CN109738653A - Combine for the antigen protein of the detection of Alzheimer's disease, diagnosis or risk profile and include its kit - Google Patents
Combine for the antigen protein of the detection of Alzheimer's disease, diagnosis or risk profile and include its kit Download PDFInfo
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Abstract
The present invention provides a kind of for detecting the kit of alzheimer's disease autoantibody in serum, the kit includes antigen protein combination, the antigen protein combination includes at least four albumen, and the albumen is selected from RABPT5, RAGE, CRYAB, SRPK1, MAPT, MBP, PLP1, PTCD2, FRMD8, POMC, DNAJC8, CENTA2, HSP60, ADARB1, ASXL1, EDRK, GDF11, P21, CCL2, IL18, VEGF, LENG1 and MRPL34.The method of the detection of purposes and Alzheimer's disease of the combination of the antigen protein the present invention also provides defined by reagent of the preparation for the detection of Alzheimer's disease, diagnosis or risk profile, diagnosis or risk profile.
Description
Technical field
The invention belongs to field of biological detection, specifically, the present invention relates to one kind for detecting in mammalian sample
The antigen combination and its application of autoantibody, to realize the early diagnosis for whether suffering from Alzheimer's disease to mammal.
Background technique
Alzheimer's disease (AD), is commonly called as senile dementia, is the unknown primary degeneration cerebral degeneration's disease of one group of cause of disease
Disease, for a lot of diseases in the senescence phase, the course of disease is slow and irreversible.The clinical manifestation of Alzheimer's disease predominantly remembers and other cognitions
Afunction, onset is slow, is not easy to be found, characterized by the pre- symptom phase that may be lasted for several years；Meanwhile patient is in clinical condition
Nerve degeneration has occurred before occurring for shape, and has the quite long course of disease after the onset.
According to World Health Organization, have 10% Alzheimer can occur in the elderly of world today's over-65s
Disease, and by 80 years old, which rises to 30% or more.The mean survival time (MST) for estimating AD patient is 10-15, is in elderly population
It is only second to the 4th cause of death of heart disease, malignant tumour and apoplexy.According to Chinese Alzheimer disease association recent statistics, I
The current AD illness population of state is already close to 8,000,000, it is contemplated that the year two thousand fifty illness population will be more than 20,000,000.Jia Jianping professor and its
Team publishes thesis " Alzheimer's disease is reappraised what China and disease worldwide bore ", the article pointed out,
Spending per capita in year for Chinese Alzheimer Disease patient is 19144.36 dollars (being roughly equal to 130,000 yuan of RMB) within 2015, China Ah
Social economical burden total value caused by Er Cihaimo disease reaches 1677.4 hundred million dollars (being roughly equal to 1,140,600,000,000 yuan of RMB).It expects
The year two thousand thirty, China's Alzheimer's disease financial burden are up to 2.54 trillion dollars and (are converted into about 17 trillion yuan of RMB.With
The aging of population and the extension of the average life span, Alzheimer's disease are just gradually becoming the important diseases of China or even other countries
Burden, causes the public health problem got worse.
This disease is so far without good treatment method, but good continuity nursing intervention can delay progression of the disease,
The remaining function of patient is prevented further to be damaged.Therefore, for the patient, disease is able to early diagnose for preventing or delaying
Disease occurs or the appearance of clinical symptoms is most important.Currently, the diagnosis goldstandard of Alzheimer's disease is starch in brain tissue
The deposition of sample albumin A β.But this diagnosis needs traumatic biopsy of brain or the dead rear postmortem of patient is just able to achieve, it is uncomfortable
It shares in clinical examination.There are also Cranial Computed Tomographies and MRI, amyloid protein PET imaging, genetic test, brain ridge for other inspection methods
Liquid amyloid protein, Tau albumen inspection etc., these indexs have usually carried out occurring clinical symptoms for many years in Alzheimer's disease
After could be detected or identify, thus be difficult to realize early diagnose.
The detection of Serum Antibody has been a kind of very mature technology, this becomes the accurate detection of autoantibody can
Energy.For example, the triumphant Borrow Biotechnology Co., Ltd in domestic corporation Hangzhou is developed using autoimmune antibody using antigen combination
It detects the first kit in the country of lung cancer, achieves extraordinary clinical effectiveness.And for Alzheimer's disease,
There are a large amount of cards it is demonstrated that human immune system can produce autoantibody early stage AD develops, this is anti-by detecting itself
Body is possibly realized to be detected or be diagnosed to Alzheimer's disease.
But due to technical restriction, many autoantibodies of Alzheimer's disease are being difficult to be detected in early days；Meanwhile
Although reporting a variety of autoantibodies and AD has correlation, which antibody has extensive clinical meaning, on the knees of the gods.
Moreover even if some autoantibodies are considered having certain correlation with AD, small-scale conceptual phase only also is stayed in, still
Lacking extensive, repeatable clinical data is proved.
Therefore, although Current Diagnostic means are continuously improving, this field still needs the new of Alzheimer's disease
Detection or diagnostic means；In particular, this field, which still needs, provides a kind of new inspection for the early diagnosis of AD and disease course prediction
Survey method.
Summary of the invention
In order to solve the above technical problem, the present invention provides a kind of for detecting the antigen protein group of Alzheimer's disease
It closes, the antigen protein combination includes at least four albumen, and the albumen is related to Alzheimer's disease specificity, therefore provides
A kind of relatively accurate early detection or diagnostic tool of Alzheimer's disease.
Therefore, it is an object of the present invention to provide a kind of kit, the kit includes antigen protein combination, described
Antigen protein combination includes at least four albumen.
It is a further object to provide the antigen protein combination preparation for Alzheimer's disease detection,
Purposes in the reagent of diagnosis or risk profile.
The method that a further object is the detection that a kind of Alzheimer's disease is provided, diagnosis or risk profile of the invention.
Technical scheme is as follows.
On the one hand, the present invention provides a kind of kit, and the kit includes antigen protein combination, the antigen protein group
Close include at least four albumen, the albumen be selected from RABPT5, RAGE, CRYAB, SRPK1, MAPT, MBP, PLP1, PTCD2,
FRMD8、POMC、DNAJC8、CENTA2、HSP60、ADARB1、ASXL1、EDRK、GDF11、P21、CCL2、IL18、VEGF、
LENG1 and MRPL34.
The antigen combination be used for detect alzheimer's disease in biological sample, such as serum autoantibody exist with
It is no.Therefore, kit provided by the invention can be used for the detection or diagnosis of alzheimer's disease, especially early detection or examine
It is disconnected；It can also be used in the risk prediction of alzheimer's disease.
The sequence of above-mentioned albumen is illustratively shown in Table 1.
The sequence for the albumen for including in the combination of 1. antigen protein of table
| Albumen | Database ID | Albumen | Database ID |
| RABPT5 | NM_004703.6 | CENTA2 | NM_018404 |
| RAGE | NM_001136.5 | HSP60 | NM_002156.5 |
| CRYAB | NM_001289807.1 | ADARB1 | NM_001112.4 |
| SRPK1 | NM_003137.5 | ASXL1 | NM_015338.5 |
| MAPT | NM_016835.4 | EDRK | NM_001199875.1 |
| MBP | NM_001025101.2 | GDF11 | NM_005811 |
| PLP1 | NM_000533.5 | P21 | NM_000389 |
| PTCD2 | NM_024754.5 | CCL2 | NM_002982.4 |
| FRMD8 | NM_031904 | IL18 | NM_001562.4 |
| POMC | NM_000939.4 | VEGF | NM_001025366.2 |
| DNAJC8 | NM_014280 | LENG1 | NM_024316.2 |
| MRPL34 | NM_023937.3 |
In kit provided by the invention, it is preferable that the albumen be selected from RAGE, HSP60, MRPL34, CCL2, MBP,
P21, CENTA2, ASXL1, ADARB1, DNAJC8, MAPT and GDF11；And optionally, selected from RABPT5, CRYAB, SRPK1,
PLP1, PTCD2, FRMD8, POMC, EDRK, IL18, VEGF and LENG1.
It is highly preferred that antigen protein combination comprising at least three kinds or at least four selected from RAGE, HSP60, MRPL34,
The albumen of CCL2, MBP, P21, CENTA2, ASXL1, ADARB1, DNAJC8, MAPT and GDF11.
Further, according to the grouping of albumen, the antigen protein combination is selected from (1) group comprising at least two below
Albumen, at least one albumen and optionally at least one albumen for being selected from (3) group selected from (2) group:
(1)RAGE,HSP60,MRPL34,CCL2,MBP,P21,CENTA2；
(2) ASXL1, ADARB1, DNAJC8, MAPT and GDF11；
(3) RABPT5, CRYAB, SRPK1, PLP1, PTCD2, FRMD8, POMC, EDRK, IL18, VEGF and LENG1.
It is highly preferred that the antigen protein combination includes at least three kinds albumen and at least one selected from described (1) group
Albumen selected from described (2) group.
Specific embodiment according to the present invention, in kit provided by the invention, the antigen protein combination includes
Albumen RAGE and DNAJC8；Preferably, the antigen protein combination further includes albumen MRPL34；It is highly preferred that described anti-
Former protein combination further includes albumen HSP60；It is further preferred that the antigen protein combination further includes albumen
ADARB1。
Specific embodiment according to the present invention, in kit provided by the invention, the antigen protein combination includes
Albumen RAGE, DNAJC8, HSP60 and MRPL34；Preferably, the antigen protein combination further includes albumin A DARB1；
Alternatively, in kit provided by the invention, the antigen protein combination comprising albumen RAGE, DNAJC8,
MRPL34 and ADARB1；Preferably, the antigen protein combination further includes albumen HSP60.
Specifically, the antigen protein combination includes albumen RAGE, DNAJC8, HSP60 and MRPL34, and also include
PROTEIN C ENTA2；Preferably, the antigen protein combination further includes PROTEIN C RYAB；Preferably, the antigen protein combination
Further include Protein S RPK1；Preferably, the antigen protein combination further includes albumen FRMD8.
It alternatively, the antigen protein combination includes albumen RAGE, DNAJC8, MRPL34 and ADARB1, and also include egg
White RABPT5；Preferably, the antigen protein combination further includes albumen PLP1；Preferably, the antigen protein combine into
One step includes albumen PTCD2；Preferably, the antigen protein combination further includes albumen POMC.
Specific embodiment according to the present invention, in kit provided by the invention, antigen protein combination can be with
Are as follows:
1)RAGE,DNAJC8,HSP60,MRPL34；
2)RAGE,DNAJC8,HSP60,MRPL34,CENTA2,CRYAB,SRPK1；
3)RAGE,DNAJC8,HSP60,MRPL34,CENTA2,CRYAB,SRPK1,FRMD8；
4)RAGE,DNAJC8,HSP60,MRPL34,CENTA2,CRYAB,SRPK1,FRMD8,EDRK；
5)RAGE,DNAJC8,HSP60,MRPL34,ADARB1,CCL2,MAPT,ASXL1,GDF11；
6)RAGE,DNAJC8,HSP60,MRPL34,ADARB1,CCL2,MAPT,MBP,GDF11；
7)RAGE,DNAJC8,MRPL34,ADARB1,RABPT5,PLP1,PTCD2；
8)RAGE,DNAJC8,MRPL34,ADARB1,RABPT5,PLP1,PTCD2,POMC；
9)RAGE,DNAJC8,MRPL34,ADARB1,RABPT5,PLP1,PTCD2,POMC,P21；Or
10)RAGE、DNAJC8、IL18、VEGF、CENTA2、CRYAB、SRPK1、LENG1。
Albumen provided by the invention can be expressed in Escherichia coli, yeast or mammalian cell and be obtained according to sequence,
And optionally, it is purified through Ni column, molecular sieve, ion column or drainage column etc..
Preferably, kit provided by the invention further includes solid phase carrier, the albumen for including in the antigen protein combination
It is individually fixed on solid phase carrier.Preferably, the solid phase carrier is, for example, micropore of enzyme marker plate, magnetic bead, affinity membrane or liquid phase core
Piece；The fixation of the albumen includes that albumen is directly fixed on solid phase carrier, or is indirectly secured to admittedly via middle interconnecting piece point
On phase carrier, such as pass through the specific reaction between biotin and Streptavidin.
Kit provided by the invention can be used for detecting the autoantibody in biological sample (such as serum), and the detection is logical
It crosses the antigen protein combination for including in kit to carry out, the albumen in the combination can be with pair in biological sample as antigen
Ag-Ab specific reaction occurs for the autoantibody (if present) answered, and the testing result of reaction can indicate biology
The presence or absence of autoantibody in sample, so indicate biological sample source (usually mammal, such as people) whether
With alzheimer's disease or with the risk for suffering from alzheimer's disease.
Therefore, correspondingly, kit provided by the invention can also include other reagents or component, other described reagents or
Component is used in using autoantibody in the antigen protein combine detection biological sample.
Specific embodiment according to the present invention, kit provided by the invention can be used for passing through enzyme linked immunosorbent assay
To detect the autoantibody in biological sample (such as serum).
For being detected by enzyme linked immunosorbent assay, kit provided by the invention can be enzyme linked immunosorbent detection examination
Agent box.For testing goal, for example, the albumen can be connected with labelled peptide, it is preferable that the labelled peptide be selected from His label,
GST label, c-Myc label, Flag label, HA label and biotin label.For another example, kit provided by the invention can also wrap
Include positive quality control product, negative quality-control product or standard items.For example, the positive quality control product or standard items are recombination human immunoglobulin(HIg)
G or its segment.Kit provided by the invention can also include immunoglobulin, coating buffer, closing with enzyme label
Liquid, 20 × cleaning solution, serum/antibody diluent, TMB color developing agent, terminate liquid etc..
It is combined based on antigen protein provided by the invention, corresponding kit can be prepared using this field routine techniques.
Specific embodiment according to the present invention can be detected with reference to disclosed in Chinese patent application open file CN103869086A
The preparation method of kit.
On the other hand, the present invention provide the kit or in which antigen protein combination in preparation for Alzheimer
Purposes in the reagent of the detection of disease, diagnosis or risk profile.
Also on the one hand, the present invention provides the method for the detection of Alzheimer's disease a kind of, diagnosis or risk profile, the side
Method includes that the antigen protein combination is made to be in contact with biological sample.
Preferably, the biological sample comes from mammal, preferably people；Preferably, the biological sample is serum.
According to reports, having had more than 30 kinds of autoantibodies and AD so far has correlation, still, which antibody has AD
Early detection or diagnosis or even risk profile have universal clinical meaning, have clinical meaning also not especially in Chinese population
It is clear, therefore, Alzheimer's disease is diagnosed even with autoantibody, there are still queries for feasibility.
In contrast, the present invention provides a kind of novel antigen protein combination, the antigen protein combination is comprising at least
Four kinds of albumen relevant to Alzheimer's disease specificity, thus detect or diagnose A Erci for high sensitivity and with high specificity
The silent disease in sea provides a kind of new tool.
Compared with the existing technology, technical solution of the present invention has the advantages that
Firstly, the present inventor screens albumen known to 50 kinds, it is found that compared to other albumen, there is 23
When kind albumen is as antigen, stronger specific reaction can occur with the autoantibody of Alzheimer Disease patient, thus prove
The feasibility of alzheimer's disease is detected or diagnosed by detecting the autoantibody of subject.
Also, the present inventor also provides a kind of antigen protein combination, which includes the 23 kinds of albumen filtered out
In at least four.Further, which can be divided into different protein groups, by from different groups of albumen
Selection forms antigen protein combination, can take into account the high sensitivity and high specific of testing result.It is demonstrated experimentally that coming from A Er
In the biological sample of Zi Haimo disease patient or health volunteer, antigen protein provided by the invention combination realize it is highly sensitive and
The detection effect of specificity, fairly accurate early detection, diagnosis or the risk for thus providing a kind of Alzheimer's disease are pre-
Survey tool.
Kit can be made in antigen protein provided by the invention combination, and can be used for related detecting method, thus by
Early screening, detection, diagnosis or the risk profile that alzheimer's disease is carried out in examination person such as people, are really realized clinically to Ah
The early screening of the silent disease in Wurz sea, has a good application prospect in terms of the diagnosing and treating of alzheimer's disease tumor.Also,
It is detected using biological sample such as autoantibodies in serum of the antigen protein to subject such as people, is a kind of non-invasive
Detection method, with advantage easy to use, subject's compliance is good.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 shows the sensibility and specificity of 23 kinds of Protein Detection autoantibodies in serum.
Fig. 2 shows the sensibility and specificity of different antigen protein combine detection autoantibodies in serum, wherein 2A is
Overall sensitivity and specificity, 2B are early stage, mid-term, the sensibility in advanced stage.
Specific embodiment
The present invention is described below with reference to specific embodiments.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, do not limit the scope of the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.Medicine as used in the following examples
Material raw material, reagent material etc. are commercially available products unless otherwise specified.
In addition to antigen protein combines, the composition of the enzyme-linked immunologic detecting kit (immobilized antigen) for detecting autoantibody
May include as needed following component:
1) it is used for the ELISA Plate of envelope antigen albumen；
2) goat anti-human immunoglobulin of horseradish peroxidase-labeled, concentration 100ng/ml；
3) positive quality control product (standard items): the anti-c-Myc label immunoglobulin G of people (is purchased from Tribioscience company)；
4) negative quality-control product；
5) sealed membrane；
6) buffer, confining liquid, 20 × cleaning solution, serum/antibody diluent, TMB color developing agent, terminate liquid are coated with, in which:
Coating buffer is PBS, preparation method: pH 7.4 takes 3.58g Na2HPO4·12H2O, 0.24g KH2PO4·
2H2O, 0.2g KCl and 8.0g NaCl, are settled to 1L after dissolving using distilled water；
The preparation method of confining liquid: 5g casein is dissolved in PBS solution, is then settled to 1L；
The preparation method of 20 × cleaning solution: 0.5%Tween20 is added in 20 × PBS, pH7.4；
Serum/antibody diluent preparation method: 1g casein and 10g bovine serum albumin(BSA) are dissolved in PBS solution, so
After be settled to 1L；
The preparation method of TMB color developing agent: 50mM imidazole buffer pH 5,7.5mM PEG3350,2.94mM hydrogen peroxide urine
Element, 1.6mM TMB；
The preparation method of terminate liquid: 2M sulfuric acid.
In the examples below, include: with the operation of ELISA detection serum sample using ELISA Plate
With coating buffer by antigen diluent to final concentration of 100nM, every hole is added on 50 μ l to ELISA Plate, 4 DEG C of coatings
Overnight；Next day outwells solution, after plate is patted dry, is washed three times with 250 μ l cleaning solutions；250 μ l confining liquids, incubation at room temperature closing is added
After 1hr, plate is drained, is washed three times with 250 μ l cleaning solutions, and drain again；
It is added and uses the diluted serum sample (dilution 1:100) of serum dilution, every hole adds 50 μ l, and room temperature is placed in microwell plate
After shaker oscillation incubation 1hr, plate is drained, is washed three times with 250 μ l cleaning solutions, and drain again, antibody diluent is used in addition
The recombination goat anti-human immunoglobulin G antibody (1:20000) of diluted horseradish peroxidase-labeled, every hole add 50 μ l, room temperature
It is placed in microwell plate shaker oscillation incubation 0.5hr, plate is drained, is washed three times with 250 μ l cleaning solutions, and drain again；
Every hole adds 50 μ l TMB color developing agents, and after color development at room temperature 15min, 50 μ l terminate liquids are added in every hole；Microplate reader is in 450nm
Read plate under wavelength, then again analyzes data.
Embodiment 1The protein screening of antigen as autoantibody
By literature search, 50 kinds of known albumen relevant to Alzheimer's disease are had chosen, are shown in Table 2.
The sequence of the albumen to be tested of table 2.
Using Human cDNA Library's (being purchased from Invitrogen company) or the DNA of full genome synthesis as template, expanded using PCR
Increase or digestion method prepares required recombinant antigen protein segment.For example, the PCR product that amplification is obtained is after purification, even
It is connected on pET28 carrier, and the appropriate labels such as HIS, c-myc is added to form fusion protein, so in antigen encoding sequences upstream
Recombinant vector is converted into bacillus coli DH 5 alpha competent cell afterwards.The recombinant plasmid of acquisition uses bacterium colony PCR, digestion and survey
Sequence identification confirmation includes correct exogenous sequences.By the recombinant plasmid transformed comprising fused antigen segment of acquisition to Escherichia coli
In BL21 (DE3) competent cell, through isopropylthio-β-D-galactoside (IPTG) inducing expression, recombinant antigen egg is obtained
White, which uses Bradford standard measure after Ni-NTA column and molecular sieve two-step purifying, anti-using SDS-PAGE identification confirmation
Former protein expression, purifying and quantitative result.
The antigen coat plate in the above way obtained, then with the operating procedure detection of above-mentioned ELISA detection serum sample
The autoantibody of correspondence every kind of antigen that 94 Alzheimer's and 94 health volunteers generate.With 94 health
Experimenter's serum is negative reference sample, calculates the average value (M) and standard deviation of the detection signal (S) of all negative reference samples
(SD), with M+3SD for CutOff value, the sample that detection signal (S) is greater than or equal to CutOff value is then set to the positive, i.e. S >=M+
3SD is then the positive, and the sample that detection signal (S) is less than CutOff value is then set to feminine gender, i.e. S < M+3SD is then negative.
Specificity and sensibility are calculated based on positive and negative findings.Wherein specificity refers to health volunteer's sample by just
Really it is determined as negative ratio, i.e., is correctly determined as negative quantity divided by negative sample sum in negative sample；
Sensibility refers to that Alzheimer's sample is correctly determined as positive ratio, i.e., is judged as in positive sample
Positive quantity is divided by positive sample sum.By the way that each tested albumen is calculated as antigen, pattern detection is carried out
When sensibility and specificity, the results are shown in Table 3.
Sensibility and specificity of 3. albumen of table as antigen test
By comparing the sensibility and specificity of each test antigen, with sensibility >=10% and specificity >=90% resists
Original work are candidate antigens.Finishing screen, which selects 23 kinds of albumen, can be used as the reason for distinguishing Alzheimer's and health volunteer
Think candidate antigens, the selection result is as shown in table 4.
The antigen protein that table 4. filters out
| Candidate antigens | Sensibility | Specificity | Candidate antigens | Sensibility | Specificity |
| RAGE | 35.1% | 94.7% | SRPK1 | 20.2% | 98.9% |
| DNAJC8 | 36.2% | 92.6% | RABPT5 | 23.4% | 98.9% |
| HSP60 | 34.0% | 98.9% | PLP1 | 13.8% | 97.9% |
| MRPL34 | 31.9% | 93.6% | PTCD2 | 16.0% | 98.9% |
| ADARB1 | 29.8% | 95.7% | FRMD8 | 19.1% | 97.9% |
| CCL2 | 28.7% | 94.7% | POMC | 21.3% | 98.9% |
| MAPT | 29.8% | 100.0% | EDRK | 18.1% | 100.0% |
| ASXL1 | 27.7% | 97.9% | P21 | 13.8% | 98.9% |
| GDF11 | 24.5% | 98.9% | IL18 | 11.7% | 97.9% |
| MBP | 21.3% | 98.9% | VEGF | 16.0% | 93.6% |
| CENTA2 | 19.1% | 97.9% | LENG1 | 12.8% | 97.9% |
| CRYAB | 21.3% | 98.9% |
Embodiment 2Candidate antigen protein combines the detected representation in Alzheimer's and health volunteer
Alzheimer's and health volunteer are pressed into simple mental state checklist (Mini-Mental State
Examination, MMSE)-Folstein editions score, score is normal person, score 21- in the tester for 27-30
26 tester is slight (early stage) patient, and the tester that score is 10-20 is moderate (mid-term) patient, and score is the survey of 0-9
Examination person is severe (advanced stage) patient.
It is scored according to MMSE, takes Alzheimer's early stage 120, mid-term 87, advanced stage 75 as detection sample
This, takes 94 MMSE to score sample of the normal health volunteer as health volunteer, and test each group antigen protein combines
Sensibility and specificity.
According to the selection result in embodiment 1, select multiple protein as antigen combination from 23 kinds of albumen, it is solid with antigen
Fixed mode uses antigen protein combine detection in Alzheimer's and health volunteer's serum by ELISA
Autoantibody.Similarly, using 94 health volunteer's serum as negative reference sample, the detection of all negative reference samples is calculated
The average value (M) and standard deviation (SD) of signal (S), with M+3SD for CutOff value, detection signal (S) is greater than or equal to CutOff
The sample of value is then set to the positive, i.e. S >=M+3SD is then the positive, and the sample that detection signal (S) is less than CutOff value is then set to yin
Property, i.e. S < M+3SD is then negative.
The combination is calculated in difference based on the respective positive and negative findings of all antigens for including in an antigen combination
Sensibility and overall sensitivity and overall specificity in period clinical samples.Wherein, the repetition that synantigen does not obtain is excluded
As a result, calculating early stage sensibility, refer to that 120 early stage Alzheimer's samples are correctly determined as positive ratio
Example is judged as positive quantity divided by early stage Alzheimer's sample in early stage Alzheimer's sample
This sum；Mid-term sensibility is calculated, refers to that 87 mid-term Alzheimer's samples are correctly determined as the positive
Ratio is judged as positive quantity divided by mid-term Alzheimer's in mid-term Alzheimer's sample
Total sample number；Advanced stage sensibility is calculated, refers to that 75 advanced stage Alzheimer's samples are correctly determined as the positive
Ratio, i.e., be late judged as positive quantity in Alzheimer's sample and suffer from divided by advanced stage alzheimer's disease
Person's total sample number.In addition, calculating overall sensitivity, refer in 120 early stages, 87 mid-terms, 75 advanced stage Alzheimers
Disease clinical samples are correctly determined as positive ratio in total, i.e., are judged as in all Alzheimer's samples
Positive quantity is divided by all Alzheimer's total sample numbers；Overall specificity is calculated, refers to that 94 health are tested
Person's sample is correctly determined as that negative ratio is judged in health volunteer's sample that is, in 94 health volunteer's samples
It is set to negative quantity divided by health volunteer's total sample number.
Sensibility and specificity is taken into account, using the antigen combination of overall sensitivity >=50% and specificity >=85% as ideal
Antigen combination, the ideal antigen protein combination filtered out are shown in Table 5.Different ideal antigen protein combinations are in detection different times
The performance of autoantibody is shown in Table 6 in Alzheimer's and health volunteer's sample.
The ideal antigen combination of table 5.
The detected representation of the different antigen protein combinations of table 6.
| Early stage sensibility | Mid-term sensibility | Advanced stage sensibility | Overall sensitivity | Overall specificity | |
| Combination 1 | 75.0% | 44.8% | 32.0% | 54.3% | 90.4% |
| Combination 2 | 75.0% | 51.7% | 44.0% | 59.6% | 89.4% |
| Combination 3 | 75.0% | 58.6% | 36.0% | 59.6% | 88.3% |
| Combination 4 | 77.5% | 58.6% | 36.0% | 60.6% | 88.3% |
| Combination 5 | 87.5% | 72.4% | 76.0% | 79.8% | 90.4% |
| Combination 6 | 87.5% | 72.4% | 82.5% | 83.0% | 90.4% |
| Combination 7 | 82.5% | 72.4% | 36.0% | 67.0% | 87.2% |
| Combination 8 | 82.5% | 75.9% | 36.0% | 68.1% | 87.2% |
| Combination 9 | 82.5% | 79.3% | 36.0% | 69.1% | 87.2% |
| Combination 10 | 72.5% | 62.1% | 48.0% | 62.8% | 90.4% |
By comparing discovery, each combined overall sensitivity is both greater than 54% and specificity both greater than 87%.Compared to
Mid-term and advanced stage, in early stage sample, each combine has higher sensibility, and both greater than 72%, combine 5 and combination 6
Sensibility all reaches 87.5%.Therefore, the present invention provides more smart for the diagnosis of Alzheimer disease, especially early diagnosis
True detection method.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deforms, and as long as it does not depart from the spirit of the invention, should belong to the model of appended claims of the present invention
It encloses.
Claims (13)
1. a kind of kit, the kit includes antigen protein combination, and the antigen protein combination includes at least four albumen,
The albumen be selected from RABPT5, RAGE, CRYAB, SRPK1, MAPT, MBP, PLP1, PTCD2, FRMD8, POMC, DNAJC8,
CENTA2, HSP60, ADARB1, ASXL1, EDRK, GDF11, P21, CCL2, IL18, VEGF, LENG1 and MRPL34.
2. kit according to claim 1, which is characterized in that the albumen be selected from RAGE, HSP60, MRPL34,
CCL2, MBP, P21, CENTA2, ASXL1, ADARB1, DNAJC8, MAPT and GDF11；And optionally, selected from RABPT5,
CRYAB, SRPK1, PLP1, PTCD2, FRMD8, POMC, EDRK, IL18, VEGF and LENG1.
3. kit according to claim 2, which is characterized in that the antigen protein combination is comprising at least three kinds or at least
Four kinds are selected from RAGE, HSP60, MRPL34, CCL2, MBP, P21, CENTA2, ASXL1, ADARB1, DNAJC8, MAPT and GDF11
Albumen.
4. kit according to claim 3, which is characterized in that antigen protein combination is comprising at least two selected from the
(1) albumen, at least one albumen and optionally at least one albumen for being selected from (3) group selected from (2) group organized:
(1)RAGE,HSP60,MRPL34,CCL2,MBP,P21,CENTA2；
(2) ASXL1, ADARB1, DNAJC8, MAPT and GDF11；
(3) RABPT5, CRYAB, SRPK1, PLP1, PTCD2, FRMD8, POMC, EDRK, IL18, VEGF and LENG1.
5. kit according to claim 4, which is characterized in that the antigen protein combination is selected from institute comprising at least three kinds
State the albumen and at least one albumen selected from described (2) group of (1) group.
6. kit according to claim 4, which is characterized in that antigen protein combination comprising albumen RAGE and
DNAJC8；
Preferably, the antigen protein combination further includes albumen MRPL34；
It is highly preferred that the antigen protein combination further includes albumen HSP60；
It is further preferred that the antigen protein combination further includes albumin A DARB1.
7. kit according to claim 6, which is characterized in that antigen protein combination comprising albumen RAGE,
DNAJC8, HSP60 and MRPL34；Preferably, the antigen protein combination further includes albumin A DARB1；
Alternatively, the antigen protein combination includes albumen RAGE, DNAJC8, MRPL34 and ADARB1；Preferably, the antigen egg
White combination further includes albumen HSP60.
8. kit according to claim 7, which is characterized in that antigen protein combination comprising albumen RAGE,
DNAJC8, HSP60 and MRPL34, and also include PROTEIN C ENTA2；
Preferably, the antigen protein combination further includes PROTEIN C RYAB；
Preferably, the antigen protein combination further includes Protein S RPK1；
Preferably, the antigen protein combination further includes albumen FRMD8.
9. kit according to claim 7, which is characterized in that antigen protein combination comprising albumen RAGE,
DNAJC8, MRPL34 and ADARB1, and also include albumen RABPT5；
Preferably, the antigen protein combination further includes albumen PLP1；
Preferably, the antigen protein combination further includes albumen PTCD2；
Preferably, the antigen protein combination further includes albumen POMC.
10. kit according to claim 1, which is characterized in that the antigen protein combination are as follows:
1)RAGE,DNAJC8,HSP60,MRPL34；
2)RAGE,DNAJC8,HSP60,MRPL34,CENTA2,CRYAB,SRPK1；
3)RAGE,DNAJC8,HSP60,MRPL34,CENTA2,CRYAB,SRPK1,FRMD8；
4)RAGE,DNAJC8,HSP60,MRPL34,CENTA2,CRYAB,SRPK1,FRMD8,EDRK；
5)RAGE,DNAJC8,HSP60,MRPL34,ADARB1,CCL2,MAPT,ASXL1,GDF11；
6)RAGE,DNAJC8,HSP60,MRPL34,ADARB1,CCL2,MAPT,MBP,GDF11；
7)RAGE,DNAJC8,MRPL34,ADARB1,RABPT5,PLP1,PTCD2；
8)RAGE,DNAJC8,MRPL34,ADARB1,RABPT5,PLP1,PTCD2,POMC；
9)RAGE,DNAJC8,MRPL34,ADARB1,RABPT5,PLP1,PTCD2,POMC,P21；Or
10)RAGE、DNAJC8、IL18、VEGF、CENTA2、CRYAB、SRPK1、LENG1。
11. the inspection that the combination of antigen protein defined in any one of claims 1 to 10 is used for Alzheimer's disease in preparation
Purposes in the reagent of survey, diagnosis or risk profile.
12. a kind of method of detection of Alzheimer's disease, diagnosis or risk profile, the method includes making the antigen egg
White combination is in contact with biological sample.
13. according to the method for claim 12, which is characterized in that the biological sample comes from mammal, preferably people；It is excellent
Selection of land, the biological sample are serum.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110850104A (en) * | 2020-01-15 | 2020-02-28 | 上海众启生物科技有限公司 | Protein antigen combination for detecting autoantibodies of Alzheimer's disease and application thereof |
| AU2019201686B2 (en) * | 2010-05-13 | 2021-05-27 | Rowan University | Diagnostic autoantibody profiles for the detection and diagnosis of neurodegenerative diseases |
| CN116064440A (en) * | 2022-09-13 | 2023-05-05 | 北京湃德智健科技有限公司 | Antigen polypeptide for detecting VKORC1 autoantibody and application thereof |
| CN116063447A (en) * | 2022-09-13 | 2023-05-05 | 北京湃德智健科技有限公司 | Antigen polypeptide for detecting ADAP autoantibody and application thereof |
| WO2024055378A1 (en) * | 2022-09-13 | 2024-03-21 | 北京湃德智健科技有限公司 | Protein-antigen combination for detecting autoantibodies in alzheimer's disease, and use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101203242A (en) * | 2005-04-22 | 2008-06-18 | 健泰科生物技术公司 | Method for treating dementia or alzheimer's disease by CD2O antibody |
| CN103154736A (en) * | 2010-05-13 | 2013-06-12 | 新泽西医科和牙科大学 | Diagnostic autoantibody profiles for the detection and diagnosis of neurodegenerative diseases |
| CN103869086A (en) * | 2014-04-14 | 2014-06-18 | 杭州凯保罗生物科技有限公司 | Serum autoantibody detection kit |
| US20180067133A1 (en) * | 2015-03-17 | 2018-03-08 | Electrophoretics Limited | Materials and methods for diagnosis and treatment of alzheimer's disease |
-
2019
- 2019-01-11 CN CN201910027394.2A patent/CN109738653B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101203242A (en) * | 2005-04-22 | 2008-06-18 | 健泰科生物技术公司 | Method for treating dementia or alzheimer's disease by CD2O antibody |
| CN103154736A (en) * | 2010-05-13 | 2013-06-12 | 新泽西医科和牙科大学 | Diagnostic autoantibody profiles for the detection and diagnosis of neurodegenerative diseases |
| CN103869086A (en) * | 2014-04-14 | 2014-06-18 | 杭州凯保罗生物科技有限公司 | Serum autoantibody detection kit |
| US20180067133A1 (en) * | 2015-03-17 | 2018-03-08 | Electrophoretics Limited | Materials and methods for diagnosis and treatment of alzheimer's disease |
Non-Patent Citations (1)
| Title |
|---|
| FEDERICA DELUNARDO ET AL.: "Screening of a microvascular endothelial cDNA library identifies rabaptin 5 as a novel autoantigen in Alzheimer´s disease", 《JOURNAL OF NEUROIMMUNOLOGY》 * |
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| AU2019201686B2 (en) * | 2010-05-13 | 2021-05-27 | Rowan University | Diagnostic autoantibody profiles for the detection and diagnosis of neurodegenerative diseases |
| US11435361B2 (en) | 2010-05-13 | 2022-09-06 | Rowan University | Diagnostic autoantibody profiles for the detection and diagnosis of neurodegenerative diseases |
| CN111471096B (en) * | 2020-01-15 | 2022-02-18 | 上海众启生物科技有限公司 | Composition containing ADARB1 protein fragment for detecting Alzheimer disease autoantibody |
| JP2022528194A (en) * | 2020-01-15 | 2022-06-08 | 上海衆啓生物科技有限公司 | Antigen combination, use of antigen combination for detection of Alzheimer's disease, kit for detecting Alzheimer's disease and antigen for detection of Alzheimer's disease |
| CN111471096A (en) * | 2020-01-15 | 2020-07-31 | 上海众启生物科技有限公司 | MAPT protein fragments for detection of alzheimer's disease autoantibodies |
| WO2021142963A1 (en) | 2020-01-15 | 2021-07-22 | 上海众启生物科技有限公司 | Protein antigen combination for detecting alzheimer's disease autoantibody and application of protein antigen combination |
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| CN111426850A (en) * | 2020-01-15 | 2020-07-17 | 上海众启生物科技有限公司 | DNAJC8 protein fragment for detecting Alzheimer's disease autoantibody |
| JP7229592B2 (en) | 2020-01-15 | 2023-02-28 | 上海衆啓生物科技有限公司 | Antigen combinations, use of antigen combinations for detecting Alzheimer's disease, kits for detecting Alzheimer's disease and antigens for detecting Alzheimer's disease |
| EP4071479A4 (en) * | 2020-01-15 | 2023-10-25 | Shanghai Zhongqi Biotechnology Co., Ltd | Protein antigen combination for detecting alzheimer's disease autoantibody and application of protein antigen combination |
| CN116063447A (en) * | 2022-09-13 | 2023-05-05 | 北京湃德智健科技有限公司 | Antigen polypeptide for detecting ADAP autoantibody and application thereof |
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