 2 are provided with six rows and three columns of circular spotting grooves 3, and the marker antibodies are fixed in the spotting grooves 3. Each row of spotting grooves 3 is respectively fixed with a marker antibody, for example Figure 1 In the embodiment, the first row is anti-Aβ(1-42)IgG, the second row is anti-Aβ(1-40)IgG, the third row is anti-T-Tau IgG, the fourth row is anti-P-Tau181 IgG, the fifth row is anti-P-Tau217 IgG, and the sixth row is anti-S100βIgG, but not limited to this order. Each chip is assigned a unique barcode, and the barcode information includes the standard curve made from the same batch of standards and the sample concentration calculation method information.

[0036] The sample diluent was 0.1 M PBS buffer containing 5% BSA and 0.5 g / L ProClin, pH 7.4.

[0037] The washing buffer is TBST buffer, which is 0.1 M PBS buffer containing 0.05% Tween 20, pH 7.4.

[0038] Goat anti-human IgG labeled with a fluorescent group, the fluorescent group used for labeling is Cy5, and the final concentration used is 1μg / mL.

[0039] The calibrators were Aβ(1-40), Aβ(1-42), T-Tau, Tau181, P-Tau217, and S100β protein solutions with different concentrations, and the concentrations were 0pg / mL, 5-100pg / mL, and 100-200pg / mL, respectively. The quality control products were Aβ(1-40), Aβ(1-42), T-Tau, Tau181, P-Tau217, and S100β protein solutions with different concentrations, and the concentrations were 5-100pg / mL and 100-200pg / mL, respectively.

[0040] A detection method of a combined detection kit for Alzheimer's disease blood markers is:

[0041] A. Dilute the serum sample with sample diluent at a ratio of 1:100, take 30 μL of the diluted sample and quality control and calibration products (optional) and add them to the spotting area, place them in a wet box and incubate at room temperature for 1 hour.

[0042] B. Take the chip out of the wet box, place it in a washing box, add about 30 ml of washing buffer, shake at 50 rpm, room temperature for 5 minutes, and repeat 3 times.

[0043] C. Take out the protein chip, shake it gently to remove excess cleaning solution, place it in a wet box, add 30 μL of 1 μg / mL Cy5 goat anti-human IgG to each spot area, and incubate in the dark for 1 hour.

[0044] D. After incubation, wash three times in the dark, then wash once with ultrapure water, centrifuge to remove excess ultrapure water, and analyze the results using a chip scanner and chip data processing software.

[0045] Example 2

[0046] A method for preparing a combined detection kit for Alzheimer's disease blood markers:

[0047] 1. Weighing ingredients and processing raw and auxiliary materials

[0048] 1.1 Protein chip preparation

[0049] 1.1.1 Human expressed anti-Aβ(1-40)IgG, anti-Aβ(1-42)IgG, anti-T-Tau IgG, anti-P-Tau181 IgG, anti-P-Tau217 IgG, and anti-S100βIgG were prepared into a solution with a concentration of 0.5 μg / mL using 0.1 M PBS buffer containing 1% BSA.

[0050] 1.1.2 According to the array arrangement of the protein chip design, a high-speed fully automatic spotter is used to fix multiple marker antibodies on the same spotting module of the protein chip substrate based on the contact spotting principle. Each spot is about 1nL, and each protein is set up as a separate detection point.

[0051] 1.1.3 After the sample is spotted, the chip is fixed at 35% RH for 2 hours at room temperature. The protein chip is placed in a blocking buffer (0.1 M PBS buffer containing 1% BSA) and shaken at 50 rpm for 1 hour at room temperature.

[0052] 1.1.4 After the sealing is completed, shake the machine at 50rpm, wash for 5 minutes, and repeat 3 times. After low-temperature drying, place the chip in the card box and seal it with tin foil.

[0053] 1.2 Preparation of buffer solutions

[0054] 1.2.1. Sample diluent formula:

[0055] Component name Content / 1000mL BSA 50g NaCl 9g <![CDATA[KH 2 AFTER 4 ]]> 0.2g <![CDATA[Na 2 HPO 4 .12H 2 Of]]> 2.9g KCl 0.2g <![CDATA[ddH 2 The]]> Add to 1000mL

[0056] 1.2.2. Concentrated washing buffer (10×) formula:

[0057] Component name Content / 1000mL NaCl 90g <![CDATA[KH 2 AFTER 4 ]]> 2g <![CDATA[Na 2 HPO 4 .12H 2 Of]]> 29g KCl 2g Tween 20 5mL <![CDATA[ddH 2 The]]> Add to 1000mL

[0058] 1.2.3. Cy5 goat anti-human IgG concentrate: Dilute the commercially available Cy5 goat anti-human IgG to 1 mg / mL using 0.1 M PBS buffer containing 1% BSA and store in a frozen state.

[0059] 1.2.4. Fill: 3 mL of sample diluent, 15 mL of blocking buffer, 15 mL of concentrated washing buffer, and 1 μL of Cy5 goatanti human IgG into 1.5 mL light-proof tubes.

[0060] 2-pack box

[0061] The buffer components and disposable wet box of the kit were placed on a foam support plate, the kit was sealed in plastic, and the Cy5 goat anti-human IgG concentrate and protein chip were stored at -20°C.

[0062] Example 3

[0063] A method for preparing a combined detection kit for Alzheimer's disease blood markers:

[0064] 1. Human expressed anti-Aβ(1-40)IgG, anti-Aβ(1-42)IgG, anti-T-Tau IgG, anti-P-Tau181IgG, anti-P-Tau217 IgG, and anti-S100βIgG were prepared into a solution with a concentration of 0.5 μg / mL using 0.1 M PBS buffer containing 1% BSA.

[0065] 2. According to the array arrangement of the protein chip design, a high-speed fully automatic spotter is used to fix multiple marker antibodies on the same spotting module of the protein chip substrate based on the contact spotting principle, with each spotting about 1nL.

[0066] 3. After the sample is spotted, the protein chip is blocked and cleaned. Specifically: after the sample is spotted, it is kept fixed at 35% RH for 2 hours at room temperature, and the protein chip is placed in a blocking buffer (0.1M PBS buffer containing 1% BSA) and shaken at 50 rpm for 1 hour at room temperature.

[0067] After the sealing is completed, shake the machine at 50 rpm, wash for 5 minutes, and repeat 3 times. After low-temperature drying, place the chip in the card box and seal it with tin foil.

[0068] 4. Add 30 μL of calibrators of different concentrations into the sample loading area according to the concentration gradient and incubate at room temperature for 1 hour.

[0069] 5. After incubation, wash three times with washing buffer.

[0070] 6. Add 30 μL of 1 μg / mL Cy5 goat anti human IgG to each loading area, incubate for 1 hour, wash three times with cleaning solution, and wash once with ultrapure water.

[0071] 7. Scan the chip with a chip scanner, generate a barcode based on the standard curve and calculation formula generated by the software, and paste it onto the plastic shell of the protein chip from the same batch.

[0072] Calculation formula for each indicator

[0073] Aβ(1-42) y=85.46x+1737.6 Aβ(1-40) y=15.396x+442.83 T-Tau y=74.676x+1429 P-Tau181 y=134.01x+2171.5 P-Tau217 y=47.253x+1044.1 S100β y=45.06x+1361.1

[0074] Experimental Example 1: Serum Sample Test

[0075] The serum sample was diluted with sample diluent at a ratio of 1:100, 30 μL was added to the spotting area, and the test was repeated 15 times. The serum sample has been tested for the concentration of each indicator using other methods. The subsequent steps are the same as steps 4-6 in Example 3, and the CV values ​​of the 15 test results are calculated. The CV values ​​of each indicator are less than 15%, which is consistent with the magnetic particle chemiluminescence method. Therefore, it is feasible to use this method to perform joint detection of AD blood markers.

[0076] Aβ(1-42) Aβ(1-40) T-Tau P-Tau181 P-Tau217 S100β Known concentration 63pg / mL 25pg / mL 166pg / mL 35pg / mL 68pg / mL 46pg / mL Test1 72pg / mL 21pg / mL 164pg / mL 28pg / mL 62pg / mL 48pg / mL Test2 57pg / mL 26pg / mL 157pg / mL 42pg / mL 72pg / mL 42pg / mL Test3 61pg / mL 21pg / mL 174pg / mL 27pg / mL 59pg / mL 49pg / mL Test4 62pg / mL 25pg / mL 171pg / mL 35pg / mL 57pg / mL 48pg / mL Test5 74pg / mL 26pg / mL 160pg / mL 27pg / mL 69pg / mL 46pg / mL Test6 79pg / mL 27pg / mL 164pg / mL 33pg / mL 63pg / mL 52pg / mL Test7 61pg / mL 28pg / mL 169pg / mL 35pg / mL 62pg / mL 58pg / mL Test8 69pg / mL 22pg / mL 174pg / mL 32pg / mL 59pg / mL 52pg / mL Test9 59pg / mL 25pg / mL 173pg / mL 42pg / mL 68pg / mL 57pg / mL Test10 69pg / mL 18pg / mL 168pg / mL 37pg / mL 71pg / mL 41pg / mL Test11 49pg / mL 20pg / mL 184pg / mL 38pg / mL 75pg / mL 39pg / mL Test12 65pg / mL 22pg / mL 159pg / mL 32pg / mL 74pg / mL 47pg / mL Test13 57pg / mL 21pg / mL 164pg / mL 38pg / mL 73pg / mL 48pg / mL Test14 68pg / mL 26pg / mL 167pg / mL 37pg / mL 78pg / mL 39pg / mL Test15 59pg / mL 24pg / mL 171pg / mL 35pg / mL 72pg / mL 37pg / mL CV(100%) 11.8 12.14 4.03 13.37 9.58 13.13

[0077] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0078] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A combined detection kit for blood markers of Alzheimer's disease, characterized in that: The invention comprises a protein chip, which comprises a substrate and marker antibodies coated on the substrate, wherein the marker antibodies comprise anti-Aβ(1-40)IgG, anti-Aβ(1-42)IgG, anti-T-Tau IgG, anti-P-Tau181 IgG, anti-P-Tau217 IgG and anti-S100βIgG.

2. The Alzheimer's disease blood marker combined detection kit according to claim 1, characterized in that: At least one spotting module is arranged on the substrate, and each spotting module is provided with a plurality of spotting grooves, in which marker antibodies are fixed.

3. The Alzheimer's disease blood marker combined detection kit according to claim 2, characterized in that: There are sixteen spotting modules, and a plurality of spotting grooves are arranged in six rows and three columns on the spotting module, and a marker antibody is fixed in each row of spotting grooves.

4. The Alzheimer's disease blood marker combined detection kit according to claim 1, characterized in that: The kit also includes a sample diluent, which is a 0.1 M PBS buffer solution containing 5% BSA and 0.5 g / L ProClin, with a pH of 7.

4.

5. The Alzheimer's disease blood marker combined detection kit according to claim 1, characterized in that: The kit also includes a washing buffer, which is a 0.1 M PBS buffer containing 0.05% Tween 20, pH 7.

4.

6. The Alzheimer's disease blood marker combined detection kit according to claim 1, characterized in that: The kit also includes goat anti-human IgG labeled with a fluorescent group, the fluorescent group used for labeling is Cy5, and the final concentration used is 1 μg / mL.

7. The Alzheimer's disease blood marker combined detection kit according to claim 1, characterized in that: The kit also includes calibrators and quality control materials. The calibrators are Aβ(1-40), Aβ(1-42), T-Tau, Tau181, P-Tau217, and S100β protein solutions with different concentrations, and the concentrations are 0pg / mL, 5-100pg / mL, and 100-200pg / mL, respectively; the quality control materials are Aβ(1-40), Aβ(1-42), T-Tau, Tau181, P-Tau217, and S100β protein solutions with different concentrations, and the concentrations are 5-100pg / mL and 100-200pg / mL, respectively.

8. A method for preparing a combined detection kit for blood markers of Alzheimer's disease according to any one of claims 1 to 7, characterized in that: The following steps are involved: Preparation of protein chip: human expressed anti-Aβ(1-40)IgG, anti-Aβ(1-42)IgG, anti-T-Tau IgG, anti-P-Tau181 IgG, anti-P-Tau217 IgG, anti-S100βIgG marker antibodies are prepared into solutions; the marker antibodies are fixed on the substrate of the protein chip.

9. The method for preparing a combined detection kit for blood markers of Alzheimer's disease according to claim 8, characterized in that: After the marker antibody is fixed on the substrate of the protein chip, it is kept fixed at room temperature under 35% RH for 2 hours. The protein chip is placed in a blocking buffer and blocked at room temperature for 1 hour on a shaker at 50 rpm.

10. The method for preparing a combined detection kit for blood markers of Alzheimer's disease according to claim 9, characterized in that: After sealing, shake at 50 rpm, wash for 5 minutes, and repeat 3 times; after treatment, place in a card box and seal with tin foil.

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