A Parkinson's disease diagnostic marker combination and its application

Abstract

The present invention discloses a Parkinson's disease diagnostic marker combination and its application, and relates to the field of biomedical technology. The Parkinson's disease diagnostic marker combination includes α-synuclein and CD81 on the neuronal extracellular vesicle membrane in serum. The present invention also provides the use of a reagent for detecting the content of the Parkinson's disease diagnostic marker combination in serum in the preparation of a diagnostic product for Parkinson's disease. The present invention has found that the ratio of α-synuclein and CD81 on the neuronal extracellular vesicle membrane can achieve an effective diagnosis of Parkinson's disease, and the AUC value in the receiver operating characteristic curve can reach 0.94. The present invention provides a new strategy for neuronal extracellular vesicle membrane protein markers for the accurate diagnosis of Parkinson's disease, showing significant advantages in improving diagnostic accuracy and efficiency.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a Parkinson's disease diagnostic marker combination and application thereof. Background Art

[0002] Parkinson's disease (PD), also known as idiopathic Parkinson's disease, paralysis agitans, shaking palsy, is a neurodegenerative disease that occurs frequently in the elderly and is also the most common extrapyramidal disease in the elderly. The prevalence rate in people over 65 years old is 1%, which increases with age, and is slightly higher in men than in women. The cause of Parkinson's disease is not yet clear, and its pathogenesis includes mitochondrial dysfunction, oxidative stress, and the toxic effects of the fast excitatory transmitter glutamate.

[0003] Extracellular vesicles are lipid bilayer membrane vesicles secreted by a variety of cells, with a diameter between 30-150nm. Extracellular vesicles contain RNA and abundant proteins, which play an important role in the process of information exchange between cells. Studies have shown that the proteins in extracellular vesicles are relatively stable in the blood, and they have great potential as clinical diagnostic markers, and are also a hot spot and breakthrough in the diagnosis of many diseases. In recent years, studies have found that the total extracellular vesicles in peripheral blood carrying α-synuclein (α-synuclein, referred to as α-Syn) content is a possible early diagnostic marker for PD, but its diagnostic value is low, and the AUC value can only reach about 0.7 (reference patent CN116840482A). Therefore, it is necessary to develop new diagnostic markers for Parkinson's disease to improve the diagnostic accuracy of Parkinson's disease. Summary of the invention

[0004] The purpose of the present invention is to provide a Parkinson's disease diagnostic marker combination and its application to solve the problems existing in the above-mentioned prior art. The Parkinson's disease diagnostic marker combination includes α-synuclein and CD81 on the neuronal extracellular vesicle membrane. The ratio of α-synuclein and CD81 on the neuronal extracellular vesicle membrane can realize effective diagnosis of Parkinson's disease, and the AUC value can reach 0.94.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a Parkinson's disease diagnostic marker combination, comprising alpha-synuclein and CD81 on neuronal extracellular vesicle membranes in serum.

[0007] Furthermore, the higher the ratio of the serum content of the α-synuclein to the CD81, the higher the probability of suffering from Parkinson's disease.

[0008] The present invention also provides the use of a reagent for detecting the content of the above Parkinson's disease diagnostic marker combination in serum in the preparation of a Parkinson's disease diagnostic product.

[0009] Furthermore, the reagent includes an antibody for immunocapturing neuronal extracellular vesicles, an antibody for recognizing the α-synuclein, and an antibody for recognizing the CD81.

[0010] Furthermore, the antibody for immunocapturing neuronal extracellular vesicles is L1CAM 13-1719-82; the antibody for recognizing the α-synuclein is Biotin α-synuclein 115-121; and the antibody for recognizing the CD81 is Biotin CD81AB239238.

[0011] Furthermore, the diagnostic product is a reagent, a test kit or a digital microfluidic chip.

[0012] The present invention also provides a diagnostic product for Parkinson's disease, comprising a reagent for detecting the content of the above Parkinson's disease diagnostic marker combination in serum.

[0013] Furthermore, the reagent includes an antibody for immunocapturing neuronal extracellular vesicles, an antibody for recognizing the α-synuclein, and an antibody for recognizing the CD81.

[0014] Furthermore, the antibody for immunocapturing neuronal extracellular vesicles is L1CAM 13-1719-82; the antibody for recognizing the α-synuclein is Biotin α-synuclein 115-121; and the antibody for recognizing the CD81 is Biotin CD81AB239238.

[0015] Furthermore, the diagnostic product is a reagent, a test kit or a digital microfluidic chip.

[0016] The present invention discloses the following technical effects:

[0017] The present invention has developed a combination of Parkinson's disease diagnostic markers, including α-synuclein and CD81 on the neuronal extracellular vesicle membrane. The ratio of α-synuclein and CD81 on the neuronal extracellular vesicle membrane can achieve effective diagnosis of Parkinson's disease, and the AUC value can reach 0.94. The present invention provides a new strategy for neuronal extracellular vesicle membrane protein markers for the accurate diagnosis of Parkinson's disease, showing significant advantages in improving diagnostic accuracy and efficiency, and providing new possibilities for the early diagnosis and treatment of Parkinson's disease. It is expected to provide more timely and effective medical intervention for Parkinson's patients, thereby improving their quality of life and delaying disease progression. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a schematic diagram of the detection principle of the droplet digital analysis chip;

[0020] Figure 2 This is a typical microscope image of proteins on the neuronal extracellular vesicle membrane detected using a droplet digital chip;

[0021] Figure 3 The results of the detection of CD9, CD81, NCAM, α-synuclein (α-Syn), aggregated α-synuclein (Aggα-Syn), or syndecan-binding protein 1 (syntenin-1, as an internal protein control) in EVs captured by L1CAM in sera of PD patients;

[0022] Figure 4 To assist in the detection of proteins on the neuronal extracellular vesicle membrane in Western blot experiments;

[0023] Figure 5 Boxplots of α-synuclein measurements for healthy controls and PD patients;

[0024] Figure 6 Box plots of CD81 measurements for healthy controls and PD patients;

[0025] Figure 7 Box plot of the ratio of α-synuclein to CD81 in healthy controls and PD patients;

[0026] Figure 8 The receiver operating characteristic curve for α-synuclein on the neuronal extracellular vesicle membrane;

[0027] Fig. 9 Receiver operating characteristic curve for the ratio of α-synuclein to CD81 on the neuronal extracellular vesicle membrane. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Example 1

[0034] 1. Methods

[0035] 1. Experimental subjects

[0036] The subjects of this experiment were divided into two groups: Parkinson's disease patients (PD) and healthy controls (HC), with 20 people in each group. All family members (legal agents) of the subjects gave informed consent, and the samples were reviewed by the ethics committee.

[0037] PD patients recruited in the PD group met the following inclusion criteria:

[0038] ① Patients who have been diagnosed with primary Parkinson's disease by a hospital and have a disease course of more than 6 months;

[0039] ② Aged between 50 and 80 years old;

[0040] ③Can stand and walk without assistance;

[0041] ④ Volunteer to participate in this study, willing to complete the assessment of relevant scales (UPDRS and MoCA) and sign the informed consent.

[0042] The healthy people recruited by HC met the above criteria of ②, ③ and ④.

[0043] Exclusion criteria:

[0044] ① Currently suffering from serious life-threatening diseases such as cardiovascular, cerebrovascular, liver, kidney, hematopoietic system diseases, malignant tumors, etc.;

[0045] ②Past or current diagnosis of schizophrenia, major depression or other psychotic disorders or cognitive dysfunction;

[0046] ③ Those who have received other clinical trials within 30 days before enrollment, which may affect the observation of the effect indicators of this study;

[0047] ④ Taking antidepressants, analgesics, antihistamines and other drugs that affect PD symptoms within 2 weeks before enrollment.

[0048] 2. Clinical data entry and blood sample processing

[0049] All clinical data of the subjects, such as gender, age, course of disease, scale score, etc., will be entered into a unified format to form a statistical table (Demographic table). The samples of the subjects will be uniformly coded with a single blind code for the inventor to conduct a single blind test (the inventor can only see the random code and is not clear which are PD and HC).

[0050] The collected venous blood of the subjects was allowed to stand at room temperature for more than 0.5 h, and then centrifuged at 3500 rpm for 10 min. The separated serum (usually accounting for 40% of the volume of the whole blood) was packaged in 250 μL per tube (the excess was stored at -80°C).

[0051] 3. Detection

[0052] The method for separating and detecting various proteins on the neuronal extracellular vesicle membrane using a digital microfluidic multi-technology detection platform is as follows:

[0053] 1. Preparation of reagents and materials

[0054] (1) Preparation of magnetic beads

[0055] Blue / yellow fluorescent magnetic beads (MBs; Bangs Laboratories, MESY002): 2.79×10 9 / mL, 20μL;

[0056] PBS: appropriate amount.

[0057] (2) Preparation of activator

[0058] EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride): 20 mg (03450, Sigma);

[0059] NHS (N-hydroxysuccinimide): 20 mg (130672, Sigma);

[0060] MES buffer (50 mM, pH 5.5): 500 μL.

[0061] (3) EVs processing of serum samples

[0062] Serum samples;

[0063] PBS;

[0064] Centrifuge tubes.

[0065] (4) Capture antibody preparation

[0066] L1CAM antibody: 40 μL (MA5-14140, Thermo Fisher Scientific);

[0067] PBS: 960 μL.

[0068] (5) Sealing agent preparation

[0069] BSA (bovine serum albumin): 30 mg (A7030, Sigma);

[0070] PBS: 1 mL.

[0071] (6) Preparation of detection antibodies

[0072] Biotin CD81 AB239238(abcam);

[0073] Biotinα-synuclein 115-121(biolegend);

[0074] Biotinα-synuclein aggregateA17183A (biolegend);

[0075] Biotin NCAM B376795(biolegend);

[0076] Biotin L1CAM 13-1719-82 (Thermo Fisher Scientific).

[0077] (7) Streptavidin-β-galactosidase conjugate (SβG) (S931, thermo);

[0078] Ultrapure water: 1.5 mL;

[0079] SβG: 20 μg / mL.

[0080] (8) Washing buffer

[0081] PBST (0.05% Tween-20);

[0082] PBS.

[0083] (II) Operation steps

[0084] (1) Sample preparation

[0085] The magnetic beads were removed and washed with PBS (500 μL PBS, twice).

[0086] The activator was prepared (20 mg EDC and 20 mg NHS added to 500 μL MES buffer).

[0087] (2) EVs processing of serum samples

[0088] Initial centrifugation: Centrifuge at 1000 g for 10 min at 4°C to remove larger precipitates (such as cell debris).

[0089] Second centrifugation: Centrifuge at 2000 g for 20 min at 4°C to remove the precipitate.

[0090] Extracellular vesicles enrichment: Extracellular vesicles were isolated by centrifugation at 10,000 g for 30 min at 4°C.

[0091] Collect the supernatant: Carefully collect the supernatant to avoid disturbing the pellet.

[0092] Sample storage: Aliquot the collected supernatant and store it at -80℃ for future use.

[0093] (3) Activation of magnetic beads

[0094] Take 500 μL of EDC and 500 μL of NHS and mix them into 1 mL. Take 100 μL of them and add them to the blue MBs. Take 10 μL of them and add them to the yellow MBs. Then add 90 μL of MES buffer and react for 0.5 hours.

[0095] (4) Linking Antibodies

[0096] After aspiration on a magnetic stand, the supernatant was removed, the cells were washed with PBS (500 μL, twice), and L1CAM antibody was added and mixed well.

[0097] (5) Closed connection

[0098] The supernatant was removed from the blue / yellow MBs and washed once with 500 μL PBS, and 500 μL BSA was added to each of them and reacted for 0.5 h.

[0099] (6) Washing the magnetic beads

[0100] The supernatant was removed from the blue / yellow MBs, and the MBs were washed once with 500 μL PBST and once with 500 μL PBS, and then 500 μL PBS was added.

[0101] (7) Grouping

[0102] The yellow MBs were divided into 5 portions (100 μL each).

[0103] (8) Add serum

[0104] Yellow 1 was used as the control group and 100 μL PBS was added, and 20 μL of the sample labeled “PD1” was added to each of Yellows 2-6 and rotated on a Ferris wheel at 4°C for 2 h to capture extracellular vesicle markers (EVs).

[0105] (9) Add detection antibody

[0106] Detection antibodies were added to groups 1-6 as shown in Table 1.

[0107] (10) Cleaning samples

[0108] (11) Remove the supernatant and wash once with 500 μL PBST (0.05% Tween-20) and once with 500 μL PBS.

[0109] (12) Connecting SβG

[0110] 20 μg / mL streptavidin-β-galactosidase conjugate (SβG), add 100 μL SβG to each of Yellow 1-6 and react for 1 hour.

[0111] (13) Cleaning samples

[0112] Wash yellow 1-6 with 500 μL PBST 6 times, changing to a new centrifuge tube each time.

[0113] (14) Final sample

[0114] The supernatant was removed and 100 μL of ultrapure water was added to Yellow 1-6 and Blue 1-6, respectively.

[0115] Note: All steps must be performed at 4°C; be careful to avoid disturbing the precipitate; be careful to prevent damage to the magnetic beads.

[0116] Using droplet digital analysis chip (schematic diagram see Figure 1) to detect CD81 protein and α-synuclein protein on the neuronal extracellular vesicle membrane as follows: take 5 μL (2.79×10 9 / mL) yellow fluorescent magnetic beads (MBs) (Bangs Laboratories, MESY002) and 5μL blue fluorescent magnetic beads (MBs) (Bangs Laboratories, MEGB002), (2)-(8) steps are the same as above, (9) blue magnetic beads are connected to biotin CD81 antibody, yellow magnetic beads are connected to biotinα-synuclein antibody, and the subsequent steps are the same as above. Among them, the oil phase used for droplet generation is mineral oil containing stabilizing surfactant 3% ABIL EM 90 (Degussa) and 0.1% w / w Triton X-100, and the two aqueous phases are respectively introduced with the above-mentioned magnetic bead mixture and fluorescein-β-D-galactoside (FDG, F1179, Thermo Fisher Scientific) substrate solution with a concentration of 2μg / mL.

[0117] After the sample is dispersed, the total number of droplets and positive droplets are counted and counted. According to the Poisson distribution, the concentration (C) can be calculated according to the following formula:

[0118]

[0119]

[0120] λ=C×V droplet ;

[0121]

[0122] In the above formula, k: the number of magnetic beads in a droplet; λ: the average number of magnetic beads in each droplet; C: the concentration of magnetic beads in the aqueous solution; D: the diameter of the droplet. Finally, the receiver operating characteristic curve was used to analyze the diagnostic value of α-synuclein protein content on the membrane of brain cell-derived exosomes in Parkinson's disease.

[0123] 2. Results

[0124] Figure 2 Results from microscopy imaging of proteins on neuronal extracellular vesicle membranes using a droplet digitization chip are presented. Figure 3The expression of CD9, CD81, NCAM, α-synuclein (α-Syn), aggregated α-synuclein (Aggα-Syn), and syntenin-1 as an internal control on L1CAM-positive extracellular vesicles (EVs) detected by immunocapture using sample serum. These results confirm the presence of CD9, CD81, NCAM, and α-synuclein and its aggregated form, which can all be detected by this chip. However, syntenin-1 showed a negative signal, consistent with its role as an internal negative control (not present on the vesicle membrane). Figure 4 The presence of these proteins on the neuronal exosome membrane was further verified by Western blot experiments, thus supporting the accuracy and consistency of this detection method.

[0125] In order to further evaluate the clinical application effect of the marker combination of the present invention, the present invention detected and statistically analyzed serum samples from 20 PD patients and 20 healthy subjects.

[0126] In the test of serum samples from PD patients, the results showed that the level of α-synuclein on the membrane of neuronal extracellular vesicles (i.e., α-synuclein of L1CAM-positive EVs) was increased in the serum of these patients. After immunocapture of L1CAM-positive EVs, dual detection of α-synuclein and CD81 was performed. The results showed that compared with healthy controls (HC), PD patients had increased expression of α-synuclein on L1CAM-positive EVs ( Figure 5 ); there was no significant difference in the expression of CD81 ( Figure 6 Interestingly, the ratio of α-synuclein to CD81 on the neuronal extracellular vesicle membrane improved the clinical differentiation of PD patients from HCs compared with α-synuclein alone ( Figure 7 These findings are consistent with previous studies showing that α-synuclein in serum L1CAM-positive EVs is elevated in clinical PD patients, whereas there were no consistent differences in the universal EV marker syntenin-1 between these groups.

[0127] In order to evaluate the clinical effectiveness of α-synuclein alone and α-synuclein / CD81 on the membrane of neuronal extracellular vesicles in diagnosing PD, the present invention conducted a receiver operating characteristic analysis. Figure 8 ; AUC was 0.89), and α-synuclein / CD81 on the neuronal extracellular vesicle membrane showed an excellent AUC value ( Fig. 9; AUC is 0.94). These results not only demonstrate that α-synuclein / CD81 on the neuronal extracellular vesicle membrane can serve as a biomarker for early and clinical PD, but also highlight the promising potential of the detection method of the present invention in the prognosis and diagnosis of PD.

[0128] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a diagnostic model for Parkinson's disease, characterized in that: Taking the content of α-synuclein on the neuronal extracellular vesicle membrane in serum and the content of CD81 on the neuronal extracellular vesicle membrane in serum as input variables, a diagnostic model for Parkinson's disease is constructed; the diagnostic model is: risk value = the content of α-synuclein on the neuronal extracellular vesicle membrane in serum / the content of CD81 on the neuronal extracellular vesicle membrane in serum; The higher the risk value, the higher the probability of suffering from Parkinson's disease.

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