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CN103983778B - Method for detecting in-vitro disease-related protein polymerization promotion capability of body fluid of subject - Google Patents

Method for detecting in-vitro disease-related protein polymerization promotion capability of body fluid of subject
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CN103983778B
CN103983778BCN201410193640.9ACN201410193640ACN103983778BCN 103983778 BCN103983778 BCN 103983778BCN 201410193640 ACN201410193640 ACN 201410193640ACN 103983778 BCN103983778 BCN 103983778B
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synuclein
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于顺
尹娜
杨巍巍
李昕
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Beijing Parcon Technology Co ltd
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Xuanwu Hospital
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Abstract

Translated fromChinese

本发明涉及一种检测α-突触核蛋白在血浆中的寡聚体形成率的方法,该方法包括将纯化的重组人α-突触核蛋白与血浆在一定温度下振荡孵育使其聚合成寡聚体,用ELISA方法测定α-突触核蛋白寡聚体含量,通过寡聚体占总α-突触核蛋白含量的比率计算获得α-突触核蛋白在血浆中的寡聚体形成率。

The invention relates to a method for detecting the oligomer formation rate of α-synuclein in plasma, the method comprising shaking and incubating purified recombinant human α-synuclein and plasma at a certain temperature to polymerize into Oligomers, the content of α-synuclein oligomers was determined by ELISA method, and the oligomer formation of α-synuclein in plasma was obtained by calculating the ratio of oligomers to the total α-synuclein content Rate.

Description

Translated fromChinese
一种检测受实验者体液体外促进疾病相关蛋白聚合能力的方法A method for detecting the ability of a subject to promote the polymerization of disease-related proteins in vitro

技术领域:Technical field:

本发明涉及一种检测α-突触核蛋白在血浆中的寡聚体形成率的方法,该方法可用于帕金森病的诊断。The invention relates to a method for detecting the oligomer formation rate of α-synuclein in plasma, which can be used for the diagnosis of Parkinson's disease.

背景技术:Background technique:

帕金森病是一种常见的神经系统退行性疾病,临床上主要表现为静止振颤、运动迟缓、肌肉僵直和姿势不稳定等运动症状[1,2]。引起这些症状的原因是中脑黑质多巴胺神经元大量死亡和由此引起的纹状体部位的多巴胺神经递质的大幅度减少。帕金森病的主要病理特征是神经细胞内出现嗜酸性包涵体——路易体(Lewybody)和路易神经索(Lewyneurite)。构成路易体和路易神经索的主要成分是纤维化的α-突触核蛋白(α-synuclein)[3]。大量研究表明,α-突触核蛋白是在帕金森病的发病中起关键作用的蛋白质。α-突触核蛋白基因点突变和多倍体变异可以引起家族性早发性帕金森病,其基因启动子多态性与帕金森病的发病风险有关[4-6]。已经证明,帕金森病人脑组织中的α-突触核蛋白存在改变,表现为该蛋白的表达量异常增高,磷酸化、硝基化、泛素化和糖基化的修饰体增加[7-9]。这些异常表达和修饰的α-突触核蛋白是引起该蛋白聚集成寡聚体的原因并进而形成纤维而沉积在路易体中。大量证据表明,异常表达、修饰和聚集的α-突触核蛋白对神经元具有毒性作用,是各种原因导致神经元退变和帕金森病的关键[10-13]。通过对帕金森病人组织的病理检查证明,路易体在病人的组织中广泛分布,除中枢神经系统的广泛区域外,还包括分布于各外周器官的神经组织,如消化道、膀胱、心脏、唾液腺等,并且认为消化道的路易体病变甚至早于中枢神经系统[14]。这些结果提示,帕金森病很可能是机体内环境改变引起的一种全身性代谢性紊乱疾病,血液作为机体内环境的重要组成部分,很可能发生某种改变,从而有利于α-突触核蛋白的聚集。这可能是以聚集的α-突触核蛋白为主要成分的路易体的广泛存在的重要原因。因此,检测血浆促进α-突触核蛋白形成能力或α-突触核蛋白在血浆中的寡聚体形成率有助于了解α-突触核蛋白在帕金森病人神经组织易于聚集的机体内在环境因素的改变。Parkinson's disease is a common degenerative disease of the nervous system, clinically manifested mainly by motor symptoms such as resting tremor, bradykinesia, muscle stiffness, and postural instability[1,2] . The cause of these symptoms is the massive death of dopamine neurons in the substantia nigra of the midbrain and the resulting substantial reduction of dopamine neurotransmitters in the striatum. The main pathological feature of Parkinson's disease is the appearance of eosinophilic inclusion bodies in nerve cells - Lewy bodies and Lewyneurite. The main component of Lewy bodies and Lewy nerve cord is fibrotic α-synuclein (α-synuclein)[3] . Numerous studies have shown that α-synuclein is a protein that plays a key role in the pathogenesis of Parkinson's disease. α-synuclein gene point mutations and polyploid mutations can cause familial early-onset Parkinson's disease, and the gene promoter polymorphisms are associated with the risk of Parkinson's disease[4-6] . It has been proved that there are changes in α-synuclein in the brain tissue of Parkinson's patients, manifested as an abnormal increase in the expression of the protein, and an increase in phosphorylation, nitration, ubiquitination and glycosylation modifications[7- 9] . These aberrantly expressed and modified α-synuclein proteins are responsible for the aggregation of the protein into oligomers, which in turn form fibers deposited in Lewy bodies. A large number of evidences show that abnormally expressed, modified and aggregated α-synuclein has toxic effects on neurons and is the key to various causes of neuron degeneration and Parkinson's disease[10-13] . The pathological examination of Parkinson's patient's tissue proves that Lewy bodies are widely distributed in the patient's tissue, in addition to the wide area of the central nervous system, it also includes nerve tissue distributed in various peripheral organs, such as the digestive tract, bladder, heart, salivary glands etc., and it is believed that Lewy body lesions in the digestive tract even precede the central nervous system[14] . These results suggest that Parkinson's disease is likely to be a systemic metabolic disorder caused by changes in the internal environment of the body. protein aggregation. This may be an important reason for the widespread presence of Lewy bodies with aggregated α-synuclein as the main component. Therefore, detecting the ability of plasma to promote the formation of α-synuclein or the oligomer formation rate of α-synuclein in plasma is helpful to understand the aggregation of α-synuclein in the nervous tissue of Parkinson's patients. Changes in environmental factors.

现有技术中对血液中α-突触核蛋白及其寡聚体的检测主要是对其含量的直接测定。所使用的方法主要是利用ELISA方法直接检测血浆或血清中α-突触核蛋白及寡聚体含量,尚未有关于检测血浆中α-突触核蛋白寡聚体形成率的文献报导。In the prior art, the detection of α-synuclein and its oligomers in blood is mainly the direct determination of its content. The method used mainly uses the ELISA method to directly detect the content of α-synuclein and oligomers in plasma or serum, and there is no literature report on the detection of the formation rate of α-synuclein oligomers in plasma.

上述方法存在以下缺陷:对血浆和血清中的α-突触核蛋白及其寡聚体含量的测定结果极其不稳定,不同文献报道的正常值相差近1000倍,对病人血浆中的α-突触核蛋白及其寡聚体含量测定未能得出一致结论。例如,FouldsPG等人的一项研究检测到帕金森病人血浆中α-突触核蛋白含量较正常对照升高[15],而LiQX等人的研究则发现帕金森病人血浆中α-突触核蛋白含量低于正常对照[16]。El-Agnaf等人的研究发现血浆中α-突触核蛋白寡聚体高于正常对照[17],而FouldsPG等人的一项研究检测到帕金森病人血浆中α-突触核蛋白寡聚体含量低于正常对照[18]。这样相互矛盾的结果可能是由于血浆中α-突触核蛋白及其寡聚体本身含量较低,不同实验室所建立的ELISA方法的灵敏度之间存在差异以及样本溶血所造成的。因此,二者(上述方法)是否可作为帕金森病诊断标记物尚需进一步验证。The above method has the following defects: the measurement results of α-synuclein and its oligomer content in plasma and serum are extremely unstable, and the normal values reported in different documents differ by nearly 1000 times. The content determination of synuclein and its oligomers failed to draw consistent conclusions. For example, a study by FouldsPG et al. detected that the content of α-synuclein in the plasma of Parkinson's patients was higher than that of normal controls[15] , while the study by LiQX et al. found that α-synuclein in the plasma of Parkinson's patients The protein content was lower than that of the normal control[16] . A study by El-Agnaf et al. found that α-synuclein oligomers in plasma were higher than normal controls[17] , while a study by FouldsPG et al. detected α-synuclein oligomers in plasma of Parkinson's patients The content was lower than that of the normal control[18] . Such conflicting results may be due to the low content of α-synuclein and its oligomers in plasma itself, the differences in the sensitivity of ELISA methods established by different laboratories, and the hemolysis of samples. Therefore, whether the two (the above methods) can be used as diagnostic markers for Parkinson's disease needs further verification.

为克服上述缺陷,本发明提供一种检测α-突触核蛋白在血浆中的寡聚体形成率的检测方法,该方法具有较好的稳定性和重复性,可以显著区分帕金森病人和正常健康人的血浆促进α-突触核蛋白寡聚体的形成能力,是筛选帕金森病高危人群和临床诊断帕金森病的重要手段。In order to overcome the above-mentioned defects, the present invention provides a method for detecting the oligomer formation rate of α-synuclein in plasma. The method has good stability and repeatability, and can significantly distinguish Parkinson's patients from normal patients. The plasma of healthy people can promote the formation of α-synuclein oligomers, which is an important means for screening high-risk groups of Parkinson's disease and clinical diagnosis of Parkinson's disease.

发明内容:Invention content:

本发明提供一种检测α-突触核蛋白在血浆中的寡聚体形成率的检测方法,该方法可以用于测试血浆促进α-突触核蛋白寡聚体的形成能力。The invention provides a detection method for detecting the oligomer formation rate of α-synuclein in plasma, and the method can be used for testing the ability of plasma to promote the formation of α-synuclein oligomers.

本发明将纯化的重组人α-突触核蛋白与血浆(人或动物)在一定温度下进行振荡孵育,使其聚合,然后利用识别人α-突触核蛋白同一抗原表位的单克隆抗体分别作为捕获抗体和检测抗体,利用抗原-抗体反应原理检测α-突触核蛋白在病人和正常健康人血浆中的寡聚体含量,并计算α-突触核蛋白寡聚体的形成率。In the present invention, purified recombinant human α-synuclein and plasma (human or animal) are shaken and incubated at a certain temperature to polymerize, and then a monoclonal antibody that recognizes the same antigenic epitope of human α-synuclein is used As the capture antibody and detection antibody respectively, the antigen-antibody reaction principle was used to detect the oligomer content of α-synuclein in the plasma of patients and normal healthy people, and the formation rate of α-synuclein oligomer was calculated.

本发明的寡聚体形成率的检测方法包括以下步骤和内容:The detection method of oligomer formation rate of the present invention comprises the following steps and content:

1)分离血浆;1) Separation of plasma;

2)加入重组人α-突触核蛋白,得到寡聚体;2) adding recombinant human α-synuclein to obtain oligomers;

3)抗人α-突触核蛋白单克隆抗体和寡聚体反应,随后加入碱性磷酸酶和显色剂,进行显色;3) Anti-human α-synuclein monoclonal antibody reacts with oligomers, and then adds alkaline phosphatase and chromogen for color development;

4)用分光光度法对显色的样品进行吸光度测定,根据标准曲线计算样品中寡聚体的形成率。4) Measure the absorbance of the colored sample by spectrophotometry, and calculate the oligomer formation rate in the sample according to the standard curve.

以下对本发明所用名称术语进行解释:The term used in the present invention is explained below:

重组人α-突触核蛋白:将携带人α-突触核蛋白基因的质粒转染至大肠杆菌,培养、提取并纯化获得的帕金森病相关蛋白。Recombinant human α-synuclein: The plasmid carrying the human α-synuclein gene was transfected into Escherichia coli, and the obtained Parkinson's disease-related protein was cultured, extracted and purified.

血浆中人α-突触核蛋白寡聚体:血浆中存在的聚合形式的α-突触核蛋白,是其主要毒性形式。Human α-synuclein Oligomers in Plasma: Aggregated form of α-synuclein present in plasma, which is its major toxic form.

抗人α-突触核蛋白单克隆抗体:本发明的抗人α-突触核蛋白单克隆抗体命名为1B2抗体,用于识别α-突触核蛋白的特异氨基酸序列的鼠抗人α-突触核蛋白单克隆抗体,可与灵长类动物的α-突触核蛋白发生反应。Anti-human α-synuclein monoclonal antibody: The anti-human α-synuclein monoclonal antibody of the present invention is named 1B2 antibody, which is used to recognize the specific amino acid sequence of α-synuclein. Synuclein monoclonal antibody reactive with primate alpha-synuclein.

碱性磷酸酶:在ELISA测定中,碱性磷酸酶的色原底物是对硝基苯磷酸盐(p-nitropheny-phosate,pNPP)。pNPP在碱性磷酸酶的作用下生成对硝基酚(pNP),其在λ=405nm处有最大吸收。Alkaline phosphatase: In the ELISA assay, the chromogen substrate of alkaline phosphatase is p-nitrophenyl phosphate (p-nitrophenyl-phosphate, pNPP). pNPP generates p-nitrophenol (pNP) under the action of alkaline phosphatase, which has a maximum absorption at λ=405nm.

亲和素标记的碱性磷酸酶:将碱性磷酸酶进行亲和素标记,亲和素可与生物素结合,起级联放大作用。Avidin-labeled alkaline phosphatase: Avidin-labeled alkaline phosphatase, avidin can be combined with biotin to play a cascade amplification role.

显色剂:硝基苯磷酸单钠盐,可简称为pNPP单钠。与pNPP一样,用于ELISA的化学显色底物。碱性磷酸酶和pNPP反应后,形成黄色水溶反应产物,该产物在405nm处有光吸光。Chromogenic agent: monosodium nitrophenylphosphate, which can be referred to as monosodium pNPP for short. Like pNPP, a chromogenic substrate for ELISA. After the reaction between alkaline phosphatase and pNPP, a yellow water-soluble reaction product is formed, which has light absorption at 405nm.

ELISA酶标板:在酶联免疫吸附试验(EnzymeLinkedImmunosorbantAssay,ELISA)中,作为载体的固相聚苯乙烯(Polystyrene)表面对抗原、抗体或抗原抗体复合物的吸附起重要作用。抗原、抗体和其它生物分子通过多种机制吸附至载体表面,这包括通过疏水键、流水/离子键的被动吸附,通过引入其它活性基团如氨基和碳基的共价结合,以及通过表面改性后的亲水键结合。ELISA plate: In EnzymeLinkedImmunosorbantAssay (ELISA), the solid-phase polystyrene (Polystyrene) surface as a carrier plays an important role in the adsorption of antigens, antibodies or antigen-antibody complexes. Antigens, antibodies, and other biomolecules are adsorbed to the carrier surface through a variety of mechanisms, including passive adsorption through hydrophobic bonds, water/ionic bonds, covalent bonding through the introduction of other active groups such as amino and carbon groups, and surface modification. After the nature of the hydrophilic bond.

PBS:磷酸缓冲液(phosphatebufferedsaline),浓度为0.01mol/L。PBS: phosphate buffered saline (phosphate buffered saline), the concentration is 0.01mol/L.

NaHCO3缓冲液:碳酸氢钠缓冲液,浓度为200mmol/L,pH9.6。NaHCO3 buffer solution: sodium bicarbonate buffer solution, the concentration is 200mmol/L, pH9.6.

封闭液:明胶含量为2.5%的PBST溶液,作用为封闭非特异结合,降低ELISA背景。Blocking solution: PBST solution with 2.5% gelatin content, used to block non-specific binding and reduce ELISA background.

PBST:在0.01mol/LPBS中含有0.05%Tween-20即为磷酸盐吐温缓冲液,PBST。PBST: 0.05% Tween-20 in 0.01mol/LPBS is phosphate Tween buffer, PBST.

标准曲线的制作方法如下:每次试验时将α-突触核蛋白进行系列稀释,终浓度分别为0.5、0.25、0.125、0.0625、0.03125、0μmol/L,与样品同时进行检测,读取相对应405nm处吸光值,做出其与浓度之间的关系曲线。The method of making the standard curve is as follows: α-synuclein is serially diluted in each test, and the final concentrations are 0.5, 0.25, 0.125, 0.0625, 0.03125, 0 μmol/L, and the samples are detected at the same time, and the readings correspond to The absorbance value at 405nm was used to make the relationship curve between it and the concentration.

本发明所述抗人α-突触核蛋白单克隆抗体属于全新的概念,为此,本发明还提供抗人α-突触核蛋白单克隆抗体作为本发明的一部分。The anti-human α-synuclein monoclonal antibody of the present invention belongs to a brand-new concept. Therefore, the present invention also provides an anti-human α-synuclein monoclonal antibody as a part of the present invention.

本发明提供抗体抗人α-突触核蛋白单克隆抗体,其制备方法如下:The present invention provides antibody anti-human α-synuclein monoclonal antibody, and its preparation method is as follows:

步骤1,重组蛋白抗原的制备:制备并纯化人全长α-突触核蛋白作为抗原。Step 1, preparation of recombinant protein antigen: prepare and purify human full-length α-synuclein as antigen.

步骤2,免疫小鼠:使用福氏完全佐剂(第一次免疫)与福氏不完全佐剂(随后的免疫)乳化重组人α-突触核蛋白,每隔2周对BALB/c雌性小鼠进行免疫(50μg抗原/次/小鼠)。抽取动物血清,利用ELISA与Westernblot方法检测其效价与特异性。Step 2. Immunization of mice: Recombinant human α-synuclein was emulsified with Freund's complete adjuvant (first immunization) and Freund's incomplete adjuvant (subsequent immunization), and BALB/c females were treated every 2 weeks. Mice were immunized (50 μg antigen/time/mouse). Animal serum was extracted, and the potency and specificity were detected by ELISA and Western blot.

步骤3,筛选杂交瘤细胞株:选取血清效价高的小鼠,取出其脾细胞,在存在聚乙二醇4000(PEG4000)的条件下与小鼠骨髓瘤细胞Sp2融合,随后使用HAT培养基对融合的产生抗体的杂交瘤细胞进行选择培养,最后用ELISA方法筛选可以产生特异性抗体的杂交瘤细胞。ELISA筛选阳性的杂交瘤细胞进一步利用免疫组化与免疫印迹法鉴定所筛选杂交瘤细胞产生抗体的特异性。经筛选,克隆号为1B2的杂交瘤细胞可以产生特异性抗人α-突触核蛋白抗体。1B2的杂交瘤细胞经过2次亚克隆进行纯化后,在冷冻保护液的保护下与液氮中长期保存。(该细胞株已经保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),编号为CGMCC9104,保藏日期2014年04月15日,分类命名:杂交瘤细胞株(1B2),地址北京市朝阳区北辰西路1号院3号)。Step 3, screening hybridoma cell lines: select mice with high serum titer, take out their splenocytes, fuse with mouse myeloma cell Sp2 under the condition of presence of polyethylene glycol 4000 (PEG4000), then use HAT medium The fused antibody-producing hybridoma cells are selected and cultured, and finally the hybridoma cells that can produce specific antibodies are screened by ELISA method. The positive hybridoma cells screened by ELISA were further identified by immunohistochemistry and immunoblotting to identify the specificity of the antibodies produced by the screened hybridoma cells. After screening, the hybridoma cell with the clone number 1B2 can produce specific anti-human α-synuclein antibody. After the hybridoma cells of 1B2 were purified by subcloning twice, they were stored in liquid nitrogen for a long time under the protection of cryoprotectant solution. (The cell line has been preserved in the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microbial Cultures (CGMCC), the number is CGMCC9104, the preservation date is April 15, 2014, the classification name is hybridoma cell line (1B2), and the address is Chaoyang District, Beijing No. 3, Courtyard No. 1, Beichen West Road).

步骤4;抗体制备:将1B2杂交瘤细胞从液氮中复苏,并用杂交瘤培养基在培养箱中大量扩增,收获细胞,将细胞由腹膜内注射入BALB/c裸鼠体内,产生腹水。收获腹水,利用proteinA亲和层析纯化法获得纯化的1B2鼠抗人α-突触核蛋白单克隆抗体,定量、分装、保存。Step 4: Antibody preparation: 1B2 hybridoma cells were recovered from liquid nitrogen, amplified in large quantities in an incubator with hybridoma medium, harvested, and injected intraperitoneally into BALB/c nude mice to produce ascites. Harvest ascites, use proteinA affinity chromatography to obtain purified 1B2 mouse anti-human α-synuclein monoclonal antibody, quantify, subpackage, and store.

本发明所述生物素化的抗人α-突触核蛋白单克隆抗体1B2,其制备方法如下:The preparation method of the biotinylated anti-human α-synuclein monoclonal antibody 1B2 of the present invention is as follows:

步骤1,用无水DMSO配制10mg/ml生物素N-羟基琥珀酰亚胺酯溶液。Step 1, prepare 10 mg/ml biotin N-hydroxysuccinimide ester solution with anhydrous DMSO.

步骤2,用硼酸盐缓冲液(0.1mol/L,pH8.8)配制浓度至少为1~3mg/ml的抗体1B2溶液,若抗体1B2储存时加入了叠氮钠,则标记前须先在硼酸盐缓冲液中充分透析以除去叠氮钠。Step 2, use borate buffer (0.1mol/L, pH8.8) to prepare antibody 1B2 solution with a concentration of at least 1-3mg/ml. If antibody 1B2 is stored with sodium azide, it must be in the Dialyze extensively against borate buffer to remove sodium azide.

步骤3,按25~100μg/mg的比率将生物素酯加入抗体1B2中,混合均匀并在室温下孵育4h。Step 3: Add biotin ester to antibody 1B2 at a ratio of 25-100 μg/mg, mix well and incubate at room temperature for 4 hours.

步骤4,每250μg生物素酯内加入20μl1mol/L的氯化铵,室温孵育10min。Step 4, add 20 μl of 1 mol/L ammonium chloride per 250 μg of biotin ester, and incubate at room temperature for 10 minutes.

步骤5,将抗体1B2溶液用PBS或其他所需的缓冲液透析,以除去未结合的生物素。Step 5, dialyze the antibody 1B2 solution against PBS or other required buffers to remove unbound biotin.

步骤6,按纯化抗体的储存方法保存标记抗体。Step 6, store the labeled antibody according to the storage method of the purified antibody.

优选的本发明的寡聚体形成率的检测方法,步骤如下:The detection method of preferred oligomer formation rate of the present invention, the steps are as follows:

步骤1,step 1,

取静脉血,必要时可以加入EDTA、肝素或枸橼酸抗凝,分离血浆层备用;Take venous blood, if necessary, add EDTA, heparin or citrate for anticoagulation, and separate the plasma layer for later use;

步骤2,Step 2,

血浆中加入重组人α-突触核蛋白,得到寡聚体;Add recombinant human α-synuclein to plasma to obtain oligomers;

步骤3,Step 3,

ELISA酶标板中加入抗人α-突触核蛋白单克隆抗体和寡聚体,亲和素标记的碱性磷酸酶,对硝基酚磷酸显色液;Add anti-human α-synuclein monoclonal antibody and oligomers, avidin-labeled alkaline phosphatase, and p-nitrophenol phosphoric acid chromogenic solution to the ELISA plate;

步骤4,Step 4,

在405nm处测定酶标板各孔的吸光度值,根据标准曲线计算寡聚体的含量以及形成率。The absorbance value of each well of the microplate plate was measured at 405 nm, and the oligomer content and formation rate were calculated according to the standard curve.

特别优选的本发明的寡聚体形成率的检测方法,步骤如下:The detection method of the particularly preferred oligomer formation rate of the present invention, the steps are as follows:

步骤1,step 1,

抽取5-10ml抗凝血(EDTA、肝素或枸橼酸抗凝)。将抗凝血上下颠倒轻轻混匀,贴壁加入至50ml离心管中,记录全血的体积,1:1加入PBS,充分混匀。Draw 5-10ml anticoagulant blood (EDTA, heparin or citrate anticoagulant). Gently mix the anticoagulated blood upside down, add it into a 50ml centrifuge tube, record the volume of whole blood, add PBS at a ratio of 1:1, and mix well.

将稀释后的全血缓慢加至淋巴细胞分离液上,400×g,离心20min。此时管内液体由上至下的顺序依次为血浆层、白膜层(外周血单个核细胞)、分离液层以及红细胞层。Slowly add the diluted whole blood to the lymphocyte separation medium, centrifuge at 400×g for 20min. At this time, the order of the liquid in the tube from top to bottom is the plasma layer, the buffy coat layer (peripheral blood mononuclear cells), the separation liquid layer and the red blood cell layer.

吸取表面的血浆层、分装,-80℃保存备用。Aspirate the plasma layer on the surface, aliquot and store at -80°C for later use.

步骤2,Step 2,

α-突触核蛋白寡聚体的制备:将血浆用PBS适当稀释,加入一定浓度的重组人α-突触核蛋白,在恒温振荡器中37℃振荡孵育一定时间,于4℃保存备用。Preparation of α-synuclein oligomers: Plasma was properly diluted with PBS, a certain concentration of recombinant human α-synuclein was added, incubated in a constant temperature shaker at 37°C for a certain period of time, and stored at 4°C for later use.

步骤3,Step 3,

包被:用NaHCO3缓冲液稀释抗人α-突触核蛋白单克隆抗体1B2至终浓度为1~2μg/mL。向酶标板各孔加入100μL该抗体稀释液,4℃孵育过夜。用PBST(其为含有吐温的PBS)冲洗酶标板各孔,根据需要确定冲洗次数和时间。Coating: Dilute anti-human α-synuclein monoclonal antibody 1B2 with NaHCO3 buffer to a final concentration of 1-2 μg/mL. Add 100 μL of the antibody dilution to each well of the microtiter plate and incubate overnight at 4°C. Rinse each well of the microtiter plate with PBST (which is PBS containing Tween), and determine the number and time of washing as required.

封闭:向酶标板各孔加入100μL封闭液(其为含有明胶和吐温的PBS),在37℃孵育1~2小时。用PBST冲洗酶标板各孔,根据需要确定冲洗次数和时间。Blocking: Add 100 μL of blocking solution (PBS containing gelatin and Tween) to each well of the microtiter plate, and incubate at 37° C. for 1 to 2 hours. Rinse each well of the microtiter plate with PBST, and determine the number and time of washing as needed.

向各孔加入100μL已制备好的α-突触核蛋白寡聚体样品稀释液,在37℃孵育1~2小时。用PBST冲洗酶标板各孔,根据需要确定冲洗次数和时间。Add 100 μL of the prepared α-synuclein oligomer sample dilution to each well, and incubate at 37°C for 1-2 hours. Rinse each well of the microtiter plate with PBST, and determine the number and time of washing as needed.

用封闭液稀释生物素化的抗人α-突触核蛋白单克隆抗体1B2抗体至终浓度为1~2μg/mL。向酶标板的各孔加入100μL该抗体稀释液,在37℃孵育1~2小时。用PBST冲洗酶标板各孔,根据需要确定冲洗次数和时间。Dilute the biotinylated anti-human α-synuclein monoclonal antibody 1B2 antibody with blocking solution to a final concentration of 1-2 μg/mL. Add 100 μL of the antibody dilution to each well of the microtiter plate, and incubate at 37°C for 1-2 hours. Rinse each well of the microtiter plate with PBST, and determine the number and time of washing as needed.

用封闭液稀释亲和素标记的碱性磷酸酶(由Sigma公司出售),向酶标板的各孔加入100μL该酶的稀释液,在37℃孵育0.5~2小时。用PBST冲洗酶标板各孔,根据需要确定冲洗次数和时间。Dilute avidin-labeled alkaline phosphatase (sold by Sigma) with blocking solution, add 100 μL of the diluted enzyme solution to each well of the microtiter plate, and incubate at 37° C. for 0.5 to 2 hours. Rinse each well of the microtiter plate with PBST, and determine the number and time of washing as needed.

向酶标板的各孔加入100μLpNPP(对硝基酚磷酸显色液,由Sigma公司出售),37℃显色10~50分钟。Add 100 μL of pNPP (p-nitrophenol phosphate chromogenic solution, sold by Sigma) to each well of the microtiter plate, and develop color at 37° C. for 10 to 50 minutes.

步骤4,Step 4,

测定405nm处酶标板各孔的吸光度值。Measure the absorbance value of each well of the microplate plate at 405nm.

根据α-突触核蛋白寡聚体标准品浓度与405nm处吸光值之间的关系曲线计算受试者血液中α-突触核蛋白寡聚体的形成量。The amount of α-synuclein oligomers formed in the blood of the subjects was calculated according to the relationship curve between the concentration of the α-synuclein oligomer standard substance and the absorbance value at 405 nm.

本发明经过研究发现,上述方法与现有技术相比具有如下优点:The present invention finds through research that said method has the following advantages compared with the prior art:

克服溶血对检测结果的影响;Overcome the influence of hemolysis on test results;

稳定性高;High stability;

以下通过比较证明本发明的优点:Prove advantage of the present invention by comparing below:

已有文献证实红细胞中存在较大量的α-突触核蛋白及寡聚体,而血浆中α-突触核蛋白及寡聚体含量较低,因此,样本溶血将影响检测结果。这也是造成目前研究结论不一致的原因之一。本方法采用外加大量α-突触核蛋白间接检测血浆中α-突触核蛋白寡聚体形成量则不受溶血的影响,并且大大提高了检测的稳定性。It has been confirmed in the literature that there are relatively large amounts of α-synuclein and oligomers in red blood cells, while the content of α-synuclein and oligomers in plasma is relatively low. Therefore, hemolysis of samples will affect the test results. This is also one of the reasons for the inconsistent conclusions of the current research. The method adopts adding a large amount of α-synuclein to indirectly detect the formation of α-synuclein oligomers in plasma, which is not affected by hemolysis, and greatly improves the stability of detection.

由于本发明效果的优越性,本发明人根据上述方法制备了一种检测试剂盒,所述试剂盒包括以下试剂:Due to the superiority of the effect of the present invention, the inventor has prepared a detection kit according to the above method, and the kit includes the following reagents:

重组人α-突触核蛋白recombinant human α-synuclein

抗人α-突触核蛋白单克隆抗体anti-human alpha-synuclein monoclonal antibody

进一步还可包括生物素化的抗人α-突触核蛋白单克隆抗体Further can include biotinylated anti-human α-synuclein monoclonal antibody

根据需要,所述试剂盒还可包括以下辅助试剂:According to needs, the kit can also include the following auxiliary reagents:

碱性磷酸酶,或亲和素标记的碱性磷酸酶Alkaline phosphatase, or avidin-labeled alkaline phosphatase

对硝基酚磷酸显色液p-Nitrophenol Phosphate Chromogenic Solution

ELISA酶标板ELISA plate

PBSPBS

NaHCO3缓冲液NaHCO3 buffer

封闭液blocking solution

PBSTPBST

其中,in,

PBS的浓度为0.01mol/L,配制方法为The concentration of PBS is 0.01mol/L, and the preparation method is

NaHCO3缓冲液的浓度为200mmol/L,pH9.6,配制方法为The concentration of NaHCO3 buffer solution is 200mmol/L, pH9.6, and the preparation method is

NaHCO30.84gNaHCO3 0.84g

20%叠氮钠(NaN3)50μl20% sodium azide (NaN3 ) 50μl

DDW50mlDDW50ml

封闭液的浓度为2.5%,配制方法为The concentration of the blocking solution is 2.5%, and the preparation method is

明胶2.5gGelatin 2.5g

PBST100mlPBST100ml

PBST为含0.05%Tween20的0.01mol/LPBS,配制方法为PBST is 0.01mol/LPBS containing 0.05% Tween20, the preparation method is

上述试剂盒的使用是以血浆中重组人α-突触核蛋白寡聚体作为标准蛋白,以该寡聚体浓度与吸光度值的关系曲线作为标准曲线,通过测定样本中的血浆-重组人α-突触核蛋白寡聚体的浓度以及形成率达到诊断帕金森病的目的。The use of the above kit is to use the recombinant human α-synuclein oligomer in plasma as a standard protein, and use the relationship curve between the oligomer concentration and absorbance value as a standard curve to determine the plasma-recombinant human α-synuclein in the sample. - The concentration and formation rate of synuclein oligomers achieves the purpose of diagnosing Parkinson's disease.

本发明的试剂盒,各试剂分别包装,优选使用包装管,每个包装管中装入试剂的量以够一个样本使用量为基本量,可以扩大到10个,100个,1000个样本的使用量。In the kit of the present invention, each reagent is packaged separately, preferably using a packaging tube, and the amount of the reagent loaded in each packaging tube is based on the amount that is enough for one sample, and can be expanded to 10, 100, or 1000 samples. quantity.

本发明的试剂盒的使用是根据上述检测血浆中重组人α-突触核蛋白寡聚体的方法进行的,使用时将试剂盒中的试剂根据需要配制成相应的浓度,按照所述方法测定即可。The use of the kit of the present invention is carried out according to the above-mentioned method for detecting recombinant human α-synuclein oligomers in plasma. That's it.

为研究本发明,还进行了以下重复性试验,精密度试验,检测方法的筛选等试验For researching the present invention, also carried out following repeatability test, precision test, tests such as the screening of detection method

相同样本重复实验3次,结果显示相对标准差RSD为0.80%。The experiment was repeated three times with the same sample, and the results showed that the relative standard deviation (RSD) was 0.80%.

将实施例3中制备的试剂盒分别取三批进行精密度实验。以实施例3中所抽取的试剂盒测定三份高、中、低值样本0.5μmol/L、0.125μmol/L和0.03125μmol/L各8次。计算测定浓度的变异系数。实施例3中的三批试剂盒的结果表明变异系数小于8.0%。Three batches of the kit prepared in Example 3 were taken respectively for precision experiment. The kit extracted in Example 3 was used to measure three high, medium and low value samples 0.5 μmol/L, 0.125 μmol/L and 0.03125 μmol/L each 8 times. Calculate the coefficient of variation for the measured concentrations. The results of the three batches of kits in Example 3 showed a coefficient of variation of less than 8.0%.

采用棋盘滴定试验确定抗体包被浓度以及生物素化抗体浓度。根据初步确定的浓度缩小间距,再做进一步棋盘滴定,确定最佳试验条件。最终确定包被浓度为1~2μg/mL,生物素化抗体浓度为1~2μg/mL。A checkerboard titration assay was used to determine the antibody coating concentration as well as the biotinylated antibody concentration. According to the initially determined concentration, narrow the distance, and then do further checkerboard titration to determine the best experimental conditions. Finally, the coating concentration was determined to be 1-2 μg/mL, and the biotinylated antibody concentration was 1-2 μg/mL.

附图说明:Description of drawings:

图1为血浆的分离过程中,不同成分分层示意图。Figure 1 is a schematic diagram of the layers of different components during the plasma separation process.

图2为92例对照与PD患者血浆中α-Syn寡聚体形成量ELISA的检测结果。Figure 2 shows the ELISA detection results of α-Syn oligomer formation in the plasma of 92 cases of controls and PD patients.

具体实施方式:detailed description:

以下通过实施例进一步说明本发明。The present invention is further illustrated by the following examples.

实施例1,Example 1,

具体步骤如下:Specific steps are as follows:

(1)血浆的分离:(1) Separation of plasma:

●抽取5-10ml抗凝血(EDTA、肝素或枸橼酸抗凝)。将抗凝血上下颠倒轻轻混匀,贴壁加入至50ml离心管中,记录全血的体积,1:1加入PBS,充分混匀。● Draw 5-10ml anticoagulant blood (EDTA, heparin or citrate anticoagulant). Gently mix the anticoagulated blood upside down, add it into a 50ml centrifuge tube, record the volume of whole blood, add PBS at a ratio of 1:1, and mix well.

●将稀释后的全血缓慢加至淋巴细胞分离液上,400×g,离心20min。此时管内液体由上至下的顺序依次为血浆层、白膜层(外周血单个核细胞)、分离液层以及红细胞层。● Slowly add the diluted whole blood to the lymphocyte separation medium, centrifuge at 400×g for 20min. At this time, the order of the liquid in the tube from top to bottom is the plasma layer, the buffy coat layer (peripheral blood mononuclear cells), the separation liquid layer and the red blood cell layer.

●吸取表面的血浆层、分装,-80℃保存备用。●Aspiration of the plasma layer on the surface, aliquoting, and storing at -80°C for later use.

(2)稳定的α-突触核蛋白寡聚体的制备:将血浆用PBS适当稀释,加入一定浓度的重组人α-突触核蛋白,在恒温振荡器中37℃振荡孵育一定时间,于4℃保存备用。(2) Preparation of stable α-synuclein oligomers: Dilute plasma appropriately with PBS, add a certain concentration of recombinant human α-synuclein, shake and incubate in a constant temperature shaker at 37°C for a certain period of time, and then Store at 4°C for later use.

(3)包被:用NaHCO3缓冲液稀释抗人α-突触核蛋白单克隆抗体1B2至终浓度为1~2μg/mL。向酶标板各孔加入100μL该抗体稀释液,4℃孵育过夜。用PBST(其为含有吐温的PBS)冲洗酶标板各孔,根据需要确定冲洗次数和时间。(3) Coating: Dilute the anti-human α-synuclein monoclonal antibody 1B2 with NaHCO3 buffer to a final concentration of 1-2 μg/mL. Add 100 μL of the antibody dilution to each well of the microtiter plate and incubate overnight at 4°C. Rinse each well of the microtiter plate with PBST (which is PBS containing Tween), and determine the number and time of washing as required.

(4)封闭:向酶标板各孔加入100μL封闭液(其为含有明胶和吐温的PBS),在37℃孵育1~2小时。用PBST冲洗酶标板各孔,根据需要确定冲洗次数和时间。(4) Blocking: add 100 μL of blocking solution (PBS containing gelatin and Tween) to each well of the microplate, and incubate at 37° C. for 1 to 2 hours. Rinse each well of the microtiter plate with PBST, and determine the number and time of washing as needed.

(5)向各孔加入100μL已制备好的α-突触核蛋白聚合体样品稀释液,在37℃孵育1~2小时。用PBST冲洗酶标板各孔,根据需要确定冲洗次数和时间。(5) Add 100 μL of the prepared α-synuclein polymer sample dilution to each well, and incubate at 37° C. for 1 to 2 hours. Rinse each well of the microtiter plate with PBST, and determine the number and time of washing as needed.

(6)用封闭液稀释生物素化的抗人α-突触核蛋白单克隆抗体1B2抗体至终浓度为1~2μg/mL。向酶标板的各孔加入100μL该抗体稀释液,在37℃孵育1~2小时。用PBST冲洗酶标板各孔,根据需要确定冲洗次数和时间。(6) Dilute the biotinylated anti-human α-synuclein monoclonal antibody 1B2 antibody with blocking solution to a final concentration of 1-2 μg/mL. Add 100 μL of the antibody dilution to each well of the microtiter plate, and incubate at 37°C for 1-2 hours. Rinse each well of the microtiter plate with PBST, and determine the number and time of washing as needed.

(7)用封闭液稀释亲和素标记的碱性磷酸酶(由Sigma公司出售),向酶标板的各孔加入100μL该酶的稀释液,在37℃孵育0.5~2小时。用PBST冲洗酶标板各孔,根据需要确定冲洗次数和时间。(7) Dilute avidin-labeled alkaline phosphatase (sold by Sigma) with blocking solution, add 100 μL of the enzyme dilution to each well of the microtiter plate, and incubate at 37° C. for 0.5 to 2 hours. Rinse each well of the microtiter plate with PBST, and determine the number and time of washing as needed.

(8)向酶标板的各孔加入100μLpNPP(对硝基酚磷酸显色液,由Sigma公司出售),37℃显色10~50分钟。(8) Add 100 μL of pNPP (p-nitrophenol phosphate chromogenic solution, sold by Sigma) to each well of the microtiter plate, and develop color at 37° C. for 10 to 50 minutes.

(9)测定405nm处酶标板各孔的吸光度值。(9) Measure the absorbance value of each well of the microplate plate at 405 nm.

(10)根据α-突触核蛋白聚合体标准品浓度与405nm处吸光值之间的关系曲线计算受试者血液中α-突触核蛋白聚合体的形成量。(10) Calculate the amount of α-synuclein polymer formed in the subject's blood according to the relationship curve between the concentration of the α-synuclein polymer standard substance and the absorbance value at 405 nm.

实施例2,Example 2,

血浆促进α-突触核蛋白聚合能力的检测Detection of the Ability of Plasma to Promote α-Synuclein Polymerization

采用相对定量ELISA法,对92例临床诊断的帕金森病人和92例健康对照血浆促进α-突触核蛋白聚合的能力进行了检测。结果显示帕金森病人血浆中α-突触核蛋白寡聚体形成量为72.09μmol/L;而正常健康对照血浆中α-突触核蛋白寡聚体形成量为54.26μmol/L。帕森病人血浆中α-突触核蛋白寡聚体形成量明显高于正常健康对照(p<0.01)。The relative quantitative ELISA method was used to detect the ability of plasma from 92 clinically diagnosed Parkinson's patients and 92 healthy controls to promote the polymerization of α-synuclein. The results showed that the formation of α-synuclein oligomers in the plasma of Parkinson's patients was 72.09 μmol/L; while the formation of α-synuclein oligomers in the plasma of normal healthy controls was 54.26 μmol/L. The amount of α-synuclein oligomers formed in the plasma of Parson's patients was significantly higher than that of normal healthy controls (p<0.01).

实施例3,Example 3,

试剂盒成分举例Examples of kit components

重组人α-突触核蛋白,10mgRecombinant human alpha-synuclein, 10mg

生物素化的抗人α-突触核蛋白单克隆抗体10μgBiotinylated anti-human α-synuclein monoclonal antibody 10 μg

碱性磷酸酶,或亲和素标记的碱性磷酸酶5μlAlkaline phosphatase, or avidin-labeled alkaline phosphatase 5 μl

对硝基酚磷酸显色液10mlp-nitrophenol phosphoric acid color solution 10ml

ELISA酶标板ELISA plate

PBS10mlPBS10ml

NaHCO3缓冲液10mlNaHCO3 buffer 10ml

封闭液20mlBlocking solution 20ml

PBST500mlPBST500ml

以下为参考文件:The following are reference documents:

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Claims (7)

Translated fromChinese
1.一种检测α-突触核蛋白在血浆中的寡聚体形成率的试剂盒,试剂盒中包括以下试剂:重组人α-突触核蛋白,抗人α-突触核蛋白单克隆抗体1B2,所述的抗人α-突触核蛋白单克隆抗体1B2为克隆号为1B2的杂交瘤细胞产生,所述的克隆号为1B2的杂交瘤细胞保藏在中国普通微生物菌种保藏管理中心,编号为CGMCC9104。1. A test kit for detecting the oligomer formation rate of α-synuclein in blood plasma, comprising following reagents in the test kit: recombinant human α-synuclein, anti-human α-synuclein monoclonal Antibody 1B2, the anti-human α-synuclein monoclonal antibody 1B2 is produced by the hybridoma cell with the clone number 1B2, and the hybridoma cell with the clone number 1B2 is preserved in the China General Microorganism Culture Collection Management Center , number CGMCC9104.2.根据权利要求1所述的试剂盒,试剂盒中还包括生物素化的抗人α-突触核蛋白单克隆抗体1B2。2. The kit according to claim 1, further comprising biotinylated anti-human α-synuclein monoclonal antibody 1B2.3.根据权利要求1所述的试剂盒,根据需要,所述试剂盒还包括以下辅助试剂:3. test kit according to claim 1, as required, described test kit also comprises following auxiliary reagent:碱性磷酸酶,或亲和素标记的碱性磷酸酶Alkaline phosphatase, or avidin-labeled alkaline phosphatase对硝基酚磷酸显色液p-Nitrophenol Phosphate Chromogenic SolutionELISA酶标板ELISA plate磷酸盐缓冲液Phosphate bufferNaHCO3缓冲液NaHCO3 buffer封闭液blocking solutionPBST。PBST.4.根据权利要求3所述的试剂盒,其中4. The test kit according to claim 3, wherein磷酸盐缓冲液的浓度为0.01mol/L,The concentration of phosphate buffer is 0.01mol/L,NaHCO3缓冲液的浓度为200mmol/L,pH9.6,The concentration of NaHCO3 buffer solution is 200mmol/L, pH9.6,封闭液的浓度为2.5%,The concentration of blocking solution is 2.5%,PBST为含0.05%Tween-20的0.01MPBS。PBST was 0.01 MPBS with 0.05% Tween-20.5.根据权利要求2所述的试剂盒,其中各试剂分别包装,每个包装装入试剂的量以够一个样本使用量为基本量。5. The kit according to claim 2, wherein each reagent is packaged separately, and the amount of reagent loaded in each package is basically the amount enough for one sample.6.根据权利要求2所述的试剂盒,其中各试剂分别包装,每个包装装入试剂的量扩大到10个,100个或1000个样本的使用量。6. The kit according to claim 2, wherein each reagent is packaged separately, and the amount of reagents loaded in each package is expanded to the usage amount of 10, 100 or 1000 samples.7.生物素化的抗人α-突触核蛋白单克隆抗体1B2,其制备方法如下:7. Biotinylated anti-human α-synuclein monoclonal antibody 1B2, its preparation method is as follows:步骤1,用无水DMSO配制10mg/ml生物素N-羟基琥珀酰亚胺酯溶液,Step 1, prepare 10mg/ml biotin N-hydroxysuccinimide ester solution with anhydrous DMSO,步骤2,用0.1mol/L,pH8.8硼酸盐缓冲液配制浓度为1~3mg/ml的抗体1B2溶液,若抗体1B2储存时加入了叠氮钠,则标记前须先在硼酸盐缓冲液中充分透析以除去叠氮钠,Step 2, use 0.1mol/L, pH8.8 borate buffer to prepare antibody 1B2 solution with a concentration of 1-3mg/ml. If antibody 1B2 is stored with sodium azide, it must be added in borate before labeling. The buffer was extensively dialyzed to remove sodium azide,步骤3,按25~100μg/mg的比率将生物素N-羟基琥珀酰亚胺酯加入抗体1B2中,混合均匀并在室温下孵育4h,Step 3: Add biotin N-hydroxysuccinimide ester to antibody 1B2 at a ratio of 25-100 μg/mg, mix well and incubate at room temperature for 4 hours,步骤4,每250μg生物素酯内加入20μl1mol/L的氯化铵,室温孵育10min,Step 4, add 20 μl of 1mol/L ammonium chloride per 250 μg of biotin ester, incubate at room temperature for 10 minutes,步骤5,将抗体1B2溶液用PBS缓冲液透析,以除去未结合的生物素,Step 5, the antibody 1B2 solution was dialyzed with PBS buffer to remove unbound biotin,步骤6,按纯化抗体的储存方法保存标记抗体,Step 6, store the labeled antibody according to the storage method of the purified antibody,所述的抗人α-突触核蛋白单克隆抗体1B2为克隆号为1B2的杂交瘤细胞产生,所述的克隆号为1B2的杂交瘤细胞保藏在中国普通微生物菌种保藏管理中心,编号为CGMCC9104。The anti-human α-synuclein monoclonal antibody 1B2 is produced by the hybridoma cell with the clone number 1B2, and the hybridoma cell with the clone number 1B2 is preserved in the China General Microorganism Culture Collection Management Center, and the number is CGMCC9104.
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