Parkinson's disease biomarker, detection reagent and application

Abstract

The invention discloses a Parkinson's disease biomarker, a detection reagent and application, and the Parkinson's disease biomarker has the advantages of high sensitivity and low misdiagnosis rate, and can be effectively used for early diagnosis of Parkinson's disease and risk prediction of adverse events. The biomarker at least comprises one of the following indexes: a concentration ratio of C16 ceramide to C24 ceramide (Ce16: 0 / Cer24: 0), a concentration ratio of C18 ceramide to C24 ceramide (Ce18: 0 / Cer24: 0), a concentration ratio of C24: 1 ceramide to C24 ceramide (Ce24: 1 / Cer24: 0), and a combined index Cer16: 0, Cer18: 0, and a ratio of the total concentration of Cer24: 1 to the concentration of Cer24: 0 (Conc (Ce16: 0 + Cer18: 0 + Cer24: 1) / Conc (Ce24: 0).

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, and in particular to a biomarker, detection reagent and application of Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is a chronic neurodegenerative disease. With the continuous aging of the population, the number of PD patients has increased year by year, bringing a heavy burden to individuals, families and society. The main pathological characteristics of PD are severe loss of dopaminergic neurons in the substantia nigra of the midbrain and reduction of dopamine neurotransmitters in the striatum. The symptoms of PD vary, mainly manifested in two types of symptoms: motor and non-motor. At present, the diagnosis of PD mainly relies on clinical manifestations, signs and auxiliary examinations, such as cerebellar signs and supranuclear gaze palsy, presynaptic dopaminergic PET imaging, etc. These methods lack sensitivity and specificity, and also have serious lags, making it impossible to predict PD in advance. Therefore, it is crucial to find biomarkers for PD in patients to predict and assess the risk of disease early and change the treatment and prevention plans for this population to reduce the occurrence of adverse events.

[0003] Ceramide, or N-acylsphingosine, is the basic unit of sphingomyelin, a component of the cell membrane. As a second messenger, ceramide plays an important regulatory role in cell signal transduction and basic life activities such as cell growth, differentiation, apoptosis, and aging. Studies have shown that disorders in ceramide metabolism may lead to α-synuclein deposition and Lewy body formation, which is related to the pathological mechanism of PD. With the update of detection methodology, liquid chromatography-mass spectrometry technology has been widely used for blood / tissue ceramide detection due to its unique detection advantages. This study detected the content distribution of various ceramides in healthy people and PD patients, analyzed them by statistical methods, and screened out PD biomarkers or indication standards for risk prediction, dynamic monitoring of progression and prognosis of PD patients, early confirmation of the disease and intervention treatment, improvement of treatment management of PD population, and significant improvement of diagnosis and treatment effects and quality of life of PD population, which has important theoretical significance and application value. Summary of the invention

[0004] In order to overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide a biomarker and diagnostic model for Parkinson's disease, which has the advantages of high sensitivity and low misdiagnosis rate, and can be effectively used for the early diagnosis of Parkinson's disease and the risk prediction of adverse events.

[0005] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions: The present invention discloses a biomarker for Parkinson's disease, which includes at least one of the following indicators: a concentration ratio of C16 ceramide to C24 ceramide (Cer16:0 / Cer24:0), a concentration ratio of C18 ceramide to C24 ceramide (Cer18:0 / Cer24:0), a concentration ratio of C24:1 ceramide to C24 ceramide (Cer24:1 / Cer24:0), and a combined indicator of a ratio of the total concentration of Cer16:0, Cer18:0 and Cer24:1 to the concentration of Cer 24:0 (Conc (Cer16:0+Cer18:0+Cer24:1) / Conc (Cer24:0)).

[0006] The independent sample t-test in SPSS 25.0 statistical analysis software was used to compare the significant differences among the groups (p<0.05). The sensitivity and specificity of biomarkers in diagnosing Parkinson's disease were analyzed by ROC curve. The area under the ROC analysis curve greater than 0.7 was considered to have good diagnostic ability. Finally, the concentration ratios of C16 ceramide, C18 ceramide, C24:1 ceramide to C24 ceramide, and the ratio of the total concentration of C16 ceramide, C18 ceramide and C24:1 ceramide to the concentration of C24 ceramide were screened as indicators for predicting the risk of Parkinson's disease in subjects.

[0007] The present invention establishes an optimal prediction model by selecting a combined biomarker evaluation index and finds the optimal critical point for evaluating the effect of diagnosing Parkinson's disease.

[0008] The biomarkers, detection reagents and kits provided by the present invention can be effectively used for the early diagnosis of Parkinson's disease, have high sensitivity and low misdiagnosis rate, and can be used for large-scale detection of Parkinson's disease.

[0009] Also provided are assays comprising biomarkers for Parkinson's disease.

[0010] Also provided are a method for preparing a detection reagent, a sample pretreatment method, and a quality control method, which include the following steps: (1) Weigh 1 mg of each of C16 ceramide, C18 ceramide, C24 ceramide, and C24:1 ceramide standard substances and dissolve them in 1 mL of methanol solution, vortex and sonicate until the sample is dissolved, and obtain a single standard stock solution of 1 mg / mL of the above standard substances. (2) Weigh 1 mg each of each of internal standard substances C16 ceramide-D7, C18 ceramide-D7, C24 ceramide-D7, and C24:1 ceramide-D7 and dissolve them in 1 mL of methanol solution, vortex and sonicate until the sample is dissolved, and obtain an internal standard stock solution with a concentration of 1 mg / mL. (3) The detection reagent also includes a substitute plasma matrix, which is a bovine serum albumin (BSA) solution produced by Shanghai Maokang Biotechnology Co., Ltd.; each ceramide single standard stock solution (1 mg / mL) is used to prepare a concentration of 10 times the highest point of the standard curve (S5×10) with methanol, and then diluted to S5 with a 4% BSA (phosphate buffer) solution, and then diluted step by step with a 4% BSA solution to finally prepare the working solution of each concentration standard curve; (4) Accurately pipette 10 μL of 1 mg / mL internal standard stock solution of C16 ceramide-D7, C18 ceramide-D7, C24 ceramide-D7, and C24:1 ceramide-D7, add 960 μL of methanol solution, and mix to obtain a 10 μg / mL mixed internal standard working solution. Take 500 μL of the mixed internal standard working solution (10 μg / mL), place it in a 5 mL volumetric flask, dilute it with methanol solution and make up to volume, and mix to obtain a 1 μg / mL internal standard working solution. (5) Accurately aspirate 50 μL of plasma sample, add 450 μL of internal standard working solution, vortex for 1 min, centrifuge at 12,000 rpm for 10 min, and take the supernatant for analysis; aspirate the plasma sample to be tested and mix it, prepare it according to the sample pretreatment method, and then analyze it on the machine for 5 consecutive days, five times a day, with 2 parallels each time, and then calculate the total mean and total standard deviation; the mean value is the target value of the quality control product, and the mean value ± 2 × standard deviation is the target value range. The measured concentration of the quality control product is within the target value range and the precision calculated after 10 repeated injections meets the coefficient of variation CV ≤ 15.0% for quality control.

[0011] The invention also provides an application of a combination of markers to assess or predict the risk of PD.

[0012] The Parkinson's disease biomarkers and diagnostic models of the present invention can be effectively used for the prediction and diagnosis of Parkinson's disease, have high sensitivity and low misdiagnosis rate, can be used for the early diagnosis of Parkinson's disease and the risk prediction of adverse events, provide a basis for clinical decision-making, and provide some references for subsequent basic research and clinical research, and have great research and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1This is a diagram of the ceramide structure analyzed in the technical solution of the present invention.

[0014] Figure 2 Based on the MRM extraction profile of ceramide in the surrogate matrix under optimized chromatographic mass spectrometry conditions (the concentrations of Cer16:0, Cer18:0, Cer24:0, and Cer24:1 were 50, 50, 200, and 200 ng / mL, respectively).

[0015] Figure 3 Standard curve of ceramide in surrogate matrix.

[0016] Figure 4 ROC curve analysis results of the biomarker ceramide Cer16:0 / Cer24:0 between healthy subjects (H) and PD patients.

[0017] Figure 5 ROC curve analysis results of the biomarker ceramide Cer18:0 / Cer24:0 between healthy subjects (H) and PD patients.

[0018] Figure 6 ROC curve analysis results of the biomarker ceramide Cer24:1 / Cer24:0 between healthy subjects (H) and PD patients.

[0019] Figure 7 ROC curve analysis results of multifactorial combined analysis of biomarker ceramide between healthy subjects (H) and PD patients.

[0020] Figure 8 ROC curve analysis results of the diagnostic model between healthy subjects (H) and PD patients. DETAILED DESCRIPTION

[0021] like Figure 1 As shown in the figure, the structural formulas of various ceramide substances involved in the biomarkers of Parkinson's disease were obtained. Through the inter-group t-test and the receiver operating characteristic curve (ROC) screening, it was obtained that the biomarkers included at least one of the following indicators: the concentration ratio of C16 ceramide to C24 ceramide (Cer16:0 / Cer24:0), the concentration ratio of C18 ceramide to C24 ceramide (Cer18:0 / Cer24:0), the concentration ratio of C24:1 ceramide to C24 ceramide (Cer24:1 / Cer24:0) and the ratio of the total concentration of the combined indicator Cer16:0, Cer18:0 and Cer24:1 to the concentration of Cer 24:0 (Conc (Cer16:0+Cer18:0+Cer24:1) / Conc (Cer24:0)).

[0022] The clinical diagnostic model of this biomarker uses the ratio of the total concentration of C16 ceramide, C18 ceramide and C24:1 ceramide to the concentration of C24 ceramide (Conc (Cer16:0 + Cer18:0 + Cer24:1) / Conc (Cer24:0)) as a combined indicator for evaluation. A series of sensitivity and 1-specificity values ​​are obtained based on the coordinate points of the ROC curve of the combined indicator. The Youden index is obtained by subtracting 1 from the sum of the sensitivity and specificity. The optimal critical point is selected at the maximum Youden index, which is the cutoff value. According to this algorithm, the optimal critical point value of the diagnostic model is 0.203.

[0023] The present invention provides a biomarker for Parkinson's disease, which has the advantages of high sensitivity and low misdiagnosis rate, and can be effectively used for early diagnosis of Parkinson's disease and risk prediction of adverse events.

[0024] Also provided are reagents comprising biomarkers for detecting Parkinson's disease.

[0025] Preferably, the detection reagents include a single standard stock solution, an internal standard stock solution, a matrix standard curve working solution, and an internal standard working solution.

[0026] Also provided are a method for preparing a detection reagent, a sample pretreatment method, and a quality control method, which include the following steps: (1) Weigh 1 mg of each of C16 ceramide, C18 ceramide, C24 ceramide, and C24:1 ceramide standard substances and dissolve them in 1 mL of methanol solution, vortex and sonicate until the sample is dissolved, and obtain a single standard stock solution of 1 mg / mL of the above standard substances. (2) Weigh 1 mg each of each of internal standard substances C16 ceramide-D7, C18 ceramide-D7, C24 ceramide-D7, and C24:1 ceramide-D7 and dissolve them in 1 mL of methanol solution, vortex and sonicate until the sample is dissolved, and obtain an internal standard stock solution with a concentration of 1 mg / mL. (3) The detection reagent also includes a substitute plasma matrix, which is a bovine serum albumin (BSA) solution produced by Shanghai Maokang Biotechnology Co., Ltd.; each ceramide single standard stock solution (1 mg / mL) is used to prepare a concentration of 10 times the highest point of the standard curve (S5×10) with methanol, and then diluted to S5 with a 4% BSA (phosphate buffer) solution, and then diluted step by step with a 4% BSA solution to finally prepare the working solution of each concentration standard curve; (4) Accurately pipette 10 μL of 1 mg / mL internal standard stock solution of C16 ceramide-D7, C18 ceramide-D7, C24 ceramide-D7, and C24:1 ceramide-D7, add 960 μL of methanol solution, and mix to obtain a 10 μg / mL mixed internal standard working solution. Take 500 μL of the mixed internal standard working solution (10 μg / mL), place it in a 5 mL volumetric flask, dilute it with methanol solution and make up to volume, and mix to obtain a 1 μg / mL internal standard working solution. (5) Accurately aspirate 50 μL of plasma sample, add 450 μL of internal standard working solution, vortex for 1 min, centrifuge at 12,000 rpm for 10 min, and take the supernatant for analysis; aspirate the plasma sample to be tested and mix it, prepare it according to the sample pretreatment method, and then analyze it on the machine for 5 consecutive days, five times a day, with 2 parallels each time, and then calculate the total mean and total standard deviation; the mean value is the target value of the quality control product, and the mean value ± 2 × standard deviation is the target value range. The measured concentration of the quality control product is within the target value range and the precision calculated after 10 repeated injections meets the coefficient of variation CV ≤ 15.0% for quality control.

[0027] Preferably, in step (5), the biological sample is serum, plasma, whole blood or dried blood spot.

[0028] The embodiments of the present invention are described in detail below.

[0029] 1. Experimental conditions 1. Sample grouping Including healthy people (H) and Parkinson's disease (PD) samples, the number of samples in each group is shown in Table 1.

[0030] Table 1 Sample component information Sample Type quantity Healthy people (H) 100 Parkinson's disease (PD) 100 2 Experimental instruments and materials CaLQuant-S liquid chromatography tandem mass spectrometry detection system with MultiQuant 3.0.3 data processing system, Hangzhou Kailaipu Precision Medical Testing Technology Co., Ltd., China; QUINTIX35-1CN electronic balance, Sartorius, Germany; MS200 multi-tube vortex mixer, Hangzhou Ruicheng Instrument Co., Ltd., China. C16 ceramide (d18:1 / 16:0) (abbreviation: Cer16:0; batch number: FS1638498), C18 ceramide (d18:1 / 18:0) (abbreviation: Cer18:0; batch number: FS1640916), C24 ceramide (d18:1 / 24:0) (abbreviation: Cer24:0; batch number: FS1638499), C24:1 ceramide (d18:1 / 24:1(15Z)) (abbreviation: Cer24:1; batch number: FS1641821), and The corresponding internal standards C16 ceramide-D7 (abbreviation: IS-Cer16:0, batch number: FS1640915), C18 ceramide-D7 (abbreviation: IS-Cer18:0, batch number: S170703), C24 ceramide-D7 (abbreviation: IS-Cer24:0, batch number: S165455), and C24:1 ceramide-D7 (abbreviation: IS-Cer24:1, batch number: FS1642644) were purchased from Tianjin Alta Technology Co., Ltd., China (the structural formulas of each ceramide standard substance are shown in Figure 1 ); Methanol (Batch No.: O234231112D), chromatographic grade, Shanghai Xingke High Purity Solvent Co., Ltd., China; Formic acid (Batch No.: G2209224), chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd., China. Bovine serum albumin (Batch No.: MP6104), Shanghai Maokang Biotechnology Co., Ltd., China; PBS buffer (Batch No.: 2023.08.02), Changde Beekman Biotechnology Co., Ltd., China.

[0031] 3 Analysis methods 3.1 Chromatographic conditions Chromatographic column: DISIGNS column-004 (50mm*2.1mm); mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid methanol solution, gradient conditions are as shown in Table 2; flow rate: 0.4 mL·min-1; column temperature: 40C; injection volume: 5 mL.

[0032] Table 2 Mobile phase gradient elution program

[0033] 3.2 Mass spectrometry conditions Ion source: electrospray ion source, positive ion detection; ion spray voltage: 5500 V; temperature: 500°C; source gas 1 (GS1, N2) pressure: 50 psi; gas 2 (GS2, N2) pressure: 50 psi.; curtain gas (N2) pressure: 20 psi; scanning mode: multiple reaction monitoring (MRM); collision gas (N2) pressure: 9 psi; MRM parameters are shown in Table 3.

[0034] Table 3 MRM parameters

[0035] Q1: precursor ion; Q3: product ion; DP: declustering potential; CE: collision energy 4 Detection Reagents 4.1 Preparation of stock solution Weigh 1 mg of C16 ceramide, C18 ceramide, C24 ceramide, and C24:1 ceramide standards respectively and dissolve them in 1 mL of methanol solution. Vortex and sonicate until the sample is dissolved to obtain 1 mg / mL single-standard stock solutions of the above standards.

[0036] 4.2 Preparation of internal standard stock solution.

[0037] Weigh 1 mg of C17 ceramide reference substance and dissolve it in 1 mL of methanol solution. Vortex and sonicate until the sample is dissolved to obtain an internal standard stock solution with a concentration of 1 mg / mL.

[0038] 4.3 Preparation of matrix standard curve working solution 4.3.1 Selection of alternative matrices Ceramide is an endogenous substance, and an alternative matrix that does not contain this substance must be selected for the preparation of the standard curve solution. If the matrix of the alternative standard is significantly different from the matrix of the serum sample, there will be differences in the matrix effect, resulting in inconsistent chromatographic mass spectrometry response signals of the analyte and its internal standard in different matrices, resulting in matrix interoperability problems, affecting the accuracy and precision of the detection method. First, a bovine serum albumin (BSA) solution that has been verified by a large number of experiments to be a substitute for the plasma matrix was selected. Through the verification of BSA products produced by 5 manufacturers, a BSA produced by Shanghai Maokang Biotechnology Co., Ltd. was screened out. The machine test showed that this BSA product did not contain the 9 ceramide substances to be tested, so it can be used as a substitute matrix for plasma.

[0039] 4.3.2 Preparation of standard curve working solution Use each ceramide single standard stock solution (1 mg / mL) to prepare a concentration 10 times the highest point of the standard curve (S5×10) with methanol, then dilute it to S5 with 4% BSA (prepared with phosphate buffer) solution, and then dilute it step by step with 4% BSA solution to finally prepare the standard curve working solutions of each concentration as shown in Table 4.

[0040] Table 4 Working solutions of ceramide standard curves

[0041] 4.4 Preparation of internal standard working solution Accurately pipette 10 μL of 1 mg / mL internal standard stock solution of C16 ceramide-D7, C18 ceramide-D7, C24 ceramide-D7, and C24:1 ceramide-D7, add 960 μL of methanol solution, and mix to obtain 10 μg / mL mixed internal standard working solution. Take 500 μL of mixed internal standard working solution (10 μg / mL), place it in a 5 mL volumetric flask, dilute and fix to volume with methanol solution, and mix to obtain 1 μg / mL internal standard working solution.

[0042] 4.5 Sample pretreatment Accurately pipette 50 μL of plasma sample, add 450 μL of internal standard working solution, vortex for 1 min, centrifuge at 12000 rpm for 10 min, and take the supernatant for analysis.

[0043] 4.6 Quality Control Sample Preparation After the plasma samples to be tested are mixed, they are prepared according to the sample pretreatment method and then analyzed on the machine for 5 consecutive days, five times a day, with 2 parallels each time, and then the total mean and total standard deviation are calculated. The mean value is the target value of the quality control product, and "mean value ± 2 × standard deviation" is the target value range. The concentration of the quality control product is within the target value range and the precision calculated by repeated injection 10 times must meet the coefficient of variation CV ≤ 15.0% for quality control.

[0044] 4.7 Data Analysis 4.7.1 Data Processing The data were quantitatively analyzed using the MultiQuant 3.0.3 data processing system configured with a CaLQuant-S liquid chromatography tandem mass spectrometry detection system.

[0045] The data analysis and result calculation are as follows: 1) Plotting the standard curve: Plot the standard curve with the target concentration of the matrix standard curve working solution as the horizontal axis (x) and the ratio of the measured peak area of ​​the standard substance with the corresponding concentration to the peak area of ​​the internal standard as the vertical axis (y).

[0046] 2) Fitting of the standard curve equation: The ratio of the measured peak area of ​​the standard to the peak area of ​​the internal standard (y) is used to perform linear regression on the labeled concentration (x) using the weighted least squares method. Fit the linear regression equation: y=ax+b, and calculate the correlation coefficient r.

[0047] 3) Quality control product data analysis: When the r of the standard curve is ≥ 0.990, substitute the ratio of the peak area of ​​the quality control product to the peak area of ​​the internal standard into the regression equation to obtain the measured concentration of the quality control product; 4) Sample result calculation: When the concentration of the quality control product is within the target value range and the precision is within the required range, substitute the ratio of the peak area of ​​the various ceramides in the sample to the peak area of ​​the internal standard into the regression equation to obtain the concentration of the various ceramides in the sample.

[0048] 5) Quality control requirements: If the results of the quantitative standard curve quality control products are not within the target value range, the test results of the analytical batch samples will be invalidated and retested.

[0049] 4.7.2 Data Analysis The independent sample t test in SPSS 25.0 statistical analysis software was used to compare the significant differences between the groups (p < 0.05). The ROC curve in SPSS 25.0 statistical analysis software was used to measure the ability of this method to identify the disease, including sensitivity (true positive rate) and misdiagnosis rate (false positive rate, 1-specificity). This method is an important means to determine the diagnostic test standard (i.e., the cutoff value for distinguishing normal from abnormal). In this study, the sensitivity and specificity of ceramide metabolites in diagnosing PD were comprehensively investigated through ROC curve analysis. In large-scale data analysis, the area under the ROC analysis curve was greater than 0.7, which was considered to have good diagnostic ability and was defined as a candidate biomarker. Select a single or combined biomarker evaluation indicator, establish the optimal prediction model, and find the optimal critical point through ROC to evaluate the effect of diagnosing Parkinson's disease.

[0050] 2. Experimental Results 1. Standard curves and linearity of each ceramide See the MRM extraction diagram of the target ceramide in the surrogate matrix sample. Figure 2 , take samples of each ceramide matrix standard curve series concentration, after pretreatment, continuously inject from low concentration to high concentration, use the target concentration of the matrix standard curve working solution as the horizontal axis (x), and the ratio of the measured peak area of ​​the corresponding concentration standard to the internal standard peak area as the vertical axis (y) to draw the standard curve. The weighted least squares method is used to perform linear regression on the labeled concentration (x) using the ratio of the measured peak area of ​​the standard to the peak area of ​​the internal standard to obtain the linear equation and linear correlation coefficient value r( Figure 3The results showed that C16 ceramide, C18 ceramide, C24 ceramide and C24:1 ceramide had good linearity within their respective concentration ranges, and the correlation coefficients r were all greater than 0.99 (Table 5).

[0051] Table 5 Standard curve equation and linear correlation coefficient of target analytes

[0052] 2. Quality Control Sample Analysis Mixed plasma samples were used as quality control samples to measure the target values ​​and ranges of each ceramide in the quality control samples as shown in Table 6. Sample tests were carried out on the machine and the concentrations of each ceramide substance were all within the target value range. The coefficient of variation CV of precision was less than 15%, which met the quality control requirements for quality control product analysis (Table 7).

[0053] Table 6 Target values ​​and ranges of ceramides in quality control samples

[0054] Table 7 Recovery rate and coefficient of variation of each ceramide in quality control samples

[0055] 3. Analysis of Human Plasma Samples The target ceramides in the plasma samples of healthy subjects (H) and Parkinson's disease patients (PD) were quantitatively analyzed, and the measured plasma concentrations of target ceramides and the concentration ratios of Cer16:0, Cer18:0, Cer 24:1 and Cer 24:0 in each group of samples are shown in Table 8.

[0056] The t-test and foldchange comparison of the concentration ratios of Cer16:0, Cer18:0, Cer 24:1 and Cer 24:0 between the groups showed that in the comparison between the PD patients and healthy controls (H), the inter-group fold changes of the concentration ratios of Cer16:0, Cer18:0, Cer 24:1 and Cer 24:0 were 1.45, 2.20 and 2.03 times, respectively; at the same time, in the comparison between the PD patients and healthy controls (H), the inter-group t-test of the concentration ratios of Cer16:0, Cer18:0, Cer 24:1 and Cer 24:0 showed significant differences (p < 0.001).

[0057] Further, the diagnostic biomarkers for PD were determined based on the ROC curve. SPSS software was used to set the group as the state variable, and the target ceramide concentration ratio was set as the test variable. The larger the test result, the more positive the test. The results showed that in the comparison between the PD patients and the healthy people (H) group, the ROC analysis area under the curve (AUC) of the Cer16:0, Cer18:0, Cer 24:1 and Cer24:0 concentration ratios were 0.787, 0.806, and 0.889, respectively. Figure 4-Figure 6 , the areas under the ROC curve were all greater than 0.75, indicating that the indicator has a high accuracy in predicting Parkinson's disease, and the concentration ratios of Cer16:0, Cer18:0, Cer 24:1 and Cer 24:0 can be used as candidate biomarker indicators for PD. The diagnostic biomarkers of PD screened by comprehensive t-test, fold change analysis and ROC analysis are Cer16:0, Cer18:0, Cer 24:1 and Cer 24:0 concentration ratios, see Table 9. At the same time, these three ceramide indicators were combined to investigate the accuracy of multi-indicator comprehensive diagnosis of PD, that is, multifactor combination analysis. From the results, it can be seen that the area under the ROC analysis curve (AUC) in the multifactor combination analysis is 0.902, see Figure 7 , further proving that these three ceramide indicators have high efficacy in predicting PD disease.

[0058] Table 8 Ceramide concentrations and concentration ratios in the healthy group (H) and the diseased group (PD)

[0059] Table 9 Analysis of PD biomarkers 4. Establishment of diagnostic model

[0060] SPSS software was used to establish a diagnostic model, and the ROC curve was used to explore the diagnostic value of ceramide alone or in combination for PD. The ratio of the total concentration of Cer16:0, Cer18:0, and Cer24:1 to the concentration of Cer 24:0 (Conc (Cer16:0+Cer18:0+Cer24:1) / Conc (Cer24:0)) was used as a combined indicator for evaluation. The higher the ROC value, the higher the accuracy of the model in predicting the disease. Figure 8This is the ROC curve analysis result of the biomarker ceramide combination index between the healthy group and the PD patient group. It can be seen from the results that the AUC corresponding to the combined index is as high as 0.905, which is higher than the AUC corresponding to the single index of Cer16:0, Cer18:0, Cer 24:1 and Cer 24:0 concentration ratio. Therefore, the prediction accuracy of PD will be higher if the combined index diagnostic model is selected. According to the coordinate points of the ROC curve of the combined index, a series of sensitivity and 1-specificity values ​​are obtained. The Youden index is obtained by subtracting 1 from the sum of the sensitivity and specificity. The optimal critical point is selected at the maximum Youden index, which is the cutoff value. According to this algorithm, the optimal critical point value of the diagnostic model is 0.203, that is, (Conc (Cer16:0 + Cer18:0 + Cer24:1) / Conc (Cer24:0)) > 0.203, predicting that the subject is at risk of PD.

[0061] 5. Application case analysis The established marker diagnostic model was applied to 80 PD patients and 50 control samples. The ratio of the total concentration of Cer16:0, Cer18:0 and Cer24:1 to the concentration of Cer 24:0 was analyzed, and then the verified diagnostic sensitivity, specificity and total coincidence rate were statistically obtained, as shown in Table 10 below. The diagnostic sensitivity was 86.3%, the specificity was 92.0%, and the total coincidence rate was 88.5%. The results showed that the prediction model has high sensitivity, high specificity, and high accuracy, and has a good ability to assess or predict the risk of PD.

[0062] Table 10 Evaluation of the effect of the prediction model in diagnosing PD

[0063] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A biomarker for Parkinson's disease, characterized by: At least one of the following indicators is included: the concentration ratio of C16 ceramide to C24 ceramide (Cer16:0 / Cer24:0), the concentration ratio of C18 ceramide to C24 ceramide (Cer18:0 / Cer24:0), the concentration ratio of C24:1 ceramide to C24 ceramide (Cer24:1 / Cer24:0) and the ratio of the total concentration of the combined indicator Cer16:0, Cer18:0 and Cer24:1 to the concentration of Cer 24:0 (Conc (Cer16:0+Cer18:0+Cer24:1) / Conc (Cer24:0)).

2. The use of a biomarker for Parkinson's disease according to claim 1, characterized in that: The biomarker is used to assess or predict the risk of a subject suffering from Parkinson's disease, and the optimal critical point value of the diagnostic model of the combined indicator (Conc (Cer16:0+Cer18:0+Cer24:1) / Conc (Cer24:0)) is further given as 0.

203.

3. The use of a biomarker for Parkinson's disease according to claim 2, characterized in that: The biological samples used are serum, plasma, whole blood or dried blood spots.

4. The use of a biomarker for Parkinson's disease according to claim 2, characterized in that: The levels and concentration ratios of various ceramides in the biological sample of the test subject are obtained by one or more of the following methods: chemical analysis, immunoassay, chromatography, and mass spectrometry.

5. A detection reagent, characterized in that: It comprises a concentration ratio of a biomarker for detecting Parkinson's disease according to claim 1.

6. The detection reagent according to claim 5, characterized in that: It includes a biomarker calibrator and a biomarker extraction solvent, wherein the extraction solvent is an internal standard organic solvent, and the organic solvent is selected from one or more of methanol, acetonitrile, and isopropanol; the internal standard organic solvent is used for pretreatment protein precipitation and marker extraction of biological samples before detection; the volume ratio of the internal standard organic solvent to the biological sample is 3-10 times.

7. A method for preparing the detection reagent according to claim 5, characterized in that: It includes the following steps: (1) Weigh 1 mg of each standard of C16 ceramide, C18 ceramide, C24 ceramide, and C24:1 ceramide and dissolve them in 1 mL of methanol solution. Vortex and sonicate until the sample is dissolved to obtain 1 mg / mL single-standard stock solutions of the above standards. (2) Weigh 1 mg each of the internal standards C16 ceramide-D7, C18 ceramide-D7, C24 ceramide-D7, and C24:1 ceramide-D7 and dissolve them in 1 mL of methanol solution. Vortex and sonicate until the sample is dissolved to obtain a single standard stock solution with a concentration of 1 mg / mL. (3) The detection reagent also includes a substitute plasma matrix, which is a bovine serum albumin (BSA) solution that has been screened experimentally. The BSA does not contain endogenous ceramide substances, making the detection result more accurate; each ceramide single-label stock solution is used to prepare a concentration of 10 times the highest point S5 of the standard curve with methanol, and then diluted to S5 with a 4% BSA solution prepared in phosphate buffer, and finally prepared into a standard curve working solution through stepwise dilution with a 4% BSA solution; (4) Accurately pipette 10 μL of 1 mg / mL internal standard stock solution of C16 ceramide-D7, C18 ceramide-D7, C24 ceramide-D7, and C24:1 ceramide-D7, add 960 μL of methanol solution, mix well to obtain 10 μg / mL mixed internal standard working solution, take 500 μL of the mixed internal standard working solution, place it in a 5 mL volumetric flask, dilute and make up to volume with methanol solution, and mix well to obtain 1 μg / mL internal standard working solution.

Images