CN118707111A - Alzheimer's disease pTau181 chemiluminescence immunoassay kit, preparation method and detection method thereof - Google Patents
Alzheimer's disease pTau181 chemiluminescence immunoassay kit, preparation method and detection method thereof Download PDFInfo
- Publication number
- CN118707111A CN118707111A CN202411177922.XA CN202411177922A CN118707111A CN 118707111 A CN118707111 A CN 118707111A CN 202411177922 A CN202411177922 A CN 202411177922A CN 118707111 A CN118707111 A CN 118707111A
- Authority
- CN
- China
- Prior art keywords
- ptau
- antibody
- magnetic
- magnetic particle
- disease
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 208000024827 Alzheimer disease Diseases 0.000 title claims abstract description 38
- 238000003018 immunoassay Methods 0.000 title claims abstract description 29
- 238000001514 detection method Methods 0.000 title abstract description 27
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 239000006249 magnetic particle Substances 0.000 claims abstract description 77
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 31
- 239000004005 microsphere Substances 0.000 claims abstract description 13
- -1 acridine ester Chemical class 0.000 claims abstract description 7
- 239000000725 suspension Substances 0.000 claims abstract description 7
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000002372 labelling Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 70
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 54
- 239000011324 bead Substances 0.000 claims description 52
- 239000000047 product Substances 0.000 claims description 42
- 238000006243 chemical reaction Methods 0.000 claims description 37
- 239000007853 buffer solution Substances 0.000 claims description 31
- 238000003908 quality control method Methods 0.000 claims description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 15
- 238000007789 sealing Methods 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 13
- 239000000427 antigen Substances 0.000 claims description 11
- 102000036639 antigens Human genes 0.000 claims description 11
- 108091007433 antigens Proteins 0.000 claims description 11
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 230000010355 oscillation Effects 0.000 claims description 10
- 230000009471 action Effects 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 8
- 210000002966 serum Anatomy 0.000 claims description 8
- 229920002582 Polyethylene Glycol 600 Polymers 0.000 claims description 7
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 5
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 5
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 5
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 239000007983 Tris buffer Substances 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- 239000006185 dispersion Substances 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 5
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 5
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 5
- 239000000600 sorbitol Substances 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical group OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 230000008569 process Effects 0.000 abstract description 5
- 238000011049 filling Methods 0.000 abstract description 2
- 239000011159 matrix material Substances 0.000 description 8
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 238000011033 desalting Methods 0.000 description 5
- 102000013498 tau Proteins Human genes 0.000 description 5
- 108010026424 tau Proteins Proteins 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000004020 luminiscence type Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 3
- 238000001641 gel filtration chromatography Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000026731 phosphorylation Effects 0.000 description 3
- 238000006366 phosphorylation reaction Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 2
- 230000006933 amyloid-beta aggregation Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 208000010877 cognitive disease Diseases 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000007863 gel particle Substances 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 239000011859 microparticle Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- LRWZZZWJMFNZIK-UHFFFAOYSA-N 2-chloro-3-methyloxirane Chemical compound CC1OC1Cl LRWZZZWJMFNZIK-UHFFFAOYSA-N 0.000 description 1
- 208000037259 Amyloid Plaque Diseases 0.000 description 1
- 208000028698 Cognitive impairment Diseases 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 230000001713 cholinergic effect Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 208000027061 mild cognitive impairment Diseases 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000002314 neuroinflammatory effect Effects 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000003557 neuropsychological effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
A chemiluminescent immunoassay kit for Alzheimer's disease pTau181 and a preparation method and a detection method thereof belong to the technical field of immunodetection. In order to solve the problems of complex operation, high cost, long detection time, low sensitivity and poor precision of the existing Alzheimer disease detection kit, the invention comprises a magnetic particle reagent, an AE-marked pTau-181 antibody and a pTau-181 calibrator, wherein 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 acridine ester. The invention has low pretreatment requirement on samples, simple pretreatment process, rapid and high-flux detection of a large number of samples, and the detection method is convenient and easy to implement, thus filling the blank in the prior art.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a chemiluminescent immunoassay kit for Alzheimer's disease pTau181, a preparation method and a detection method thereof.
Background
Alzheimer's Disease (AD) is a neurological degenerative disease with hidden onset and slow progressive exacerbation, commonly seen in elderly people over 65 years of age, with significant increases in incidence with age. The pathogenic hypothesis that changes in the pathology of AD are currently widely accepted is: abeta aggregation hypothesis, tau protein abnormality hypothesis, cholinergic hypothesis, neuroinflammatory hypothesis, etc., wherein phosphorylation modification of Tau protein is critical in the course of the disease, tau and APP can undergo phosphorylation modification at multiple sites, and are largely classified into T-Tau, pTau-181, pTau-217, 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.
Current methods of diagnosis of AD are mainly combined diagnosis, comprising: neuropsychological assessment, cognitive impairment testing; PET scanning of senile plaques and Tau proteins of the brain; brain nuclear Magnetic Resonance (MRI) and cerebrospinal fluid (CSF) markers, aβ deposition, phosphorylated tau detection, and the like. However, since early symptoms of AD are not obvious, when the disease is clearly diagnosed, the patient reaches late stage of the disease course, most neurons die, if the disease can be rapidly diagnosed in early stage of the disease of the patient or the potential patient is found in advance to be at risk, the disease course of the patient can be retained in a slight mental injury stage (mild cognitive impairment, MCI) to slow down the deterioration, so that the life quality of the patient is ensured and the social burden is lightened. Therefore, how to provide a detection reagent and a detection method for pTau-181 is a problem to be solved by those skilled in the art.
Disclosure of Invention
The invention aims to solve the problems of complex operation, high cost, long detection time, low sensitivity, poor precision, lack of accurate prediction capability for Alzheimer's disease and the like of the existing Alzheimer's disease detection kit, and provides a Alzheimer's disease pTau181 chemiluminescence immunoassay kit, a preparation method thereof and a detection method thereof.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
The kit comprises a magnetic particle reagent, an AE-marked pTau-181 antibody and a pTau-181 calibrator, wherein 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 acridine ester.
Further, the particle size of the magnetic particles in the magnetic particle reagent is 1-3 mu m, and the surfaces of the magnetic particles are coated with magnetic microspheres with carboxyl active groups;
When the magnetic microsphere suspension of the pTau-181 antibody is prepared, 0.5-2.5mg of the pTau-181 antibody is added for coating every 50mg of magnetic microspheres, the mass ratio of the pTau-181 antibody to the carboxyl magnetic beads in the magnetic particle reagent is 1:20-100, the temperature of the connection reaction is room temperature, and the time of the connection reaction is 16-20 h.
Further, the mass ratio of the pTau-181 antibody to the acridine ester in the AE-labeled pTau-181 antibody is 1:1.
A preparation method of a chemiluminescent immunoassay kit for Alzheimer's disease pTau181 comprises the following steps:
Step1, preparing a magnetic particle reagent;
Step 1.1, dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer solution to obtain a dispersed carboxyl magnetic bead solution;
Step 1.2, carrying out a connection reaction on the dispersed carboxyl magnetic bead solution and the pTau-181 antibody to obtain a connection product;
Step 1.3, placing the connection product in a magnetic field, and flushing with a magnetic particle buffer solution to prepare a magnetic particle solution connected with the pTau-181 antibody;
Step 2, preparing AE marked pTau-181 antibody;
step 2.1, preparing AE into AE solution with concentration of 5mg/mL by using 0.15M sodium bicarbonate buffer solution;
Step 2.2, mixing the mouse monoclonal anti-pTau-181 antibody and AE solution according to the mass ratio of 1:1, and carrying out a linking reaction to obtain a linking product of the pTau-181 antibody and AE;
Step 2.3, separating and purifying the linking product of the pTau-181 antibody and AE by using carbonate buffer solution with pH of 8-9 to obtain AE marked pTau-181 antibody;
step 3, preparing a pTau-181 calibrator: the pTau-181 calibrator is Tris buffer containing pTau-181 antigen, BSA with the mass percentage of 0.5% and prolin300,300 with the mass percentage of 0.05%; the concentration of pTau-181 antigen was 20pg/mL and 200pg/mL, respectively.
Further, in the step 1.1, the carboxyl magnetic bead concentrated solution is placed in a magnetic field for 1 to 3 minutes, the supernatant is removed after all carboxyl magnetic beads are settled, then the carboxyl magnetic bead concentrated solution is added into a magnetic particle buffer solution for oscillation, and the supernatant is removed after the carboxyl magnetic bead concentrated solution is placed in the magnetic field for 1 to 3 minutes; the oscillation time is set to 20-30 min; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:2-5, and the dispersion treatment times are 3 times.
Further, the method for implementing the intercommunication in the step 1.2 comprises the following steps:
Step 1.2.1, according to the mass ratio of 100:5-100: 1, weighing a dispersed carboxyl magnetic bead solution and pTau-181 antibody, wherein the concentration of the dispersed carboxyl magnetic bead solution is 10-50 mg/mL, the temperature of the connection reaction is set to be room temperature, and the time of the connection reaction is 16-20 h;
S1.2.2 after the connection reaction is finished, adding a sealing liquid for sealing, wherein the formula of the sealing liquid is as follows: PBS, 3wt% BSA, 1wt% Tween-20, 1wt% maltose, 5wt% sorbitol, 2wt% sucrose, 3wt% glycerol, 0.1-0.8 wt% PEG 600.
Further, the ligation product in step 1.3 was subjected to a magnetic field for 5 to 10 minutes, and then washed with a magnetic particle buffer solution to prepare a magnetic particle solution to which pTau-181 antibody was ligated.
A detection method of a chemiluminescent immunoassay kit for Alzheimer's disease pTau181 comprises the following steps:
S1, preparing a magnetic particle reagent and an AE-marked pTau-181 antibody;
S2, adding the serum sample and the AE-marked pTau-181 antibody prepared in the step S1 into a magnetic particle reagent for incubation;
And S3, after the incubation is completed and the magnetic particle reagent is settled, removing supernatant, cleaning, adding a substrate solution, shaking and mixing uniformly, and detecting the relative luminous intensity.
Further, the volume ratio of the serum sample, AE-labeled pTau-181 antibody and magnetic particle reagent in step S2 is 2:2:1.
Further, after detecting the relative luminous intensity in the step S3, calibrating and calibrating by using a high-value calibration material and a low-value calibration material, and fitting to generate a new working curve for sample measurement; and taking the pTau-181 quality control product as a sample, performing concentration fitting by using a working curve to obtain the corresponding quality control product concentration, and observing that the fitting quality control product concentration is +/-30% of a standard concentration value.
The invention has the beneficial effects that:
The invention relates to a chemiluminescent immunoassay kit for Alzheimer's disease pTau181, which uses magnetic beads as a solid phase, so that the immune reaction is closer to a liquid phase, the reaction is more complete and rapid, the combined immune complex is easier to separate, and the nonspecific adsorption is reduced. The AE labeled antibody reagent used in the invention is a monoclonal antibody mixture, so that the affinity of immune reaction is higher, the difference between production batches of the monoclonal antibody is relatively small, and the stability between batches of the product is easier to ensure.
The invention provides possibility for rapid and accurate pTau-181 detection, has low pretreatment requirement on samples, simple pretreatment process, and can rapidly and high-flux detect a large number of samples, and the detection method is convenient and easy to implement, thereby filling the blank in the prior art.
The chemiluminescent immunoassay kit for Alzheimer's disease pTau181 combines chemiluminescent technology with immunomagnetic particles, provides a nearly homogeneous reaction system, has higher detection sensitivity, specificity, accuracy and precision, and achieves better performance parameters. The magnetic particle reagent, the AE labeled antibody and the calibrator in the kit are optimal formulas under the reaction system, and provide powerful guarantee for the service life and detection performance of the kit.
Drawings
FIG. 1 is a standard curve data chart of example 1 of the present invention;
FIG. 2 is a graph of standard curve data for example 2 of the present invention;
FIG. 3 is a graph of standard curve data for example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail below with reference to the accompanying drawings and detailed description. It should be understood that the embodiments described herein are for purposes of illustration only and are not intended to limit the invention, i.e., the embodiments described are merely some, but not all, of the embodiments of the invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein can be arranged and designed in a wide variety of different configurations, and the present invention can have other embodiments as well.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.
For further understanding of the invention, the following detailed description is to be taken in conjunction with fig. 1-3, in which the following detailed description is given, of the invention:
example 1:
a preparation method of a chemiluminescent immunoassay kit for Alzheimer's disease pTau181 comprises the following steps:
Step1, preparing a magnetic particle reagent;
Step 1.1, dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer solution to obtain a dispersed carboxyl magnetic bead solution;
Further, in the step 1.1, the carboxyl magnetic bead concentrated solution is placed in a magnetic field for 1min, the supernatant is removed after all carboxyl magnetic beads are settled, then the carboxyl magnetic bead concentrated solution is added into a magnetic particle buffer solution for oscillation, and the carboxyl magnetic bead concentrated solution is placed in the magnetic field for 1min, and then the supernatant is removed; the oscillation time is set to 20-30 min; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:2, and the dispersion treatment times are 3 times;
Step 1.2, carrying out a connection reaction on the dispersed carboxyl magnetic bead solution and the pTau-181 antibody to obtain a connection product;
further, the method for implementing the intercommunication in the step 1.2 comprises the following steps:
Step 1.2.1, weighing the dispersed carboxyl magnetic bead solution and the pTau-181 antibody according to the mass ratio of 100:5, wherein the concentration of the dispersed carboxyl magnetic bead solution is 10mg/mL, setting the temperature of the connection reaction to be room temperature, and the time of the connection reaction to be 16h;
S1.2.2 after the connection reaction is finished, adding a sealing liquid for sealing, wherein the formula of the sealing liquid is as follows: a mixed solution of PBS, 3wt% BSA, 1wt% Tween-20, 1wt% maltose, 5wt% sorbitol, 2wt% sucrose, 3wt% glycerol, 0.1 wt% PEG 600;
Step 1.3, placing the connection product in a magnetic field, and flushing with a magnetic particle buffer solution to prepare a magnetic particle solution connected with the pTau-181 antibody;
Further, the connection product in the step 1.3 is placed in a magnetic field for 1-3min, and then washed by a magnetic particle buffer solution to prepare a magnetic particle solution connected with the pTau-181 antibody;
further, the time of placing the connection product in a magnetic field is 1-3min, and the washing times are 3 times; the concentration of the magnetic particle solution connected with the pTau-181 antibody is 10mg/mL;
Step 2, preparing AE marked pTau-181 antibody;
step 2.1, preparing AE into AE solution with concentration of 5mg/mL by using 0.15M sodium bicarbonate buffer solution;
Step 2.2, mixing the mouse monoclonal anti-pTau-181 antibody and AE solution according to the mass ratio of 1:1, and carrying out a linking reaction to obtain a linking product of the pTau-181 antibody and AE;
Step 2.3, separating and purifying the linking product of the pTau-181 antibody and AE by using carbonate buffer solution with pH of 8to obtain an AE-marked pTau-181 antibody;
step 3, preparing a pTau-181 calibrator: the pTau-181 calibrator is Tris buffer containing pTau-181 antigen, BSA with the mass percentage of 0.5% and prolin300,300 with the mass percentage of 0.05%; the concentration of pTau-181 antigen was 20pg/mL and 200pg/mL, respectively.
Further, the manufacturer and model of the pTau-181 antigen is RDP002;
Further, the mouse monoclonal anti-pTau-181 antibody was purchased from the manufacturer and model number of the same were manufactured by West Bao Biotechnology (Shanghai) Inc., EKN0456A.
Example 2:
a preparation method of a chemiluminescent immunoassay kit for Alzheimer's disease pTau181 comprises the following steps:
Step1, preparing a magnetic particle reagent;
Step 1.1, dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer solution to obtain a dispersed carboxyl magnetic bead solution;
Further, in the step 1.1, the carboxyl magnetic bead concentrated solution is placed in a magnetic field for 2min, the supernatant is removed after all carboxyl magnetic beads are settled, then the carboxyl magnetic bead concentrated solution is added into a magnetic particle buffer solution for oscillation, and after the carboxyl magnetic bead concentrated solution is placed in the magnetic field for 2min, the supernatant is removed; the oscillation time was set to 20min; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:3, and the dispersion treatment times are 3 times;
Step 1.2, carrying out a connection reaction on the dispersed carboxyl magnetic bead solution and the pTau-181 antibody to obtain a connection product;
further, the method for implementing the intercommunication in the step 1.2 comprises the following steps:
Step 1.2.1, weighing the dispersed carboxyl magnetic bead solution and the pTau-181 antibody according to the mass ratio of 100:3, wherein the concentration of the dispersed carboxyl magnetic bead solution is 10mg/mL, setting the temperature of the connection reaction to be room temperature, and the time of the connection reaction to be 18h;
S1.2.2 after the connection reaction is finished, adding a sealing liquid for sealing, wherein the formula of the sealing liquid is as follows: a mixed solution of PBS, 3wt% BSA, 1wt% Tween-20, 1wt% maltose, 5wt% sorbitol, 2wt% sucrose, 3wt% glycerol, 0.5 wt% PEG 600;
Step 1.3, placing the connection product in a magnetic field, and flushing with a magnetic particle buffer solution to prepare a magnetic particle solution connected with the pTau-181 antibody;
Further, the connection product in the step 1.3 is placed in a magnetic field for 1-3min, and then washed by a magnetic particle buffer solution to prepare a magnetic particle solution connected with the pTau-181 antibody;
further, the time of placing the connection product in a magnetic field is 1-3min, and the washing times are 3 times; the concentration of the magnetic particle solution connected with the pTau-181 antibody is 10mg/mL;
Step 2, preparing AE marked pTau-181 antibody;
step 2.1, preparing AE into AE solution with concentration of 5mg/mL by using 0.15M sodium bicarbonate buffer solution;
Step 2.2, mixing the mouse monoclonal anti-pTau-181 antibody and AE solution according to the mass ratio of 1:1, and carrying out a linking reaction to obtain a linking product of the pTau-181 antibody and AE;
Step 2.3, separating and purifying the linking product of the pTau-181 antibody and AE by using carbonate buffer solution with pH of 8to obtain an AE-marked pTau-181 antibody;
step 3, preparing a pTau-181 calibrator: the pTau-181 calibrator is Tris buffer containing pTau-181 antigen, BSA with the mass percentage of 0.5% and prolin300,300 with the mass percentage of 0.05%; the concentration of pTau-181 antigen was 20pg/mL and 200pg/mL, respectively.
Example 3:
a preparation method of a chemiluminescent immunoassay kit for Alzheimer's disease pTau181 comprises the following steps:
Step1, preparing a magnetic particle reagent;
Step 1.1, dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer solution to obtain a dispersed carboxyl magnetic bead solution;
Further, in the step 1.1, the carboxyl magnetic bead concentrated solution is placed in a magnetic field for 3min, the supernatant is removed after all carboxyl magnetic beads are settled, then the carboxyl magnetic bead concentrated solution is added into a magnetic particle buffer solution for oscillation, and the carboxyl magnetic bead concentrated solution is placed in the magnetic field for 3min, and the supernatant is removed; the oscillation time was set to 30min; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:5, and the dispersion treatment times are 3 times;
Step 1.2, carrying out a connection reaction on the dispersed carboxyl magnetic bead solution and the pTau-181 antibody to obtain a connection product;
further, the method for implementing the intercommunication in the step 1.2 comprises the following steps:
Step 1.2.1, according to the mass ratio of 100:1, weighing a dispersed carboxyl magnetic bead solution and pTau-181 antibody, wherein the concentration of the dispersed carboxyl magnetic bead solution is 10mg/mL, the temperature of the connection reaction is set to be room temperature, and the time of the connection reaction is 20h;
S1.2.2 after the connection reaction is finished, adding a sealing liquid for sealing, wherein the formula of the sealing liquid is as follows: a mixed solution of PBS, 3wt% BSA, 1wt% Tween-20, 1wt% maltose, 5wt% sorbitol, 2wt% sucrose, 3wt% glycerol, 0.8 wt% PEG 600;
Step 1.3, placing the connection product in a magnetic field, and flushing with a magnetic particle buffer solution to prepare a magnetic particle solution connected with the pTau-181 antibody;
Further, the connection product in the step 1.3 is placed in a magnetic field for 1-3min, and then washed by a magnetic particle buffer solution to prepare a magnetic particle solution connected with the pTau-181 antibody;
further, the time of placing the connection product in a magnetic field is 1-3min, and the washing times are 3 times; the concentration of the magnetic particle solution connected with the pTau-181 antibody is 10mg/mL;
Step 2, preparing AE marked pTau-181 antibody;
step 2.1, preparing AE into AE solution with concentration of 5mg/mL by using 0.15M sodium bicarbonate buffer solution;
Step 2.2, mixing the mouse monoclonal anti-pTau-181 antibody and AE solution according to the mass ratio of 1:1, and carrying out a linking reaction to obtain a linking product of the pTau-181 antibody and AE;
Step 2.3, separating and purifying the linking product of the pTau-181 antibody and AE by using carbonate buffer solution with pH of 8to obtain an AE-marked pTau-181 antibody;
step 3, preparing a pTau-181 calibrator: the pTau-181 calibrator is Tris buffer containing pTau-181 antigen, BSA with the mass percentage of 0.5% and prolin300,300 with the mass percentage of 0.05%; the concentration of pTau-181 antigen was 20pg/mL and 200pg/mL, respectively.
Example 4:
An alzheimer's disease pTau181 chemiluminescent immunoassay kit prepared based on the preparation method of the alzheimer's disease pTau181 chemiluminescent immunoassay kit described in examples 1-3, the kit comprising a magnetic microparticle reagent, an AE-labeled pTau-181 antibody, a pTau-181 calibrator, the magnetic microparticle reagent being 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 acridine ester.
Further, the particle size of the magnetic particles in the magnetic particle reagent is 1-3 mu m, and the surfaces of the magnetic particles are coated with magnetic microspheres with carboxyl active groups;
When the magnetic microsphere suspension of the pTau-181 antibody is prepared, 0.5-2.5mg of the pTau-181 antibody is added for coating every 50mg of magnetic microspheres, the mass ratio of the pTau-181 antibody to the carboxyl magnetic beads in the magnetic particle reagent is 1:20-100, the temperature of the connection reaction is room temperature, and the time of the connection reaction is 16-20 h.
Further, the mass ratio of the pTau-181 antibody to the acridine ester in the AE-labeled pTau-181 antibody is 1:1.
Example 5:
The application of the Alzheimer's disease pTau181 chemiluminescent immunoassay kit prepared based on the preparation method of the Alzheimer's disease pTau181 chemiluminescent immunoassay kit described in the embodiment 1 to the embodiment 3 comprises the following steps:
S1, preparing a magnetic particle reagent and an AE-marked pTau-181 antibody;
S2, adding the serum sample and the AE-marked pTau-181 antibody prepared in the step S1 into a magnetic particle reagent for incubation;
Further, the volume ratio of the serum sample, AE-labeled pTau-181 antibody and magnetic particle reagent in step S2 is 2:2:1.
And S3, after the incubation is completed and the magnetic particle reagent is settled, removing supernatant, cleaning, adding a substrate solution, shaking and mixing uniformly, and detecting the relative luminous intensity.
Further, after detecting the relative luminous intensity in the step S3, calibrating and calibrating by using a high-value calibration material and a low-value calibration material, and fitting to generate a new working curve for sample measurement; and taking the pTau-181 quality control product as a sample, performing concentration fitting by using a working curve to obtain the corresponding quality control product concentration, and observing that the fitting quality control product concentration is +/-30% of a standard concentration value.
Furthermore, the AE labeled antibody reagent is an acridinium ester solution connected with the pTau-181 antibody, and because the linking reaction time is too long and the efficiency is too low, nonspecific binding is easy to generate during on-machine test, further purification treatment is needed, the reaction time can be effectively shortened, and the efficiency is improved.
The treatment method comprises the following steps: dispersing the acridinium ester solution of the labeled antibody under the action of carbonate buffer solution to obtain diluent; and adding the diluted solution into a G25 gel separation column for separation and purification.
The G-25 desalting column is one simple, fast and efficient pre-packed instant chromatographic column for separating large molecular weight matter from small molecular weight matter with G-25 matrix, and is used mainly in eliminating salt ion, detergent, small molecular dye, buffering agent and other impurity from protein, nucleic acid, polypeptide, polysaccharide and other sample.
1. The packing used for the desalting column is Beyodex ™ G-25 Medium, and the matrix is a high molecular polymer of three-dimensional network mesh structure formed by crosslinking Dextran (Dextran) through an epoxy chloropropane crosslinking agent through ether bonds, and the performance of the high molecular polymer is basically consistent with that of Sephadex G-25 Medium. The filler has a particle size of 70-250 mu m, can separate protein or other macromolecular samples with molecular weight of more than 5kDa, has a column bed volume of 8.3ml and a recommended loading amount of 1-2.5ml, and has functions and using methods consistent with those of PD-10 Desalting Column of Cytiva, and is suitable for a gravity method or a centrifugal method.
2. When a sample is separated by the desalting column, large molecules cannot enter a matrix mesh sieve mesh and are blocked outside matrix gel, and the desalting column is eluted out of the column firstly along with the movement of a flowing solution along gaps among matrix gel particles with higher downward movement speed; molecules or ions of smaller volume can enter the mesh openings of the matrix and thus the gel, and as the flowing solution moves within the mesh openings of the gel particles, the small molecular weight substances stay longer in the matrix and move down more slowly, and then are eluted out of the column, in a separation and purification manner called Size exclusion chromatography (Size-exclusion chromatography, SEC), also commonly called molecular sieve chromatography (Molecular sieve chromatography, MSC) or gel filtration chromatography (Gel filtration chromatography, GFC)
The detection method of pTau-181 antibody detection cassette is the same as in example 1.
The performance index of the magnetic particle chemiluminescence immunoassay kit for Alzheimer's disease prepared by the preparation method of the Alzheimer's disease pTau181 chemiluminescence immunoassay kit disclosed by the embodiments 1-3 of the invention is tested:
1. Linearity of
The linear range (0-500) pg/mL, and the correlation coefficient r is more than or equal to 0.9900.
The operation method is as follows:
Serum or serum substitutes near the upper limit of the linear range are diluted into five concentrations by a sample diluent according to a certain proportion, 5 concentration samples before and after dilution are respectively repeatedly measured for 3 times by using a chemiluminescent analyzer, an average value is calculated, a four-parameter fitting curve is used according to the average value and the dilution proportion, and a linear correlation coefficient r (obtained by X/Y axis straight line fitting of a column of 1-1/100 and RLU average values) is calculated, wherein the result is more than or equal to 0.9900. The specific values tested are shown in table 1:
Table 1:
According to the table, the luminescence value of example 1 is too low in detection of a low-value sample, and the phenomenon caused by incorrect formulation when the antibody coating is blocked is suspected, so that the concentration of PEG600 is increased in example 2, and the result is ideal, the concentration of PEG600 is continuously increased in example 3, the luminescence value of a blank background is obviously increased, and the assumption is that the over-concentration non-specific binding is serious. The method can obtain: the scheme of the embodiment 2 has stable curve rising and better trend in a linear range, and meets the requirements best.
The linear correlation coefficients (r) of the reaction curves of examples 1-3 were determined as follows: 0.9944, 0.9994, 0.9957, all > 0.9900, are significantly superior to the prior art, with example 2 performing best.
2. Accuracy (recovery rate)
The recovery rate in examples 1-3 in the present invention was in the range of 85 to 115%.
The operation method is as follows:
Sample A of known concentration pTau-181 is added to sample B identical to the prescribed sample matrix to be tested, the volume of added A not exceeding 10% of the total volume (A+B) being preferably measured in parallel 3 times, calculated according to formula (1):
（1）
Wherein:
R: recovery rate;
v: the volume of sample A;
v 0 volume of sample B;
C: detecting the concentration of the sample B after the solution A is added;
c 0: concentration of sample B fluid;
Cs: concentration of sample a.
Data cases (v=0.1 mL, V 0＝0.9mL,C0 =0 pg/mL, cs=200 pg/mL, where V and V 0 are 1:9 in relation to the above method, and C 0 and Cs are the concentrations of the high concentration point and the low concentration point in the calibrator respectively): the test results are shown in table 2:
TABLE 2
The accuracy (recovery) of examples 1-3 were measured as: 97.00%, 86.40% and 100.17% are all in the range of 85-115%, which meets the requirements. Of these, example 2 is preferred.
3. Minimum detection limit (sensitivity)
The lowest detection limit is less than or equal to 5pg/mL.
The operation method is as follows:
The measurement was repeated 20 times using zero concentration calibrator or sample diluent as the sample to be measured. Obtaining RLU values (relative luminescence values) of 20 measurement results, calculating an average value (M) and a Standard Deviation (SD) of the RLU values, obtaining m+2sd, performing two-point regression fitting according to concentration-RLU value results between a zero concentration calibrator and an adjacent calibrator to obtain a primary equation, substituting the RLU values of m+2sd into the equation, and obtaining a corresponding concentration value, namely the lowest detection limit, as shown in table 3:
Table 3:
The average luminescence value m=267.80, sd=44.80 of the pTau-181 sample dilutions measured in example 2, brings the value of (m+2sd) into this reaction curve, giving the sensitivity: 2.55pg/mL (< 5 pg/mL) is satisfactory.
4. Repeatability of
The coefficient of variation CV is less than or equal to 8 percent. The operation method is as follows:
Taking a quality control product 1 (20 pg/mL) and a quality control product 2 (200 ng/mL) as samples to be detected, and respectively carrying out 10 repeated determinations on the pTau-181 content in the samples to be detected by using a chemiluminescent determinator. The Coefficient of Variation (CV) is calculated according to equation (2).
......................................... （2）
Wherein: Average value of secondary test results;
standard deviation of SD-10 test results;
CV-coefficient of variation;
The data for example 1 are shown in table 4:
TABLE 4 Table 4
Example 2 data are shown in table 5:
TABLE 5
Example 3 data are shown in table 6:
TABLE 6
Analysis: examples 1 to 3 the quality control product 1.2 was tested ten times each, and CV was calculated. The quality control 1SD measured in the protocol of example 1 is: 1.24, CV is: 6.00%; quality control 2SD is: 6.00, CV is: 2.95%; the quality control 1SD measured in the protocol of example 2 is: 0.70, CV is: 3.35%; quality control 2SD is: 3.47, CV is: 1.70%; example 3 scheme the quality control 1SD was determined as: 1.37, CV is: 6.32%; quality control 2SD is: 5.78, CV is: 2.90%. All CVs are < 8%.
The following conclusions were drawn: all CVs were < 8% and comparison with SD found that example 2 protocol was more reproducible, less biased and satisfactory.
In conclusion, the magnetic particle chemiluminescence immunoassay kit for Alzheimer's disease pTau181 disclosed by the invention has higher detection sensitivity, specificity, accuracy and precision, and achieves better performance parameters. The magnetic particle reagent, the AE labeled antibody and the calibrator in the kit are the optimal formulas under the reaction system, and powerful guarantee is provided for the detection performance of the kit.
It is noted that relational terms such as "first" and "second", and the like, are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
Although the application has been described above with reference to specific embodiments, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the application. In particular, the features of the disclosed embodiments may be combined with each other in any manner so long as there is no structural conflict, and the exhaustive description of these combinations is not given in this specification solely for the sake of brevity and resource saving. Therefore, it is intended that the application not be limited to the particular embodiments disclosed herein, but that the application will include all embodiments falling within the scope of the appended claims.
Claims (10)
1. The chemiluminescent immunoassay kit for Alzheimer's disease pTau181 is characterized by comprising a magnetic particle reagent, an AE-marked pTau-181 antibody and a pTau-181 calibrator, wherein 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 acridine ester.
2. The Alzheimer's disease pTau181 chemiluminescent immunoassay kit of claim 1 is characterized in that the particle size of 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;
When the magnetic microsphere suspension of the pTau-181 antibody is prepared, 0.5-2.5mg of the pTau-181 antibody is added for coating every 50mg of magnetic microspheres, the mass ratio of the pTau-181 antibody to the carboxyl magnetic beads in the magnetic particle reagent is 1:20-100, the temperature of the connection reaction is room temperature, and the time of the connection reaction is 16-20 h.
3. The Alzheimer's disease pTau181 chemiluminescent immunoassay kit according to claim 2 wherein the mass ratio of pTau-181 antibody to acridinium ester in the AE-labeled pTau-181 antibody is 1:1.
4. A method for preparing the alzheimer's disease pTau181 chemiluminescent immunoassay kit of claims 1-3 comprising the steps of:
Step1, preparing a magnetic particle reagent;
Step 1.1, dispersing the carboxyl magnetic bead solution under the action of a magnetic particle buffer solution to obtain a dispersed carboxyl magnetic bead solution;
Step 1.2, carrying out a connection reaction on the dispersed carboxyl magnetic bead solution and the pTau-181 antibody to obtain a connection product;
Step 1.3, placing the connection product in a magnetic field, and flushing with a magnetic particle buffer solution to prepare a magnetic particle solution connected with the pTau-181 antibody;
Step 2, preparing AE marked pTau-181 antibody;
step 2.1, preparing AE into AE solution with concentration of 5mg/mL by using 0.15M sodium bicarbonate buffer solution;
Step 2.2, mixing the mouse monoclonal anti-pTau-181 antibody and AE solution according to the mass ratio of 1:1, and carrying out a linking reaction to obtain a linking product of the pTau-181 antibody and AE;
Step 2.3, separating and purifying the linking product of the pTau-181 antibody and AE by using carbonate buffer solution with pH of 8-9 to obtain AE marked pTau-181 antibody;
step 3, preparing a pTau-181 calibrator: the pTau-181 calibrator is Tris buffer containing pTau-181 antigen, BSA with the mass percentage of 0.5% and prolin300,300 with the mass percentage of 0.05%; the concentration of pTau-181 antigen was 20pg/mL and 200pg/mL, respectively.
5. The method for preparing a chemiluminescent immunoassay kit for Alzheimer's disease pTau181 according to claim 4, wherein in step 1.1, the carboxyl magnetic bead concentrate is placed in a magnetic field for 1-3 min, supernatant is removed after all carboxyl magnetic beads settle, and then added into a magnetic particle buffer solution for oscillation, and the supernatant is removed after being placed in the magnetic field for 1-3 min; the oscillation time is set to 20-30 min; the volume ratio of the carboxyl magnetic beads to the magnetic particle buffer is 1:2-5, and the dispersion treatment times are 3 times.
6. The method for preparing the Alzheimer's disease pTau181 chemiluminescent immunoassay kit according to claim 5 is characterized in that the method for realizing the intercommunication of step 1.2 comprises the following steps:
Step 1.2.1, according to the mass ratio of 100:5-100: 1, weighing a dispersed carboxyl magnetic bead solution and pTau-181 antibody, wherein the concentration of the dispersed carboxyl magnetic bead solution is 10-50 mg/mL, the temperature of the connection reaction is set to be room temperature, and the time of the connection reaction is 16-20 h;
S1.2.2 after the connection reaction is finished, adding a sealing liquid for sealing, wherein the formula of the sealing liquid is as follows: PBS, 3wt% BSA, 1wt% Tween-20, 1wt% maltose, 5wt% sorbitol, 2wt% sucrose, 3wt% glycerol, 0.1-0.8 wt% PEG 600.
7. The method for preparing a chemiluminescent immunoassay kit for Alzheimer's disease pTau-181 according to claim 6, wherein the time for which the ligation product is subjected to a magnetic field in step 1.3 is 5-10 min, and then the ligation product is washed with a magnetic particle buffer solution to obtain a magnetic particle solution to which the pTau-181 antibody is ligated.
8. A method for detecting the alzheimer's disease pTau181 chemiluminescent immunoassay kit of claims 1-3 comprising the steps of:
S1, preparing a magnetic particle reagent and an AE-marked pTau-181 antibody;
S2, adding the serum sample and the AE-marked pTau-181 antibody prepared in the step S1 into a magnetic particle reagent for incubation;
And S3, after the incubation is completed and the magnetic particle reagent is settled, removing supernatant, cleaning, adding a substrate solution, shaking and mixing uniformly, and detecting the relative luminous intensity.
9. The method for detecting the Alzheimer' S disease pTau181 chemiluminescent immunoassay kit according to claim 8 wherein the volume ratio of the serum sample, the AE-labeled pTau-181 antibody and the magnetic particle reagent in step S2 is 2:2:1.
10. The method for detecting the Alzheimer' S disease pTau181 chemiluminescent immunoassay kit according to claim 9 is characterized in that after the relative luminous intensity is detected in the step S3, calibration is carried out by a high-value and low-value two-point calibrator, and a new working curve is generated by fitting for sample determination; and taking the pTau-181 quality control product as a sample, performing concentration fitting by using a working curve to obtain the corresponding quality control product concentration, and observing that the fitting quality control product concentration is +/-30% of a standard concentration value.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411177922.XA CN118707111A (en) | 2024-08-27 | 2024-08-27 | Alzheimer's disease pTau181 chemiluminescence immunoassay kit, preparation method and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411177922.XA CN118707111A (en) | 2024-08-27 | 2024-08-27 | Alzheimer's disease pTau181 chemiluminescence immunoassay kit, preparation method and detection method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118707111A true CN118707111A (en) | 2024-09-27 |
Family
ID=92822239
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411177922.XA Pending CN118707111A (en) | 2024-08-27 | 2024-08-27 | Alzheimer's disease pTau181 chemiluminescence immunoassay kit, preparation method and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118707111A (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009031062A (en) * | 2007-07-25 | 2009-02-12 | Siliconbio:Kk | Asbestos detection method and asbestos detecting kit |
| RU2009120888A (en) * | 2009-06-01 | 2010-12-10 | Учреждение Российской академии наук Институт биохимии и физиологии растений и микроорганизмов РАН (ИБФРМ РАН) (RU) | METHOD FOR DETERMINING BACTERIAL LIPOPOLYSACCHARIDE ANTIGENS IN SOIL |
| WO2019169797A1 (en) * | 2018-03-07 | 2019-09-12 | 深圳市伯劳特生物制品有限公司 | Composition for enzyme-linked immunosorbent assay kit, anti-nuclear antibody repertoire detection kit and preparation method therefor |
| CN113311154A (en) * | 2021-05-28 | 2021-08-27 | 宁波瑞源生物科技有限公司 | Coupling method, diluent and application of immunomagnetic particles |
| CN113419059A (en) * | 2021-06-30 | 2021-09-21 | 迈克生物股份有限公司 | Blocking agent for chemiluminescence immune analysis method |
| CN114034872A (en) * | 2022-01-12 | 2022-02-11 | 山东大学齐鲁医院 | Kit for early diagnosis of Alzheimer's disease and application thereof |
| CN114778856A (en) * | 2022-05-30 | 2022-07-22 | 苏州宇测生物科技有限公司 | Phosphorylated tau protein detection kit |
| CN115112905A (en) * | 2022-07-26 | 2022-09-27 | 浙江瑞硕生物技术有限公司 | RANKL magnetic particle chemiluminescence immunoassay kit and detection method thereof |
| US20240085436A1 (en) * | 2022-09-14 | 2024-03-14 | Hangzhou Cellgene Biotech Co.,Ltd | Test kit for testing biomarkers of alzheimer disease and the method thereof |
-
2024
- 2024-08-27 CN CN202411177922.XA patent/CN118707111A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009031062A (en) * | 2007-07-25 | 2009-02-12 | Siliconbio:Kk | Asbestos detection method and asbestos detecting kit |
| RU2009120888A (en) * | 2009-06-01 | 2010-12-10 | Учреждение Российской академии наук Институт биохимии и физиологии растений и микроорганизмов РАН (ИБФРМ РАН) (RU) | METHOD FOR DETERMINING BACTERIAL LIPOPOLYSACCHARIDE ANTIGENS IN SOIL |
| WO2019169797A1 (en) * | 2018-03-07 | 2019-09-12 | 深圳市伯劳特生物制品有限公司 | Composition for enzyme-linked immunosorbent assay kit, anti-nuclear antibody repertoire detection kit and preparation method therefor |
| CN113311154A (en) * | 2021-05-28 | 2021-08-27 | 宁波瑞源生物科技有限公司 | Coupling method, diluent and application of immunomagnetic particles |
| CN113419059A (en) * | 2021-06-30 | 2021-09-21 | 迈克生物股份有限公司 | Blocking agent for chemiluminescence immune analysis method |
| CN114034872A (en) * | 2022-01-12 | 2022-02-11 | 山东大学齐鲁医院 | Kit for early diagnosis of Alzheimer's disease and application thereof |
| CN114778856A (en) * | 2022-05-30 | 2022-07-22 | 苏州宇测生物科技有限公司 | Phosphorylated tau protein detection kit |
| CN115112905A (en) * | 2022-07-26 | 2022-09-27 | 浙江瑞硕生物技术有限公司 | RANKL magnetic particle chemiluminescence immunoassay kit and detection method thereof |
| US20240085436A1 (en) * | 2022-09-14 | 2024-03-14 | Hangzhou Cellgene Biotech Co.,Ltd | Test kit for testing biomarkers of alzheimer disease and the method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 北京农业大学: "《果品贮藏加工学》", vol. 2, 31 October 1990, 农业出版社, pages: 221 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108362688B (en) | Detection kit for chemiluminescence of 25-hydroxy vitamin D magnetic particles | |
| US6162645A (en) | Determination of % glycated hemoglobin | |
| CN108761088B (en) | Composition, kit and method for separating and detecting abnormal sugar chain protein and application | |
| JPH01503808A (en) | Affinity separation using immobilized flocculants | |
| WO2021258618A1 (en) | Biological sample testing method and test kit | |
| CN111381027A (en) | Immunocapture composition, preparation method and application thereof, and immunoassay kit | |
| Zhang et al. | Rapid determination of aflatoxin B1 by an automated immunomagnetic bead purification sample pretreatment method combined with high‐performance liquid chromatography | |
| CN101046479B (en) | Process of preparing human serum base matter containing no target protein | |
| Peng et al. | Magnetic quantitative analysis for multiplex glycoprotein with polymer-based elemental tags | |
| CN118707111A (en) | Alzheimer's disease pTau181 chemiluminescence immunoassay kit, preparation method and detection method thereof | |
| CN116840487B (en) | Method for detecting sugar-deficient transferrin | |
| EP3885362A1 (en) | Antibody conjugate | |
| CN113030497A (en) | Magnetic particle chemiluminescence immunoassay kit for procalcitonin and detection method thereof | |
| CN115112905A (en) | RANKL magnetic particle chemiluminescence immunoassay kit and detection method thereof | |
| CN111896743A (en) | Fluorescence immunochromatography test strip and preparation method and application thereof | |
| CN116559333B (en) | Absolute quantification method for exosome surface proteins and application thereof | |
| CN112858687B (en) | Serum amyloid A detection reagent and preparation method thereof | |
| CN116819072B (en) | Antigen protein composition for detecting anti-AQP 4 autoantibody and chemiluminescent detection kit | |
| CN117647606B (en) | Kit suitable for LC-MS/MS and capable of detecting multiple proteins simultaneously | |
| CN113686841B (en) | Kit for quantitatively detecting thyroxine binding force, preparation method and detection method thereof | |
| Li et al. | A novel time-resolved fluoroimmunoassay based on magnetic microspheres method for detecting antibodies against the phospholipase A2 receptor | |
| CN110907548B (en) | Method for detecting biapenem and/or related substances | |
| CN118259015A (en) | Kit for determining GP73 content of human and preparation method thereof | |
| CN114236115A (en) | Kit for determining alpha tumor necrosis factor by magnetic particle chemiluminescence method and preparation method thereof | |
| CN110275011A (en) | The sensitization detection method of colloidal gold immunity chromatography and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |