CN109596842A - A kind of alzheimer's disease detection reagent and its application in alzheimer's disease detection - Google Patents
A kind of alzheimer's disease detection reagent and its application in alzheimer's disease detection Download PDFInfo
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- CN109596842A CN109596842A CN201910038773.1A CN201910038773A CN109596842A CN 109596842 A CN109596842 A CN 109596842A CN 201910038773 A CN201910038773 A CN 201910038773A CN 109596842 A CN109596842 A CN 109596842A
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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
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
Abstract
The present invention provides a kind of alzheimer's disease detection reagents, including AD1:A to mix with B according to volume 1:1, wherein A:5.0%SDS, pH 8.3, the NP-40 of the PNGaseF and 3.33% of B:2.2U/ μ L 1:20 mixed preparing by volume；AD2: the NaCNBH3 of the APTS and 1M of the 20mM of same volume are mixed；NH4AC, 2mU/ μ L sialidase and the hydrogen peroxide of AD3:100mM is mixed according to volume 5:1:14；AD4: buffer.The present invention also provides a kind of application of composition in alzheimer's disease detection reagent, the composition is made of NGA2F and NA2, and the composition detects alzheimer's disease by the ratio of NGA2F/NA2.The present invention using high sensitivity, it is easy to operate, only need micro-example, the detection method that repeatability is high, stability is good and high-throughput, compared with prior art, accuracy reaches 87.7%, hence it is evident that better than existing detection method.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of alzheimer's disease detection reagent and its in A Er
Application in the detection of Zi Haimo disease.
Background technique
Alzheimer's disease is exactly the senile dementia being commonly called as, and is a kind of degenerative change of brain, is mainly shown as progress
Property memory and cognition dysfunction.The generally acknowledged main pathological change of alzheimer's disease is intracerebral beta-amyloyd patch at present
Form the neurofibrillary tangles mediated with Protein tau.Alzheimer's disease disease incidence significantly increases with the age.It typically occurs in 65 years old
After, onset is not noticeable, develops slowly, and earliest period is often started with the amnesia gradually aggravated, often stages alzheimer's disease
Tendency.It is mainly shown as the psychoneurals disease such as gradual memory obstacle, cognition dysfunction, personality change and aphasis
Shape seriously affects social, occupation and vital function.Alzheimer disease is the brain diseases of the elderly, the brain cell meeting of patient
Sharp degradation, but be not normal aging course.
According to international 2015 annual report of Alzheimer's disease association, dementia example 9,900,000 of newly staring blankly every year, to the year two thousand fifty, the whole world
1.315 hundred million people will be increased to from 46,000,000 current people with dull-witted number, 68% growth will occur in low middle income state
Family.Currently, dementia is after cardiovascular disease, cerebrovascular disease and malignant tumour " the fourth-largest killer " of senior health and fitness；Be
The maximum disease of the cost being number three after cancer and heart disease.Alzheimer's disease have become seriously endanger elderly population health and
One of the principal disease for influencing China's sustainable development, how to carry out effective early diagnosis and therapy to it is reply aging
One significant challenge of society.
In April, 2018, " 2018 China dull-witted and cognitive disorder diagnosis and treatment guide " are formally delivered, the second part of guide to Ah
The diagnosis of Alzheimer's disease proposes suggestion:Clinical AD diagnosis can be according to NINCDS-ADRDA or 2011 edition NIA-AA in 1984
The AD diagnostic criteria of proposition is diagnosed.It has ready conditions when carrying out the inspection of AD molecular image and cerebrospinal fluid and detecting, it can be according to 2011
NIA-AA or 2014 edition IWG-2 diagnostic criteria of version carries out AD diagnosis.The consciousness of the diagnosis to the AD that is not true to type should be improved.Thus may be used
See, the diagnosis of alzheimer's disease is still sufficiently complex, clinically mostly using the questionnaire test of detection cognitive function, cerebrospinal fluid
It chemically examines and is diagnosed in conjunction with iconography.Extract cerebrospinal fluid have it is larger traumatic, currently on the market without pass through blood test, just
It can be with the reagent of auxiliary diagnosis alzheimer's disease.
Summary of the invention
The N- oligosaccharide of glycoprotein is synthesized by complicated between hundreds of enzyme, transcription factor, ion channel and albumen
It interacts and is formed.N- oligosaccharide participates in different kinds of molecules process, such as protein folding, cell adherence, molecular migration, signal turn
It leads, adjusts receptor active etc..Therefore the change of N- oligosaccharide is closely related with disease.
It is found by numerous studies early period, in Alzheimer's serum, N- oligosaccharide
Anagalactosylated, core-a-1,6-fucosylated biantennary glycan (NGA2F),
Bigalactosylated, biantennary glycan (NA2) relative amount has occurred obvious compared in normal serum
Variation, therefore can be used as the marker of detection alzheimer's disease.
For in the detection of existing alzheimer's disease, the detection of cognitive function questionnaire and iconography detection are specific and accurate
It spends not high.Cerebrospinal fluid detection wound is larger, there is no other blood testings and pathological examination method, and the present invention provides a kind of apply
Alzheimer's disease detection reagent in alzheimer's disease detection.
The technical solution adopted by the invention is as follows:
A kind of alzheimer's disease detection reagent, including AD1:A are mixed with B according to volume 1:1, and wherein A:5.0% SDS, pH are
The NP-40 of the PNGaseF and 3.33% of 8.3, B:2.2U/ μ L 1:20 mixed preparing by volume；AD2: the 20mM of same volume is mixed
APTS and 1M NaCNBH3；NH4AC, 2mU/ μ L sialidase and the hydrogen peroxide of AD3:100mM is mixed according to volume 5:1:14
It closes；AD4: buffer.
It is 2 μ L, AD3 volumes be 2 μ L, AD4 volumes is 200 μ L that the volume of the AD1, which is the volume of 4 μ L, AD2,.
Reagent preparation:
AD1:A is mixed with B according to volume 1:1.
A:SDS(ammonium lauryl sulfate) it is dissolved in the NH4HCO3 of 10mM, it is configured to the solution for being 8.3 containing 5% SDS, pH
The NP-40 of the PNGaseF and 3.33% of B:2.2U/ μ L 1:20 mixed preparing by volume
AD2: the NaCNBH3 of the APTS and 1M of the 20mM of same volume are mixed.
NH4AC, sialidase (neuraminidase, 2mU/ μ L) and the hydrogen peroxide of AD3:100mM is according to volume 5:1:
14
Mixing.
AD4: buffer.
The present invention also provides a kind of application of composition in alzheimer's disease detection reagent, the composition by
NGA2F and NA2 composition, the composition detect alzheimer's disease by the ratio of NGA2F/NA2.
The detection of N- oligonucleotide chain map
1. the preparation of oligonucleotide chain: it takes the 2 μ L of serum inactivated at 95 DEG C, is added 4 μ L AD1,37 DEG C
It is dried after reaction 3h.
2. the label of oligonucleotide chain: be added the reagent A D2 of 2 μ L in sample after the drying, at 65 DEG C into
After row fluorescent marker 3h, the AD4 that 200 μ L are added terminates reaction.
3. label post-processing: taking labeled good 2 μ L of sample, be added the AD3 of 2 μ L, at 45 DEG C
3h is reacted, the AD4 for finally adding 200 μ L terminates reaction.
4. oligosaccharides chain separation is analyzed: taking 10 μ L of fluid sample 3., carried out under ABI3500dx instrument
N- oligosaccharides chain separation, to obtain N- sugar group map
5. Data Management Analysis: obtained N- sugar group map being carried out peak value quantization, with each peak
Peak value is compared with the summation of the height at all peaks, to quantitatively calculate the relative amount at each peak, then carry out into
The statistical analysis of one step carries out further statistical analysis by function NGA2F/NA2, to detect alzheimer's disease.
Beneficial effects of the present invention:
(1) present invention be alzheimer's disease detection reagent, using high sensitivity, it is easy to operate, only need it is micro-
Sample is measured, the detection method that repeatability is high, stability is good and high-throughput, according to 408 sample (alzheimer's disease samples
70,338, normal healthy controls sample) the N- oligonucleotide chain test map of serum established, obtain the N- oligomerization in experimenter's serum
Sugared NGA2F, NA2 relative amount detect subject by function NGA2F/ NA2.Reagent of the invention and detection side
Method, compared with prior art, accuracy reach 87.7%, hence it is evident that are better than existing detection method.
(2) reagent and detection method of the invention are used, the crowd of numerous high risks can be allowed to receive conventional, noninvasive inspection
It surveys, helps doctor and patient to monitor generation and the disease progression of alzheimer's disease in time, be expected to promote the use of in clinic.
Detailed description of the invention
A shown in Fig. 1 is using the typical map of this detection reagent detection alzheimer's disease sample, and b is the inspection of this reagent
Survey the typical map of healthy control group serum sample；
Fig. 2 is that detection sample passes through ROC indicatrix made by NGA2F/ NA2；Detect total sample number be 408, wherein Ah
Wurz sea is write from memory 70, disease sample, and 338, healthy control group sample；Compare alzheimer's disease patient using function NGA2F/NA2
The N- oligonucleotide chain test map peak value of (n=70) non-alzheimer's disease (n=338), obtains area under the curve AUC=0.877.
Specific embodiment
Combined with specific embodiments below and this reagent and method is described in further detail in attached drawing.It should be noted that following
Embodiment is merely to illustrate rather than limits the scope of the invention.The experiment side of actual conditions is not specified in following embodiment
Method is tested usually according to normal condition, or the condition suggested according to manufacturer, and reagent is all that cell culture is dedicated.
Embodiment one:
(1) experimental facilities:
ABI3500dx analyzer (Applied Biosystems), PCR, centrifuge.
(2) prepared by reagent:
AD1:A is mixed with B according to volume 1:1.
A:SDS(ammonium lauryl sulfate) it is dissolved in the NH4HCO3 of 10mM, it is configured to the solution for being 8.3 containing 5% SDS, pH
The NP-40 of the PNGaseF and 3.33% of B:2.2U/ μ L 1:20 mixed preparing by volume
AD2: the NaCNBH3 of the APTS and 1M of the 20mM of same volume are mixed.
NH4AC, sialidase (neuraminidase, 2mU/ μ L) and the hydrogen peroxide of AD3:100mM is according to volume 5:1:
14
Mixing.
AD4: buffer.
(3) N- oligonucleotide chain map detects
A, the preparation of oligonucleotide chain: taking the 2 μ L of serum inactivated at 95 DEG C, and 4 μ L AD1,37 DEG C of reactions are added
It is dried after 3h.
B, the label of oligonucleotide chain: the reagent A D2 of 2 μ L is added in sample after the drying, is carried out at 65 DEG C
After fluorescent marker 3h, the AD4 that 200 μ L are added terminates reaction.
C, label post-processing: taking labeled good 2 μ L of sample, is added the AD3 of 2 μ L, at 45 DEG C
3h is reacted, the AD4 for finally adding 200 μ L terminates reaction.
D, oligosaccharides chain separation is analyzed: being taken 10 μ L of fluid sample 3., is carried out under ABI3500dx instrument
N- oligosaccharides chain separation, to obtain N- sugar group map
E, Data Management Analysis: obtained N- sugar group map is subjected to peak value quantization, with the peak at each peak
High level is compared with the summation of the height at all peaks, to quantitatively calculate the relative amount at each peak, then carries out into one
The statistical analysis of step carries out further statistical analysis by function NGA2F/NA2, to detect alzheimer's disease.
It detects sample and passes through ROC indicatrix made by NGA2F/ NA2；Detect total sample number be 408, wherein Ah
Wurz sea is write from memory 70, disease sample, and 338, healthy control group sample；Compare alzheimer's disease patient using function NGA2F/NA2
The N- oligonucleotide chain test map peak value of (n=70) non-alzheimer's disease (n=338), obtains area under the curve AUC=0.877.
Reagent and detection method of the invention, compared with prior art, accuracy reaches 87.7%, hence it is evident that better than existing
Detection method.Using reagent and detection method of the invention, the crowd of numerous high risks can be allowed to receive conventional, Non-invasive detection,
It helps doctor and patient to monitor generation and the disease progression of alzheimer's disease in time, is expected to promote the use of in clinic.
The specific embodiment in conjunction with described in attached drawing above carries out the purpose of the present invention, technical scheme and beneficial effects
It is further described, it should be understood that the above is only a specific embodiment of the present invention, but not to the present invention
The limitation of protection scope, those skilled in the art should understand that, all within the spirits and principles of the present invention, do not need to pay
Any modification, equivalent substitution, improvement and etc. that creative work can be made, should all be included in the protection scope of the present invention.
Claims (3)
1. a kind of alzheimer's disease detection reagent, it is characterised in that: mixed with B according to volume 1:1 including AD1:A, wherein A:
The NP-40 of the PNGaseF and 3.33% of 5.0% SDS, pH 8.3, B:2.2U/ μ L 1:20 mixed preparing by volume；AD2: mixed
The NaCNBH3 of the APTS and 1M of the 20mM of contract volume；NH4AC, 2mU/ μ L sialidase and the hydrogen peroxide of AD3:100mM according to
Volume 5:1:14 mixing；AD4: buffer.
2. alzheimer's disease detection reagent according to claim 1, it is characterised in that: the volume of the AD1 is 4 μ L,
It is 2 μ L, AD4 volumes is 200 μ L that the volume of AD2, which is 2 μ L, AD3 volumes,.
3. a kind of application of composition in alzheimer's disease detection reagent, the composition are made of NGA2F and NA2, institute
Composition is stated by the ratio of NGA2F/NA2 to detect alzheimer's disease.
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Cited By (1)
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| CN113527459A (en) * | 2021-06-28 | 2021-10-22 | 广州睿徕医学诊断技术有限公司 | Extracting agent and preparation method and application thereof |
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Effective date of registration: 20200201 Address after: 225312 Jiangsu city of Taizhou province China pharmaceutical Road East, Xinyang City Road on the north side of building six on the eastern side of G26 Applicant after: Jiangsu first star Biological Technology Co., Ltd. Applicant after: Xiansida (Nanjing) Biotechnology Co., Ltd. Address before: 225312 Jiangsu city of Taizhou province China pharmaceutical Road East, Xinyang City Road on the north side of building six on the eastern side of G26 Applicant before: Jiangsu first star Biological Technology Co., Ltd. Applicant before: Xiansida (Nanjing) Biotechnology Co., Ltd. Applicant before: Changzhou Ji Tai Biotechnology Co., Ltd. |
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Inventor after: Chen Cuiying Inventor after: Tan Zongnan Inventor after: Zhu Hongzhang Inventor after: Yang Xin Inventor before: Chen Cuiying Inventor before: Tan Zongnan Inventor before: Zhu Hongzhang Inventor before: Yang Xin |
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