Diagnostic marker for Parkinson's disease, application of diagnostic marker and heteropolymer detection method

Abstract

The invention discloses a diagnostic marker for Parkinson's disease, application of the diagnostic marker and a heteropolymer detection method, and relates to the technical field of biology. The diagnostic marker is a protein heterozygous aggregate in a brain-derived exosome, and the protein heterozygous aggregate comprises alpha-synuclein and amylin. The invention also provides application of a reagent for detecting the diagnostic marker in preparation of a diagnostic product for Parkinson's disease. According to the present invention, the research results show that the important biomarker protein aggregate in the Parkinson's disease diagnosis is not the aggregate simply formed by polymerizing the alpha-Synuclein (aS) protein, but is doped with the other easily aggregated protein-amylin (Amylin); a heteropolymer composed of aS and Amylin is adopted as a biomarker, and the diagnosis efficiency on the Parkinson's disease can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a diagnostic marker for Parkinson's disease and an application thereof and a heteropolymer detection method. Background Art

[0002] Many neurodegenerative diseases have a pathological feature, that is, the aggregates of some specific proteins appear in brain tissue. These specific proteins are often classified as amyloidogenic proteins, that is, a class of aggregation-prone proteins. It is generally believed that each disease is involved in a certain aggregation-prone protein, but recently people have noticed that different aggregation-prone proteins may aggregate with each other to form aggregates. This hybrid aggregation phenomenon is believed to have previously unnoticed pathological effects, and the detection of protein hybrid aggregates (co-aggregates, hereinafter referred to as heteroaggregates) also has important diagnostic significance.

[0003] For a long time, protein aggregates of α-synuclein (α-Syn, aS) have been regarded as important biomarkers for Parkinson's disease, but their diagnostic value is currently limited. There is an urgent need to develop new biomarkers to improve the diagnostic accuracy of Parkinson's disease. Summary of the invention

[0004] The purpose of the present invention is to provide a diagnostic marker for Parkinson's disease and its application and a heteropolymer detection method to solve the problems existing in the above-mentioned prior art. The diagnostic marker provided by the present invention can effectively improve the diagnostic efficacy of Parkinson's disease.

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

[0006] The present invention provides a diagnostic marker for Parkinson's disease. The diagnostic marker is a protein hybrid aggregate in brain-derived exosomes. The protein hybrid aggregate includes alpha-synuclein and amylin.

[0007] The present invention also provides the use of a reagent for detecting the above-mentioned diagnostic marker in the preparation of a diagnostic product for Parkinson's disease.

[0008] Furthermore, the diagnostic product is a kit.

[0009] The present invention also provides a diagnostic product for Parkinson's disease, comprising a reagent for detecting the above-mentioned diagnostic marker.

[0010] Furthermore, the diagnostic product is a kit.

[0011] Furthermore, it includes α-synuclein antibodies and amylin antibodies.

[0012] Furthermore, the α-synuclein antibody is the sc-12767 antibody; and the amylin antibody is the sc-377530 antibody.

[0013] Furthermore, the sc-12767 antibody is labeled with phycoerythrin; and the sc-377530 antibody is labeled with phycocyanin.

[0014] The present invention also provides a method for detecting protein hybrid aggregates, wherein the protein hybrid aggregates include a first protein and a second protein;

[0015] The detection method comprises the following steps:

[0016] Adding the second antibody to the biological sample to be detected, performing a first incubation, then adding the first antibody, performing a second incubation, and then performing microscopic imaging, reading the number of overlapping fluorescent spots on two fluorescent channels through an image recognition system, substituting the number of fluorescent spots into the standard curves of the first protein and the second protein, respectively, to calculate the content of the first protein and the second protein in the biological sample to be detected;

[0017] The standard curve of the first protein is constructed by using different concentrations of the first protein;

[0018] The standard curve of the second protein is constructed by using different concentrations of the second protein;

[0019] The first antibody is an antibody that specifically recognizes the first protein;

[0020] The second antibody is an antibody that specifically recognizes the second protein;

[0021] The first antibody and the second antibody are labeled with different fluorescent markers.

[0022] Furthermore, the first antibody and the second antibody are labeled with phycoerythrin and phycocyanin, respectively.

[0023] The present invention discloses the following technical effects:

[0024] The present invention found that the important biomarker protein aggregate in the diagnosis of Parkinson's disease is not a simple aggregate of α-Synuclein (aS) protein, but is doped with another aggregation-prone protein, amylin. Using a heteropolymer composed of aS and Amylin as a biomarker can effectively improve the diagnostic efficiency of Parkinson's disease.

[0025] The present invention develops a method for detecting protein heteropolymers, which can ensure high sensitivity and high specificity in detecting heteropolymer aggregates, and at the same time, has the advantages of reasonable cost and rapid detection. The present invention uses two reliable antibodies for identification, uses the principle of immune sandwich to enhance selectivity, uses different fluorophores marked on two antibodies to generate fluorescent point signals, and then reads and calculates the co-localized (overlapping) sites through an image recognition algorithm to confirm that the molecule is indeed a heteropolymer. The present invention uses the strongest fluorescent protein to achieve signal amplification, introduces a more sensitive fluorescence microscope, and can have a tracer detection limit. The present invention can simultaneously know the amount of each of the two components through one detection, thereby knowing the hybridization of the heteropolymer, rather than only being able to check one of the components. The detection method of the present invention uses simple and reliable operating conditions. In essence, it belongs to a sandwich fluorescent immunoprecipitation method, which is almost the same as the common ELISA method in operation, except that a fluorescence microscope is required instead of an enzyme marker to read it out. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 The comparison diagram of absorption / emission spectra of R-PE and APC;

[0028] Figure 2 It is the immune sandwich configuration diagram;

[0029] Figure 3 is the standard curve graph of aS aggregates;

[0030] Figure 4 It is the standard curve graph of Amylin aggregates;

[0031] Figure 5 The images of aS, Amylin channels and the superposition of the heteropolymer (co-aggregate); A is the image of the aS channel; B is the image of the Amylin channel; C is the image of the co-aggregate;

[0032] Figure 6 This is a statistical diagram of the fluorescence quantity of α-Syn protein in the serum of Parkinson's disease patients and healthy people;

[0033] Figure 7 It is a statistical diagram of the fluorescence quantity of heteropolymers in the serum of Parkinson's disease patients and healthy people;

[0034] Figure 8 The receiver operating characteristic curves for serum α-Syn protein and heteropolymers are shown;

[0035] Fig. 9 This is a statistical diagram of the fluorescence quantity of α-Syn protein in brain-derived exosomes from Parkinson's disease patients and healthy people;

[0036] Fig.10 The figure shows the statistical diagram of heteropolymer fluorescence in brain-derived exosomes from Parkinson's disease patients and healthy subjects;

[0037] Fig.11 Receiver operating characteristic curves for brain-derived exosomal α-Syn protein and heteropolymers;

[0038] Fig.12 This is a correlation analysis diagram between the number of heteropolymers in serum and the unified Parkinson's disease rating scale (UPDRS) score;

[0039] Fig.13 This is a correlation analysis chart between the number of heteropolymers in brain-derived exosomes and UPDRS scores. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] The protein aggregates detected in the present invention are important biomarkers in the diagnosis of Parkinson's disease. They have long been considered to be aggregates formed by the aggregation of α-Synuclein (aS) protein alone, but have recently been found to be doped with another aggregation-prone protein, amylin.

[0046] Example 1

[0047] Establishment of heteropolymer detection method:

[0048] The present invention uses the antibody of the aggregate body as the capture antibody, and uses the same antibody as the first detection antibody. On this basis, the dopant antibody is used as the second detection antibody to develop a detection method for heteropolymers. For protein aggregates, the sites recognized by most antibodies are unique on the monomers of the protein. The recognition sites of monomeric proteins will be occupied by the capture antibody in the capture step, so they will not be recognized again by the detection antibody; and since the aggregate is composed of many monomers, each aggregate can carry more than one recognition site, so it will be recognized again by the same antibody. Therefore, the present invention can use the capture antibody / first recognition antibody as a pair of immune sandwiches to achieve detection of the heteropolymer body.

[0049] For heteropolymers, after the main body is detected by the above-mentioned antibody pair, the impurity should also be detected. Therefore, an antibody for the impurity is used to identify it and confirm its spatial position relationship with the main body.

[0050] In order to read out the signals of the main body and the adulterant respectively, the present invention uses different fluorophores to label the first detection antibody / second detection antibody respectively. The first detection antibody (aS antibody sc-12767 produced by Santa Cruz) is labeled with phycoerythrin (R-PE), and the second detection antibody (Amylin antibody sc-377530 produced by Santa Cruz) is labeled with phycocyanin (APC). These two fluorophores are the fluorophores with the highest (brightest) known fluorescence quantum yields and can provide better sensitivity. There is little spectral overlap between the two, and crosstalk (such as fluorescence resonance energy transfer effect) is not likely to occur, which can provide better discrimination ( Figure 1). Fluorophores and their labeling methods come from Elabscience (E-FN-S102 is an R-PE product, and E-FN-S103 is an APC product). If you do not perform fluorescent labeling yourself, you can purchase sc-12767PE and sc-377530Alexa Fluor 647 produced by Santa Cruz as detection antibodies for aS and Amylin. These are finished products labeled with fluorophores, and the Alexa Fluor 647 fluorophore can be regarded as a substitute for APC. The immunosandwich configuration is shown in the figure below. Figure 2 shown.

[0051] Detection operation: Based on the common ELISA paradigm, it goes through five steps: "colonization of capture antibody", "blocking", "sample loading and capture", "detection antibody loading", and "signal reading". Cleaning is required between each step. The cleaning method is unified as follows: wash twice with 100μL PBST buffer using a multistepper, wash twice with PBS buffer and pat dry.

[0052] The method reported in “Vashist, SK, Schneider, EM & Luong, JHT Rapid sandwich ELISA-based in vitro diagnostic procedure for the highly-sensitive detection of human fetuin A. Biosens. Bioelectron. 67, 73–78 (2015)” was used to colonize the aS antibody sc-12767 produced by Santa Cruz Biotechnology Co., Ltd. in the United States on WP384-4BCCSH or WP96-4BCCSH produced by Qingdao Jindian Biotechnology Co., Ltd., and the aS antibody sc-12767 produced by Thermo Scientific TM Pierce sold by the company TM Block with protein-free blocking buffer (37572) according to the manufacturer's instructions. Once blocking and washing are complete, the test wells are ready.

[0053] Add 20 μL of biological sample to the sealed test well. Biological samples can be exosomes, tissue or cell lysate, artificially prepared aggregate solution or diluted serum. The loading conditions of various samples need to be adjusted after trial. After loading, incubate at 37°C for 90 minutes, and complete the loading and capture after washing.

[0054] When loading the detection antibody, it is necessary to load the antibody of the dopant first. In the present invention, the labeled sc-377530 second detection antibody is loaded first, and after adjusting to a concentration of 2ng / mL, 50μL is added to each test well and incubated for 1 hour (room temperature 25°C). Thereafter, on this basis, the labeled sc-12767 first detection antibody is loaded, and after adjusting to 1ng / mL, 50μL is added to each test well, and then incubated for 2 hours (room temperature 25°C). After the incubation is completed and the detection antibody is loaded after cleaning. From the beginning of this step, it is necessary to avoid light as much as possible and store it in aluminum foil to avoid light.

[0055] The signal was read out using the BZ-X800 microscope platform of Keyence, Japan, and the bottom of the well plate was imaged microscopically under a 60× or 100× oil-immersion objective. The R-PE fluorescence that identifies aS was sampled using the TRITC channel with an exposure time of 0.5s; the APC fluorescence that identifies Amylin was sampled using the Cy5 channel with an exposure time of 2s. In the autofocus and autopositioning modes, random sampling was performed, and a total area of ​​0.5×0.5mm was scanned, which was used as the region of interest (ROI) for calculation. After sampling, the image appears in the form of many light spots. Using the image recognition algorithm of OpenCV, the light spot counts of each sample were batch calculated and further quantified.

[0056] The standard curve method was used for quantification. Commercial aS aggregate standard (Abcam, ab218819, 2 mg / mL) and Amylin aggregate standard (2 mg / mL) were diluted 1000 times with TBS as the initial concentration, and then gradient diluted by 5 times, and 9 gradients including the initial concentration plus 1 blank standard test solution were prepared (i.e., a total of 10 samples). The two types of standard test solutions were added to the prepared 384-well plate according to the above method for measurement, and two standard curves (i.e., aS and Amylin) were obtained for the number of light spots, see Figure 3 and Figure 4 The actual concentration of the target substance in the biological sample is calculated based on this curve.

[0057] Preparation method of Amylin aggregate standard: Amylin monomer (purchased from Beijing Bioson Biotechnology Co., Ltd., product number Y-0158) is dissolved in pure water to form a 1 mg / mL solution, and allowed to stand at room temperature for 30 minutes to automatically form aggregates. The solution at this time is the Amylin aggregate standard.

[0058] Using OpenCV-based image recognition algorithm programming, after optimizing the image, the light spots of the two channels are recognized separately (see the image example Figure 5 ). The number of overlapping spots on the two channels was identified and counted as confirmed heteropolymer results.

[0059] The present invention utilizes a specific immune sandwich reaction in combination with an accurate and efficient signal readout method to establish for the first time a method for detecting protein heteropolymers, which has the advantages of high throughput and low cost, and the detection limit can be as low as 1 pg / mL, as shown in Table 1.

[0060] Table 1 Comparison of detection methods

[0061] Technology Platform Dynamic range Detection limit applicability Detection Methods ELISA Unknown 0.02ng / mg Heteropolymer Technique 5 Spot analysis <![CDATA[7.5×10-1.5×10 6 ]]> 1fM Common aggregate Techniques 1-4 Spot analysis Undetermined 1pg / mL Heteropolymers and normal aggregates The present invention

[0062] Note: 1.Kass, B. et al. Aβ oligomer concentration in mouse and human brain and its drug-induced reduction ex vivo. Cell Rep. Med. 3, 100630 (2022).

[0063] 2. Herrmann, Y. et al. sFIDA automation yields sub-femtomolar limit of detection for Aβ aggregates in body fluids. Clin. Biochem. 50, 244-247 (2017).

[0064] 3. Kulawik, A., Heise, H., Zafiu, C., Willbold, D. & Bannach, O. Advancements of the sFIDA method for oligomer-based diagnostics of neurodegenerative diseases. FEBS Lett. 592, 516-534 (2018).

[0065] 4.Kühbach,K.et al.Application of an Amyloid Beta Oligomer Standard in the sFIDA Assay.Front.Neurosci.10,(2016).

[0066] 5. Kotiya, D. et al. Rapid, scalable assay of amylin-βamyloid co-aggregation in brain tissue and blood. J. Biol. Chem. 299, (2023).

[0067] In order to avoid the false positive results produced by traditional protein amplification, the detection method of the present invention does not use the protein aggregate amplification method, and by pre-occupying the recognition site, it eliminates the possibility of false aggregate signals generated by monomers. At the same time, compared with other recognition molecules, the use of reliable antibodies for target recognition also reduces the possibility of misidentification.

[0068] The detection method of the present invention will reduce the cost of use. Technologies 1-4 in Table 1 are relatively expensive: 1. In terms of materials, Greiner Bio-one microplates are used, which are relatively expensive (500 yuan / piece), and require the use of complex surface modification methods to colonize antibodies, which has high costs, long operating cycles, and poor robustness. The present invention uses domestically produced well plates and rapid colonization methods, which are low in cost and fast in speed. 2. In terms of operation, the total internal reflection microscope (Tirfm) or confocal microscope (confocal) required by the aforementioned technologies 1-4 has a relatively high cost of use. The present invention only uses a fluorescence microscope, and can edit autofocus and automatically select point sampling, and the instrument has a high universality.

[0069] The present invention has achieved a conceptual breakthrough. The technique 5 in Table 1 has a conceptual flaw and its sensitivity is very low, and it is not a reliable means for heteropolymer detection.

[0070] Example 2

[0071] Shantou University Medical College recruited 21 Parkinson's disease patients and 21 corresponding healthy controls (see Table 2 for details) to conduct the following research experiments:

[0072] Table 2 Information of 21 Parkinson's disease patients and their corresponding 21 healthy subjects

[0073]

[0074] Note: MoCA: Montreal Cognitive Assessment; NA: not applicable; UPDRS III: Movement Disorder Society Unified Parkinson's Disease Rating Scale part III; ns: not significant (or “not significant”).

[0075] 1. Detection of heteropolymers and α-Syn protein content in serum and analysis of diagnostic efficacy

[0076] Blood was drawn from patients and healthy subjects respectively, and serum was collected after natural standing for 1 hour. The serum was centrifuged at 10000g for 10 minutes, and the intermediate clear liquid was collected to remove possible impurities such as precipitated cell debris and floating lipids. The number of fluorescent spots of heteropolymers and α-Syn protein in the serum of patients and healthy subjects was detected respectively, wherein the detection method adopted the method established in Example 1.

[0077] The results of the fluorescence spot detection of serum α-Syn protein and heteropolymer are shown in Figure 6 and Figure 7 The receiver operating characteristic curves of serum α-Syn protein and heteropolymer were drawn separately. The results are shown in Figure 8 The results showed that the diagnostic performance of serum-based heteroaggregates was not much different from that of the more traditional pure α-Syn aggregate detection method.

[0078] 2. Brain exosomes were extracted from the serum of patients and healthy subjects respectively, and then the number of fluorescent spots of heteropolymers and α-Syn proteins was detected. The method was the same as that in “1. Detection of heteropolymers and α-Syn protein content in serum”.

[0079] Extraction method of brain exosomes: "Anti-L1CAM antibodies" are coupled to magnetic beads to prepare immunomagnetic beads, which are then added to 1 ml of 100-fold diluted serum to allow specific adsorption, indicating the presence of L1CAM exosomes, and the adsorbed magnetic beads are collected by magnetic force. After brain exosomes (i.e., L1CAM exosomes) are enriched on the immunomagnetic beads, 30 μL of lysis solution is used to lyse them at room temperature for 15 minutes. The lysate obtained is the extract containing brain exosomes, which can be directly used for subsequent detection.

[0080] Lysis buffer formula: 50 mM Tris-HCl, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% (v / v) Triton X-100 (Triton X-100) and 0.1% (w / v) sodium dodecyl sulfate (SDS), pH 7.0.

[0081] The results of the fluorescence spot detection of α-Syn protein and heteropolymers in brain exosomes are shown in Fig. 9 and Fig.10 The receiver operating characteristic curves of brain exosome α-Syn protein and heteropolymer were drawn separately. The results are shown in Fig.11 The results showed that compared with the more traditional pure α-Syn aggregate detection method, the diagnostic efficacy of brain-derived exosome-based heteropolymers was higher, with AUC = 0.87, which is a high diagnostic accuracy.

[0082] 3. Correlation analysis between the number of heteropolymers and UPDRS score

[0083] Currently, symptom-based scales are still the most commonly used diagnostic criteria. Fig.12 As shown in Figure 2, there was no significant correlation between the number of heteropolymers in serum and the commonly used Parkinson's rating scale (UPDRS) score (r=0.128, p=0.707). However, the number of heteropolymers in brain-derived exosome samples was positively correlated with the UPDRS score (r=0.582, p<0.01). Fig.13This suggests that heteroaggregates in brain-derived exosome samples have significant diagnostic value in the progression of Parkinson's disease.

[0084] 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 diagnostic marker for Parkinson's disease, characterized in that The diagnostic marker is a protein hybrid aggregate in brain-derived exosomes, and the protein hybrid aggregate includes α-synuclein and amylin.

2. Use of a reagent for detecting the diagnostic marker according to claim 1 in the preparation of a diagnostic product for Parkinson's disease.

3. The use according to claim 2, characterized in that: The diagnostic product is a kit.

4. A diagnostic product for Parkinson's disease, characterized in that: Comprising a reagent for detecting the diagnostic marker according to claim 1.

5. The diagnostic product according to claim 4, characterized in that The diagnostic product is a kit.

6. The diagnostic product according to claim 4, characterized in that These include antibodies to α-synuclein and amylin.

7. The diagnostic product according to claim 6, characterized in that The α-synuclein antibody is sc-12767 antibody; the amylin antibody is sc-377530 antibody.

8. The diagnostic product according to claim 7, characterized in that The sc-12767 antibody is labeled with phycoerythrin; the sc-377530 antibody is labeled with phycocyanin.

9. A method for detecting protein hybrid aggregates, characterized in that: The protein hybrid aggregate comprises a first protein and a second protein; The detection method comprises the following steps: Adding the second antibody to the biological sample to be detected, performing a first incubation, then adding the first antibody, performing a second incubation, and then performing microscopic imaging, reading the number of overlapping fluorescent spots on two fluorescent channels through an image recognition system, substituting the number of fluorescent spots into the standard curves of the first protein and the second protein, respectively, to calculate the content of the first protein and the second protein in the biological sample to be detected; The standard curve of the first protein is constructed by using different concentrations of the first protein; The standard curve of the second protein is constructed by using different concentrations of the second protein; The first antibody is an antibody that specifically recognizes the first protein; The second antibody is an antibody that specifically recognizes the second protein; The first antibody and the second antibody are labeled with different fluorescent markers.

10. The detection method according to claim 9, characterized in that: The first antibody and the second antibody are labeled with phycoerythrin and phycocyanin, respectively.

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