CN110907640A - Neurofilament protein in-vitro diagnosis kit - Google Patents
Neurofilament protein in-vitro diagnosis kit Download PDFInfo
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- 238000000338 in vitro Methods 0.000 title claims abstract description 10
- 102000008763 Neurofilament Proteins Human genes 0.000 title claims description 11
- 108010088373 Neurofilament Proteins Proteins 0.000 title claims description 11
- 238000003745 diagnosis Methods 0.000 title description 4
- 238000005406 washing Methods 0.000 claims abstract description 16
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 238000009007 Diagnostic Kit Methods 0.000 claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 claims abstract description 11
- 102000004190 Enzymes Human genes 0.000 claims abstract description 11
- 238000011161 development Methods 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims abstract description 9
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 8
- 230000007131 anti Alzheimer effect Effects 0.000 claims abstract description 8
- 238000003908 quality control method Methods 0.000 claims abstract description 8
- 238000007865 diluting Methods 0.000 claims abstract description 7
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000007789 gas Substances 0.000 claims abstract description 4
- 230000009972 noncorrosive effect Effects 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 239000012089 stop solution Substances 0.000 claims description 6
- 238000002835 absorbance Methods 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 4
- 238000010009 beating Methods 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000009941 weaving Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 230000001537 neural effect Effects 0.000 abstract description 4
- 238000007689 inspection Methods 0.000 abstract description 3
- 208000024827 Alzheimer disease Diseases 0.000 description 16
- 230000018109 developmental process Effects 0.000 description 7
- 210000002700 urine Anatomy 0.000 description 6
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 3
- 210000002569 neuron Anatomy 0.000 description 3
- 206010012289 Dementia Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 208000000044 Amnesia Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 208000036110 Neuroinflammatory disease Diseases 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 102000007982 Phosphoproteins Human genes 0.000 description 1
- 108010089430 Phosphoproteins Proteins 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 208000010877 cognitive disease Diseases 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000003619 fibrillary effect Effects 0.000 description 1
- 230000002962 histologic effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 231100000863 loss of memory Toxicity 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000002682 neurofibrillary tangle Anatomy 0.000 description 1
- 230000003959 neuroinflammation Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a neural thread protein in vitro diagnostic kit, which is suitable for an enzyme-labeled analyzer with 450nm wavelength, and comprises: the AD7C-NTP micropore plate is coated with an anti-AD 7C-NTP monoclonal antibody; AD7C-NTP calibrator with concentrations of 0,1.0,2.0,5.0 and 10ng/ml respectively; AD7C-NTP negative quality control, target value: 0.82 +/-0.18 ng/ml; AD7C-NTP positive quality control, target value: 2.32 plus or minus 0.46 ng/ml; an enzyme label containing an anti-AD 7C-NTP monoclonal antibody marked by horseradish peroxidase (HRP); the color developing liquid A comprises the main component of hydrogen peroxide; the color development liquid B mainly comprises TMB; concentrating the washing solution by 10 times, and diluting before use; stopping solution mainly containing dilute H2SO4(ii) a 2-8 ℃, the validity period is 270 days, and after being unsealed: storing in a non-corrosive gas refrigerator at the temperature of 2-8 ℃ for 60 days. Shorten and examineMeasuring time; and can dock the automated inspection platform, improve the detection precision of this product.
Description
Technical Field
The invention relates to the field of diagnostic kits, in particular to a neurofilament protein in-vitro diagnostic kit.
Background
Alzheimer's Disease (AD) is the most common degenerative disease of the central nervous system in humans in the elderly, and the most common cause of dementia. Clinically, it is manifested as chronic impairment of the intelligence level and chronic loss of memory. With the aging of the global population, the incidence of AD is increasing day by day, and because of the difficulty in early diagnosis and poor treatment effect, AD becomes an important problem to be solved urgently in the fields of the old medical science and the neuroscience. The current progression of AD is divided into three phases: in the early stage of clinic; a period of mild cognitive dysfunction; a dementia period. Biomarkers are a class of indicators that can be objectively measured for assessing physiological, pathological processes or some characteristic response for treatment and prognosis.
Alzheimer's disease associated neurofilament protein (AD7c-NTP) is a transmembrane phosphoprotein expressed in neuronal cells and having a molecular weight of about 41 kDa. Present in axons emanating from nerve cells; and are present in large numbers in neurofibrillary tangles (NFTs) in the brains of AD patients. The de la Monte professor in general Hospital of Massachusetts in 1996 found that AD7c-NTP has an increased expression level in the brain of AD patients, coexists with tau-1 protein, is positively correlated with p-tau protein level, and appears in degenerated neurons with histologic integrity, and the increased expression of AD7c-NTP protein is an early event of AD neurodegeneration. A number of subsequent studies have shown that: AD7c-NTP is present in Neuronal Fibrillary Tangles (NFTs), and is primarily involved in the induction of neuroinflammation and cell death. There is a selective increase in both brain tissue and cerebrospinal fluid in AD patients. Thus, it was also determined that AD7c-NTP can be used as a biomarker for diagnosing AD.
The molecular mass of AD7C-NTP in urine sample of AD patient is the same as AD7c-NTP in human cerebrospinal fluid and brain tissue, and the detection result is similar. Thus, the detection of AD7C-NTP in urine samples has been established for the diagnosis or large-scale screening of Alzheimer's disease. The detection of the level of AD7C-NTP in the urine sample has the same significance as the detection of the level of AD7C-NTP in the cerebrospinal fluid sample. The detection of urine samples is a non-invasive test. Is easier to be accepted by patients and is easy to be popularized in clinic.
The similar products on the market at present adopt: an Alzheimer's disease-associated neurofilament protein (AD7c-NTP) polypeptide antigen; polypeptides are the primary structure of proteins and are less biologically active than proteins.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the in-vitro diagnosis kit for the neurofilament protein, which shortens the detection time; and can dock the automated inspection platform, improve the detection precision of this product.
In order to achieve the aim of the invention, the invention adopts the specific scheme that:
an in vitro diagnostic kit of neural thread protein, which is suitable for an enzyme-labeled analyzer with 450nm wavelength, comprises: the AD7C-NTP micropore plate is coated with an anti-AD 7C-NTP monoclonal antibody; AD7C-NTP calibrator with concentrations of 0,1.0,2.0,5.0 and 10ng/ml respectively; AD7C-NTP negative quality control, target value: 0.82 +/-0.18 ng/ml; AD7C-NTP positive quality control, target value: 2.32 plus or minus 0.46 ng/ml; an enzyme label containing an anti-AD 7C-NTP monoclonal antibody marked by horseradish peroxidase (HRP); the color developing liquid A comprises the main component of hydrogen peroxide; the color development liquid B mainly comprises TMB; concentrating the washing solution by 10 times, and diluting before use; stopping solution mainly containing dilute H2SO4(ii) a 2-8 ℃, the validity period is 270 days, and after being unsealed: storing in a non-corrosive gas refrigerator at the temperature of 2-8 ℃ for 60 days.
Further, the use steps of the diagnostic kit are as follows:
(1) preparation of a washing solution: diluting the concentrated washing liquid with distilled water or deionized water according to the proportion of 1: 10;
(2) after the kit is balanced to room temperature, taking out the required microporous strips, fixing the microporous strips on a plate frame, and weaving the microporous strips in sequence;
(3) sample adding: respectively adding the calibrator and 100ul of a sample to be tested into corresponding holes;
(4) adding 50ul enzyme label into each reaction hole, and incubating for 1 hour at 37 ℃;
(5) washing the plate for 5 times, beating to dry, and avoiding generating bubbles in the washing process;
(6) adding color development solution, adding 50u1 of each solution of developer A and B into each well (or adding 100ul of color development solution into each well after mixing at 1:1 ratio before use), mixing, and standing at room temperature in dark place for 20 min;
(7) adding stop solution, adding 50ul stop solution into each hole as soon as possible, and fully and uniformly mixing;
(8) detecting, namely selecting a wavelength of 450nm by using an enzyme-labeling instrument, and measuring the OD value of each hole;
(9) if the AD7C-NTP highest concentration calibrator 0D is more than or equal to 0.8, the test result is effective; taking the concentration of the calibrator as an abscissa and the absorbance as an ordinate, and making a standard curve in a best fitting mode; the detection value of the sample will be calculated from the standard curve.
Further, the water culture is that the reaction plate is placed in a water culture box and floats on the water surface.
The invention has the beneficial effects that:
the detection time is shortened; and can dock the automated inspection platform, improve the detection precision of this product.
Detailed Description
The present invention is further described below by way of specific examples, but the present invention is not limited to only the following examples. Variations, combinations, or substitutions of the invention, which are within the scope of the invention or the spirit, scope of the invention, will be apparent to those of skill in the art and are within the scope of the invention.
An in vitro diagnostic kit of neural thread protein, which is suitable for an enzyme-labeled analyzer with 450nm wavelength, comprises: the AD7C-NTP micropore plate is coated with an anti-AD 7C-NTP monoclonal antibody; AD7C-NTP calibrator with concentrations of 0,1.0,2.0,5.0 and 10ng/ml respectively; AD7C-NTP negative quality control, target value: 0.82 +/-0.18 ng/ml; AD7C-NTP positive quality control, target value: 2.32 plus or minus 0.46 ng/ml; an enzyme label containing an anti-AD 7C-NTP monoclonal antibody marked by horseradish peroxidase (HRP); the color developing liquid A comprises the main component of hydrogen peroxide; the color development liquid B mainly comprises TMB; concentrating the washing solution by 10 times, and diluting before use; stopping solution mainly containing dilute H2SO4(ii) a 2-8 ℃, the validity period is 270 days, and after being unsealed: storing in a non-corrosive gas refrigerator at the temperature of 2-8 ℃ for 60 days.
The diagnostic kit comprises the following steps:
(1) preparation of a washing solution: diluting the concentrated washing liquid with distilled water or deionized water according to the proportion of 1: 10;
(2) after the kit is balanced to room temperature, taking out the required microporous strips, fixing the microporous strips on a plate frame, and weaving the microporous strips in sequence;
(3) sample adding: respectively adding the calibrator and 100ul of a sample to be tested into corresponding holes;
(4) adding 50ul enzyme label into each reaction hole, and incubating for 1 hour at 37 ℃;
(5) washing the plate for 5 times, beating to dry, and avoiding generating bubbles in the washing process;
(6) adding color development solution, adding 50u1 of each solution of developer A and B into each well (or adding 100ul of color development solution into each well after mixing at 1:1 ratio before use), mixing, and standing at room temperature in dark place for 20 min;
(7) adding stop solution, adding 50ul stop solution into each hole as soon as possible, and fully and uniformly mixing;
(8) detecting, namely selecting a wavelength of 450nm by using an enzyme-labeling instrument, and measuring the OD value of each hole;
(9) if the AD7C-NTP highest concentration calibrator 0D is more than or equal to 0.8, the test result is effective; taking the concentration of the calibrator as an abscissa and the absorbance as an ordinate, and making a standard curve in a best fitting mode; the detection value of the sample will be calculated from the standard curve.
The water culture is to place the reaction plate in a water culture box and float on the water surface.
And (3) checking the principle: the kit applies an enzyme-linked immunosorbent assay (ELISA) principle to quantitatively detect the concentration of Alzheimer disease related neurofilament protein (AD7C-NTP) in a human urine sample. The kit selects human recombinant Alzheimer disease related neurofilament protein (AD7C-NTP) with independent intellectual property rights; a pair of monoclonal antibodies is prepared and screened. Are respectively used for coating a microporous plate and marking horseradish peroxidase. Formed during the reaction: double antibody sandwich format of anti (AD7C-NTP) antibody (mab) -anti (AD7C-NTP) -anti (AD7C-NTP) (mab) -HRP. The absorbance of the sample was measured at a wavelength of 450nm by color development. The content of AD7C-NTP in the human urine sample is in positive correlation with the absorbance.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (3)
1. A neurofilament protein in-vitro diagnostic kit is suitable for an enzyme-labeled analyzer with 450nm wavelength, and is characterized by comprising: the AD7C-NTP micropore plate is coated with an anti-AD 7C-NTP monoclonal antibody; AD7C-NTP calibrator with concentrations of 0,1.0,2.0,5.0 and 10ng/ml respectively; AD7C-NTP negative quality control, target value: 0.82 +/-0.18 ng/ml; AD7C-NTP positive quality control, target value: 2.32 plus or minus 0.46 ng/ml; an enzyme label containing an anti-AD 7C-NTP monoclonal antibody marked by horseradish peroxidase (HRP); the color developing liquid A comprises the main component of hydrogen peroxide; the color development liquid B mainly comprises TMB; concentrating the washing solution by 10 times, and diluting before use; stopping solution mainly containing dilute H2SO4(ii) a 2-8 ℃, the validity period is 270 days, and after being unsealed: storing in a non-corrosive gas refrigerator at the temperature of 2-8 ℃ for 60 days.
2. The in vitro neurofilament protein diagnostic kit according to claim 1, wherein the diagnostic kit is prepared by the following steps:
(1) preparation of a washing solution: diluting the concentrated washing liquid with distilled water or deionized water according to the proportion of 1: 10;
(2) after the kit is balanced to room temperature, taking out the required microporous strips, fixing the microporous strips on a plate frame, and weaving the microporous strips in sequence;
(3) sample adding: respectively adding the calibrator and 100ul of a sample to be tested into corresponding holes;
(4) adding 50ul enzyme label into each reaction hole, and incubating for 1 hour at 37 ℃;
(5) washing the plate for 5 times, beating to dry, and avoiding generating bubbles in the washing process;
(6) adding color development solution, adding 50u1 of each solution of developer A and B into each well (or adding 100ul of color development solution into each well after mixing at 1:1 ratio before use), mixing, and standing at room temperature in dark place for 20 min;
(7) adding stop solution, adding 50ul stop solution into each hole as soon as possible, and fully and uniformly mixing;
(8) detecting, namely selecting a wavelength of 450nm by using an enzyme-labeling instrument, and measuring the OD value of each hole;
(9) if the AD7C-NTP highest concentration calibrator 0D is more than or equal to 0.8, the test result is effective; taking the concentration of the calibrator as an abscissa and the absorbance as an ordinate, and making a standard curve in a best fitting mode; the detection value of the sample will be calculated from the standard curve.
3. The in vitro neurofilament protein diagnostic kit as claimed in claim 2, wherein the incubation is performed by placing the reaction plate in an incubator, floating on the water surface.
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| CN201911296997.9A CN110907640A (en) | 2019-12-17 | 2019-12-17 | Neurofilament protein in-vitro diagnosis kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111579799A (en) * | 2020-06-23 | 2020-08-25 | 江苏省荣军医院 | Preparation method of fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine |
| CN113281522A (en) * | 2021-05-24 | 2021-08-20 | 迪瑞医疗科技股份有限公司 | Chemiluminescence immunoassay kit for Alzheimer related neurofilament protein and preparation method thereof |
Citations (2)
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|---|---|---|---|---|
| CN101215323A (en) * | 2007-12-28 | 2008-07-09 | 首都医科大学宣武医院 | Human AD 7C-NTP antigenic determinant polypeptide, antibody and application thereof in diagnostic kit |
| CN105085628A (en) * | 2015-08-12 | 2015-11-25 | 浙江聚康生物工程有限公司 | Human AD7C-NTP epitope polypeptide, human AD7C-NTP antibody and human AD7C-NTP in-vitro diagnosis reagent kit with human AD7C-NTP antibody |
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2019
- 2019-12-17 CN CN201911296997.9A patent/CN110907640A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215323A (en) * | 2007-12-28 | 2008-07-09 | 首都医科大学宣武医院 | Human AD 7C-NTP antigenic determinant polypeptide, antibody and application thereof in diagnostic kit |
| CN105085628A (en) * | 2015-08-12 | 2015-11-25 | 浙江聚康生物工程有限公司 | Human AD7C-NTP epitope polypeptide, human AD7C-NTP antibody and human AD7C-NTP in-vitro diagnosis reagent kit with human AD7C-NTP antibody |
Non-Patent Citations (2)
| Title |
|---|
| WILLIAM J. JORDAN ET AL: "Enzyme-Linked Immunosorbent Assay", 《MEDICAL BIOMETHODS HANDBOOK》 * |
| 张炙萍等: "AD7C-NTP在老年痴呆患者及不同种属动物脑内的存在和意义", 《首都医科大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111579799A (en) * | 2020-06-23 | 2020-08-25 | 江苏省荣军医院 | Preparation method of fluorescence immunochromatographic test paper for detecting Alzheimer disease related neurofilament protein in urine |
| CN113281522A (en) * | 2021-05-24 | 2021-08-20 | 迪瑞医疗科技股份有限公司 | Chemiluminescence immunoassay kit for Alzheimer related neurofilament protein and preparation method thereof |
| CN113281522B (en) * | 2021-05-24 | 2024-01-09 | 迪瑞医疗科技股份有限公司 | Alzheimer related neurofilament protein chemiluminescence immunoassay kit and preparation method thereof |
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