CN115754297A - Application of biomarker in preparation of kit for detecting Parkinson's disease - Google Patents
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Abstract
The invention belongs to the field of clinical medicine, and particularly relates to application of a biomarker in preparation of a kit. Use of a biomarker comprising an intracellular alpha-synuclein (alpha-syn) oligomer and an intracellular total protein in a red blood cell in the manufacture of a kit for assessing or judging the risk of a subject for developing parkinson's disease. The invention discloses application of alpha-syn oligomer in erythrocytes in early judging Parkinson's disease. The method takes human peripheral blood as a sample, is convenient to collect biological samples, has low price, short detection period and small wound on patients, can judge the occurrence of the Parkinson disease at early stage by detecting markers in erythrocytes, is a novel detection system which is convenient, quick and high in accuracy, can be used as a rapid detection technology of high-throughput and large samples, can screen the Parkinson disease patients as early as possible, and has wide application prospect for early starting intervention pretreatment.
Description
Technical Field
The invention belongs to the field of clinical medicine, and particularly relates to application of a biomarker in preparation of a kit.
Background
Parkinson's Disease (PD) is a common degenerative disease of the nervous system in the elderly, with a prevalence of 1.7% in people over the age of 65, characterized by degenerative loss of nigral dopaminergic neurons and formation of lewy bodies within the surviving neurons. Early diagnosis is of great significance in improving the quality of life of patients and preventing the deterioration of disease conditions.
The diagnosis of PD relies on major motor symptoms including resting tremor, muscle rigidity, bradykinesia, and abnormal gait in posture. However, diagnosis based on motor symptoms has limitations, requires evaluation by a specialist due to complexity and overlap of clinical manifestations, and the evaluation results are susceptible to human subjective factors, failing to accurately distinguish PD from other diseases with similar clinical manifestations of PD, and failing to detect pathological changes in PD. Therefore, identification of PD-specific biomarkers is crucial for early diagnosis of PD and early treatment. However, a convenient, rapid, high-throughput detection method for early identification of PD is still lacking.
There is a large body of evidence that α -synuclein (α -syn) plays a key role in the pathogenesis of PD. Fibrotic alpha-syn oligomers have been identified as a major component of the lewy body, a major pathological feature of sporadic and familial PD. Furthermore, point mutations and abnormal amplification of the alpha-syn gene are associated with familial PD. Furthermore, polymorphisms in the alpha-syn gene promoter are associated with increased risk of developing PD. Therefore, detecting changes in alpha-syn in cerebrospinal fluid and plasma of PD patients is considered to be an ideal biomarker for PD diagnosis.
However, numerous studies have shown that there is significant inconsistency in the variation of α -syn concentration in cerebrospinal fluid, and additionally the detection of α -syn in plasma has proven unreliable.
Disclosure of Invention
The invention aims to provide application of a biomarker in preparation of a PD detection kit.
Use of a biomarker according to a particular embodiment of the invention, comprising alpha-syn oligomers in erythrocytes and total protein in erythrocytes, in the preparation of a kit for assessing or judging the risk of a subject for developing PD.
Use of a biomarker according to a particular embodiment of the invention, in the preparation of a kit, said biomarker being the concentration of alpha-syn oligomers and the concentration of total protein in peripheral red blood cells, in particular the biomarker being the concentration of alpha-syn oligomers/the concentration of total protein in peripheral red blood cells, i.e. the ratio of the concentration of alpha-syn oligomers to the concentration of total protein.
The invention finds that hemolysis affects the accurate assessment of alpha-syn changes in cerebrospinal fluid and plasma, and contamination by hemolysis is unavoidable. Therefore, the invention has higher accuracy by using the content/total protein concentration of alpha-syn oligomer in peripheral red blood cells as a marker.
Use of a biomarker according to a particular embodiment of the invention in the preparation of a kit, said assessment or judgment comprising the steps of:
(1) Determining the concentration of alpha-syn oligomer and the concentration of total protein within erythrocytes in a blood sample of the subject;
(2) Comparing the concentration of the alpha-syn oligomer protein and the concentration of the total protein determined in the step (1) with reference values to judge the risk of the subject to develop PD.
According to the application of the biomarker in the preparation of the kit of the embodiment of the present invention, in the step (2), the reference value is 15.03ng/mg, and when the ratio of alpha-syn oligomer/total protein in peripheral red blood cells is higher than 15.03ng/mg, the subject can be judged to be PD.
The use of the biomarkers according to embodiments of the invention in the preparation of kits uses enzyme-linked immunosorbent assay (ELISA) to determine the concentration of alpha-syn oligomers.
Use of a biomarker according to a particular embodiment of the invention in the preparation of a kit, the ELISA comprising the steps of:
coating a 96-well ELISA plate with a non-biotinylated 3D5 monoclonal antibody alpha-syn; biotinylation of the 3D5 antibody as a detection antibody; coupling the Extravidin alkaline phosphatase with a detection antibody; adding substrate p-nitrophenyl phosphate (pNPP) for color development; the absorbance values at 405nm and 603nm were measured. The specific operation steps are as follows:
s1, coating a non-biotinylated 3D5 monoclonal anti-alpha-syn antibody on an ELISA plate, washing the ELISA plate by using a Phosphate Buffer Solution (PBST) containing 0.05% Tween 20, and blocking the ELISA plate by using a PBST solution containing 2.5% gelatin;
s2, diluting a blood sample of a subject and an alpha-syn oligomer standard to 500nM by using a Phosphate Buffered Saline (PBS) solution, respectively adding the diluted sample and the alpha-syn oligomer standard to an ELISA plate, and incubating;
s3, adding 1 mu g/ml of biotinylated 3D5 antibody to an ELISA plate, and then incubating;
s4, washing the ELISA plate by using a PBST solution, adding alkaline phosphatase, and incubating;
s5, adding pNPP to each reaction well of the ELISA plate, and measuring absorbance values at 405nm and 603 nm.
More specifically, the present invention is directed to a method for producing,
non-biotinylated 3D5 mouse monoclonal anti-alpha-syn antibody was coated on 96-well ELISA plates and, after washing with PBST solution, blocked with PBST solution containing 2.5% gelatin.
100 μ l of red blood cell samples and prepared alpha-syn oligomer standards were diluted to 500nM with PBS solution, added to 96-well ELISA plates, respectively, and incubated at 37 ℃ for 2 hours.
After washing with PBST, 100. Mu.l of biotinylated 3D5 antibody diluted to 1. Mu.g/ml with blocking buffer was added, followed by incubation at 37 ℃ for 2 hours.
The 96-well ELISA plates were washed with PBST solution and incubated with ExtrAvidin alkaline phosphatase diluted to 1.
The 96-well ELISA plates were washed 4 times with PBST solution, 100. Mu.l of pNPP was added to each well, and absorbance values at 405nm and 603nm were measured using an ELISA detector (Bio-Tek, winooski, VT, USA).
In the present invention, the coating antibody and the detection antibody are the same antibody recognizing the same surface antigen of α -syn. The surface antigens of amino acids 115-121 of human alpha-syn were recognized using 3D5 monoclonal antibodies. In the case where α -syn is a monomer, when the surface antigen is bound by the coated antibody, the α -syn monomer will have no excess surface antigen sites to be bound by the detection antibody. The detection antibody is biotinylated, recognizing the same surface antigenic sites as the coating antibody. In the case of the alpha-syn oligomer, since several alpha-syn monomers are stacked together, when the surface antigen of one of the alpha-syn monomers in the alpha-syn oligomer is bound by the coating antibody, the surface antigens of the other alpha-syn monomers are still bound by the detection antibody. Thus, in the ELISA assay of the invention, only a-syn oligomers can emit a signal to be detected.
Wherein, the Extravidin alkaline phosphatase can be combined with biotinylated 3D5 monoclonal antibody, the pNPP is an acting substrate of the alkaline phosphatase, when the alkaline phosphatase reacts with the pNPP, a yellow water-soluble product is formed, the water-soluble product can be detected by an ELISA detector, the absorbance values at 405nm and 603nm are only required to be detected, and the height of the absorbance value represents the concentration of the detected alpha-syn oligomer.
According to the application of the biomarker in the preparation of the kit, in step S2, the preparation method of the alpha-syn oligomer standard comprises the following steps: by dissolving the α -syn sample with PBS to a final concentration of 100 μ M and incubating at 37 ℃ for 48 hours with continuous shaking at a speed of 500-2000rpm, two or more α -syn monomers were aggregated to form α -syn oligomers, and thus α -syn oligomers had multiple surface antigenic sites recognized by 3D5 monoclonal antibodies.
Use of a biomarker according to a specific embodiment of the invention in the preparation of a kit, wherein the PD comprises grades 1, 2, 3 and 4 according to the H-Y scale.
The invention has the beneficial effects that:
in one aspect, the present invention identifies a reliable biomarker for the early diagnosis of PD, i.e., alpha-syn oligomer/total protein in peripheral red blood cells of PD patients as a biomarker for the diagnosis of PD. 100 PD patients and 102 healthy controls were collected, the accuracy of α -syn oligomer/total protein as a biomarker for PD was verified, and further, it was suggested that PD could be judged when the ratio of the concentration between α -syn oligomer/total protein in peripheral red blood cells was higher than 15.03 ng/mg.
On the other hand, in the alpha-syn oligomer detection, the 3D5 monoclonal antibody is used as a coating antibody and a detection antibody at the same time, the surface antigen of 115-121 amino acids of the human alpha-syn is recognized, in the case of an alpha-syn monomer, the surface antigen is combined by the coating antibody, and the alpha-syn monomer does not have redundant surface antigen sites to be combined by the detection antibody; in the case of the α -syn oligomer, since a plurality of α -syn monomers are stacked together, when the surface antigen of one of the monomers is bound by the coating antibody, the surface antigens of the other α -syn monomers are still bound by the detection antibody. Thus, the method of the invention improves the accuracy of the detection of alpha-syn oligomers.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows alpha-syn oligomers in erythrocytes versus total erythrocyte protein for PD patients and healthy controls; wherein, control is healthy Control group; PD: (ii) the parkinson group;
FIG. 2 shows the alpha-syn oligomer/total protein levels in erythrocytes of PD patients of different H-Y grades;
FIG. 3 shows the correlation of alpha-syn oligomer/total protein in erythrocytes of PD patients with (A) age of onset, (B) course of disease, (C) age, (D) Unified Parkinson's Disease Rating Scale (UPDRS) motor Scale score;
FIG. 4 shows ROC analysis of alpha-syn oligomer/total protein levels in erythrocytes as a biomarker for PD.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
UPDRS motion score: is used for comprehensively evaluating the motor function evaluation scale of PD patients.
H-Y grading: the H-Y scale suggests the severity of the disease in PD patients: level 1: patients have unilateral limb illness; 1.5 level: the single limb is sick and affects the muscles of the trunk, or the other limb is suspicious affected; and 2, stage: bilateral limbs are diseased, but balance is not compromised; 2.5 level: the balance of the patient is influenced to a certain degree, and the patient can automatically restore the balance when a back pull test is carried out; and 3, level: the patient has a posture balance disorder; 4, level: patients have serious dyskinesia, but can stand or walk by themselves; and 5, stage: patients cannot stand and walk without help from outsiders, get up or live on wheelchairs. Wherein grades 1-2.5 are early stage PD and grades 3-5 are middle and late stage PD.
Alpha-syn standard: sigma, st. louis, MO, USA;
ExtrAvidin alkaline phosphatase: sigma, st. louis, MO, USA;
yellow p-nitrophenyl phosphate (pNPP): sigma, st.
The embodiment of the invention mainly comprises the following steps:
1) Collecting clinical data of PD patients and healthy controls;
2) Detecting the concentration of alpha-syn oligomer and the ratio of the concentration of the alpha-syn oligomer to the concentration of total protein in peripheral red blood cells of PD patients and healthy controls by a modified ELISA method;
3) Analyzing the correlation between alpha-syn oligomer/total protein in erythrocytes of PD patients and patient age, disease course, UPDRS score, and disease severity;
4) The optimal cut-off for PD was determined based on the results of the alpha-syn oligomer/total protein assay in erythrocytes. The study protocol was approved by the ethical committee of the beijing tiantan hospital, affiliated to the university of capital medicine.
All subjects participated voluntarily, were informed for experimental purposes and signed informed consent.
Example 1
1.1 collecting PD patient clinical data:
PD patients and healthy contrast patients were collected from the department of neurology outpatient service in the Tiantan hospital. Patients with PD met the clinical diagnostic criteria for PD introduced in the international society for parkinson and Movement Disorders (MDS) 2015. Exclusion criteria: secondary parkinsonism, parkinsonism plus syndrome; severe cognitive impairment, language impairment, etc. can not be matched with the examiner; chronic consumptive diseases such as severe cardiopulmonary disease, tumor, and liver and kidney diseases; those with mental disorders. The healthy control people are required to have no disease history.
All subjects received a general clinical data survey including: sex, age of onset of PD, course of disease, history of present disease, past history, family history, etc. The subjects were asked for diagnosis, diagnosed, graded for H-Y, and assessed for UPDRS by 2 dyskinesias specialist, see table 1.
TABLE 1 clinical data for PD patients and healthy controls
1.2 collecting erythrocyte samples of PD patients and healthy controls
Subject whole blood (5 ml) was collected in EDTA-coated tubes and aliquoted. Erythrocytes were separated from plasma by centrifugation at 2200 Xg for 20 minutes at 4 ℃.
Precipitating red blood cells in the absence of Ca 2+ /Mg 2+ Washed three times in PBS buffer consisting of 137.5mM phosphate and 150mM NaCl (pH 7.4) and centrifuged on ice for 10 minutes.
The supernatant was removed and the erythrocyte pellet was diluted in ice-cold PBS 1:1 before the erythrocyte samples were stored in a-80 ℃ freezer and thawed only at the time of analysis.
1.3 preparation of alpha-syn oligomer standards:
purified alpha-syn samples were dissolved with sterile PBS to a final concentration of 100 μ M. Eppendorf tubes containing 100. Mu.l of alpha-syn solution were sealed with a sealing film, incubated at 37 ℃ for 48 hours and continuously shaken at 1000 rpm. The resulting alpha-syn oligomers were aliquoted and stored at-80 ℃.
1.4 establishment of ELISA method for detection of alpha-syn oligomers Using 3D5 antibodies
Non-biotinylated 3D5 mouse monoclonal anti-alpha-syn antibody was coated on 96-well ELISA plates and, after washing with PBST solution, blocked with PBST solution containing 2.5% gelatin.
100 μ l of red blood cell samples and prepared alpha-syn oligomer standards were diluted to 500nM with PBS solution, added to 96-well ELISA plates, respectively, and incubated at 37 ℃ for 2 hours.
After washing with PBST, 100. Mu.l of biotinylated 3D5 antibody diluted to 1. Mu.g/ml with blocking buffer was added, followed by incubation at 37 ℃ for 2 hours.
The 96-well ELISA plates were washed with PBST solution and incubated for 1 hour at 37 ℃ with Extravidin alkaline phosphatase diluted to 1 with blocking buffer.
The 96-well ELISA plate was washed 4 times with PBST solution, 100. Mu.l of pNPP was added to each reaction well, and absorbance values at 405nm and 603nm were measured with an ELISA detector.
And comparing the absorbance value with that of the alpha-syn oligomer standard to judge the concentration of the alpha-syn oligomer in the erythrocyte sample.
And (3) placing the ELISA plate on an enzyme-linked immunosorbent assay (ELISA) instrument, reading the light absorption value of each hole, subtracting the light absorption value of the blank control hole respectively, carrying out zero setting, making a standard curve according to a standard sample, and calculating the antibody content of each sample respectively.
1.5 measurement of protein concentration in erythrocytes:
the absorbance values of the red blood cell samples at 562nm were measured using a bicinchoninic acid (BCA) protein assay kit (Sigma, st. Louis, MO, USA), and the total protein concentration in the red blood cell samples was determined by comparing the absorbance values of the protein standards.
Subtracting the light absorption value of the blank control hole from the light absorption values of the standard substance and the sample, combining the known concentration of the protein standard substance and the corrected light absorption value, using Microsoft excel as a scatter diagram, setting the mass of protein as x and the light absorption value as y, and making a standard curve according to the standard substance to obtain a standard curve equation y = ax; and calculating the protein concentration according to the corrected light absorption value of each protein sample by referring to the standard curve.
1.6 analysis of the concentration ratio of alpha-syn oligomer to Total protein in erythrocytes of PD patients and healthy controls
The present invention takes into account the individual differences in the number of peripheral red blood cells and normalizes the concentration of alpha-syn oligomers to the concentration of total red blood cell protein.
Alpha-syn oligomers were detected in erythrocytes of both PD patients and healthy controls, and the levels of alpha-syn oligomers were significantly higher in PD patients than in healthy controls (29.0 + -19.8 ng/mgvs.15.4 + -7.4 ng/mg, P < 0.001) relative to total erythrocyte protein, as shown in FIG. 1.
1.7 alpha-syn oligomers in erythrocytes as biomarkers for the early determination of PD
The present invention analyzes the levels of alpha-syn oligomers in erythrocytes of PD patients of different H-Y grades, wherein 22 PD patients are classified in the H-Y grade 1-1.5, 58 PD patients are classified in the grade 2-2.5, 16 PD patients are classified in the grade 3, and 4 PD patients are classified in the grade 4. The levels of alpha-syn oligomers in erythrocytes of different H-Y fractionated PD patients were calculated separately as described above.
As shown in FIG. 2, alpha-syn oligomers/total protein in erythrocytes were essentially identical in PD patients at each stage, 29.9. + -. 19.3ng/mg (grade 1-1.5), 28.2. + -. 19.7ng/mg (grade 2-2.5), 28.6. + -. 19.5ng/mg (grade 3-3.5) and 26.9. + -. 20.7ng/mg (grade 4-4.5), respectively (P = 0.893). Indicating that alpha-syn oligomers are detectable in erythrocytes of PD patients (grade 1-4.5) at various stages of the disease course and are not subject to changes in disease severity, and that alpha-syn oligomers/total protein can serve as a very reliable peripheral blood biomarker for early determination of PD occurrence.
1.8 analysis of the correlation between alpha-syn oligomer/Total protein and age, course, patient age, UPDRS movement score in erythrocytes from PD patients
The Pearson correlation method analyzed the correlation of alpha-syn oligomers/total protein to various clinical characteristics of the subjects.
Firstly, determining whether variables (alpha-syn oligomer/total protein, onset age, disease course, patient age and UPDRS movement score) conform to normal distribution by using SPSS 17.0 statistical software, if so, applying Pearson correlation analysis in SPSS software, wherein the Pearson correlation coefficient range is (-1, + 1), and the correlation coefficient is closer to 0 and the correlation relationship is weaker; the closer to-1 or +1, the stronger the correlation.
The results are shown in fig. 3, where α -syn oligomer/total protein in erythrocytes of PD patients was not correlated with age of onset (r = -0.142, p = 0.117), course of disease (r = -0.003, p = 0.970), age of patient (r = -0.136, p = 0.140) and UPDRS movement score (r = -0.056, p = 0.573).
The invention uses multiple linear regression for further testing, uses SPSS 17.0 statistical software to perform multiple linear regression analysis by using alpha-syn oligomer/total protein in erythrocytes as dependent variable and disease age, disease course, patient age and UPDRS movement score as independent variable, and judges whether linear relation exists between the independent variable and the dependent variable.
The present invention demonstrates that there is no correlation between alpha-syn oligomers/total proteins and patient age (P = 1.361), course (P = 0.830), UPDRS score (P = 2.801).
Combining the above results, alpha-syn oligomer/total protein in erythrocytes of PD patients is not affected by the patient's age, course and UPDRS motor score, and can be used as a stable and reliable peripheral blood biomarker.
1.9 differentiation of PD patients from healthy controls by ROC Curve
To assess the role of the alpha-syn oligomer/total protein ratio in peripheral red blood cells in distinguishing between PD and healthy controls, ROC analysis was performed.
ROC curve analysis was performed using SPSS 17.0 statistical software, the area under the curve is called AUC, the true and false positive rates of PD were calculated at different alpha-syn oligomer/total protein ratios, and the best cut-off was found using the john index (sensitivity + specificity-1) max.
As shown in FIG. 4, the cutoff value of α -syn oligomer/total protein of 15.03ng/mg is the optimal cutoff value for distinguishing PD patients from the control group, and the sensitivity is 79.0% (95% confidence interval is 70.9% -87.1%), the specificity is 64.7% (55.3% -74.1%), and the area under the ROC curve is 0.76, so that when the ratio of α -syn oligomer/total protein in peripheral red blood cells is higher than 15.03ng/mg, PD can be judged.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily think of the changes or substitutions within the technical scope of the present invention, and shall cover the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (8)
1. Use of a biomarker comprising an intra-erythrocyte a-syn oligomer and intra-erythrocyte total protein in the manufacture of a kit for assessing or judging the risk of a subject for developing parkinson's disease.
2. The use according to claim 1, wherein the biomarker is the concentration of alpha-syn oligomer/the concentration of total protein within erythrocytes.
3. The use according to claim 1, wherein said evaluating or determining comprises the steps of:
(1) Determining the concentration of alpha-syn oligomers and the concentration of total protein within erythrocytes in a blood sample of the subject;
(2) Comparing the concentration of the alpha-syn oligomer protein and the concentration of the total protein determined in the step (1) with reference values to judge the risk of the subject to develop the Parkinson's disease.
4. The use according to claim 3, wherein in step (2), the subject is judged to have Parkinson's disease when the ratio of the concentration of alpha-syn oligomers to the concentration of total protein in erythrocytes is higher than 15.03 ng/mg.
5. Use according to claim 1 or 3, characterized in that the concentration of alpha-syn oligomers is determined by enzyme-linked immunosorbent assay.
6. Use according to claim 5, wherein the enzyme-linked immunosorbent assay comprises the following steps:
s1, coating a non-biotinylated 3D5 monoclonal anti-alpha-syn antibody on an ELISA plate, washing the ELISA plate by using a phosphate buffer solution containing 0.05% Tween 20, and blocking the ELISA plate by using a PBST solution containing 2.5% gelatin;
s2, diluting a blood sample of a subject and an alpha-syn oligomer standard to 500nM by using a phosphate buffer solution, respectively adding the diluted samples to an ELISA plate, and incubating;
s3, adding 1 mu g/ml of biotinylated 3D5 antibody to an ELISA plate, and then incubating;
s4, washing the ELISA plate by using a PBST solution, adding alkaline phosphatase, and incubating;
s5, adding p-nitrophenyl phosphate into each reaction hole of the ELISA plate, and measuring absorbance values at 405nm and 603 nm.
7. The use according to claim 6, wherein in step S2, the α -syn oligomer standard is formulated by: alpha-syn samples were dissolved with PBS to a final concentration of 100. Mu.M and incubated at 37 ℃ for 48 hours with continuous shaking at 500-2000 rpm.
8. The use of claim 1, wherein said parkinson's disease comprises H-Y grades 1, 2, 3 and 4, on the H-Y grade.
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