WO2023185871A1 - α-突触核蛋白在辅助诊断神经退行性疾病中的用途 - Google Patents

α-突触核蛋白在辅助诊断神经退行性疾病中的用途 Download PDF

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WO2023185871A1
WO2023185871A1 PCT/CN2023/084456 CN2023084456W WO2023185871A1 WO 2023185871 A1 WO2023185871 A1 WO 2023185871A1 CN 2023084456 W CN2023084456 W CN 2023084456W WO 2023185871 A1 WO2023185871 A1 WO 2023185871A1
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syn
protein
antibodies
oral mucosal
disease
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French (fr)
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冯涛
郑元初
于震维
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首都医科大学附属北京天坛医院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease

Definitions

  • the present disclosure relates to the field of biomedical technology, and specifically to the use of a product for detecting ⁇ -Syn protein in oral mucosa in preparing reagents or kits for assisting in the diagnosis of neurodegenerative diseases.
  • ⁇ -Synuclein is a small protein encoded by the 4q21-22SNCA gene. It has a molecular weight of 19kDa and consists of 140 amino acids. There is a dynamic balance between normal, misfolding and oligomerization of ⁇ -synuclein. When this balance is broken, the fibrils rapidly aggregate into macromolecules and insoluble thin fibers; It will show many forms under influencing factors, including stretched state, pre-dissolved globular form, ⁇ -helical state (membrane-bound), ⁇ -sheet state, dimer state, oligomer state, and insoluble amorphous state.
  • Parkinson’s disease is a common degenerative disease of the central nervous system. PD diagnosis is mainly based on medical history and physical examination, and the accuracy of diagnosis in the early stage of the disease is limited.
  • the neuropathological characteristics of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta and abnormal deposition of ⁇ -synuclein ( ⁇ -Syn).
  • ⁇ -Syn ⁇ -synuclein
  • Studies have shown that abnormal ⁇ -Syn deposition not only exists in the central nervous system, but also occurs in body fluids and peripheral tissues, including blood, olfactory mucosa, skin, salivary glands, retina, adrenal medulla, heart, gastrointestinal tract and cerebrospinal fluid (Eusebi, Paolo, et al.
  • the purpose of the present disclosure is to provide a biological specimen that can be collected non-invasively and has a short update cycle for diagnosing neurodegenerative diseases.
  • One aspect of the present disclosure provides the use of a product for detecting oral mucosal proteins in the preparation of reagents or kits for assisting in the diagnosis of neurodegenerative diseases.
  • the collection process of oral mucosa is non-invasive and easily accepted by patients and normal people.
  • oral mucosal epithelial cells are renewed every 7-21 days, making it easy to repeatedly sample within a short period of time.
  • the auxiliary diagnosis includes assisting other neurodegenerative disease diagnostic techniques commonly used in this field.
  • Other neurodegenerative disease diagnostic techniques include medical history, clinical symptoms, physical and physical signs diagnosis, etc.
  • the neurodegenerative disease includes cerebral ischemia (CI), brain injury (BI), epilepsy; the brain injury includes Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease, etc.
  • CI cerebral ischemia
  • BI brain injury
  • epilepsy the brain injury includes Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease, etc.
  • the oral mucosal protein is ⁇ -Syn protein
  • the neurodegenerative disease is Parkinson's disease
  • the product for detecting ⁇ -Syn protein includes a reagent for detecting the ⁇ -Syn protein.
  • the product for detecting ⁇ -Syn protein includes reagents for detecting ⁇ -Syn oligomer protein and phosphorylated ⁇ -Syn protein.
  • the phosphorylated ⁇ -Syn protein is phosphorylated ⁇ -Syn protein at position 129 (pS129).
  • the product for detecting oral mucosal proteins includes ⁇ -Syn protein antibodies.
  • the product for detecting oral mucosal proteins is selected from one or more of ⁇ -Syn protein antibodies, ⁇ -Syn protein oligomer antibodies, or anti-phosphorylated ⁇ -Syn protein antibodies.
  • the detection is based on immunofluorescence analysis.
  • the intracellular expression of oral mucosal proteins can be visually displayed through immunofluorescence staining.
  • the oral mucosal protein is ⁇ -Syn protein.
  • the detection is based on electrochemiluminescence immunoassay, which allows the ⁇ -Syn antibody to bind to the analyte in the electron-rich well, and then generates an electrochemiluminescence signal.
  • the detection range can be accurate to 5-10 pg/ ml.
  • the product for detecting oral mucosal proteins includes capture antibodies and observation antibodies, wherein the capture antibodies include antibodies selected from ⁇ -Syn protein antibodies, ⁇ -Syn protein oligomer antibodies, or anti-phosphorylated ⁇ - One or more Syn protein antibodies, the observation antibody is a Sulfo-TAG labeled ⁇ -Syn antibody.
  • a reagent or kit is provided to assist in the diagnosis of Parkinson's disease, the reagent or kit comprising a reagent for detecting oral mucosal proteins.
  • the oral mucosal protein is ⁇ -Syn protein.
  • the reagent for detecting ⁇ -Syn protein includes one or more of ⁇ -Syn antibody, ⁇ -Syn oligomer antibody and phosphorylated ⁇ -Syn antibody. .
  • the reagent for detecting ⁇ -Syn protein includes recombinant anti- ⁇ -Syn MJFR-1 (ab138501, Abcam, Cambridge, MA, USA), recombinant anti- ⁇ -Syn oligo Polymeric MJFR-14 (ab209538, Abcam, Cambridge, MA, USA) and anti-phosphorylated ⁇ -Syn (pS129,825701, BioLegend, San Diego, CA, USA) antibodies.
  • the kit further includes a Parkinson's Disease Rating Scale.
  • the Parkinson's disease rating scale is selected from one or more of the H-Y staging scale, the Mini-Mental State Examination (MMSE), and the Montreal Cognitive Assessment (MOCA) scale. .
  • the reagent for detecting ⁇ -Syn protein includes ⁇ -Syn antibodies and/or ⁇ -Syn oligomer antibodies, and the Parkinson's disease scoring scale is an H-Y staging scale.
  • a method for diagnosing Parkinson's disease which includes:
  • the standard concentration is the concentration of ⁇ -Syn protein in the oral mucosa of healthy people.
  • the ⁇ -Syn protein includes one or more of ⁇ -Syn protein, ⁇ -Syn protein oligomers, and phosphorylated ⁇ -Syn protein.
  • the method for detecting ⁇ -Syn protein concentration in the oral mucosa of a subject is an electrochemiluminescence immunoassay method.
  • the inventor uses a non-invasive and convenient oral mucosal exfoliated cell technology to non-invasively collect oral mucosal cells. Then, the expression of ⁇ -SYN in oral mucosal cells was qualitatively and quantitatively detected through immunofluorescence staining and electrochemiluminescence immunoassay technology. A new PD diagnosis technology based on the detection of pathological ⁇ -SYN in oral mucosal cells is provided.
  • Figure 1 shows ⁇ -Syn immunoreaction signals in oral mucosal cells of PD patients and controls.
  • Figure 2 shows the subcellular distribution of ⁇ -Syn in oral mucosal cells of PD patients.
  • Figure 3 shows the results of quantitative analysis of ⁇ -Syn in oral mucosa cells.
  • Figure 4 shows the results of ROC analysis of oral mucosal cells.
  • Figure 5 shows the standard curve of the electrochemiluminescence immunoassay.
  • oligomer refers to a short polymer composed of a small number of monomers connected repeatedly by covalent bonds, including short polymers of amino acids, sugars, and nucleotides. The number of monomers Below 20.
  • phosphorylated ⁇ -Syn refers to ⁇ -Syn protein that is phosphorylated at serine residue 129.
  • immunofluorescence analysis refers to labeling an antibody (or antigen) with a fluorescent dye that does not affect the activity of the antigen and antibody. After binding to its corresponding antigen (or antibody), it will display specific fluorescence under a fluorescence microscope for detection and analysis. .
  • electrochemical immunoassay refers to an immunoassay technology that uses ruthenium terpyridine to label antigens or antibodies and uses the principle of chemiluminescence generated by electrochemistry.
  • the basic principle is that tripropylamine and ruthenium terpyridine in the immune reaction complex lose electrons around the electrode to form trivalent free radicals.
  • the excited ruthenium terpyridine is deexcited, it emits photons with a wavelength of 620nm, and generates them repeatedly on the electrode surface. More photons are generated, and measuring these photons enhances the signal of the detected object.
  • capture antibody refers to the specific binding to the target protein to be detected in electrochemiluminescence immunoassay, immobilizing it and forming the molecule to be detected.
  • observation antibody refers to an antibody labeled with Sulfo-TAG ( Why Electrochemiluminescence Meso Scale Discovery ), which can form a complex with the capture antibody and the antigen to be tested, and is used to display the concentration of the antigen to be tested.
  • patient and “subject” are used interchangeably and refer to human or other mammalian patients and subjects, and include any individual who is examined or treated using the methods of the present disclosure. However, it should be understood that “patient” does not mean the presence of symptoms.
  • kit refers to any delivery system used to deliver a substance.
  • delivery systems include storing, transporting, or transporting reactive reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and/or support materials (e.g., buffers, instructions for performing the assay, etc.) from a location.
  • support materials e.g., buffers, instructions for performing the assay, etc.
  • a kit contains one or more enclosures (eg, boxes) containing relevant reaction reagents and/or support materials.
  • Exclusion criteria for the PD group included those diagnosed with atypical or secondary parkinsonism, severe head trauma, history of stroke, severe mental illness, severe systemic disease, and oral mucosal disease.
  • Exclusion criteria for the control group included those diagnosed with PD or other movement disorder diseases, family history of movement disorder diseases, severe head trauma, history of stroke, severe mental illness, severe systemic disease and oral mucosal disease.
  • Demographic and clinical data were collected from all participants, including age, gender, education, Mini-Mental State Examination (MMSE), and Montreal Cognitive Assessment (MOCA).
  • MMSE Mini-Mental State Examination
  • MOCA Montreal Cognitive Assessment
  • MDS-UPDRS III Movement Disorder Society Unified Parkinson's Disease Rating Scale Part III
  • a histocyte is brushed onto a silane-coated microscope slide and a thin smear is made for immunofluorescence staining.
  • Slides were stored at -20°C.
  • Another cell brush was immersed in a 1.5 ml centrifuge tube containing 200 ⁇ l RIPA buffer (Applygen, cat. no. C1053+) for electrochemiluminescence (ECL) immunoassay. Vortex and mix for 1 minute, then discard the cell brush.
  • ECL electrochemiluminescence
  • Use an ultrasonic cell disrupter to sonicate the remaining lysate in the centrifuge tube for 1 minute, and place the centrifuge tube on ice during the process.
  • Bicinchoninic acid (BCA) protein analysis kit (Pierce/Thermo Fisher Scientific, Rockford, IL, USA) was used as a protein standard, and the protein concentration in oral mucosal samples was evaluated at absorbance at 562 nm.
  • the slides were fixed in 4% paraformaldehyde for 10 minutes and then washed three times with phosphate buffer solution (PBS). After washing, slides were permeabilized in 1% Triton X-100 for 10 minutes. After washing three times, the slides were blocked in 5% bovine serum albumin (BSA) blocking solution for 1 h.
  • PBS phosphate buffer solution
  • Recombinant anti- ⁇ -Syn MJFR-1 (ab138501, Abcam, Cambridge, MA, USA), recombinant anti- ⁇ -Syn oligomer MJFR-14 (ab209538, Abcam, Cambridge, MA, USA) and anti-phosphorylated ⁇ -Syn (pS129, 825701, BioLegend, San Diego, CA, USA) antibody was diluted 1:1000 in blocking solution and the slides were incubated overnight at 4°C.
  • Use PBST buffer PBS+0.05% 20
  • goat anti-mouse Alexa Fluor 488 (1:1000 dilution
  • goat anti-rabbit Alexa Fluor 488 (1:1000 dilution
  • Standard proteins recombinant ⁇ -Syn monomer (RP-001, Proteos, Inc.), recombinant phosphorylated ⁇ -Syn (RP-004, Proteos, Inc.) and ⁇ -Syn oligomer (RP-002, Proteos ,Inc.).
  • the standard protein concentration was detected using a NanoDrop OneC spectrophotometer, diluted to 1 ⁇ g/ml in Diluent 35 (D35, MSD, Rockville, MD, USA), and then 1:3 gradient dilution was used to prepare a standard curve.
  • biotinylated recombinant anti- ⁇ -Syn-MJFR-1 (ab138501, Abcam, Cambridge, MA, USA)
  • biotinylated recombinant anti- ⁇ -Syn oligomer MJFR-14 (ab209538, Abcam, Cambridge, MA, USA) MA, USA
  • biotinylated anti-pS129 antibody (825701, BioLegend, San Diego, CA, USA).
  • Statistical analysis was performed using SPSS 22.0 software and GraphPad Prism 8. Prior to analysis, the concentrations of ⁇ -Syn, pS129, and ⁇ -Syn oligomers were normalized based on the total protein concentration of oral mucosal cell protein extracts. The non-parametric Mann-Whitney U test was used to compare ⁇ -Syn levels between PD and healthy control groups, and the Spearman rank correlation coefficient was used to analyze the correlation between ⁇ -Syn levels and disease severity. Binary logistic regression was used to establish a multivariable logistic regression model including ⁇ -Syn, pS129 and ⁇ -Syn oligomers in oral mucosal cells for PD diagnosis. The area under the receiver operating characteristic curve (ROC) curve (AUC) was used, and the Youden index (sensitivity + specificity -1) was calculated to obtain the optimal cutoff value for PD and control groups.
  • ROC receiver operating characteristic curve
  • Table 1 presents the demographic and clinical data of all subjects. There was no difference in the average gender, age, MMSE scale, and MOCA scale distribution between PD patients and controls.
  • the median disease duration of PD patients is 5 years (range, 7 months to 16 years), the median off-stage H-Y score is stage 3 (range, 1-5 stages), and the median off-stage MDS-UPDRS III score is 41.5 points (range 5-74 points).
  • Immunofluorescence staining results showed that immunoreactive signals of ⁇ -Syn, pS129, and ⁇ -Syn oligomers (Figure 1) could be detected in oral mucosal cells of PD patients and controls. Compared with controls, the immune response signals of ⁇ -Syn, pS129, and ⁇ -Syn oligomers in oral mucosal cells of PD patients were significantly increased.
  • ⁇ -Syn was mainly detected in the nucleus and cytoplasm of oral mucosal cells in PD patients ( Figure 2A)
  • pS129 was mainly distributed in the cytoplasm of oral mucosal cells ( Figure 2B)
  • ⁇ -Syn oligomers were found in the nucleus of oral mucosal cells. with higher expression levels in perinuclear cytoplasm (Fig. 2C).
  • Fig. 2C Three-dimensional reconstruction of Z-stack confocal images shows that ⁇ -Syn, pS129 and ⁇ -Syn oligomers have different subcellular distribution characteristics in oral mucosal cells.
  • ⁇ -Syn was mainly detected in the nucleus and cytoplasm of oral mucosal cells in PD patients ( Figure 2A)
  • pS129 was mainly distributed in the cytoplasm of oral mucosal cells
  • ⁇ -Syn oligomers were found in the nucleus of oral mucosal cells.
  • ⁇ -Syn, pS129, and ⁇ -Syn oligomer concentrations were calculated based on oral Mucosal samples were normalized for total protein concentration.
  • ROC analysis was used to analyze the efficacy of ⁇ -Syn, pS129 and ⁇ -Syn oligomers in oral mucosal cells in the diagnosis of PD.
  • the sensitivity of using ⁇ -Syn in oral mucosal cells to diagnose PD is 60.8%, and the specificity is 77.6%.
  • AUC Absolute Under Curve, ROC curve and coordinate axis area
  • the Cut off value is 25.09pg/mg.
  • the AUC of pS129 in distinguishing PD from the control group was 0.674, the sensitivity was 45.3%, the specificity was 88.0%, and the cut off value was 99.98pg/mg.
  • the AUC of ⁇ -Syn oligomer to distinguish PD from the control group was 0.641, the sensitivity was 74.1%, the specificity was 50.0%, and the cut off value was 35.94pg/mg.

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Abstract

本公开提供一种检测口腔黏膜蛋白的产品在制备用于辅助诊断神经退行性疾病的试剂或试剂盒中的用途，口腔黏膜可无创收集、更新周期短，易于在短时间内重复取样。

Description

α-突触核蛋白在辅助诊断神经退行性疾病中的用途 技术领域
本公开涉及生物医学技术领域，具体涉及检测口腔黏膜α-Syn蛋白的产品在制备用于辅助诊断神经退行性疾病的试剂或试剂盒中的用途。
背景技术
α-突触核蛋白是位于4q21-22SNCA基因编码的一个小分子蛋白质，分子量为19kDa，由140个氨基酸构成。α-突触核蛋白正常、错误折叠及其寡聚化之间存在动态平衡，当这种平衡被打破后原纤维迅速聚集成大分子、不溶性的细纤维；α-突触核蛋白在不同的影响因素下会表现出许多种形态，包括舒展态、溶解前球型态、α-螺旋态(膜结合)，β-片层态、二聚体态、寡聚体态、以及不可溶的无定型态和纤维态；α-突触核蛋白的点突变导致的结构改变、胞内含量的增加、大量蛋白分子的堆积、结构序列的截短、胞内阴离子及盐类的浓度(PH值的改变)、神经毒性分子(重金属，有机溶剂，一氧化碳，MPTP，杀虫剂和除草剂)、翻译后的修饰(氧化，磷酸化和硝基化)等等都可以促进α-突触核蛋白聚集形成难溶纤维。
帕金森病(Parkinson’s disease,PD)是常见的中枢神经系统退行性疾病，PD诊断主要依据病史和体格检查，在疾病早期诊断准确率有限。PD的神经病理学特征为黑质致密部多巴胺能神经元的丢失以及α-突触核蛋白(α-Syn)异常沉积。研究表明，α-Syn异常沉积不仅存在于中枢神经系统，还出现于体液和外周组织中，包括血液、嗅黏膜、皮肤、唾液腺、视网膜、肾上腺髓质、心脏、胃肠道和脑脊液(Eusebi,Paolo,et al.Diagnostic utility of cerebrospinal fluidα-synuclein in Parkinson's disease:A systematic review and meta-analysis.Mov Disord 32(10),1389-1400.doi:10.1002/mds.27110.)。通过体液或外周组织α-Syn检测可实现帕金森病病理诊断。但是目前体液及外周组织采样多为有创性操作，且采样难度大、患者依从性不佳。目前亟需一种无创性、采样便捷的检测手段。
发明内容
为了解决现有技术的不足，本公开的目的在于提供一种可无创收集、更新周期短的诊断神经退行性疾病的生物检体。
具体来说，本公开提出了如下技术方案：
本公开的一方面，提供一种检测口腔黏膜蛋白的产品在制备用于辅助诊断神经退行性疾病的试剂或试剂盒中的用途。口腔黏膜的收集过程是无创的，患者和正常人都很容易接受。此外，口腔黏膜上皮细胞每7-21天更新一次，易于在短时间内重复取样。
所述辅助诊断包括辅助其他本领域常用神经退行性疾病诊断技术，其他神经退行性疾病诊断技术包括病史、临床症状、体格和体征诊断等。
在一实施方式中，所述神经退行性疾病包括脑缺血(CI)、脑损伤(BI)、癫痫；所述脑损伤包括阿尔茨海默病(AD)、帕金森病(PD)、亨廷顿病(HD)、肌萎缩性侧索硬化(ALS)、不同类型脊髓小脑共济失调(SCA)、Pick病等。
在一实施方式中，所述口腔黏膜蛋白为α-Syn蛋白，所述神经退行性疾病为帕金森病。
在一实施方式中，所述检测α-Syn蛋白的产品包括检测所述α-Syn蛋白的试剂。
在一实施方式中，所述检测α-Syn蛋白的产品包括检测α-Syn寡聚体蛋白、磷酸化α-Syn蛋白的试剂。
在一优选的实施方式中，所述磷酸化α-Syn蛋白为第129位磷酸化α-Syn蛋白(pS129)。
在一实施方式中，所述检测口腔黏膜蛋白的产品包括α-Syn蛋白抗体。
在一实施方式中，所述检测口腔黏膜蛋白的产品选自α-Syn蛋白抗体、α-Syn蛋白寡聚体抗体或抗磷酸化α-Syn蛋白抗体中的一种或多种。
在一实施方式中，所述检测是基于免疫荧光分析。可以通过免疫荧光染色直观的显示口腔黏膜蛋白的细胞内表达情况。
在一实施方式中，所述口腔黏膜蛋白为α-Syn蛋白。
在一实施方式中，所述检测是基于电化学发光免疫分析，可使得α-Syn抗体与分析物在电子富集孔内结合，随后产生电化学发光信号，检测范围可精确至5-10pg/ml。
在一实施方式中，所述检测口腔黏膜蛋白的产品包括捕获抗体和观察抗体，其中，所述捕获抗体包括选自α-Syn蛋白抗体、α-Syn蛋白寡聚体抗体或抗磷酸化α-Syn蛋白抗体中的一种或多种，所述观察抗体为Sulfo-TAG标记的α-Syn抗体。
本公开的再一方面，提供一种辅助诊断帕金森病的试剂或试剂盒，所述试剂或试剂盒包含检测口腔黏膜蛋白的试剂。在一实施方式中，所述口腔黏膜蛋白为α-Syn蛋白。
在一实施方式中，在上述试剂或试剂盒中，所述检测α-Syn蛋白的试剂包括α-Syn抗体、α-Syn寡聚体抗体以及磷酸化α-Syn抗体中的一种或多种。
在一实施方式中，所述试剂或试剂盒中，所述检测α-Syn蛋白的试剂包括重组抗α-Syn MJFR-1(ab138501,Abcam,Cambridge,MA,USA)、重组抗α-Syn寡聚体MJFR-14(ab209538,Abcam,Cambridge,MA,USA)和抗磷酸化α-Syn(pS129,825701,BioLegend,San Diego,CA,USA)抗体。
在一实施方式中，所述试剂盒还包含帕金森病评分量表。
在一实施方式中，所述帕金森病评分量表选自H-Y分期量表、简易智力状态检查量表(MMSE)、蒙特利尔认知评估量表(MOCA)量表中的一种或两种以上。
在一实施方式中，所述检测α-Syn蛋白的试剂包括α-Syn抗体和/或α-Syn寡聚体抗体，所述帕金森病评分量表为H-Y分期量表。
本公开的再一方面，提供一种诊断帕金森病的方法，其包括：
测定受试者口腔黏膜中的α-Syn蛋白浓度；
将测得的受试者口腔黏膜中的α-Syn蛋白浓度与标准浓度进行比较；
判断受试者患有帕金森症的风险。
在一实施方式中，所述标准浓度为健康人群的口腔黏膜α-Syn蛋白浓度。
在一实施方式中，所述α-Syn蛋白包括α-Syn蛋白、α-Syn蛋白寡聚体、磷酸化α-Syn蛋白中的一种或多种。
在一实施方式中，所述检测受试者口腔黏膜中的α-Syn蛋白浓度的方法为电化学发光免疫分析方法。
本发明人采用一种无创、便捷的口腔黏膜脱落细胞技术对口腔黏膜细胞进行无创采集。再通过免疫荧光染色和电化学发光免疫测定技术对口腔黏膜细胞中α-SYN表达情况进行定性及定量检测。提供了一种基于口腔黏膜细胞病理性α-SYN检测的PD诊断新技术。
附图说明
图1示出了PD患者和对照组的口腔黏膜细胞中的α-Syn免疫反应信号。
图2示出了α-Syn在PD患者口腔黏膜细胞内的亚细胞分布。
图3示出了口腔黏膜细胞内α-Syn定量分析结果。
图4示出了口腔黏膜细胞ROC分析结果。
图5示出了电化学发光免疫分析法的标准曲线。
具体实施方式
为了使本技术领域的人员更好地理解本公开方案，下面将结合本公开附图，对本公开实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本公开一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
除非另外定义，否则本文所用的所有科学技术术语的含义与本公开所属领域的一般技术人员通常所了解的含义相同。虽然与本文中所述的方法和材料类似或等同的任何方法和材料都可以用于本公开实践或测试中，但是描述了优选方法和材料。为了本公开的目的，以下术 语定义如下。
术语“寡聚体”指的是由数量较少的单体以共价键重复的连接而成的短多聚体，包括氨基酸、糖、核苷酸的短多聚体，其单体的数目在20以下。
术语“磷酸化α-Syn”指的是在129位丝氨酸残基磷酸化修饰的α-Syn蛋白。
术语“免疫荧光分析”指的是将不影响抗原抗体活性的荧光色素标记在抗体(或抗原)上，与其相应的抗原(或抗体)结合后，在荧光显微镜下呈现特异性荧光用于检测分析。
术语“电化学发光免疫分析”指的是用三联吡啶钌标记抗原或抗体，利用电化学产生的化学发光原理进行的免疫分析技术。基本原理是免疫反应复合物中的三丙胺和三联吡啶钌在电极周围失去电子，形成三价自由基，激发态的三联吡啶钌退激时发射波长620nm的光子，并在电极表面反复始进行产生更多光子，测定这些光子使被检测物的信号得以加强。
术语“捕获抗体”指的是在电化学发光免疫分析中与待测目标蛋白特异性结合，使之固定并形成待测分子。
术语“观察抗体”指的是带有Sulfo-TAG标记(Why Electrochemiluminescence Meso Scale  Discovery)，可与捕获抗体及待测抗原形成复合物结合的抗体，用于显示待测抗原浓度。
术语“患者”和“受试者”可互换使用，是指人类或其他哺乳动物的患者和受试者，并且包括使用本公开的方法进行检查或治疗的任何个体。但是，应当理解，“患者”并不意味着存在症状。
术语“试剂盒”是指用来递送物质的任何递送系统。在反应测定中，这类递送系统包括将反应试剂(例如适当容器中的寡核苷酸、酶等)和/或支持材料(例如缓冲液、进行测定的说明书等)贮存、从一个位置转运或递送到另一个位置的系统。例如，试剂盒包含一个或多个外壳(例如盒子)，其中包含有关的反应试剂和/或支持材料。
实施例
下面，参考具体实施例更详细地描述本公开，然而，实施例仅用于说明目的，对于本公开不具有限制作用。下述实施例中所述试剂和生物材料，如无特殊说明，均可从商业途径获得。
实施例1
1.材料和方法
1.1研究对象
2021年3月至2021年10月期间，从首都医科大学附属北京天坛医院运动障碍性疾病科招募57例原发性PD患者和51例年龄匹配的健康对照。所有PD患者均符合国际运动障碍协会2015年版PD临床诊断标准。
PD组的排除标准包括诊断为非典型或继发性帕金森综合征、严重头部创伤、卒中史、严重精神疾病、严重系统性疾病和口腔黏膜疾病者。对照组的排除标准包括诊断为PD或其他运动障碍性疾病、有运动障碍性疾病家族史、严重头部创伤、卒中史、严重精神疾病、严重系统性疾病和口腔黏膜疾病者。
收集所有参与者的人口学和临床数据，包括年龄、性别、教育程度、简易精神状态检查量表(MMSE)和蒙特利尔认知评估量表(MOCA)。对于PD患者，使用病程、关期H-Y分期量表和运动障碍协会统一帕金森病评定量表第三部分(MDS-UPDRSⅢ)来评估疾病的严重程度。横断面研究的方案由首都医科大学附属北京天坛医院伦理委员会审查和批准，并获得了所有参与者书面知情同意。
1.2口腔黏膜细胞样本采集和制备
参与者在采样前用生理盐水充分漱口，以避免口腔食物残留和唾液污染。漱口后立即使用两个刷头长2厘米的组织细胞刷分别从两侧颊部采集口腔黏膜细胞样本。旋转刮拭一侧口腔颊部内侧壁30次。
完成采样后，将一个组织细胞刷在硅烷涂层的显微镜玻片上进行薄涂片，用于免疫荧光染色。载玻片在零下20℃下保存。另一个细胞刷浸入装有200μl RIPA缓冲液(Applygen,cat.no.C1053+)的1.5ml的离心管中，用于电化学发光(ECL)免疫分析。Vortex漩涡混合器振荡混匀1分钟后，弃掉细胞刷。使用超声波细胞破碎仪将离心管内剩余裂解液超声1min，过程中离心管置于冰上。超声结束后12000×g离心力4℃下离心10分钟。将含有上清液的口腔黏膜细胞提取蛋白转移到新的离心管中，然后在-80℃下储存，进行分析时再解冻样本。
使用二喹啉甲酸(BCA)蛋白质分析试剂盒(Pierce/Thermo Fisher Scientific,Rockford,IL,USA)作为蛋白质标准品，562nm吸光度下评估口腔黏膜样品中的蛋白质浓度。
1.3免疫荧光染色分析与口腔黏膜细胞三维重建
将载玻片放在4％多聚甲醛中固定10分钟，然后用磷酸盐缓冲溶液(PBS)洗3次。洗涤后，将载玻片在1％的Triton X-100中通透10分钟。洗涤3次后，将载玻片在5％牛血清白蛋白(BSA)封闭溶液中封闭1h。将重组抗α-Syn MJFR-1(ab138501,Abcam,Cambridge,MA,USA)、重组抗α-Syn寡聚体MJFR-14(ab209538,Abcam,Cambridge,MA,USA)和抗磷酸化α-Syn(pS129,825701,BioLegend,San Diego,CA,USA)抗体用封闭溶液进行1:1000的稀释，并在4℃下孵育载玻片过夜。用PBST缓冲液(PBS+0.05％20)洗涤3次后，使用山羊抗鼠Alexa Fluor 488(1:1000稀释)和山羊抗兔Alexa Fluor 488(1:1000稀释)在室温下孵育1h。细胞核用DAPI(0.2μg/mL)染色5分钟。用罗丹明标记鬼笔环肽(1:200稀释)显示口 腔黏膜细胞的细胞骨架范围。荧光淬灭封片剂封片后，使用蔡司LSM 700共焦显微镜的40×目镜观察玻片。通过Z-stacks图像模式以40×的放大倍数进行口腔黏膜细胞三维重建，用于可视化α-Syn的细胞内定位。
1.4电化学发光免疫分析法
标准蛋白：重组α-Syn单体(RP-001,Proteos,Inc.)、重组磷酸化的α-Syn(RP-004,Proteos,Inc.)和α-Syn寡聚体(RP-002,Proteos,Inc.)。标准蛋白使用NanoDrop OneC分光光度计进行浓度检测，在Diluent 35(D35,MSD,Rockville,MD,USA)中稀释至1μg/ml，然后1:3梯度稀释制备标准曲线。
观察抗体：Sulfo-TAG标记的抗α-Syn抗体(624096,BD Bioscience,San Jose,CA,USA)。
捕获抗体：生物素化的重组抗α-Syn-MJFR-1(ab138501,Abcam,Cambridge,MA,USA)、生物素化的重组抗α-Syn寡聚体MJFR-14(ab209538,Abcam,Cambridge,MA,USA)、生物素化的抗pS129抗体(825701,BioLegend,San Diego,CA,USA)。
在室温下，将三种捕获抗体溶液分别加在Meso Scale Discovery(MSD,Rockville,MD,USA)U-Plex平板上，以600rpm的转速振荡孵育1小时，使捕获抗体与电化学发光孔连接。用150μl洗涤缓冲液(MSD,Rockville,MD,USA)洗涤3次后，在室温下用150μl的Diluent 35(D35,MSD,Rockville,MD,USA)以600rpm转速震荡封闭1小时，然后再洗涤3次。将含有口腔黏膜细胞蛋白提取物样品在D35中以1:2.5比例稀释，并在600rpm转速下孵育1小时。洗涤3次后，加入D35稀释后的观察抗体溶液(1μg/ml)并在600rpm转速下孵育1小时。孵育结束后，用洗涤缓冲液冲洗3次，然后加入150μl的2×读取缓冲液(MSD,Rockville,MD,USA)，立即在Sector Imager 6000检测平台读板分别分析α-Syn、α-Syn寡聚体、抗磷酸化α-Syn的检测结果。
1.5统计分析
使用SPSS 22.0软件和GraphPad Prism 8进行统计分析。在分析之前，将α-Syn、pS129和α-Syn寡聚体的浓度基于口腔黏膜细胞蛋白提取液总蛋白浓度进行标准化。非参数Mann-Whitney U检验用于比较PD及健康对照组组间α-Syn水平，斯皮尔曼等级相关系数用于分析α-Syn水平与疾病严重程度之间的相关性。采用二元logistic回归用于建立包含口腔黏膜细胞α-Syn、pS129和α-Syn寡聚体的多变量logistic回归模型，用于PD诊断。使用受试者工作特征曲线(ROC)曲线下的面积(AUC)，并计算约登指数(灵敏度+特异度-1)得出PD和对照组的最佳临界值。
2.研究结果
2.1人口学及临床特征
本研究共纳入了57名PD患者和51名年龄匹配的健康(HC)对照。表1为所有受试者的人口学和临床数据。PD患者和对照组的平均性别、年龄、MMSE量表、MOCA量表分布无差异。PD患者的病程中位数为5年(范围为7个月-16年)，关期H-Y评分中位数为3期(范围为1-5期)，关期MDS-UPDRSⅢ评分中位数为41.5分(范围为5-74分)。
表1人口学及临床数据
2.2 PD及健康对照组口腔黏膜细胞中α-Syn表达情况
免疫荧光染色结果显示，PD患者和对照组的口腔黏膜细胞中均可检测到α-Syn、pS129和α-Syn寡聚体(图1)的免疫反应信号。与对照组相比，PD患者口腔黏膜细胞中的α-Syn、pS129和α-Syn寡聚体的免疫反应信号显著增加。
Z-stack共聚焦图像的三维重建显示，α-Syn、pS129和α-Syn寡聚体在口腔黏膜细胞内具有不同的亚细胞分布特点。α-Syn主要在PD患者口腔黏膜细胞的细胞核和细胞质中检测到(图2A)，pS129主要分布于口腔黏膜细胞的细胞质中(图2B)，而α-Syn寡聚体在口腔黏膜细胞的细胞核与核周细胞质中表达水平较高(图2C)。但在PD患者和对照组之间，α-Syn在细胞内的分布情况没有差异。
PD患者和对照组的口腔黏膜细胞中α-Syn、pS129和α-Syn寡聚体存在差异性表达。免疫荧光结果显示PD患者口腔黏膜细胞中α-Syn、pS129和α-Syn寡聚体表达水平高于对照组，PD患者口腔黏膜细胞中存在异常的α-Syn沉积。与对照组相比，PD患者口腔黏膜样本中的α-Syn、pS129和α-Syn寡聚体的水平显著升高。这一差异性表达特点表明口腔黏膜细胞中α-Syn可以作为PD的生物标记物。
2.3口腔黏膜细胞内α-Syn定量分析
在进行口腔黏膜细胞α-Syn定量分析前，将α-Syn、pS129和α-Syn寡聚体浓度基于口腔 黏膜样品中总蛋白浓度进行标准化。
与HC对照组相比，PD患者口腔黏膜细胞中的α-Syn、pS129和α-Syn寡聚体水平均显著升高(表2，图3)。
在PD组中，口腔黏膜细胞中的α-Syn和α-Syn寡聚体水平均与关期H-Y评分显著相关(α-Syn r＝0.495，p＝0.001；α-Syn寡聚体r＝0.324，p＝0.03)。然而，口腔黏膜细胞pS129水平与关期H-Y评分无显著相关性。此外，口腔黏膜细胞α-Syn、pS129和α-Syn寡聚体的水平与病程和关期MDS-UPDRSⅢ评分等疾病严重程度指标无显著相关性。
表2口腔黏膜细胞内α-Syn定量分析结果
以重组α-Syn单体为标准蛋白，按1.4电化学发光免疫分析法所述步骤进行检测得到如图5所示的标准曲线，从图5可得，本公开基于电化学发光免疫分析检测α-Syn蛋白时，其检测范围可精确至5-10pg/ml。
2.4 ROC曲线分析
采用ROC分析对口腔黏膜细胞中α-Syn、pS129和α-Syn寡聚体在PD诊断中的效力进行分析。如图4所示，电化学发光免疫分析法中，使用口腔黏膜细胞α-Syn诊断PD的敏感性为60.8％，特异性为77.6％，AUC(Area Under Curve,ROC曲线下与坐标轴围成的面积)为0.684，Cut off值为25.09pg/mg。pS129区别PD与对照组的AUC为0.674，敏感性为45.3％，特异性为88.0％，Cut off值为99.98pg/mg。α-Syn寡聚体区别PD与对照组的AUC为0.641，敏感性为74.1％，特异性为50.0％，Cut off值为35.94pg/mg。
基于口腔黏膜样本中α-Syn、pS129和α-Syn寡聚体的定量结果，采用二元logistic回归建立了多变量PD诊断模型口腔黏膜样本中α-Syn、pS129和α-Syn寡聚体水平的多变量logistic回归PD诊断模型。该诊断模型的AUC为0.749，敏感性为66.7％，特异性为72.5％，总体敏感性中等(图4)。
表3 ROC分析结果

Claims (12)

  1. 检测口腔黏膜蛋白的产品在制备用于辅助诊断神经退行性疾病的试剂或试剂盒中的用途。
  2. 根据权利要求1所述的用途，所述口腔黏膜蛋白为α-Syn蛋白，所述神经退行性疾病为帕金森病；
    优选地，所述检测α-Syn蛋白的产品包括检测所述α-Syn蛋白的试剂。
  3. 根据权利要求1或2所述的用途，所述检测口腔黏膜蛋白的产品包括α-Syn蛋白抗体。
  4. 根据权利要求1-3任一项所述的用途，所述检测口腔黏膜蛋白的产品选自α-Syn蛋白抗体、α-Syn蛋白寡聚体抗体或抗磷酸化α-Syn蛋白抗体中的一种或多种。
  5. 根据权利要求1-4任一项所述的用途，所述检测是基于免疫荧光分析。
  6. 根据权利要求1-4任一项所述的用途，所述检测是基于电化学发光免疫分析。
  7. 根据权利要求6所述的用途，所述检测口腔黏膜蛋白的产品包括捕获抗体和观察抗体，其中，所述捕获抗体包括选自α-Syn蛋白抗体、α-Syn蛋白寡聚体抗体或抗磷酸化α-Syn蛋白抗体中的一种或多种，所述观察抗体为Sulfo-TAG标记的α-Syn抗体。
  8. 一种辅助诊断帕金森病的试剂或试剂盒，所述试剂或试剂盒包含检测口腔黏膜蛋白的试剂；其中，所述口腔黏膜蛋白为α-Syn蛋白。
  9. 根据权利要求7所述的试剂或试剂盒，其中，所述检测α-Syn蛋白的试剂包括α-Syn抗体、α-Syn寡聚体抗体以及磷酸化α-Syn抗体中的一种或多种。
  10. 根据权利要求8或9所述的试剂或试剂盒，其中，所述检测是基于免疫荧光分析或电化学发光免疫分析的检测。
  11. 根据权利要求8-10任一项所述的试剂盒，其中，所述试剂盒还包含帕金森病评分量表；优选地，所述帕金森病评分量表选自H-Y分期量表、简易智力状态检查量表(MMSE)、蒙特利尔认知评估量表(MOCA)量表中的一种或两种以上。
  12. 一种诊断帕金森病的方法，其包括：
    测定受试者口腔黏膜中的α-Syn蛋白浓度；
    将测得的受试者口腔黏膜中的α-Syn蛋白浓度与标准浓度进行比较；
    判断受试者患有帕金森症的风险；
    优选地，所述标准浓度为健康人群的口腔黏膜α-Syn蛋白浓度。
PCT/CN2023/084456 2022-03-28 2023-03-28 α-突触核蛋白在辅助诊断神经退行性疾病中的用途 WO2023185871A1 (zh)

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