CN114034872A - Kit for early diagnosis of Alzheimer's disease and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biological detection, in particular to a kit for early diagnosis of Alzheimer's disease and application thereof. The kit achieves the purpose of early diagnosis of the Alzheimer's disease by detecting the combined detection of T-Tau, p-Tau-181, p-Tau-217 and p-Tau-231 in serum and plasma. The kit has the advantages of wide detection range, high sensitivity, good precision, suitability for a full-automatic chemiluminescence apparatus, simple operation, suitability for the requirements of clinical detection of a large amount of serum and plasma, and capability of providing more accurate and personalized guidance suggestions for early diagnosis of the Alzheimer's disease.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a kit for early diagnosis of Alzheimer's disease and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Alzheimer's Disease (AD), also known as primary senile dementia, is a fatal neurodegenerative Disease, the most common type of dementia, with about 50% to 70% of dementia belonging to this class of dementia; its main symptoms are hypomnesis, cognitive decline, behavior decline, life decline, and so on, and it may also cause depression, anxiety and other complications with great harm. Currently, the main diagnostic means of AD are neuropsychological scales, imaging examinations, biomarker detection, and the like. The neuropsychological scale is used for evaluating the cognitive function of the testee, and the scales such as a simple mental state scale, a Terier cognitive evaluation scale, a ChangYuchuan dementia scale and the like are mainly adopted at present, but the test result has large fluctuation due to different education degrees and individual comprehension abilities of the testee; the neuroimaging detection mainly comprises Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), Computer Tomography (CT) and the like, which are strong evidences for AD diagnosis, wherein the CT can directly display the brain atrophy phenomenon of AD patients, but the value of the CT is limited due to poor soft tissue resolution, and the MRI and the PET are expensive and are not suitable for population screening.
Cerebrospinal fluid (CSF) biomarkers are currently accepted sources of AD markers, and cerebrospinal fluid examination can be used to rule out rare, reversible cognitive impairment diseases, and also to aid in positive molecular diagnosis of AD. CSF isolation and invasive procedures, however, limit the use of these biomarkers in the diagnosis of early AD. Current research on blood biomarkers involved in the pathogenesis of AD involves multiple markers, including Α β, Tau protein, P-Tau protein, etc.; these biomarkers can also be measured more quickly, less in plasma, and can provide an important basis for the development of early diagnosis of alzheimer's disease.
However, the inventor finds that the detection kit for the alzheimer disease in the prior art often has the following problems: the kit has the problems of complex operation procedure, high cost, long detection time, low sensitivity, poor precision, narrow linear range, incapability of realizing automatic and batch measurement, incapability of realizing specific detection, lack of accurate prediction capability on the Alzheimer disease and the like; but also is not suitable for clinical diagnosis at all, and has very limited clinical application significance.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a kit for early diagnosis of Alzheimer's disease and application thereof, wherein magnetic particles and a chemiluminescence technology are combined in the kit, so that the problems of complex operation, high cost, long detection time, low sensitivity, poor precision, lack of accurate prediction capability on Alzheimer's disease and the like of the existing kit for detecting Alzheimer's disease are solved, and the kit has important application value and significance for large-range early screening of Alzheimer's disease.
In order to achieve the above object, the technical solution of the present invention is as follows:
in a first aspect of the present invention, there is provided a kit for early diagnosis of alzheimer's disease, the kit comprising: reagent A, reagent B and substrate solution;
the reagent A comprises: a1: a biotin-labeled monoclonal antibody for recognizing the T-Tau site;
a2: a monoclonal antibody labeled with biotin for recognizing the p-Tau-181 site;
a3: a biotin-labeled monoclonal antibody for recognizing the p-Tau-217 site;
a4: a biotin-labeled monoclonal antibody for recognizing the p-Tau-231 site;
the reagent B comprises: the monoclonal antibody which is used for identifying the Tau protein N1-17 sites and is marked with acridine ester;
wherein, the biotin-labeled monoclonal antibody is combined with streptavidin magnetic beads.
In one or more embodiments, the substrate solution includes a substrate solution a and a substrate solution B, the substrate solution a is a hydrogen peroxide solution, and the substrate solution B is a sodium hydroxide solution.
The working principle is as follows: and quantitatively determining the contents of T-Tau, p-Tau-181, p-Tau-217 and p-Tau-231 in the sample by a double-antibody sandwich method and a magnetic particle chemiluminescence immunoassay method. The technical principle of the reaction is as follows: the biotin-labeled monoclonal antibody (recognizing the T-Tau, p-Tau-181, p-Tau-217 or p-Tau-231 sites, respectively) is bound to streptavidin magnetic beads, and forms an antibody-antigen-antibody sandwich complex with a calibrator, a quality control product or a substance to be detected (T-Tau, p-Tau-181, p-Tau-217 or p-Tau-231) in a sample and the labeled acridinium ester monoclonal antibody (recognizing the Tau protein N1-17 sites), and the antibody-antigen-antibody sandwich complex is directly precipitated in an external magnetic field, so that the complex formed by immunoreaction is separated from other unbound substances. After removing the supernatant, the precipitated complex is washed and added with chemiluminescent substrate solutions A and B. The acridinium ester is catalytically cracked under alkaline conditions to form an unstable excited state intermediate, and when the excited state intermediate returns to a ground state, photons are emitted to form a luminescence reaction, namely, a luminometer is used for detecting the luminescence intensity of the reaction. The luminous intensity is in direct proportion to the content of the detected substance in the sample, and the concentration of the detected substance in the sample can be calculated by using four-parameter Logistic equation fitting.
In a second aspect of the invention, there is provided the use of the kit of the first aspect in the early diagnosis of alzheimer's disease.
In a third aspect of the invention, a system for early diagnosis of alzheimer's disease is provided, comprising a detection module, a data processing module and a result display module; the detection module is used for detecting the content of the substance to be detected in the sample by using the kit of the first aspect, and the data processing module is internally provided with a logistic regression algorithm for calculating the probability of the patient suffering from the Alzheimer's disease based on the detection result of the marker and the patient information, and the result display module is used for outputting a calculation result heat map.
The fourth aspect of the invention provides a combined detection method of T-Tau, p-Tau-181, p-Tau-217 and p-Tau-231, which comprises the detection by adopting the kit.
The specific embodiment of the invention has the following beneficial effects:
the invention provides a kit for early diagnosis of Alzheimer's disease, wherein the first kit adopts a joint detection mode of multiple markers such as T-Tau, p-Tau-181, p-Tau-217, p-Tau-231 and the like to improve the specificity and sensitivity of early diagnosis and screening of Alzheimer's disease; the kit and the analysis method have higher detection sensitivity, specificity and stability by adopting the high affinity and multi-stage amplification effect of biotin and streptavidin; the method of pre-coating streptavidin magnetic beads and antibody-biotin labels in advance is adopted, so that the detection process and time are simplified; the stability of the reagent and the repeatability of detection are improved by adopting a unique buffer system.
The kit for detecting the human serum sample realizes small wound, easy operation, accurate detection, automation and more suitability for crowd screening and early diagnosis; the magnetic particles are combined with a chemiluminescence technology, the detection range is wide, the sensitivity is high, the precision is good, the method is suitable for a full-automatic chemiluminescence apparatus, the operation is simple, and the method is suitable for the requirement of clinical detection of a large amount of serum.
The kit result can be combined with a large amount of long-term clinical information (such as the age of a patient, the APOE carrying condition, the education degree, the mutation condition of APP protein and gamma secretase), and provides more accurate and personalized guidance for early diagnosis of the Alzheimer's disease.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In one embodiment of the present invention, there is provided a kit for early diagnosis of alzheimer's disease, comprising: reagent A, reagent B and substrate solution; the reagent A comprises: a1: a biotin-labeled monoclonal antibody for recognizing the T-Tau site; a2: a monoclonal antibody labeled with biotin for recognizing the p-Tau-181 site; a3: a biotin-labeled monoclonal antibody for recognizing the p-Tau-217 site; a4: a biotin-labeled monoclonal antibody for recognizing the p-Tau-231 site; the reagent B comprises: the monoclonal antibody which is used for identifying the Tau protein N1-17 sites and is marked with acridine ester; the biotin-labeled monoclonal antibody was bound to streptavidin magnetic beads.
In one or more embodiments of the present invention, the substrate solution comprises a substrate solution a and a substrate solution B; the substrate solution A is hydrogen peroxide solution; the substrate solution B is NaOH solution and provides an alkaline hydrogen peroxide environment.
In one or more embodiments of the invention, the kit further comprises a calibrator;
the calibrator is Tau protein which is fully phosphorylated, is diluted by calibrator buffer solution, and is subjected to assignment by using T-Tau, p-Tau-181, p-Tau-217 and p-Tau-231 respectively and then is subjected to freeze-drying, so as to prepare the composite universal calibrator.
In one embodiment of the present invention, there is provided a method for preparing the above kit, the method comprising:
the preparation method of the reagent A comprises the following steps: uniformly mixing the T-Tau, p-Tau-181, p-Tau-217 and p-Tau-231 monoclonal antibodies with biotin in a molar ratio of 1:10-1:40, uniformly mixing the mixture at room temperature for reaction for 1-4 h, and then placing the reactant in a PBS buffer solution for dialysis to obtain a biotin-labeled monoclonal antibody; adding the biotin-labeled monoclonal antibody into a buffer system of streptavidin magnetic microspheres, wherein the coupling ratio of biotinylated protein to the streptavidin magnetic microspheres is 10-30 mug: 1 mg.
The preparation method of the reagent B comprises the following steps: uniformly mixing the monoclonal antibody of the Tau protein N1-17 site with acridinium ester according to the molar ratio of 1:10-1:40, reacting for 2-4 h at room temperature, adding lysine to terminate the coupling reaction, and placing the reaction product in PBS buffer solution for dialysis to obtain the product.
Preferably, the monoclonal antibody of the Tau protein N1-17 site is dissolved by borate buffer solution, and the concentration is 1-3 mg/ml.
Preferably, the concentration of the acridinium ester is 2-10 mg/ml.
Preferably, the concentration of the lysine is 100-500 mg/ml, and the input amount is 5-40 times of the antibody amount.
Preferably, the time for mixing after adding lysine is 15 min-2 h.
Preferably, the dialysis frequency of the reaction product is 2-4 times.
In an embodiment of the present invention, the method for using the kit for early diagnosis of alzheimer's disease comprises: mixing a sample to be treated with the reagent A, incubating for 5-15 min, adding the reagent B into the reaction system after cleaning by using a cleaning solution, continuing incubating for 5-15 min, cleaning by an external magnetic field, adding the substrate solution A and the substrate solution B, and detecting a luminescent signal.
The invention will be further explained and illustrated with reference to specific examples.
Example 1
In the present embodiment, a kit for early diagnosis of alzheimer's disease is provided, which comprises: the kit comprises a reagent A1 (T-Tau magnetic microsphere working solution), a2 (p-Tau-181 magnetic microsphere working solution), A3 (p-Tau-231 magnetic microsphere working solution), A4 (four kinds of p-Tau-217 magnetic microsphere working solution), a reagent B1 (acridinium ester working solution prepared by monoclonal antibodies for recognizing Tau protein N1-17 sites), and a calibrator (a universal composite calibrator for establishing a standard curve).
The method comprises the following steps: preparation of buffer solution
1. Preparation method of buffer system of magnetic microspheres
Adjusting the pH value to 7.3 in a 0.15M PBS buffer system, adding 3% BSA, 2% trehalose, 0.05% magnetic microsphere dispersant, 0.25% Tween 20 and 0.05% Proclin 300, fully and uniformly mixing until the mixture is completely dissolved, adding a proper amount of purified water for quantification, stirring and uniformly mixing for 1 h, and adjusting the pH value to 7.3. The mixture was filtered using a filter and stored at 4 ℃ for further use.
2. Preparation method of acridine ester buffer solution
Adjusting the pH value to 6.0 in a 0.05M MES buffer system, adding 0.8% BSA, 2% sucrose, 0.25% Tween 20 and 0.05% Proclin 300, fully and uniformly mixing until completely dissolving, adding a proper amount of purified water for quantification, stirring and uniformly mixing for 1 h, and verifying whether the pH value is 6.0 or not. The mixture was filtered using a filter and stored at 4 ℃ for further use.
3. Preparation method of calibrator buffer solution
Adding 5% saccharide, 1.5% BSA, 10 mM EDTA and 1.5% mannitol into a 0.15M PBS buffer system, mixing for 1 h, adjusting pH to 8.2, adding 0.1% surfactant and 1 ‰ preservative, mixing for 1 h, verifying pH to 8.2, filtering with a filter, and storing at 4 deg.C.
Step two: preparation of reagents A1, A2, A3, A4
1. Biotin was coupled to T-Tau, p-Tau-181, p-Tau-217 and p-Tau-231 monoclonal antibodies using the following procedure
Diluting the T-Tau, p-Tau-181, p-Tau-217 and p-Tau-231 monoclonal antibodies into 2 mg/mL by using CB buffer solution, uniformly mixing the diluted monoclonal antibodies with biotin with the prepared concentration of 35 mg/mL according to the molar ratio of 1:20, uniformly mixing the diluted monoclonal antibodies and the biotin at room temperature for reaction for 2 hours, finally placing reactants in PBS buffer solution by using a dialysis bag for dialysis, changing the dialysate every two hours, repeating the steps for 3 times, and storing the solution obtained by dialysis at 4 ℃ for later use.
2. Streptavidin magnetic beads and biotinylated T-Tau, p-Tau-181, p-Tau-217 and p-Tau-231 monoclonal antibodies were coupled using the following steps:
washing the streptavidin magnetic microspheres for 2 times by using Tris-HCl buffer solution, removing supernatant, carrying out redissolution by using Tris-HCl buffer solution, adding biotinylated T-Tau, p-Tau-181, p-Tau-217 and p-Tau-231 monoclonal antibodies, wherein the coupling ratio of biotinylated protein and the streptavidin magnetic microspheres is 20 mu g:1mg, and uniformly mixing at room temperature for 1.5 h. The reacted magnetic microspheres were washed 3 times with Tris-HCl buffer, the supernatant removed, and reconstituted with PBS buffer containing protein to a storage concentration of 3 mg/mL.
3. Preparation method of reagent A1, A2, A3 and A4-magnetic bead working solution
The mother liquor of the magnetic microspheres coated with the T-Tau, the p-Tau-181, the p-Tau-217 and the p-Tau-231 monoclonal antibodies is respectively diluted to ensure that the final concentration of the T-Tau, the p-Tau-181, the p-Tau-217 and the p-Tau-231 monoclonal antibodies is 0.75 mg/mL, and the mother liquor is stored for later use at 4 ℃.
Step three: preparation of reagent B
The monoclonal antibody for identifying the Tau protein N1-17 sites is coupled with acridinium ester by adopting the following steps:
diluting the monoclonal antibody for identifying the Tau protein N1-17 site to 2 mg/mL by using borate buffer solution, uniformly mixing the monoclonal antibody with the acridine ester with the preparation concentration of 6 mg/mL according to the molar ratio of 1:20, and uniformly mixing at room temperature for reacting for 3 hours; then adding prepared lysine stop solution of 300 mg/mL for stopping, uniformly mixing the lysine with the antibody amount of 20 times of that of the lysine, reacting for 1 h, finally putting the reactant into PBS buffer solution for dialysis by using a dialysis bag, changing the dialysate every two hours, repeating the dialysis for 3 times, and storing the solution obtained by dialysis at 4 ℃ for later use.
Step four: preparation of calibrator
The calibrator is total phosphorylated Tau protein, is diluted by calibrator buffer solution, is subjected to assignment by using T-Tau, p-Tau-181, p-Tau-217 and p-Tau-231 respectively, and is then lyophilized to prepare the composite universal calibrator.
Step five: the use method of the kit for detecting the contents of T-Tau, p-Tau-181, p-Tau-217 and p-Tau-231
The full-automatic magnetic particle chemiluminescence apparatus takes 60 ul of magnetic microsphere working solution into an incubation cup, then 60 ul of calibrator, quality control material or sample is added, incubation is carried out for 10 min at 37 ℃, cleaning is carried out for 3 times by 400 ul of cleaning solution, 70 ul of acridinium ester working solution is added, incubation is carried out for 10 min at 37 ℃, cleaning is carried out for 3 times by 400 ul of cleaning solution, and finally, 100 ul of chemiluminescence substrates A and B are added, luminescence values are detected, the concentrations of T-Tau, p-Tau-181, p-Tau-217 and p-Tau-231 are respectively calculated according to a calibrator curve, and finally, the risk coefficient of Alzheimer's disease is calculated through a data processing module.
In this embodiment, the accuracy of the kit for early diagnosis of alzheimer's disease is examined, the examination items include precision, detection limit, and sensitivity, and the examination results are as follows:
(1) precision: the kit for jointly detecting the early detection of the Alzheimer's disease by using three batches of kits is used for respectively testing samples with high and low levels, the parallel determination is carried out for 10 times, and the determination result is calculated, wherein the precision CV among batches is less than 10%.
TABLE 1 results of precision between batches
As can be seen from the data in the table, the precision of the same sample measured by the three batches of kits is less than 10% between batches, which shows that the difference between the kits of different batches is very small, and the measurement result has repeatability
(2) Linear range: according to the specification requirements of the kit, linear gradient samples of each item are respectively detected on a full-automatic chemiluminescence instrument, the samples with each concentration are repeatedly detected for three times, the average value is calculated, and the correlation coefficient in the linear range of each item is calculated to be more than or equal to 0.99.
TABLE 2 Linear Range determination of Experimental results
As shown by the linearity of the dose-response curve calculated in Table 2, the correlation coefficient of each item is more than or equal to 0.99 in the linear range, and the correlation coefficient meets the requirement.
(3) The lowest detection limit is: low concentration samples are selected for multiple dilution to prepare a series of low concentration samples, and the batch-to-batch CV of each concentration sample is determined and calculated, with the analyte content of the sample with the CV closest to 20% being the functional sensitivity.
As can be seen from the following table 3, the minimum detection limit of the kit is not more than 0.05 pg/mL, and each item can be accurately detected.
TABLE 3 sensitivity test results
(4) Clinical validation of the kit of this example
In order to determine the applicability of the kit of this embodiment for the detection of early stage alzheimer's disease, 228 samples from hospital normal persons, 47 sera from confirmed alzheimer's disease patients, and 41 sera from confirmed cognitive impairment patients were taken and detected by the kit of this embodiment, and the detection results are shown in table 4;
the verification result shows that the kit in the embodiment has a positive rate of 97.87% for the Alzheimer disease patients, 1.32% for the healthy people and 2.44% for the cognitive impairment, and is obviously superior to other combined detection items. The kit of the embodiment can highlight the differential diagnosis of the Alzheimer disease patient and has better clinical practicability.
TABLE 4 results of clinical sample determination
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A kit for early diagnosis of alzheimer's disease, comprising: reagent A, reagent B and substrate solution; the reagent A comprises: a1: a biotin-labeled monoclonal antibody for recognizing the T-tau site; a2: a monoclonal antibody labeled with biotin for recognizing the p-Tau-181 site; a3: a biotin-labeled monoclonal antibody for recognizing the p-Tau-217 site; a4: a biotin-labeled monoclonal antibody for recognizing the p-Tau-231 site;
the reagent B comprises: monoclonal antibodies labeled with acridinium esters for recognizing tau protein N1-17 sites;
wherein, the biotin-labeled monoclonal antibody is combined with streptavidin magnetic beads.
2. The kit for early diagnosis of alzheimer's disease according to claim 1, wherein said substrate solution comprises substrate solution a and substrate solution B; the substrate solution A is hydrogen peroxide solution, and the substrate solution B is sodium hydroxide solution.
3. Use of the kit of claim 1 or 2 for the early diagnosis of alzheimer's disease.
4. A method for the combined detection of T-Tau, p-Tau-181, p-Tau-217, p-Tau-231 in serum and plasma, said method comprising the use of a kit according to claim 1 or 2 for the detection.
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| CN118707111A (en) * | 2024-08-27 | 2024-09-27 | 浙江瑞硕生物技术有限公司 | Alzheimer's disease pTau181 chemiluminescence immunoassay kit, preparation method and detection method thereof |
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