A chemiluminescent immunoassay kit for Alzheimer's disease pTau181 and its preparation method and detection method

Abstract

A pTau181 chemiluminescent immunoassay kit for Alzheimer's disease and its preparation method and detection method belong to the field of immunoassay technology. In order to solve the problems of complex operation, high cost, long detection time, low sensitivity and poor precision of the existing Alzheimer's disease detection kit, the present invention includes a magnetic particle reagent, an AE-labeled pTau-181 antibody, and a pTau-181 calibrator. The magnetic particle reagent is a magnetic microsphere suspension coated with a pTau-181 antibody; the AE-labeled pTau-181 antibody is obtained by labeling the pTau-181 antibody with an acridinium ester. The present invention has low sample pretreatment requirements, a simple pretreatment process, can quickly and high-throughput detect a large number of samples, and the inspection method is convenient and easy, filling the gap in the prior art.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and specifically relates to an Alzheimer's disease pTau181 chemiluminescent immunoassay kit, a preparation method thereof, and a detection method thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with an insidious onset and a slow and progressive worsening. It is common in people over 65 years old, and the incidence rate increases significantly with age. The widely accepted pathogenic hypotheses for the characteristic pathological changes of AD include: Aβ aggregation hypothesis, Tau protein abnormality hypothesis, cholinergic hypothesis, neuroinflammation hypothesis, etc. Among them, the phosphorylation modification of Tau protein is crucial in the progression of the disease. Tau and APP can be phosphorylated at multiple sites, and are mainly divided into T-tau, pTau-181, pTau-217, and pTau-231 according to different phosphorylation sites. These biomarkers can also be measured faster and less frequently in plasma, and can provide an important basis for the early diagnosis of Alzheimer's disease.

[0003] At present, the diagnostic method of AD is mainly combined diagnosis, including: neuropsychological assessment, cognitive impairment test; brain senile plaque and Tau protein PET scan; brain magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) markers, Aβ deposition, phosphorylated tau protein detection, etc. However, since the early symptoms of AD are not obvious, when a clear diagnosis is made, most patients have reached the late stage of the disease, and most neurons have died. If a rapid diagnosis can be made in the early stage of the patient's onset or the potential patient can discover the risk in advance, targeted treatment or prevention can be carried out, which can help the patient's disease course to remain in the mild cognitive impairment (MCI) stage and slow down the deterioration, thereby ensuring the patient's quality of life and reducing the social burden. Therefore, how to provide a detection reagent and detection method for pTau-181 is a problem that technicians in this field urgently need to solve. Summary of the invention

[0004] The problem to be solved by the present invention is that the existing Alzheimer's disease detection kits are complicated to operate, expensive, take a long time to detect, have low sensitivity, poor precision, and lack the ability to accurately predict Alzheimer's disease. An Alzheimer's disease pTau181 chemiluminescent immunoassay kit, a preparation method thereof, and a detection method thereof are proposed.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] A pTau181 chemiluminescent immunoassay kit for Alzheimer's disease, the kit comprising a magnetic particle reagent, an AE-labeled pTau-181 antibody, and a pTau-181 calibrator. The magnetic particle reagent is a magnetic microsphere suspension coated with the pTau-181 antibody; the AE-labeled pTau-181 antibody is obtained by labeling the pTau-181 antibody with an acridinium ester.

[0007] Furthermore, the particle size of the magnetic particles in the magnetic particle reagent is 1-3 μm, and the surface of the magnetic particles is coated with magnetic microspheres with carboxyl active groups;

[0008] When preparing the magnetic microsphere suspension of the pTau-181 antibody, 0.5-2.5 mg of pTau-181 antibody is added to every 50 mg of magnetic microspheres for coating. The mass ratio of pTau-181 antibody to carboxyl magnetic beads in the magnetic microparticle reagent is 1: 20-100. The temperature of the connection reaction is room temperature and the time of the connection reaction is 16 to 20 hours.

[0009] Furthermore, the mass ratio of pTau-181 antibody to acridinium ester in the AE-labeled pTau-181 antibody is 1:1.

[0010] A method for preparing a chemiluminescent immunoassay kit for Alzheimer's disease pTau181 comprises the following steps:

[0011] Step 1. preparing magnetic particle reagent;

[0012] Step 1.1. Dispersing the carboxyl magnetic bead solution in a magnetic particle buffer to obtain a dispersed carboxyl magnetic bead solution;

[0013] Step 1.2. The dispersed carboxyl magnetic bead solution and the pTau-181 antibody are subjected to a connection reaction to obtain a connection product;

[0014] Step 1.3. Place the ligated product in a magnetic field and wash it with a magnetic particle buffer to obtain a magnetic particle solution connected with the pTau-181 antibody;

[0015] Step 2. Prepare AE-labeled pTau-181 antibody;

[0016] Step 2.1. Prepare AE solution with a concentration of 5 mg / mL using 0.15 M sodium bicarbonate buffer;

[0017] Step 2.2. The mouse monoclonal anti-pTau-181 antibody and the AE solution were mixed at a mass ratio of 1:1 to perform a linking reaction to obtain a linking product of the pTau-181 antibody and AE;

[0018] Step 2.3. The linkage product of the pTau-181 antibody and AE is separated and purified using a carbonate buffer having a pH of 8 to 9 to obtain an AE-labeled pTau-181 antibody;

[0019] Step 3. Prepare pTau-181 calibrator: The pTau-181 calibrator is a Tris buffer containing pTau-181 antigen, 0.5% by mass of BSA, and 0.05% by mass of prolin300; the concentrations of the pTau-181 antigen are 20 pg / mL and 200 pg / mL, respectively.

[0020] Furthermore, in step 1.1, the carboxyl magnetic bead concentrate is placed in a magnetic field for 1 to 3 minutes, the supernatant is removed after all the carboxyl magnetic beads are precipitated, and then added to the magnetic particle buffer and oscillated, and then placed in a magnetic field for 1 to 3 minutes, and then the supernatant is removed; the oscillation time is set to 20 to 30 minutes; the volume ratio of the carboxyl magnetic beads and the magnetic particle buffer is 1:2 to 5, and the number of dispersion treatments is 3 times.

[0021] Furthermore, the intercommunication implementation method of step 1.2 includes the following steps:

[0022] Step 1.2.1. Weigh the dispersed carboxyl magnetic bead solution and pTau-181 antibody at a mass ratio of 100:5 to 100:1, the concentration of the dispersed carboxyl magnetic bead solution is 10 to 50 mg / mL, the connection reaction temperature is set to room temperature, and the connection reaction time is 16 to 20 hours;

[0023] S1.2.2. After the connection reaction is completed, add blocking solution for blocking. The formula of the blocking solution is: a mixed solution of PBS, 3wt% BSA, 1wt% Tween-20, 1wt% maltose, 5wt% sorbitol, 2wt% sucrose, 3wt% glycerol, and 0.1~0.8wt% PEG600.

[0024] Furthermore, the connection product in step 1.3 is placed in the magnetic field for 5 to 10 minutes and then rinsed with a magnetic particle buffer to obtain a magnetic particle solution connected with the pTau-181 antibody.

[0025] A detection method of a chemiluminescent immunoassay kit for Alzheimer's disease pTau181 comprises the following steps:

[0026] S1. Prepare magnetic particle reagents and AE-labeled pTau-181 antibody;

[0027] S2. Add the serum sample and the AE-labeled pTau-181 antibody prepared in step S1 to the magnetic particle reagent for incubation;

[0028] S3. After the incubation is completed and the magnetic particle reagent has settled, remove the supernatant, wash, then add the substrate solution and shake to mix, and detect the relative luminescence intensity.

[0029] Furthermore, the volume ratio of the serum sample, AE-labeled pTau-181 antibody and magnetic particle reagent in step S2 is 2:2:1.

[0030] Furthermore, after the relative luminescence intensity is detected in step S3, calibration is performed using two-point calibration products of high and low values, and a new working curve is generated by fitting for sample measurement; the pTau-181 quality control product is used as a sample, and the concentration is fitted using the working curve to obtain the corresponding quality control product concentration, and it is observed that the fitted quality control product concentration is ±30% of the standard concentration value.

[0031] Beneficial effects of the present invention:

[0032] The pTau181 chemiluminescent immunoassay kit for Alzheimer's disease described in the present invention uses magnetic beads as a solid phase, which makes the immune response closer to the liquid phase, the reaction is more complete and rapid, and the bound immune complexes are easier to separate, reducing nonspecific adsorption. The AE-labeled antibody reagent used in the present invention is a mixture of monoclonal antibodies, which makes the affinity of the immune response higher, and the production batch difference of the monoclonal antibody is relatively small, which makes it easier to ensure the batch stability of the product.

[0033] A chemiluminescent immunoassay kit for pTau181 for Alzheimer's disease provides the possibility for rapid and accurate pTau-181 detection. The invention has low requirements for sample pretreatment, a simple pretreatment process, can rapidly and high-throughput detect a large number of samples, and the detection method is convenient and easy, thus filling the gap in the prior art.

[0034] A chemiluminescent immunoassay kit for Alzheimer's disease pTau181 combines chemiluminescent technology with immunomagnetic particles to provide a near-homogeneous reaction system with higher detection sensitivity, specificity, precision and accuracy, and achieves better performance parameters. The magnetic particle reagent, AE-labeled antibody, and calibrator in the kit are the optimal formula for this reaction system, providing a strong guarantee for the shelf life and detection performance of the kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a standard curve data diagram of Example 1 of the present invention;

[0036] Figure 2 This is a standard curve data diagram of Example 2 of the present invention;

[0037] Figure 3 This is a standard curve data diagram of Example 3 of the present invention. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention, that is, the specific embodiments described are only part of the embodiments of the present invention, rather than all of the specific embodiments. The components of the specific embodiments of the present invention described and shown in the drawings herein can be arranged and designed in various different configurations, and the present invention can also have other embodiments.

[0039] Therefore, the following detailed description of the specific embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected specific embodiments of the present invention. Based on the specific embodiments of the present invention, all other specific embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0040] In order to further understand the content, features and effects of the present invention, the following specific implementation methods are given as examples, and the attached Figure 1 -Attached Figure 3 The detailed instructions are as follows:

[0041] Embodiment 1:

[0042] A method for preparing a chemiluminescent immunoassay kit for Alzheimer's disease pTau181 comprises the following steps:

[0043] Step 1. preparing magnetic particle reagent;

[0044] Step 1.1. Dispersing the carboxyl magnetic bead solution in a magnetic particle buffer to obtain a dispersed carboxyl magnetic bead solution;

[0045] Furthermore, in step 1.1, the carboxyl magnetic bead concentrate is placed in a magnetic field for 1 minute, the supernatant is removed after all the carboxyl magnetic beads are completely precipitated, and then added to the magnetic particle buffer for oscillation, and then placed in a magnetic field for 1 minute, and the supernatant is removed; the oscillation time is set to 20 to 30 minutes; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:2, and the number of dispersion treatments is 3 times;

[0046] Step 1.2. The dispersed carboxyl magnetic bead solution and the pTau-181 antibody are subjected to a connection reaction to obtain a connection product;

[0047] Furthermore, the intercommunication implementation method of step 1.2 includes the following steps:

[0048] Step 1.2.1. Weigh the dispersed carboxyl magnetic bead solution and pTau-181 antibody at a mass ratio of 100:5. The concentration of the dispersed carboxyl magnetic bead solution is 10 mg / mL. Set the temperature of the ligation reaction to room temperature and the ligation reaction time to 16 h.

[0049] S1.2.2. After the ligation reaction is completed, a blocking solution is added for blocking. The formula of the blocking solution is: a mixed solution of PBS, 3wt% BSA, 1wt% Tween-20, 1wt% maltose, 5wt% sorbitol, 2wt% sucrose, 3wt% glycerol, and 0.1wt% PEG600;

[0050] Step 1.3. Place the ligated product in a magnetic field and wash it with a magnetic particle buffer to obtain a magnetic particle solution connected with the pTau-181 antibody;

[0051] Furthermore, the connection product in step 1.3 is placed in the magnetic field for 1-3 minutes, and then washed with a magnetic particle buffer to obtain a magnetic particle solution connected with the pTau-181 antibody;

[0052] Furthermore, the connection product was placed in the magnetic field for 1-3 minutes and washed three times; the concentration of the magnetic particle solution connected with the pTau-181 antibody was 10 mg / mL;

[0053] Step 2. Prepare AE-labeled pTau-181 antibody;

[0054] Step 2.1. Prepare AE solution with a concentration of 5 mg / mL using 0.15 M sodium bicarbonate buffer;

[0055] Step 2.2. The mouse monoclonal anti-pTau-181 antibody and the AE solution were mixed at a mass ratio of 1:1 to perform a linking reaction to obtain a linking product of the pTau-181 antibody and AE;

[0056] Step 2.3. The linkage product of pTau-181 antibody and AE was separated and purified using a carbonate buffer solution at pH 8 to obtain AE-labeled pTau-181 antibody;

[0057] Step 3. Prepare pTau-181 calibrator: The pTau-181 calibrator is a Tris buffer containing pTau-181 antigen, 0.5% by mass of BSA, and 0.05% by mass of prolin300; the concentrations of the pTau-181 antigen are 20 pg / mL and 200 pg / mL, respectively.

[0058] Furthermore, the pTau-181 antigen was purchased from Suzhou Renduan Biopharmaceutical Technology Co., Ltd., RDP002;

[0059] Furthermore, the mouse monoclonal anti-pTau-181 antibody was purchased from Xibao Biotechnology (Shanghai) Co., Ltd., EKN0456A.

[0060] Embodiment 2:

[0061] A method for preparing a chemiluminescent immunoassay kit for Alzheimer's disease pTau181 comprises the following steps:

[0062] Step 1. preparing magnetic particle reagent;

[0063] Step 1.1. Dispersing the carboxyl magnetic bead solution in a magnetic particle buffer to obtain a dispersed carboxyl magnetic bead solution;

[0064] Furthermore, in step 1.1, the carboxyl magnetic bead concentrate is placed in a magnetic field for 2 minutes, the supernatant is removed after all the carboxyl magnetic beads are completely precipitated, and then added to the magnetic particle buffer for oscillation, and then placed in a magnetic field for 2 minutes, and the supernatant is removed; the oscillation time is set to 20 minutes; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:3, and the number of dispersion treatments is 3 times;

[0065] Step 1.2. The dispersed carboxyl magnetic bead solution and the pTau-181 antibody are subjected to a connection reaction to obtain a connection product;

[0066] Furthermore, the intercommunication implementation method of step 1.2 includes the following steps:

[0067] Step 1.2.1. Weigh the dispersed carboxyl magnetic bead solution and pTau-181 antibody at a mass ratio of 100:3. The concentration of the dispersed carboxyl magnetic bead solution is 10 mg / mL. Set the temperature of the ligation reaction to room temperature and the ligation reaction time to 18 h.

[0068] S1.2.2. After the ligation reaction is completed, a blocking solution is added for blocking. The formula of the blocking solution is: a mixed solution of PBS, 3wt% BSA, 1wt% Tween-20, 1wt% maltose, 5wt% sorbitol, 2wt% sucrose, 3wt% glycerol, and 0.5wt% PEG600;

[0069] Step 1.3. Place the ligated product in a magnetic field and wash it with a magnetic particle buffer to obtain a magnetic particle solution connected with the pTau-181 antibody;

[0070] Furthermore, the connection product in step 1.3 is placed in the magnetic field for 1-3 minutes, and then washed with a magnetic particle buffer to obtain a magnetic particle solution connected with the pTau-181 antibody;

[0071] Furthermore, the connection product was placed in the magnetic field for 1-3 minutes and washed three times; the concentration of the magnetic particle solution connected with the pTau-181 antibody was 10 mg / mL;

[0072] Step 2. Prepare AE-labeled pTau-181 antibody;

[0073] Step 2.1. Prepare AE solution with a concentration of 5 mg / mL using 0.15 M sodium bicarbonate buffer;

[0074] Step 2.2. The mouse monoclonal anti-pTau-181 antibody and the AE solution were mixed at a mass ratio of 1:1 to perform a linking reaction to obtain a linking product of the pTau-181 antibody and AE;

[0075] Step 2.3. The linkage product of pTau-181 antibody and AE was separated and purified using a carbonate buffer at pH 8 to obtain AE-labeled pTau-181 antibody;

[0076] Step 3. Prepare pTau-181 calibrator: The pTau-181 calibrator is a Tris buffer containing pTau-181 antigen, 0.5% by mass of BSA, and 0.05% by mass of prolin300; the concentrations of the pTau-181 antigen are 20 pg / mL and 200 pg / mL, respectively.

[0077] Embodiment 3:

[0078] A method for preparing a chemiluminescent immunoassay kit for Alzheimer's disease pTau181 comprises the following steps:

[0079] Step 1. preparing magnetic particle reagent;

[0080] Step 1.1. Dispersing the carboxyl magnetic bead solution in a magnetic particle buffer to obtain a dispersed carboxyl magnetic bead solution;

[0081] Furthermore, in step 1.1, the carboxyl magnetic bead concentrate is placed in a magnetic field for 3 minutes, the supernatant is removed after all the carboxyl magnetic beads are completely precipitated, and then added to the magnetic particle buffer for oscillation, and then placed in a magnetic field for 3 minutes, and then the supernatant is removed; the oscillation time is set to 30 minutes; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:5, and the number of dispersion treatments is 3 times;

[0082] Step 1.2. The dispersed carboxyl magnetic bead solution and the pTau-181 antibody are subjected to a connection reaction to obtain a connection product;

[0083] Furthermore, the intercommunication implementation method of step 1.2 includes the following steps:

[0084] Step 1.2.1. Weigh the dispersed carboxyl magnetic bead solution and pTau-181 antibody at a mass ratio of 100:1. The concentration of the dispersed carboxyl magnetic bead solution is 10 mg / mL. Set the temperature of the ligation reaction to room temperature and the ligation reaction time to 20 h.

[0085] S1.2.2. After the ligation reaction is completed, a blocking solution is added for blocking. The formula of the blocking solution is: a mixed solution of PBS, 3wt% BSA, 1wt% Tween-20, 1wt% maltose, 5wt% sorbitol, 2wt% sucrose, 3wt% glycerol, and 0.8wt% PEG600;

[0086] Step 1.3. Place the ligated product in a magnetic field and wash it with a magnetic particle buffer to obtain a magnetic particle solution connected with the pTau-181 antibody;

[0087] Furthermore, the connection product in step 1.3 is placed in the magnetic field for 1-3 minutes, and then washed with a magnetic particle buffer to obtain a magnetic particle solution connected with the pTau-181 antibody;

[0088] Furthermore, the connection product was placed in the magnetic field for 1-3 minutes and washed three times; the concentration of the magnetic particle solution connected with the pTau-181 antibody was 10 mg / mL;

[0089] Step 2. Prepare AE-labeled pTau-181 antibody;

[0090] Step 2.1. Prepare AE solution with a concentration of 5 mg / mL using 0.15 M sodium bicarbonate buffer;

[0091] Step 2.2. The mouse monoclonal anti-pTau-181 antibody and the AE solution were mixed at a mass ratio of 1:1 to perform a linking reaction to obtain a linking product of the pTau-181 antibody and AE;

[0092] Step 2.3. The linkage product of pTau-181 antibody and AE was separated and purified using a carbonate buffer at pH 8 to obtain AE-labeled pTau-181 antibody;

[0093] Step 3. Prepare pTau-181 calibrator: The pTau-181 calibrator is a Tris buffer containing pTau-181 antigen, 0.5% by mass of BSA, and 0.05% by mass of prolin300; the concentrations of the pTau-181 antigen are 20 pg / mL and 200 pg / mL, respectively.

[0094] Embodiment 4:

[0095] An Alzheimer's disease pTau181 chemiluminescent immunoassay kit is prepared based on the preparation method of an Alzheimer's disease pTau181 chemiluminescent immunoassay kit described in Examples 1 to 3, the kit comprising a magnetic particle reagent, an AE-labeled pTau-181 antibody, and a pTau-181 calibrator, the magnetic particle reagent being a magnetic microsphere suspension coated with a pTau-181 antibody; the AE-labeled pTau-181 antibody is obtained by labeling the pTau-181 antibody with an acridinium ester.

[0096] Furthermore, the particle size of the magnetic particles in the magnetic particle reagent is 1-3 μm, and the surface of the magnetic particles is coated with magnetic microspheres with carboxyl active groups;

[0097] When preparing the magnetic microsphere suspension of the pTau-181 antibody, 0.5-2.5 mg of pTau-181 antibody is added to every 50 mg of magnetic microspheres for coating. The mass ratio of pTau-181 antibody to carboxyl magnetic beads in the magnetic microparticle reagent is 1:20-100. The temperature of the connection reaction is room temperature and the time of the connection reaction is 16 to 20 hours.

[0098] Furthermore, the mass ratio of pTau-181 antibody to acridinium ester in the AE-labeled pTau-181 antibody is 1:1.

[0099] Embodiment 5:

[0100] The use of an Alzheimer's disease pTau181 chemiluminescent immunoassay kit prepared based on the preparation method of an Alzheimer's disease pTau181 chemiluminescent immunoassay kit described in Examples 1 to 3 comprises the following steps:

[0101] S1. Prepare magnetic particle reagents and AE-labeled pTau-181 antibody;

[0102] S2. Add the serum sample and the AE-labeled pTau-181 antibody prepared in step S1 to the magnetic particle reagent for incubation;

[0103] Furthermore, the volume ratio of the serum sample, AE-labeled pTau-181 antibody and magnetic particle reagent in step S2 is 2:2:1.

[0104] S3. After the incubation is completed and the magnetic particle reagent has settled, remove the supernatant, wash, then add the substrate solution and shake to mix, and detect the relative luminescence intensity.

[0105] Furthermore, after the relative luminescence intensity is detected in step S3, calibration is performed using two-point calibration products of high and low values, and a new working curve is generated by fitting for sample measurement; the pTau-181 quality control product is used as a sample, and the concentration is fitted using the working curve to obtain the corresponding quality control product concentration, and it is observed that the fitted quality control product concentration is ±30% of the standard concentration value.

[0106] Furthermore, the AE-labeled antibody reagent is an acridinium ester solution connected to the pTau-181 antibody. Since the linking reaction time is too long and the efficiency is too low, non-specific binding is easily generated during the on-machine test. Therefore, it needs to be further purified, which can effectively shorten the reaction time and improve efficiency.

[0107] The treatment method is as follows: dispersing the acridinium ester solution of the labeled antibody under the action of a carbonate buffer to obtain a diluent; adding the diluent to a G25 gel separation column for separation and purification.

[0108] 1. G-25 desalting column is a pre-packaged, ready-to-use chromatography column that uses G-25 matrix to separate large molecular weight substances from small molecular weight substances in a simple, rapid and efficient manner. It is commonly known as a desalting column and is mainly used to remove salt ions, detergents, small molecule dyes, buffers and other impurities from samples such as proteins, nucleic acids, polypeptides and polysaccharides.

[0109] 1. The filler used in the desalting column is Beyodex™ G-25 Medium, which is a high molecular polymer with a three-dimensional mesh structure formed by cross-linking dextran with an ether bond through an epichlorohydrin cross-linking agent, and has basically the same performance as Sephadex G-25 Medium. The filler particle size is 70-250μm, and it can separate proteins or other macromolecular samples with a molecular weight >5kDa. The column bed volume is 8.3ml, and the recommended sample volume is 1-2.5ml. The function and use of this product are consistent with Cytiva's PD-10 Desalting Column, and it is suitable for gravity method or centrifugation method.

[0110] 2. When the desalting column separates samples, large molecules cannot enter the matrix mesh pores and are blocked outside the matrix gel. As the flowing solution moves along the gaps between the matrix gel particles, they move down faster and are eluted out of the column first; smaller molecules or ions can enter the matrix mesh pores and thus enter the gel. As the flowing solution moves in the mesh pores of the gel particles, small molecular weight substances stay in the matrix longer, move down slower, and are eluted out of the column later. This separation and purification method is called size exclusion chromatography (SEC), also often called molecular sieve chromatography (MSC) or gel filtration chromatography (GFC).

[0111] The detection method of the pTau-181 antibody detection kit is the same as that in Example 1.

[0112] The performance indicators of the magnetic microparticle chemiluminescent immunoassay kit for Alzheimer's disease prepared by the preparation method of a pTau181 chemiluminescent immunoassay kit for Alzheimer's disease described in Examples 1-3 of the present invention were tested:

[0113] 1. Linear

[0114] The linear range was (0-500) pg / mL, and the correlation coefficient was r≥0.9900.

[0115] Here’s how to do it:

[0116] Serum or serum substitute close to the upper limit of the linear range is diluted into five concentrations with sample diluent at a certain ratio. The five concentration samples before and after dilution are measured three times using a chemiluminescence analyzer, and the average value is calculated. A four-parameter fitting curve is used based on the average value and the dilution ratio, and the linear correlation coefficient r (obtained by the X / Y axis linear fitting of the column of 1 to 1 / 100 and the RLU average value) is calculated. The result should be ≥ 0.9900. The specific values ​​of the test are shown in Table 1:

[0117] Table 1:

[0118]

[0119] According to the above table, the luminescence value of Example 1 is too low when detecting low-value samples. It is speculated that this is caused by incorrect formulation when the antibody is coated and blocked. Therefore, the concentration of PEG600 is increased in Example 2, and the result is relatively ideal. The concentration of PEG600 is further increased in Example 3, and the blank background luminescence value increases significantly. It is speculated that the non-specific binding is serious due to excessive concentration. It can be obtained that the curve of Example 2 rises steadily and has a good trend in the linear range, which best meets the requirements.

[0120] The measured linear correlation coefficients (r) of the reaction curves of Examples 1-3 were 0.9944, 0.9994, and 0.9957, respectively, all >0.9900, which were significantly better than the prior art, and Example 2 had the best effect.

[0121] 2. Accuracy (recovery rate)

[0122] The recovery rates in Examples 1-3 of the present invention are in the range of 85 to 115%.

[0123] Here’s how to do it:

[0124] Sample A with known concentration of pTau-181 was added to sample B with the same matrix as the sample to be tested. The volume of A added should not exceed 10% of the total volume (A+B). The measurement was repeated three times and calculated according to formula (1):

[0125] (1)

[0126] Where:

[0127] R : recovery rate;

[0128] V : The volume of sample A solution;

[0129] V 0: Volume of sample B solution;

[0130] C : The detection concentration of sample B solution after adding A solution;

[0131] C 0: concentration of sample B solution;

[0132] Cs : Concentration of sample A solution.

[0133] Data Case( V =0.1mL, V 0=0.9mL, C 0 = 0 pg / mL, Cs =200 pg / mL, where V and V0 According to the above method, the ratio is 1:9. C 0 and Cs The test results are shown in Table 2:

[0134] Table 2

[0135]

[0136] The accuracy (recovery rate) of Examples 1-3 measured was 97.00%, 86.40%, and 100.17%, respectively, all within the range of 85-115%, meeting the requirements. Among them, Example 2 was the best.

[0137] 3. Minimum detection limit (sensitivity)

[0138] The minimum detection limit is ≤5pg / mL.

[0139] Here’s how to do it:

[0140] Use zero-concentration calibrator or sample dilution as the sample to be tested, and repeat the measurement 20 times. Obtain the RLU value (relative luminescence value) of the 20 measurement results, calculate its mean value (M) and standard deviation (SD), and obtain M+2SD. Perform two-point regression fitting based on the concentration-RLU value results between the zero-concentration calibrator and the adjacent calibrator to obtain a linear equation. Substitute the RLU value of M+2SD into the above equation to obtain the corresponding concentration value, which is the minimum detection limit, as shown in Table 3:

[0141] Table 3:

[0142]

[0143] The average luminescence value of the pTau-181 sample dilution measured in Example 2 is M=267.80, SD=44.80. Substituting the value of (M+2SD) into this reaction curve, the sensitivity is obtained as: 2.55pg / mL (<5pg / mL), which meets the requirements.

[0144] 4. Repeatability

[0145] The coefficient of variation CV≤8%. The operation method is as follows:

[0146] Quality control product 1 (20 pg / mL) and quality control product 2 (200 ng / mL) were taken as the samples to be tested, and the pTau-181 content in the samples to be tested was measured 10 times using a chemiluminescence analyzer. The coefficient of variation (CV) was calculated according to formula (2).

[0147] ......................................... (2)

[0148] Where: The average of the test results;

[0149] SD —Standard deviation of 10 test results;

[0150] CV—coefficient of variation;

[0151] The data of Example 1 are shown in Table 4:

[0152] Table 4

[0153]

[0154] The data of Example 2 are shown in Table 5:

[0155] Table 5

[0156]

[0157] The data of Example 3 are shown in Table 6:

[0158] Table 6

[0159]

[0160] Analysis: Examples 1-3 tested the quality control product 1.2 ten times each, and calculated the CV. The quality control product 1SD measured by the scheme of Example 1 was 1.24, and the CV was 6.00%; the quality control product 2SD was 6.00, and the CV was 2.95%; the quality control product 1SD measured by the scheme of Example 2 was 0.70, and the CV was 3.35%; the quality control product 2SD was 3.47, and the CV was 1.70%; the quality control product 1SD measured by the scheme of Example 3 was 1.37, and the CV was 6.32%; the quality control product 2SD was 5.78, and the CV was 2.90%. All CVs were <8%.

[0161] The following conclusions were drawn: All CVs were <8%, and comparison of SD revealed that the scheme in Example 2 had more stable repeatability, smaller deviation, and met the requirements.

[0162] In summary, the Alzheimer's disease pTau181 magnetic microparticle chemiluminescence immunoassay kit disclosed in the present invention has higher detection sensitivity, specificity, precision and accuracy, and achieves better performance parameters. The magnetic microparticle reagent, AE labeled antibody and calibrator in the kit are the optimal formula under the reaction system, which provides a strong guarantee for the detection performance of the kit.

[0163] It should be noted that relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0164] Although the present application has been described above with reference to specific embodiments, various modifications may be made thereto and parts thereof may be replaced with equivalents without departing from the scope of the present application. In particular, as long as there is no structural conflict, the various features in the specific embodiments disclosed in the present application may be used in combination with each other in any manner, and the fact that these combinations are not exhaustively described in this specification is only for the sake of omitting space and saving resources. Therefore, the present application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for preparing a pTau181 chemiluminescent immunoassay kit for Alzheimer's disease, the kit comprising a magnetic particle reagent, an AE-labeled pTau-181 antibody, and a pTau-181 calibrator, wherein the magnetic particle reagent is a magnetic microsphere suspension coated with a pTau-181 antibody; the AE-labeled pTau-181 antibody is obtained by labeling the pTau-181 antibody with an acridinium ester; The particle size of the magnetic particles in the magnetic particle reagent is 1-3 μm, and the surface of the magnetic particles is coated with magnetic microspheres with carboxyl active groups; The mass ratio of pTau-181 antibody to acridinium ester in the AE-labeled pTau-181 antibody is 1:1; It is characterized in that The steps include: Step 1. preparing magnetic particle reagent; Step 1.

1. Dispersing the carboxyl magnetic bead solution in a magnetic particle buffer to obtain a dispersed carboxyl magnetic bead solution; In step 1.1, the carboxyl magnetic bead concentrate is placed in a magnetic field for 1 to 3 minutes, and after all the carboxyl magnetic beads are completely precipitated, the supernatant is removed, and then added to the magnetic particle buffer for oscillation, and then placed in a magnetic field for 1 to 3 minutes, and then the supernatant is removed; the oscillation time is set to 20 to 30 minutes; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:2 to 5, and the number of dispersion treatments is 3 times; Step 1.

2. The dispersed carboxyl magnetic bead solution and the pTau-181 antibody are subjected to a connection reaction to obtain a connection product; The intercommunication implementation method of step 1.2 includes the following steps: Step 1.2.

1. Weigh the dispersed carboxyl magnetic bead solution and pTau-181 antibody at a mass ratio of 100:5 to 100:1, the concentration of the dispersed carboxyl magnetic bead solution is 10 to 50 mg / mL, the connection reaction temperature is set to room temperature, and the connection reaction time is 16 to 20 hours; S1.2.

2. After the ligation reaction is completed, a blocking solution is added for blocking. The formula of the blocking solution is: a mixed solution of PBS, 3wt% BSA, 1wt% Tween-20, 1wt% maltose, 5wt% sorbitol, 2wt% sucrose, 3wt% glycerol, and 0.5wt% PEG600; Step 1.

3. Place the ligated product in a magnetic field and wash it with a magnetic particle buffer to obtain a magnetic particle solution connected with the pTau-181 antibody; The connection product in step 1.3 is placed in the magnetic field for 5 to 10 minutes, and then washed with a magnetic particle buffer to obtain a magnetic particle solution connected with the pTau-181 antibody; Step 2. Prepare AE-labeled pTau-181 antibody; Step 2.

1. Prepare AE solution with a concentration of 5 mg / mL using 0.15 M sodium bicarbonate buffer; Step 2.

2. The mouse monoclonal anti-pTau-181 antibody and the AE solution were mixed at a mass ratio of 1:1 to perform a linking reaction to obtain a linking product of the pTau-181 antibody and AE; Step 2.

3. The linkage product of the pTau-181 antibody and AE is separated and purified using a carbonate buffer having a pH of 8 to 9 to obtain an AE-labeled pTau-181 antibody; Step 3. Prepare pTau-181 calibrator: The pTau-181 calibrator is a Tris buffer containing pTau-181 antigen, 0.5% by mass of BSA, and 0.05% by mass of prolin300; the concentrations of the pTau-181 antigen are 20 pg / mL and 200 pg / mL, respectively.

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