WO2023087616A1 - 一种原发性胆汁性胆管炎诊断用分子标志物及其应用 - Google Patents

一种原发性胆汁性胆管炎诊断用分子标志物及其应用 Download PDF

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WO2023087616A1
WO2023087616A1 PCT/CN2022/087926 CN2022087926W WO2023087616A1 WO 2023087616 A1 WO2023087616 A1 WO 2023087616A1 CN 2022087926 W CN2022087926 W CN 2022087926W WO 2023087616 A1 WO2023087616 A1 WO 2023087616A1
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human
antibody
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pbc
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French (fr)
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柴进
王惠雯
李茜
潘琼
廖敏
肖金滔
过妍
毛秀茹
彭仕芳
刘小伟
汤参娥
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中国人民解放军陆军军医大学第一附属医院
中南大学湘雅医院
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Priority to US18/281,804 priority Critical patent/US20240159748A1/en
Priority to EP22893985.6A priority patent/EP4379381A1/en
Publication of WO2023087616A1 publication Critical patent/WO2023087616A1/zh

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis

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  • the invention belongs to the technical field of molecular biology, and relates to a serum anti-human multimeric immunoglobulin receptor antibody, specifically, a serum anti-human multimeric immunoglobulin receptor antibody used for the diagnosis of primary biliary cholangitis Molecular markers and their applications.
  • PBC Primary biliary cholangitis
  • the human multimeric immunoglobulin receptor a member of the Fc receptor family, is a core member of the mucosal immune system. It is widely expressed in mucosal epithelial cells and is usually induced and upregulated by pro-inflammatory cytokines during viral or bacterial infection. Human multimeric immunoglobulin receptors can specifically bind to poly-IgA and poly-IgM, transport them from the basolateral membrane of epithelial cells to the apical membrane through transcellular transport, and finally secrete them into the exocrine fluid to form a defense against infection first line of defense.
  • liver fibrosis which may be related to hepatitis virus infection and hepatic stellate cell transdifferentiation.
  • Human multimeric immunoglobulin receptors can also Promote the metastasis of human hepatocellular carcinoma (Hepatocellular carcinoma, HCC), but whether human multimeric immunoglobulin receptor is involved in the occurrence and development of PBC and the value of anti-human multimeric immunoglobulin receptor antibody in the diagnosis of PBC have not yet been seen According to reports, this indicator has not yet been applied clinically.
  • Serum AMAs and their M2 subtypes are currently important auxiliary diagnostic indicators for PBC in clinical practice.
  • diagnosis of PBC still mainly relies on liver biopsy. Finding simple and reliable diagnostic methods and screening sensitive molecular indicators is an important problem to be solved in clinical early diagnosis of PBC.
  • the present invention detects AMA-M2 positive PBC, AMA-M2 negative PBC, normal control (Control, CTR), obstruction Human multimeric immunoglobulin receptors in liver tissues of patients with Obstructive cholestasis (OC), secondary sclerosing cholangitis (SSC) and nonalcoholic steatohepatitis (NASH)
  • CTR normal control
  • OC Obstructive cholestasis
  • SSC secondary sclerosing cholangitis
  • NASH nonalcoholic steatohepatitis
  • human multimeric immunoglobulin receptors may be the specific antigen that mediates immune injury during the pathogenesis of PBC.
  • the present invention uses enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA) to detect the serum of patients with CTR, PBC, and OC, and confirms that whether AMA-M2 is positive or negative, anti-human multimeric immunoglobulin receptor The antibodies were all significantly increased, suggesting that the anti-human polymeric immunoglobulin receptor antibody mediates the damage of cholangiocytes by targeting the human polymeric immunoglobulin receptor antigen on the small and medium bile ducts in the liver, so the serum anti-human polymeric immunoglobulin receptor Human immunoglobulin receptor antibody can be used as a molecular marker for the diagnosis of PBC.
  • enzyme-linked immunosorbent assay Enzyme linked immunosorbent assay, ELISA
  • the positive detection of anti-AMA-M2 antibody is regarded as one of the criteria for diagnosing PBC clinically, which easily leads to missed diagnosis of anti-AMA-M2 antibody-negative PBC patients. Therefore, the detection of anti-human multimeric immunoglobulin receptor antibody is helpful for the diagnosis of PBC, especially the diagnosis of AMA-M2 negative PBC patients.
  • the purpose of the present invention is to provide a molecular marker for PBC diagnosis and its application, That is, the serum anti-human multimeric immunoglobulin receptor antibody is used as a molecular marker for the diagnosis of PBC.
  • a molecular marker for the diagnosis of primary biliary cholangitis which is an anti-human multimeric immunoglobulin receptor antibody.
  • the invention also provides the application of an anti-human multimer immunoglobulin receptor antibody in the preparation of reagents for diagnosing PBC patients.
  • the PBC patients include anti-AMA-M2 antibody-positive PBC patients and anti-AMA-M2 antibody-negative PBC patients.
  • the reagent for diagnosing PBC patients is used to detect the expression level of anti-human multimeric immunoglobulin receptor antibody in a sample, and the sample is a human serum sample.
  • the reagent for diagnosing PBC patients is an ELISA kit.
  • the ELISA kit includes microwell microtiter plate, standard, sample diluent, horseradish peroxidase-labeled mouse anti-human multimer immunoglobulin monoclonal antibody (detection antibody-HRP), 20x washing buffer solution, substrate A, substrate B, stop solution, sealing film, ziplock bag.
  • the detection steps of the ELISA kit include:
  • Serum is extracted from whole blood specimens as the sample to be tested;
  • detection antibody mouse anti-human multimer immunoglobulin monoclonal antibody
  • HRP human anti-human multimer immunoglobulin monoclonal antibody
  • the present invention finds that the anti-human multimeric immunoglobulin receptor antibody can be used as a biomarker for the diagnosis of PBC.
  • Serum AMA-M2 is a specific indicator for the diagnosis of PBC. About 90-95% of PBC patients are positive for AMA-M2. Type, AMA-M2 negative PBC clinical lack of specific diagnostic markers, often lead to missed diagnosis.
  • the present invention finds that the anti-human multimer immunoglobulin receptor antibody level in the serum of PBC patients (no matter AMA-M2 positive or negative) significantly increases, which provides a new method for the diagnosis of PBC, especially for the diagnosis of AMA-M2 negative PBC patients. of markers.
  • the anti-human polymeric immunoglobulin receptor antibody a biomarker used in the diagnosis of PBC discovered in the present invention, is used in clinical application as the peripheral blood of the subject, which is simple, less traumatic to the subject, and easy to promote.
  • the present invention reveals a common pathogenesis of AMA-M2 positive and negative PBC patients, that is, small and medium-sized intrahepatic bile duct damage mediated by anti-human multimeric immunoglobulin receptor antibodies, which can greatly reduce the missed diagnosis of PBC And misdiagnosis, especially the missed diagnosis and misdiagnosis of AMA-M2 negative PBC patients.
  • Figure 1 Multiple immunohistochemical staining to confirm the expression and localization of human multimeric immunoglobulin receptor in the liver of normal control patients;
  • Figure 2 Multiple immunohistochemical staining to confirm the expression and localization of human multimeric immunoglobulin receptor in the liver of AMA-M2 positive patients with primary biliary cholangitis;
  • Figure 3 Multiplex immunohistochemical staining to confirm the expression and localization of human multimeric immunoglobulin receptor in the liver of patients with primary biliary cholangitis AMA-M2 negative;
  • Figure 4 Immunofluorescence staining to confirm the expression and localization of human multimeric immunoglobulin receptor in the liver of patients with obstructive cholestasis;
  • Figure 5 Immunofluorescence staining to confirm the expression and localization of human multimeric immunoglobulin receptor in the liver of patients with secondary sclerosing cholangitis;
  • Figure 7 Enzyme-linked immunosorbent assay to measure the level of anti-human multimer immunoglobulin receptor antibody in the serum of normal controls, primary biliary cholangitis, and patients with obstructive cholestasis.
  • liver tissue and serum samples of the research subjects were obtained from the First affiliated Hospital of the Army Military Medical University (Southwest Hospital), and the acquisition of the samples was approved by the First affiliated Hospital of the Army Military Medical University.
  • Example 1 To clarify the expression and localization of human multimeric immunoglobulin receptors in the liver tissues of CTR, PBC, OC, SSC and NASH patients.
  • liver tissue samples remaining after surgical resection or clinicopathological diagnosis of patients with CTR, PBC, OC, SSC and NASH in the inpatient department of Southwest Hospital was collected.
  • the liver tissue samples were fixed with paraformaldehyde, dehydrated and transparent, embedded in wax and then sliced. The thickness of the sliced tissue was 4um.
  • Multiple immunohistochemical fluorescence technology was used to detect the expression and localization of human multimeric immunoglobulin receptors in the liver tissue of the subject.
  • the reagent used was a multiple immunohistochemical fluorescence kit (Absin, abs50014). The specific steps were as follows:
  • Endogenous peroxidase blocking add endogenous peroxidase blocking agent (Zhongshan Jinqiao, SP-9000) dropwise, incubate at room temperature for 10 minutes; soak in TBST for 3 minutes and repeat twice.
  • endogenous peroxidase blocking agent Zhongshan Jinqiao, SP-9000
  • Secondary antibody incubation drop the secondary antibody working solution solution, immerse the sample area, and incubate at room temperature for 10 minutes. Afterwards, soak and wash the slides with TBST for 3 min, and repeat 2 times.
  • Antigen restoration Tris-EDTA microwave restoration with high heat for 2 minutes, medium-high heat for 13 minutes, airing for about 30 minutes to room temperature; soaking in sterilized water for 3 minutes; soaking in TBST for 3 minutes.
  • the present invention proves by above experimental procedure, compared with normal control (Fig. 1), regardless of AMA-M2 positive (Fig. 2) or negative (Fig. 3) in patients with primary biliary cholangitis, human multimeric immunoglobulin is affected.
  • the expression of the body in the small and medium bile ducts in the liver was significantly reduced, and the patients with obstructive cholestasis (Figure 4), secondary sclerosing cholangitis ( Figure 4) and nonalcoholic fatty liver disease ( Figure 6) were similar to normal controls , human multimeric immunoglobulin receptors are expressed in biliary epithelial cells.
  • Example 2 Determining the expression of serum anti-human multimeric immunoglobulin receptor antibodies in patients with CTR, PBC, and OC.
  • Peripheral blood samples from 12 CTR, 17 PBC, and 15 OC patients were collected from the inpatient department of Southwest Hospital, and the expression of anti-human multimeric immunoglobulin receptor antibody in the serum of the subjects was detected by enzyme-linked immunosorbent assay (ELISA) , the reagent used is human multimer immunoglobulin receptor antibody ELISA kit (Lun Changshuo Bio, 100712), and the specific steps are:
  • Microwell microtiter plate standard, sample diluent, detection antibody-HRP, 20 ⁇ wash buffer, substrate A, substrate B, stop solution, sealing film, microplate reader (450nm), high-precision loading Device and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL, 37°C thermostat, distilled or deionized water.
  • Dilution of 20 ⁇ washing buffer distilled water was diluted 1:20, that is, 1 part of 20 ⁇ washing buffer plus 19 parts of distilled water.
  • HRP horseradish peroxidase
  • the present invention proves through the above experimental steps that compared with normal controls and patients with obstructive cholestasis, regardless of whether AMA-M2 is positive or negative, the anti-human multimer immunoglobulin receptor antibody in the serum of patients with primary biliary cholangitis Significantly increased ( Figure 7), suggesting that the anti-human multimeric immunoglobulin receptor antibody mediates the damage of cholangiocytes by targeting the human multimeric immunoglobulin receptor antigen on the small and medium bile ducts in the liver, so the serum anti-human Multimeric immunoglobulin receptor antibody can be used as a molecular marker for the diagnosis of primary biliary cholangitis.

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Abstract

一种原发性胆汁性胆管炎诊断用分子标志物及其应用，将抗人多聚体免疫球蛋白受体抗体作为原发性胆汁性胆管炎诊断用分子标志物，以此来制备诊断用试剂。在PBC的发病中，血清抗人多聚体免疫球蛋白受体抗体特异性靶向肝内中小胆管上的人多聚体免疫球蛋白受体，从而导致中小胆管的损伤。通过检测血清抗人多聚体免疫球蛋白受体抗体水平为PBC的特异性诊断提供了新的手段。

Description

一种原发性胆汁性胆管炎诊断用分子标志物及其应用 技术领域
本发明属于分子生物学技术领域，涉及一种血清抗人多聚体免疫球蛋白受体抗体，具体来说是血清抗人多聚体免疫球蛋白受体抗体作为原发性胆汁性胆管炎诊断用分子标志物及其应用。
背景技术
原发性胆汁性胆管炎(Primary biliary cholangitis,PBC)是一种慢性胆汁淤积性肝病，其特征为中小型肝内胆管自身免疫性破坏，导致进行性胆汁淤积，可缓慢发展为肝硬化和肝衰竭。目前本病全世界每年新诊断病例超过10万例，其主要发生于女性，40岁以上的女性中患病率可能高达0.1％。既往研究表明，遗传和环境因素、免疫失调等多种因素参与了PBC的发病机制，其中线粒体抗原免疫耐受的丧失以及随后出现的涉及体液和细胞免疫的自身免疫反应在PBC发病中发挥至关重要的作用。PBC的自身免疫性特征在于B细胞对线粒体丙酮酸脱氢酶复合物E2亚单位(PDC-E2)的耐受性丧失，导致抗线粒体自身抗体(Antimitochondrial antibodies,AMAs)的产生及自身反应性CD4+与CD8+T激活，而胆管上皮细胞是免疫攻击的目标。然而，一些PBC患者AMA-M2亚型呈阴性，提示PBC并不总是依赖于AMAs介导的特异性免疫反应，且PDC-E2亦在肝细胞中广泛表达，无论是否存在AMAs，为什么自身免疫反应会优先破坏PBC患者中小型肝内胆管目前尚未明确。
Fc受体家族成员人多聚体免疫球蛋白受体是粘膜免疫系统的核心成员,其在黏膜上皮细胞中广泛表达，通常在病毒或细菌感染时被促炎细胞因子诱导上调。人多聚体免疫球蛋白受体可与多聚IgA和多聚IgM特异性结合,通过穿胞转运,将它们从上皮细胞基底侧膜转运到顶膜,并最终分泌到外分泌液中，形成抵抗感染的第一道防线。近年来许多研究表明肝脏人多聚体免疫球蛋白受体高表达是肝纤维化的危险因素，可能与肝炎病毒感染和肝星状细胞转分化有关，人多聚体免疫球蛋白受体还可促进人肝细胞癌(Hepatocellular carcinoma,HCC)的转移，然而人多聚体免疫球蛋白受体是否参与PBC的发生发展及抗 人多聚体免疫球蛋白受体抗体对PBC诊断的价值目前尚未见报道，该指标亦尚未在临床上得到应用。
血清AMAs及其M2亚型是目前临床上PBC重要的辅助诊断指标，然而该指标存在一定的误诊与漏诊比例，故PBC的诊断仍然主要依赖肝组织活检。寻找取材简单可靠的诊断手段与筛选敏感的分子指标是临床上早期确诊PBC亟待解决的重要问题。
发明内容
本发明通过多重免疫组化荧光技术，以胆管细胞标志物细胞角蛋白19(Cytokeratin 19,CK19)为参照，检测AMA-M2阳性PBC、AMA-M2阴性PBC、正常对照(Control,CTR)、梗阻性胆汁淤积(Obstructive cholestasis,OC)、继发性硬化性胆管炎(Secondary sclerosing cholangitis,SSC)及非酒精性脂肪性肝炎(Nonalcoholic steatohepatitis,NASH)患者肝组织中人多聚体免疫球蛋白受体的表达与定位，发现在CTR、OC、SSC及NASH患者中人多聚体免疫球蛋白受体均表达于胆管细胞，但对于PBC患者，无论AMA-M2阳性还是阴性，人多聚体免疫球蛋白受体在肝内中小胆管上的表达均显著降低，基于以上发现，我们猜想在PBC发病过程中，人多聚体免疫球蛋白受体可能为介导免疫损伤的特异性抗原。本发明运用酶联免疫吸附实验(Enzyme linked immunosorbent assay,ELISA)检测CTR、PBC、OC患者的血清，证实了无论AMA-M2阳性还是阴性，PBC患者血清中抗人多聚体免疫球蛋白受体抗体均显著升高，提示抗人多聚体免疫球蛋白受体抗体通过靶向肝内中小胆管上的人多聚体免疫球蛋白受体抗原介导胆管细胞的损伤，故血清抗人多聚体免疫球蛋白受体抗体可作为PBC诊断用分子标志物。目前临床上将检测抗AMA-M2抗体阳性作为诊断PBC的标准之一，易导致抗AMA-M2抗体阴性PBC患者漏诊。故检测抗人多聚体免疫球蛋白受体抗体有助于PBC诊断，特别是AMA-M2阴性PBC患者的诊断。
鉴于现有对患者创伤小且可靠的诊断手段的缺乏，特别是针对抗AMA-M2抗体阴性PBC患者漏诊或误诊的情况，本发明的目的是提供一种PBC诊断用分子标志物及其应用，即血清抗人多聚体免疫球蛋白受体抗体作为PBC诊断用分子标志物。
为了实现上述目的，本发明采用的技术方案是：
一种原发性胆汁性胆管炎诊断用分子标志物，为抗人多聚体免疫球蛋白受体抗体。
本发明还提供一种抗人多聚体免疫球蛋白受体抗体在制备用于诊断PBC患者的试剂中的应用。
进一步，所述PBC患者包括抗AMA-M2抗体阳性PBC患者和抗AMA-M2抗体阴性PBC患者。
进一步，所述诊断PBC患者的试剂用于检测样本中抗人多聚体免疫球蛋白受体抗体的表达水平，所述样本为人血清样本。
进一步，所述诊断PBC患者的试剂为ELISA试剂盒。所述ELISA试剂盒包括微孔酶标板，标准品，样本稀释液，辣根过氧化物酶标记的小鼠抗人多聚体免疫球蛋白单克隆抗体(检测抗体-HRP)，20x洗涤缓冲液，底物A，底物B，终止液，封板膜，自封袋。
进一步，所述ELISA试剂盒的检测步骤包括：
从全血标本中提取血清，作为待测样本；
设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50μL，样本孔中加入待测样本50μL；空白孔不加；
除空白孔外，标准品孔和样本孔中每孔加入检测抗体(小鼠抗人多聚体免疫球蛋白单克隆抗体)-HRP 100μL，用封板膜封住反应孔，37℃温育60min；
用洗涤液洗板5次；
每孔加入底物A、B各50μL，37℃避光孵育15min；
每孔加入终止液50μL，15min内，在450nm波长处测定各孔的OD值；
以所测标准品的OD值为横坐标，标准品的浓度值为纵坐标，绘制标准曲线，并得到直线回归方程，将样品的OD值代入方程，计算出样品的浓度。
与现有技术比较本发明的有益效果是：
1.本发明发现抗人多聚体免疫球蛋白受体抗体可作为PBC诊断用的生物标志物。血清AMA-M2是诊断PBC的特异性指标，约90-95％PBC患者AMA-M2阳性；根据AMA-M2是否阳性，PBC分为 AMA-M2阳性的PBC与AMA-M2阴性的PBC两种亚型，AMA-M2阴性的PBC临床缺乏特异性诊断标志物，常导致漏诊。本发明发现PBC患者(无论AMA-M2阳性还是阴性)血清中抗人多聚体免疫球蛋白受体抗体水平显著升高，为PBC的诊断，特别是AMA-M2阴性PBC患者的诊断提供了新的标志物。
2.本发明发现的用于诊断PBC的生物标志物抗人多聚体免疫球蛋白受体抗体在临床应用中取材为受试者外周血，取材简单，对受试者创伤小，容易推广。
3.本发明揭示了一种AMA-M2阳性及阴性PBC患者的共同的发病机制，即抗人多聚体免疫球蛋白受体抗体介导的中小型肝内胆管损伤，可以大大减少PBC的漏诊和误诊，特别是AMA-M2阴性PBC患者的漏诊和误诊。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1多重免疫组化荧光染色明确正常对照患者肝脏人多聚体免疫球蛋白受体的表达与定位；
图2多重免疫组化荧光染色明确原发性胆汁性胆管炎AMA-M2阳性患者肝脏人多聚体免疫球蛋白受体的表达与定位；
图3多重免疫组化荧光染色明确原发性胆汁性胆管炎AMA-M2阴性患者肝脏人多聚体免疫球蛋白受体的表达与定位；
图4免疫荧光染色明确梗阻性胆汁淤积患者肝脏人多聚体免疫球蛋白受体的表达与定位；
图5免疫荧光染色明确继发性硬化性胆管炎患者肝脏人多聚体免疫球蛋白受体的表达与定位；
图6免疫荧光染色明确非酒精性脂肪性肝炎肝脏人多聚体免疫球蛋白受体的表达与定位；
图7酶联免疫吸附实验测定正常对照、原发性胆汁性胆管炎、梗 阻性胆汁淤积患者血清中抗人多聚体免疫球蛋白受体抗体的水平。
具体实施方式
下面将结合本发明实施例及说明书附图，对本发明中的相关技术进行清楚、完整的描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，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，都属于本发明保护的范围。
在以下实施例中，研究对象的肝组织与血清样本均来自陆军军医大学附属第一医院(西南医院)，样本的获取均通过陆军军医大学附属第一医院伦理审批。
实施例1：明确CTR、PBC、OC、SSC及NASH患者肝组织中人多聚体免疫球蛋白受体的表达与定位。
收集西南医院住院部CTR、PBC、OC、SSC及NASH患者手术切除或临床病理诊断后剩余的肝脏组织样本各1例。对肝组织样本进行多聚甲醛固定、脱水透明、浸蜡包埋后进行切片，切片组织厚度为4um。采用多重免疫组化荧光技术检测受试者肝组织中人多聚体免疫球蛋白受体的表达与定位，所用试剂为多重免疫组化荧光试剂盒(Absin，abs50014)，具体步骤为：
(1)脱蜡至水：石蜡切片65度烘箱烤片1h；新鲜二甲苯浸片5min,重复3次；梯度乙醇浸片：100％乙醇10min重复2次，95％乙醇10min重复2次；灭菌水洗片5min,重复两次。
(2)抗原修复：Tris-EDTA微波修复高火2min，中高火13min，晾30min左右至室温；灭菌水浸洗3min；TBST浸洗3min。
(3)内源性过氧化物酶阻断：滴加内源性过氧化物酶阻断剂(中杉金桥，SP-9000)，室温孵育10min；TBST浸洗3min重复2次。
(4)封闭：8％山羊血清(Absin，abs933)室温孵育10min。
(5)一抗孵育：抗细胞角蛋白19抗体(Abcam，ab52625)用8％山羊血清按1:500稀释，室温保湿孵育1h。TBST浸洗玻片3min,重复2次。
(6)一抗孵育期间将二抗工作液平衡至室温。
(7)二抗孵育：滴加二抗工作液溶液，浸没样本区域，室温保 湿孵育10min。后用TBST浸洗玻片3min，重复2次。
(8)荧光染色放大信号：用信号放大液按1:100稀释HRP 520染料，用移液器在玻片上滴加染料浸没样本区域，室温保湿孵育10min。后用TBST浸洗玻片，室温浸片3min重复3次。
(9)抗原修复：Tris-EDTA微波修复高火2min，中高火13min，晾30min左右至室温；灭菌水浸洗3min；TBST浸洗3min。
(10)封闭：8％山羊血清室温孵育10min。
(11)一抗孵育：抗人多聚体免疫球蛋白受体抗体(Proteintech,22024-1-AP)用8％山羊血清按1:500稀释，室温保湿孵育1h；用TBST浸洗玻片3min,重复2次。一抗孵育期间将二抗工作液平衡至室温。
(12)二抗孵育：滴加二抗工作液溶液，浸没样本区域，室温保湿孵育10min；后用TBST浸洗玻片3min重复2次。
(13)荧光染色放大信号：用信号放大液按1:100稀释HRP 570染料，用移液器在玻片上滴加染料浸没样本区域。室温保湿孵育10min。TBST浸洗玻片，室温浸片3min重复3次
(14)染核及封片：滴加1×DAPI(用ddH2O稀释按1：100)工作液到样品上，室温孵育5min。用TBST浸洗玻片3次，每次2min。滴加抗荧光淬灭封片剂，用盖玻片封片。对染色后的组织片在共聚焦显微镜下观察并分析。
本发明通过以上实验步骤证明，与正常对照(图1)相比，原发性胆汁性胆管炎患者无论AMA-M2阳性(图2)还是阴性(图3)，人多聚体免疫球蛋白受体在肝内中小胆管上的表达均显著降低，而梗阻性胆汁淤积(图4)、继发性硬化性胆管炎(图4)及非酒精性脂肪性肝病(图6)患者与正常对照相似，人多聚体免疫球蛋白受体均表达于胆管上皮细胞。
实施例2：明确CTR、PBC、OC患者血清抗人多聚体免疫球蛋白受体抗体的表达。
收集西南医院住院部12例CTR、17例PBC、15例OC患者的外周血标本，采用酶联免疫吸附试验(ELISA)检测受试者血清中抗人多聚体免疫球蛋白受体抗体的表达，所用试剂为人多聚体免疫球蛋白受体抗体ELISA试剂盒(仑昌硕生物，100712)，具体步骤为：
(1)取血清：
将收集于血清分离管的全血标本在室温放置2小时或4℃过夜，然后1000×g离心20分钟，取上清即可，或将上清置于-20℃或-80℃保存，避免反复冻融。
(2)试剂及器材准备：
微孔酶标板，标准品，样本稀释液，检测抗体-HRP，20×洗涤缓冲液，底物A，底物B，终止液，封板膜，酶标仪(450nm)，高精度加样器及枪头：0.5-10uL、2-20uL、20-200uL、200-1000uL，37℃恒温箱，蒸馏水或去离子水。
20×洗涤缓冲液的稀释：蒸馏水按1：20稀释，即1份20×洗涤缓冲液加19份蒸馏水。
(3)操作步骤
从室温平衡20min后的铝箔袋中取出所需板条，剩余板条用自封袋密封放回4℃。
设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50μL，样本孔中加入待测样本50μL；空白孔不加。
除空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶(HRP)标记的检测抗体(小鼠抗人多聚体免疫球蛋白单克隆抗体)-HRP 100μL，用封板膜封住反应孔，37℃水浴锅或恒温箱温育60min。
弃去液体，吸水纸上拍干，每孔加满洗涤液(350μL)，静置1min，甩去洗涤液，吸水纸上拍干，如此重复洗板5次(也可用洗板机洗板)。
每孔加入底物A、B各50μL，37℃避光孵育15min。
每孔加入终止液50μL，15min内，在450nm波长处测定各孔的OD值。
(4)结果计算
以所测标准品的OD值为横坐标，标准品的浓度值为纵坐标，绘制标准曲线，并得到直线回归方程，将样品的OD值代入方程，计算出样品的浓度。
本发明通过以上实验步骤证明，与正常对照和梗阻性胆汁淤积患者相比，无论AMA-M2阳性还是阴性，原发性胆汁性胆管炎患者血清中抗人多聚体免疫球蛋白受体抗体均显著升高(图7)，提示抗人 多聚体免疫球蛋白受体抗体通过靶向肝内中小胆管上的人多聚体免疫球蛋白受体抗原介导胆管细胞的损伤，故血清抗人多聚体免疫球蛋白受体抗体可作为原发性胆汁性胆管炎诊断用分子标志物。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思做出诸多修改变化。凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等，均包含在本发明的保护范围内。

Claims (8)

  1. 一种原发性胆汁性胆管炎诊断用分子标志物，其特征在于：所述标志物为抗人多聚体免疫球蛋白受体抗体。
  2. 权利要求1所述抗人多聚体免疫球蛋白受体抗体在制备用于诊断PBC患者的试剂中的应用。
  3. 根据权利要求2所述应用，其特征在于：所述PBC患者包括抗AMA-M2抗体阳性PBC患者和抗AMA-M2抗体阴性PBC患者。
  4. 根据权利要求2或3所述应用，其特征在于：所述诊断PBC患者的试剂用于检测样本中抗人多聚体免疫球蛋白受体抗体的表达水平。
  5. 根据权利要求4所述应用，其特征在于：所述样本为人血清样本。
  6. 根据权利要求4所述应用，其特征在于：所述诊断PBC患者的试剂为ELISA试剂盒。
  7. 根据权利要求6所述应用，其特征在于：所述ELISA试剂盒包括微孔酶标板，标准品，样本稀释液，辣根过氧化物酶标记的小鼠抗人多聚体免疫球蛋白单克隆抗体(检测抗体-HRP)，20x洗涤缓冲液，底物A，底物B，终止液，封板膜，自封袋。
  8. 根据权利要求7所述应用，其特征在于：所述ELISA试剂盒的检测步骤包括：
    从全血标本中提取血清，作为待测样本；
    设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50μL，样本孔中加入待测样本50μL；空白孔不加；
    除空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶标记的检测抗体(小鼠抗人多聚体免疫球蛋白单克隆抗体)100μL，用封板膜封住反应孔，37℃温育60min；
    用洗涤液洗板5次；
    每孔加入底物A、B各50μL，37℃避光孵育15min；
    每孔加入终止液50μL，15min内，在450nm波长处测定各孔的OD值；
    以所测标准品的OD值为横坐标，标准品的浓度值为纵坐标，绘 制标准曲线，并得到直线回归方程，将样品的OD值代入方程，计算出样品的浓度。
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