CN109738653B - Antigen-protein combination for detection, diagnosis or risk prediction of alzheimer's disease and kit comprising same - Google Patents
Antigen-protein combination for detection, diagnosis or risk prediction of alzheimer's disease and kit comprising same Download PDFInfo
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Abstract
The invention provides a kit for detecting autoantibodies to alzheimer's disease in serum, comprising a combination of antigenic proteins comprising at least four proteins selected from the group consisting of RABPT5, RAGE, CRYAB, SRPK1, MAPT, MBP, PLP1, PTCD2, FRMD8, POMC, DNAJC8, CENTA2, HSP60, ADARB1, ASXL1, EDRK, GDF11, P21, CCL2, IL18, VEGF, LENG1 and MRPL 34. The invention also provides the application of the defined antigen-protein combination in preparing a reagent for detecting, diagnosing or predicting the risk of the Alzheimer disease, and a method for detecting, diagnosing or predicting the risk of the Alzheimer disease.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to an antigen combination for detecting autoantibodies in a mammal sample and application thereof, so as to realize early diagnosis on whether a mammal suffers from Alzheimer's disease.
Background
Alzheimer's Disease (AD), commonly known as senile dementia, is a group of primary degenerative brain degenerative diseases with unknown etiology, which mostly occur in the elderly and have a slow and irreversible course. The clinical manifestations of alzheimer's disease are mainly memory and other cognitive function deficits, slow onset, not easy to be found, characterized by a pre-symptomatic period that may last for years; meanwhile, the patient has neurodegeneration before clinical symptoms appear, and has a rather long course after onset.
According to the world health organization, alzheimer's disease occurs in 10% of the elderly over 65 years of age in the world today, and by the age of 80, this proportion rises to over 30%. The mean survival of AD patients is estimated to be 10-15 years, the fourth leading cause of death in the elderly population, second only to heart disease, malignancy and stroke. According to the latest statistics of the Chinese Alzheimer's disease society, the AD population of China is nearly 800 million at present, and the AD population is estimated to exceed 2000 million by 2050. Professor jia hei and his team published a paper "re-assessment of disease burden of alzheimer's disease in china and worldwide", which indicated that in 2015, alzheimer's disease patients spend 19144.36 dollars per year (about 13 ten thousand yuan for renminbi), and the total social and economic burden of alzheimer's disease in china amounts to 1677.4 billion dollars (about 11406 billion yuan for renminbi). By 2030, the economic burden of Alzheimer's disease in China will reach $ 2.54 trillion (which translates to about 17 trillion yuan in Renminbi. with the aging of the population and the extension of the per-capita life, Alzheimer's disease is gradually becoming an important disease burden in China and other countries, causing increasingly serious public health problems.
There is no good treatment method for the disease, but good continuous nursing measures can delay the disease development and prevent the residual functions of the patients from being further damaged. Therefore, early diagnosis of a disease is critical for a patient to prevent or delay the onset of disease or the onset of clinical symptoms. Currently, the diagnostic gold standard for alzheimer's disease is the deposition of amyloid a β in brain tissue. However, this diagnosis requires a traumatic brain biopsy or necropsy after death of the patient, and is not suitable for clinical examination. Other examination methods include head CT and MRI, amyloid PET imaging, gene detection, cerebrospinal amyloid, Tau protein examination, etc., which are usually detected or identified after Alzheimer's disease has been clinically manifested for many years, and thus early diagnosis is difficult.
The detection of antibodies in serum is a well established technique that enables the accurate detection of autoantibodies. For example, the national company of Kyozhou Karpura Biotechnology Limited utilizes autoimmune antibodies to develop a first national kit for detecting lung cancer by adopting antigen combination, and has good clinical effect. In the case of alzheimer's disease, there is a lot of evidence that the human immune system can produce autoantibodies in the early stage of AD development, which makes it possible to detect or diagnose alzheimer's disease by detecting autoantibodies.
However, due to technical limitations, many autoantibodies to alzheimer's disease are difficult to detect at an early stage; meanwhile, although various autoantibodies have been reported to have a correlation with AD, which antibodies have a wide clinical significance are not known. Moreover, even though some autoantibodies are thought to have some relevance to AD, they remain in a small field of study and lack large-scale, reproducible clinical data to demonstrate.
Thus, while current diagnostic tools are continually improving, there remains a need in the art for new means of detecting or diagnosing alzheimer's disease; in particular, there remains a need in the art to provide a new detection method for early diagnosis and prediction of the course of AD.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides an antigenic protein combination for detecting alzheimer's disease, said antigenic protein combination comprising at least four proteins, which are all specifically associated with alzheimer's disease, thereby providing a relatively accurate early detection or diagnosis tool for alzheimer's disease.
It is therefore an object of the present invention to provide a kit comprising a combination of antigenic proteins comprising at least four proteins.
Another object of the present invention is to provide the use of said antigenic protein combination for the preparation of a reagent for the detection, diagnosis or risk prediction of alzheimer's disease.
It is a further object of the present invention to provide a method for the detection, diagnosis or risk prediction of alzheimer's disease.
The technical scheme of the invention is as follows.
In one aspect, the invention provides a kit comprising an antigenic protein combination comprising at least four proteins selected from the group consisting of RABPT5, RAGE, CRYAB, SRPK1, MAPT, MBP, PLP1, PTCD2, FRMD8, POMC, DNAJC8, CENTA2, HSP60, ADARB1, ASXL1, EDRK, GDF11, P21, CCL2, IL18, VEGF, LENG1 and MRPL 34.
The antigen combination is used to detect the presence or absence of autoantibodies to alzheimer's disease in a biological sample, such as serum. Therefore, the kit provided by the invention can be used for the detection or diagnosis of the Alzheimer's disease, especially for early detection or diagnosis; can also be used for predicting the risk of Alzheimer's disease.
The sequences of the above proteins are exemplarily shown in table 1.
TABLE 1 sequences of proteins comprised in antigenic protein combinations
| Protein | Database ID | Protein | 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 the kit provided by the present invention, preferably, the protein is selected from RAGE, HSP60, MRPL34, CCL2, MBP, P21, CENTA2, ASXL1, ADARB1, DNAJC8, MAPT, and GDF 11; and optionally, selected from RABPT5, CRYAB, SRPK1, PLP1, PTCD2, FRMD8, POMC, EDRK, IL18, VEGF, and LENG 1.
More preferably, the antigenic protein combination comprises at least three or at least four proteins selected from RAGE, HSP60, MRPL34, CCL2, MBP, P21, CENTA2, ASXL1, ADARB1, DNAJC8, MAPT and GDF 11.
Further, the grouping of proteins according to the following grouping of antigenic protein combinations comprises at least two proteins selected from group (1), at least one protein selected from group (2) and optionally at least one protein selected from group (3):
(1)RAGE、HSP60、MRPL34、CCL2、MBP、P21、CENTA2；
(2) ASXL1, ADARB1, DNAJC8, MAPT and GDF 11;
(3) RABPT5, CRYAB, SRPK1, PLP1, PTCD2, FRMD8, POMC, EDRK, IL18, VEGF, and LENG 1.
More preferably, said antigenic protein combination comprises at least three proteins selected from said group (1) and at least one protein selected from said group (2).
According to a particular embodiment of the invention, in the kit provided herein, the antigenic protein combination comprises the proteins RAGE and DNAJC 8; preferably, the antigenic protein combination further comprises the protein MRPL 34; more preferably, the antigenic protein combination further comprises the protein HSP 60; further preferably, the antigenic protein combination further comprises the protein ADARB 1.
According to a particular embodiment of the invention, in the kit provided by the invention, the antigenic protein combination comprises the proteins RAGE, DNAJC8, HSP60 and MRPL 34; preferably, the antigenic protein combination further comprises the protein ADARB 1;
alternatively, in the kits provided herein, the antigenic protein combination comprises the proteins RAGE, DNAJC8, MRPL34, and ADARB 1; preferably, the antigenic protein combination further comprises the protein HSP 60.
In particular, the antigenic protein combination comprises the proteins RAGE, DNAJC8, HSP60 and MRPL34, and further comprises the protein CENTA 2; preferably, the antigenic protein combination further comprises the protein CRYAB; preferably, the antigenic protein combination further comprises the protein SRPK 1; preferably, the antigenic protein combination further comprises the protein FRMD 8.
Alternatively, the antigenic protein combination comprises the proteins RAGE, DNAJC8, MRPL34 and ADARB1, and further comprises the protein RABPT 5; preferably, the antigenic protein combination further comprises the protein PLP 1; preferably, the antigenic protein combination further comprises the protein PTCD 2; preferably, the antigenic protein combination further comprises the protein POMC.
According to a specific embodiment of the present invention, in the kit provided by the present invention, the antigen-protein combination may be:
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。
The protein provided by the invention can be obtained by expression in escherichia coli, yeast or mammalian cells according to the sequence, and is optionally purified by a Ni column, a molecular sieve, an ion column or a hydrophobic column and the like.
Preferably, the kit provided by the invention further comprises solid phase carriers, and the proteins contained in the antigen-protein combination are respectively fixed on the solid phase carriers. Preferably, the solid phase carrier is, for example, a microplate microwell, a magnetic bead, an affinity membrane or a liquid phase chip; immobilization of the protein includes immobilization of the protein directly on a solid support, or indirectly via an intermediate linking moiety, for example by a specific reaction between biotin and streptavidin.
The kit provided by the invention can be used for detecting autoantibodies in a biological sample (such as serum), the detection is carried out by an antigen-protein combination contained in the kit, the protein in the combination can be used as an antigen to perform antigen-antibody specific reaction with corresponding autoantibodies (if existing) in the biological sample, and the detection result of the reaction can indicate the existence or nonexistence of the autoantibodies in the biological sample, and further indicate whether the source (generally a mammal, such as a human) of the biological sample has the Alzheimer's disease or has the risk of developing the Alzheimer's disease.
Accordingly, the kits provided herein may also include other reagents or components for use in detecting autoantibodies in a biological sample using the combination of antigenic proteins.
According to a specific embodiment of the present invention, the kit provided by the present invention can be used for detecting autoantibodies in biological samples (e.g., serum) by enzyme-linked immunosorbent assay.
For detection by an enzyme-linked immunosorbent assay, the kit provided by the invention can be an enzyme-linked immunosorbent assay kit. For detection purposes, for example, the protein may have attached a tag peptide, preferably selected from the group consisting of a His tag, a GST tag, a c-Myc tag, a Flag tag, an HA tag, and a biotin tag. For another example, the kit provided by the invention can further comprise a positive quality control substance, a negative quality control substance or a standard substance. For example, the positive quality control substance or standard substance is recombinant human immunoglobulin G or a fragment thereof. The kit provided by the invention can also comprise immunoglobulin with an enzyme label, coating buffer solution, confining liquid, 20X washing solution, serum/antibody diluent, TMB color developing agent, stop solution and the like.
Based on the antigen protein combination provided by the invention, the corresponding kit can be prepared by adopting the conventional technology in the field. According to the specific embodiment of the present invention, reference may be made to the preparation method of the detection kit disclosed in chinese patent application publication CN 103869086A.
In another aspect, the invention provides the use of the kit or the combination of antigenic proteins therein for the preparation of a reagent for the detection, diagnosis or risk prediction of alzheimer's disease.
In yet another aspect, the invention provides a method for the detection, diagnosis or risk prediction of alzheimer's disease, said method comprising contacting said antigenic protein combination with a biological sample.
Preferably, the biological sample is from a mammal, preferably a human; preferably, the biological sample is serum.
According to reports, more than 30 kinds of autoantibodies have been correlated with AD so far, but those antibodies have common clinical significance in early detection or diagnosis of AD and even risk prediction, and particularly in chinese population, the clinical significance is not clear, and therefore, even though the autoantibodies are used for diagnosing alzheimer disease, the feasibility is questionable.
In contrast, the present invention provides a novel antigen-protein combination comprising at least four proteins specifically associated with alzheimer's disease, thereby providing a novel tool for detecting or diagnosing alzheimer's disease with high sensitivity and high specificity.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
first, the inventors of the present invention screened 50 known proteins and found that 23 proteins as antigens can produce a strong specific reaction with autoantibodies of alzheimer's disease patients compared to other proteins, thereby demonstrating the feasibility of detecting or diagnosing alzheimer's disease by detecting autoantibodies of subjects.
Furthermore, the present inventors also provide an antigenic protein combination comprising at least four of the 23 selected proteins. Furthermore, the 23 proteins can be divided into different protein groups, and the antigen protein combination is formed by selecting from the different protein groups, so that the high sensitivity and the high specificity of the detection result can be considered. Experiments prove that the antigen protein combination provided by the invention realizes high sensitivity and specificity detection effect in biological samples from Alzheimer patients or healthy subjects, thereby providing a rather accurate early detection, diagnosis or risk prediction tool of Alzheimer's disease.
The antigen protein combination provided by the invention can be prepared into a kit and can be used for a related detection method, so that the early screening, detection, diagnosis or risk prediction of the Alzheimer disease is carried out in a subject such as a human, the clinical early screening of the Alzheimer disease is truly realized, and the kit has good application prospect in the diagnosis and treatment of Alzheimer disease tumor. In addition, the method for detecting the autoantibodies in the biological samples of the subjects such as human blood serum by adopting the antigen protein is a non-invasive detection method, and has the advantages of convenient use and good compliance of the subjects.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the sensitivity and specificity of autoantibodies in 23 protein assay sera.
FIG. 2 shows the sensitivity and specificity of autoantibodies in serum detected by different antigen protein combinations, where 2A is overall sensitivity and specificity and 2B is early, mid, late sensitivity.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.
In addition to the antigen-protein combination, the composition of the enzyme-linked immunoassay kit (immobilized antigen) for detecting autoantibodies may include the following components as required:
1) an ELISA plate used for coating antigen protein;
2) horse radish peroxidase-labeled goat anti-human immunoglobulin with the concentration of 100 ng/ml;
3) positive quality control (standard): human anti-c-Myc tag immunoglobulin G (available from Tribioscience);
4) negative quality control products;
5) sealing films;
6) coating buffer solution, confining liquid, 20 × washing solution, serum/antibody diluent, TMB color developing agent and stop solution, wherein:
coating buffer solution is PBS, pH7.4, and the preparation method comprises the following steps: 3.58g of Na are taken2HPO4·12H2O，0.24g KH2PO4·2H2Dissolving O, 0.2g of KCl and 8.0g of NaCl by using double distilled water, and then fixing the volume to 1L;
the preparation method of the sealing liquid comprises the following steps: dissolving 5g of casein in a PBS solution, and then fixing the volume to 1L;
20 × method for preparing washing solution: 20 XPBS, pH7.4, adding 0.5% Tween 20;
the preparation method of the serum/antibody diluent comprises the following steps: dissolving 1g of casein and 10g of bovine serum albumin in a PBS solution, and then fixing the volume to 1L;
the preparation method of the TMB color developing agent comprises the following steps: 50mM imidazole buffer pH 5, 7.5mM PEG3350, 2.94mM urea hydrogen peroxide, 1.6mM TMB;
the preparation method of the stop solution comprises the following steps: 2M sulfuric acid.
In the following examples, the procedure for the detection of serum samples by ELISA using microplate comprises:
diluting the antigen to a final concentration of 100nM by using a coating buffer solution, adding 50 μ l of the antigen to each well of the ELISA plate, and coating the antigen overnight at 4 ℃; the next day, the solution was poured off, the plate was patted dry and washed three times with 250. mu.l of wash solution; adding 250 μ l of blocking solution, incubating at room temperature for 1hr, draining the plate, washing with 250 μ l of washing solution for three times, and draining again;
adding a serum sample diluted by a serum diluent (the dilution is 1:100), adding 50 mu l of the serum sample into each well, placing the mixture in a microplate shaker at room temperature for shaking incubation for 1hr, draining the plate, washing the plate for three times by using 250 mu l of a washing solution, draining the plate again, adding a horseradish peroxidase-labeled recombinant goat anti-human immunoglobulin G antibody (1:20000) diluted by an antibody diluent, adding 50 mu l of the serum sample into each well, placing the plate in the microplate shaker at room temperature for shaking incubation for 0.5hr, draining the plate, washing the plate for three times by using 250 mu l of the washing solution, and draining the plate again;
adding 50 μ l TMB color developing agent into each well, developing at room temperature for 15min, and adding 50 μ l stop solution into each well; the plate was read by the microplate reader at a wavelength of 450nm and the data was then analysed.
Example 1Protein screening as antigen for autoantibodies
Through literature search, 50 known proteins related to Alzheimer's disease are selected, and are shown in Table 2.
TABLE 2 sequences of proteins to be tested
The desired recombinant antigenic protein fragments were prepared by PCR amplification or enzymatic cleavage using a human cDNA library (purchased from Invitrogen) or whole-gene synthesized DNA as a template. For example, the PCR product obtained by amplification is purified, ligated to pET28 vector, and an appropriate tag such as HIS, c-myc, etc. is added upstream of the antigen-encoding sequence to form a fusion protein, and then the recombinant vector is transformed into E.coli DH 5. alpha. competent cells. The obtained recombinant plasmid is verified to contain correct exogenous fragments by colony PCR, enzyme digestion and sequencing identification. The obtained recombinant plasmid containing the fusion antigen fragment is transformed into competent cells of escherichia coli BL21(DE3), and is induced and expressed by isopropyl thio-beta-D-galactoside (IPTG) to obtain recombinant antigen protein, the protein is purified by two steps of Ni-NTA column and molecular sieve and then quantified by a Bradford method, and the expression, purification and quantification results of the antigen protein are confirmed by SDS-PAGE identification.
The antigen-coated plate obtained by the above method was subjected to the above procedure for ELISA to detect serum samples, and then 94 patients with Alzheimer's disease and 94 healthy subjects were tested for autoantibodies against each antigen. The serum of 94 healthy subjects was used as negative reference samples, and the mean value (M) and Standard Deviation (SD) of the detection signals (S) were calculated for all the negative reference samples, and M +3SD was used as a CutOff value, and samples having a detection signal (S) of greater than or equal to the CutOff value were determined to be positive, i.e., samples having a detection signal (S) of greater than or equal to the CutOff value were determined to be positive, and samples having a detection signal (S) of less than the CutOff value were determined to be negative, i.e., S < M +3SD was determined to be negative.
Specificity and sensitivity were calculated based on positive and negative results. Wherein specificity refers to the proportion of healthy subject samples that are correctly judged to be negative, i.e., the number of samples that are correctly judged to be negative in negative samples divided by the total number of negative samples; sensitivity refers to the proportion of samples of patients with alzheimer's disease that are correctly judged to be positive, i.e., the number of samples judged to be positive in a positive sample divided by the total number of positive samples. The sensitivity and specificity of each tested protein as antigen in sample detection were calculated and the results are shown in table 3.
TABLE 3 sensitivity and specificity of protein as antigen test
By comparing the sensitivity and specificity of each test antigen, antigens with the sensitivity of more than or equal to 10 percent and the specificity of more than or equal to 90 percent are taken as candidate antigens. The finally screened 23 proteins can be used as ideal candidate antigens for distinguishing patients with Alzheimer's disease from healthy subjects, and the screening results are shown in Table 4.
TABLE 4 selected antigenic proteins
| Candidate antigens | Sensitivity of the composition | Specificity of | Candidate antigens | Sensitivity of the composition | Specificity of |
| 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％ |
Example 2Detection performance of candidate antigen-protein combination in Alzheimer's disease patients and healthy subjects
Patients with Alzheimer's disease and healthy subjects were scored according to the simple Mental State Examination (MMSE) -Folstein edition with scores ranging from 27-30 for normal subjects, 21-26 for mild (early) subjects, 10-20 for moderate (mid) subjects, and 0-9 for severe (late) subjects.
According to the MMSE score, 120 cases in the early stage, 87 cases in the middle stage and 75 cases in the late stage of the Alzheimer disease patients are taken as detection samples, 94 cases of healthy subjects with normal MMSE score are taken as samples of the healthy subjects, and the sensitivity and the specificity of each group of antigen-protein combinations are tested.
According to the screening results in example 1, a plurality of proteins were selected from 23 proteins as antigen combinations, and autoantibodies in the sera of alzheimer's disease patients and healthy subjects were detected by ELISA using the antigen-protein combinations in an antigen-immobilized manner. Similarly, the mean (M) and Standard Deviation (SD) of the detection signals (S) were calculated for all the negative reference samples using the sera of 94 healthy subjects as negative reference samples, and M +3SD as the CutOff value, samples having a detection signal (S) of greater than or equal to the CutOff value were determined to be positive, that is, S.gtoreq.M +3SD was positive, and samples having a detection signal (S) of less than the CutOff value were determined to be negative, that is, S < M +3SD was negative.
The sensitivity, as well as the overall sensitivity and the overall specificity of a combination in patient samples at different times, were calculated based on the respective positive and negative results for all antigens contained in the combination. Wherein, excluding the repeated results obtained from different antigens, calculating early sensitivity, which is the proportion of 120 samples of early Alzheimer's disease patients correctly judged to be positive, i.e. the number of samples of early Alzheimer's disease patients judged to be positive is divided by the total number of samples of early Alzheimer's disease patients; calculating mid-term sensitivity, which is the proportion of 87 mid-term Alzheimer's disease patient samples correctly judged to be positive, namely dividing the number judged to be positive in the mid-term Alzheimer's disease patient samples by the total number of the mid-term Alzheimer's disease patient samples; calculating the late sensitivity, which is the proportion of 75 samples of patients with late Alzheimer's disease that are correctly judged to be positive, i.e. the number of samples judged to be positive in the samples of patients with late Alzheimer's disease is divided by the total number of samples of patients with late Alzheimer's disease. In addition, an overall sensitivity was calculated, which is the proportion of the samples of 120 early, 87 middle and 75 late patients with alzheimer's disease that were correctly judged positive in total, i.e., the number of samples judged positive in all the samples of patients with alzheimer's disease was divided by the total number of samples of all the patients with alzheimer's disease; the overall specificity, which is the proportion of 94 samples of healthy subjects correctly judged to be negative, was calculated, i.e., the number of samples of healthy subjects judged to be negative in 94 samples of healthy subjects was divided by the total number of samples of healthy subjects.
The sensitivity and the specificity are considered, the antigen combination with the overall sensitivity of more than or equal to 50 percent and the specificity of more than or equal to 85 percent is taken as an ideal antigen combination, and the screened ideal antigen-protein combination is shown in Table 5. The behavior of autoantibodies in different combinations of ideal antigenic proteins in samples tested for different periods of time in patients with alzheimer's disease and in healthy subjects is shown in table 6.
TABLE 5 Ideal antigen combinations
TABLE 6 test Performance of different combinations of antigenic proteins
| Early sensitivity | Sensitivity to metaphase | Late sensitivity | Overall sensitivity | General 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％ |
| Assembly 10 | 72.5％ | 62.1％ | 48.0％ | 62.8％ | 90.4％ |
The overall sensitivity and specificity of each combination was found to be greater than 54% and 87% by comparison. In the early samples, each combination had a higher sensitivity, greater than 72%, and the sensitivity reached 87.5% for both combination 5 and combination 6, compared to the mid and late samples. Therefore, the invention provides a more accurate detection method for the diagnosis, especially the early diagnosis of the Alzheimer disease.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
Claims (3)
1. A kit comprising an antigenic protein combination comprising at least four proteins selected from the group consisting of RABPT5, RAGE, CRYAB, SRPK1, MAPT, MBP, PLP1, PTCD2, FRMD8, POMC, DNAJC8, CENTA2, HSP60, ADARB1, ASXL1, EDRK, GDF11, P21, CCL2, IL18, VEGF, LENG1, and MRPL34, and said antigenic protein combination is one of:
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。
2. the kit of claim 1, wherein the amino acid sequence of RAGE is set forth in NM _ 001136.5; the amino acid sequence of DNAJC8 is shown in NM — 014280; the amino acid sequence of HSP60 is shown in NM — 002156.5; the amino acid sequence of MRPL34 is shown in NM-023937.3; the amino acid sequence of CENTA2 is shown in NM-018404; the amino acid sequence of CRYAB is shown in NM-001289807.1; the amino acid sequence of SRPK1 is shown in NM — 003137.5; the amino acid sequence of FRMD8 is shown in NM-031904; the amino acid sequence of EDRK is shown in NM-001199875.1; the amino acid sequence of ADARB1 is shown in NM — 001112.4; the amino acid sequence of CCL2 is shown in NM — 002982.4; the amino acid sequence of MAPT is shown in NM-016835.4; the amino acid sequence of ASXL1 is shown in NM-015338.5; the amino acid sequence of GDF11 is shown in NM — 005811; the amino acid sequence of MBP is shown in NM-001025101.2.
3. Use of the antigenic protein combination as defined in claim 1 or 2 for the preparation of a reagent for the detection, diagnosis or risk prediction of alzheimer's disease.
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| 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 |
| CN116063447B (en) * | 2022-09-13 | 2023-11-03 | 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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