血清抗线粒体抗体亚型检测对PBC患者的诊断价值

目的:探讨原发性胆汁性肝硬化（PBC）患者出现的抗线粒体抗体（AMA）亚型与PBC患者肝功能、免疫检查的相关性。方法:应用间接免疫荧光法检测47例PBC患者血清中AMA抗体,用免疫印迹法检测AMA-M₂、M₄、M₉抗体。应用全自动生化分析仪检测ALT等生化指标。结果::①47例PBC患者AMA与AMA-M₂阳性率100%,M₂+M₄阳性率43.8%,M₂+M₉阳性率16.7%,M₂+M₄+M₉阳性率13.5%。②PBC患者ALP、γ-GT、GLB等生化指标发生明显变化（P<0.05）。③M₄阳性与M₄阴性PBC患者比较ALT、AST、GLB等指标明显升高（P<0.05）。M₉阳性与M₉阴性PBC患者比较ALT、AST、GLB等指标明显降低（P<0.05）。结论:AMA-M₂亚型抗体检测对PBC有诊断价值,生化、免疫等实验室检查有助于诊断PBC。M₄、M₉抗体的检测,对于PBC的病情判断有意义。     

原发性胆汁性肝硬化患者血清抗线粒体抗体与抗核抗体分析及其临床意义

目的 探讨线粒体抗体(AMA)阴性和阳性的PBC患者抗线粒体抗体M2亚型(AMA-M2)阳性率及其相伴抗核抗体(ANA)核型,抗核抗体谱的不同免疫球蛋白抗体分布频率.方法 收集北京地坛医院2014年12月至2016年2月就诊的122例AMA阳性(AMA+)和46例AMA阴性(AMA-)PBC患者及224例非PBC患者(48例自身免疫性肝炎(AIH)、83例慢性乙型肝炎(CHB)、80例慢性丙型肝炎、13例酒精性肝病),采用间接免疫荧光法检测抗核抗体,免疫印迹法检测抗核抗体谱,免疫斑点法检测AMA-M2.结果 122例AMA(+)PBC患者中AMA-M2阳性率96.7％;46例AMA(-) PBC患者中AMA-M2阳性率17.4％.AMA阳性PBC患者的核膜型,核点型,着丝点型,核颗粒型阳性率分别是20.5％,7.4％,21.3％,40.2％;高于非PBC组的1.8％,0.8％,2.2％,13.8％(P均＜0.01);AMA阴性PBC患者的核膜型,核点型,着丝点型,核颗粒型阳性率分别是17.4％,10.8％,19.5％,45.6％;高于非PBC组的1.8％,0.8％,2.2％,13.8％(P均＜0.01).结论 AMA与AMA-M2及ANA部分核型的检测有助于PBC特别是AMA阴性PBC患者的诊断.     

原发性胆汁肝硬化患者血清中抗线粒体抗体及其亚型和抗核抗体的联合检测的临床价值

为了分析原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)患者的抗线粒体抗体(anti-mitochondria antibody,AMA)及其M2、M4、M9亚型、抗核抗体(antinuclear antibody,ANA)的阳性率。应用间接免疫荧光法检测ANA、AMA,免疫斑点法检测AMA M2、M4、M9。结果显示91例PBC患者中AMA有89例为阳性。其中96.7%(88/91)的患者M2型阳性,45.1%(41/91)M4型阳性,2.2%(2/91)M9型阳性。39/91例患者ANA阳性,其中21例为核膜型。ANA、AMA及其M2、M4、M9亚型联合检测对于PBC患者的诊断有重要价值。     

中国西部自身免疫性肝病患者自身抗体的分布特征

目的: 探讨中国西部自身免疫性肝病患者中相关自身抗体的存在状况及特征.方法: 57例自身免疫性肝病患者分为3组: 自身免疫性肝炎(AIH)12例、原发性胆汁性肝硬化(PBC)32例、原发性硬化性胆管炎(PSC)13例.用间接免疫荧光法检测抗核抗体(ANA)、平滑肌抗体(SMA)、抗肝肾微粒抗体1型抗体(anti-LKM1)、抗线粒体抗体(AMA)和抗中性粒细胞胞质抗体(ANCA),Western blot检测抗肝细胞胞溶质抗原1型抗体(anti-LC1)、抗可溶性肝抗原/肝胰抗原抗体(anti-SLA/LP)、抗肝肾微粒抗体1型(anti-LKM1)、 AMA-M2亚型等多种肝抗原自身抗体.结果: 57例中ANA、AMA、M-2、pANCA阳性率在组间有统计学差异(P<0.01).PBC中AMA、M-2阳性检出率均为100%, PSC中pANCA阳性检出率为53.8%, Fisher精确检验在α'=0.002水准与其他各组比较有统计学差异.AIH与PBC的ANA阳性率分别为100%和50%,Fisher精确检验在α'=0.002水准二者无统计学意义,与其他各组比较有明显差异.在AIH组SMA阳性率为25%,LKM-1、LC-1、SLA/LP阳性率均为8.3%, 与其他组无统计学意义,可能与病例少有关.PBC中分别有1例患者ANA、SMA以及ANA、LKM-1同时阳性, PSC中有1例ANA、SLA/LP同时阳性,此3例患者结合性别、生化、自身抗体等资料符合AIH诊断条件;AIH中有1例M-2阳性综合各项资料符合PBC(重叠综合征).结论: 肝抗原自身抗体、ANA、AMA及M-2亚型的检测有助于自身免疫性肝病的诊断.对肝炎病毒血清标志物阴性的肝功能异常者应该行肝抗原自身抗体检测协助诊断.     

新型的抗线粒体抗体M2亚型抗体的检测方法在原发性胆汁性肝硬化诊断中的可靠性分析

目的研究以天然M2抗原和BPO融合蛋白M2-3E（BPO）为靶抗原的ELISA法（抗-M2-3EELISA）检测抗线粒体抗体M2亚型（AMA-M2）抗体在原发性胆汁化肝硬化（PBC）诊断中的可靠性。方法分别用间接免疫荧光法（IFL）、以丙酮酸脱氢酶复合体（PDC）为靶抗原的ELISA法（抗-PDCELISA）、以三联体（BPO）为靶抗原的ELISA法（抗-3EELISA）、以天然M2抗原和BPO融合蛋白M2-3E（BPO）为靶抗原的ELISA法（抗-M2-3EELISA）检测107例PBC、167例疾病对照和30例正常对照血清中的AMA-M2抗体。结果107例PBC患者用抗-M2-3EELISA、抗-3EELISA、抗-PDCELISA和IFL4种方法检测AMA-M2的检出率（敏感性）分别为99/107（92.5%）、94/107（87.9%）、78/107（72.9%）、87/107（81.3%）,特异性分别为97.3%、98.5%、98.5%、97.3%。抗-M2-3EELISA法AMA-M2的检出率（92.5%）显著高于抗-PDCELISA法（72.9%）（P〈0.001）、IFL法（81.3%,P〈0.001）,高于抗-3EELISA（87．9％）。在20例IFL检测AMA—M2为阴性的PBC患者中,用抗-M2—3EELISA法AMA—M2的检出率为11／20（55％）。抗-M2-3EELISA和IFL的重叠度为85．0％（91／107）。结论抗-M2-3EELISA法具有比IFL、抗-PDCELISA法和抗-3EELISA更高的敏感性,特异性达97-3％。因此,抗-M2-3EELISA法可作为第一轮AMA-M2的筛查,但不可以单独用于诊断PBC的标记。     

原发性胆汁性肝硬化患者共刺激分子B7-H4的检测及临床意义

目的 探讨原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)患者共刺激因子B7-1t4的表达及其与疾病发病机制的关系.方法 分别采用荧光实时定量PCR法(real-time PCR)、酶联免疫吸附试验(ELISA)及流式细胞术(FCM)检测65名PBC患者外周血单个核细胞(PBMC)B7-H4mRNA表达水平、血清IL-2水平以及CD4+、CD8+ T细胞亚群和T细胞表面B7-H4表达百分率,同时监测抗线粒体抗体(anti-mitochondrial antibody,AMA)及临床各项生化指标的关系.结果 (1)PBC患者PBMC B7-144 mRNA水平及T细胞表面B7-H4表达百分率显著低于非PBC肝硬化组及健康对照组(P<0.01).(2)活化72 h后各实验组及对照组IL-2含量及CD4+、CD8+、CD4+ CD8+T淋巴细胞表达水平均低于活化前,以非PBC肝硬化组与健康对照组降低显著(P<0.05);PBC组IL-2含量及CD4+、CD4+ CD8+ T淋巴细胞表达水平高于非PBC肝硬化组与健康对照组(P<0.01).(3)AMA-M2阳性患者血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)及γ-谷氨酰转肽酶(GGT)水平升高,其中ALP及GGT升高显著(P<0.05);AMA-M2阳性患者与阴性患者T细胞表面B7-H4表达百分率差异无统计学意义(P>0.05).结论 共刺激因子B7-H4对PBC患者体内T细胞活化增殖及细胞因子分泌的抑制作用减弱,为研究PBC的发生发展和阐明PBC的发病机制提供了试验资料.     

抗核抗体和抗线粒体抗体对干燥综合征和原发性胆汁性肝硬化鉴别诊断分析

目的分析干燥综合征和原发性胆汁性肝硬化患者抗核抗体核型和抗线粒体抗体核型分布特征。方法收集143例干燥综合征(SS)和120例原发性胆汁性肝硬化(PBC)患者血清;抗核抗体(ANA)和抗线粒体抗体(AMA)采用间接免疫荧光法检测。结果 SS患者中ANA阳性率(83.9%)明显高于PBC(72.5%)(P0.05),而PBC中AMA阳性率(88.3%)明显高于SS(14.0%)(P0.05)。斑点型和着丝点型(阳性率分别为55.9%和18.2%)是SS主要核型,对SS阳性预测值分别为86%和48%,阴性预测值分别为63%和56%;着丝点型和核模型(阳性率分别为23.3%和20.8%)是PBC主要核型,对PBC阳性预测值为52%和100%,阴性预测值均为60%。高滴度(≥1∶1 000)的ANA在ANA阳性的SS中占57.5%,PBC中占42.5%,两者差异显著(P0.05),而高滴度的AMA在AMA阳性PBC患者(67.0%)明显高于SS(40%)(P0.05)。结论 ANA和AMA的检测可用于对SS和PBC的鉴别诊断。     

抗Ro-52抗体在自身免疫性肝病中的检测

目的 探讨抗Ro-52抗体对自身免疫性肝病(autoimmune liver disease,AILD)的临床意义.方法 对采用免疫印迹法检测抗Ro-52抗体的115例AILD患者的临床资料进行回顾性分析,比较抗Ro-52阳性和阴性MLD患者肝功和免疫学指标,对可能有相关性的血清学指标进行诊断试验一致性评价.结果 抗Ro-52抗体在自身免疫性肝炎(autoimmune hepatitis,AIH)(37例)、原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)(57例)、MH/PBC重叠综合征组(21例)的阳性率分别为32.43%、24.56%、33.33%,差异无统计学意义(x2=0.949,P>0.05).抗可溶性肝抗原/肝胰抗原抗体(anti-soluble liver antigen/liver-pancreas,anti-SLA/LP)在抗Ro-52阳性AIH组频率(58.33%)高于阴性组(16.00%)(x2=6.955,P<0.05),抗SLA/LP抗体在抗R0-52阳性AIH/PBC重叠综合征组频率(85.71%)高于阴性组(28.57%)(x2=6.109,P<0.05).抗Ro-52抗体和抗SLA/LP抗体结果有一致性(κ=0.466,P<0.05).AIH/PBC重叠综合征组抗Ro-52阳性患者IgG水平高于阴性患者(t=2.508,P<0.05).结论 抗Ro-52抗体在AIH、PBC和MH/PBC重叠综合征中的分布没有差别;抗Ro-52抗体与抗SLA/LP抗体检测结果有一致性;抗Ro-52抗体阳性MH/PBC重叠综合征患者IgG水平高于抗体阴性者.     

原发性胆汁性肝硬化患者抗着丝点抗体的检测

本文以大鼠肝组织切片和人喉头癌细胞HE_p-2作为核标本,用间接荧光抗体法(IF)检测原发性胆汁性肝硬化(PBC)患者血清中抗核抗体(ANA),发现同其他疾病比较,PBC血清中抗着丝点抗体(ACA)阳性率高。实验以21例PBC患者为对象,对照组为类狼疮肝炎(LH)9例、肝硬化(LC)9例、全身性红斑狼疮(SLE)7例、混合性结缔组织病(MCTD)10例、健康人10例。PBC等肝病患者抗线粒体抗体(AMA)均为阳性。ANA用IF法检测,抗     

58例原发性胆汁性肝硬化患者自身抗体特征分析

目的：探讨自身抗体对原发性胆汁性肝硬化（PBC）患者的诊断应用价值。方法：用间接免疫荧光、免疫印迹法检测了58例PBC患者抗核抗体（ANA）、抗平滑肌抗体（SMA）、抗线粒体抗体（AMA），并对AMAM2亚型及抗可溶性肝抗原/肝胰抗原（SLA/LP）、抗肝肾微粒体Ⅰ型（LKM-1）和抗肝特异性胞浆Ⅰ型抗体（LC-1）等肝脏疾病相关的自身抗体进行检测。结果：PBC患者自身抗体以AMA和AMAM2亚型为主。其阳性率分别为96.5%和93.1%。患者的抗体滴度均大于1∶100，其中有8例出现ANA和SMA，1例出现AMA和SLA/LP同时阳性，表现与Ⅰ型和Ⅲ型自身免疫性肝炎重叠。另有19例AMAM2阳性患者进行肝穿病理检查时，12例（63.7%）患者病理提示符合PBC诊断。结论：自身抗体对PBC有诊断意义，注重自身抗体的检测对明确自身免疫性肝病有重要的临床意义。     

Mitochondrial antigens and autoantibodies: from anti-M1 to anti-M9

Summary The specificity and clinical relevance of nine antimitochondrial antibodies (AMA) — anti-M1 to anti-M9 — are described. All nine AMA types react with antigens which are associated either with inner (M1, M2, M7) our outer mitochondrial membranes (M3, M4, M5, M6, M8, M9) derived from rat liver or beef heart mitochondria. These antigens can be clearly distinguished by their different physical and chemical properties. Anti-M1 to anti-M9 can be related to distinct clinical entities: anti-M1, anti-M5, and anti-M7 are found in nonhepatic disorders, such as syphilis (anti-M1), undefined collagen diseases (anti-M5), and some forms of cardiac diseases (anti-M7). Anti-M3 and anti-M6 are detected in drug-induced disorders, such as phenopyrazon-induced pseudolupus syndrome (PLE; anti-M3) and iproniazid-induced hepatitis (anti-M6). Anti-M2, anti-M4, anti-M8, and anti-M9 are confined to primary biliary cirrhosis (PBC).Anti-M2 is a specific marker for the diagnosis of PBC; 96% of PBC patients (n=752) were anti-M2 positive. Anti-M4 and anti-M8 seem to reflect disease activity. Anti-M9 antibodies occur preferentially in early PBC. The clinical course of PBC was analyzed with respect to four different AMA profiles: profile A: only anti-M9 positive in the ELISA; profile B: anti-M9 and anti-M2 positive in the ELISA; profile C: anti-M2 positive in ELISA and complement fixation test (CFT), but anti-M4 and anti-M8 positive only in the ELISA; and profile D: anti-M2, anti-M4, anti-M8 positive in ELISA and CFT. Patients with profile A und B were found to have a rather benign course while those patients with profile C and D showed a rather progressive course when followed over a period of 6–15 years.Considering the similarities between bacterial and mitochondrial membranes, it is suggested that the formation of AMA of different specificities in PBC, especially of the anti-M2 type, may be induced by cross-reacting antigens.Abbreviations ALAT Alanine aminotransferase - AMA Antimitochondrial antibodies - ANT Adenine nucleotide translocator - AP Alkaline phosphatase - ASAT Aspartate aminotransferase - ATPase Adenosinetriphosphatase - CAH Chronic active hepatitis - CFT Complement fixation test - DNA Deoxyribonucleic acid - ELISA Enzyme linked immunosorbent assay - ID Immunodiffusion - IFL Immunofluorescence test - PBC Primary biliary cirrhosis - PLE Pseudolupus syndrome - RNA Ribonucleic acid - SLE Systemic lupus erythematosus - SMP Submitochondrial particles - VDRL Veneral Disease Research Laboratory Dedicated to Prof. H.D. Waller on the occasion of his 60th birthday     

Characterization of a new mitochondrial antigen-antibody system (M9/anti-M9) in patients with anti-M2 positive and anti-M2 negative primary biliary cirrhosis.

A new antimitochondrial antibody (AMA) against an outer membrane associated antigen on liver mitochondria was detected by ELISA in sera from patients with primary biliary cirrhosis (PBC). This antibody was named anti-M9. There is evidence that it is a partial organ-specific antibody as shown by absorption studies using submitochondrial particles prepared from heart, liver and kidney. A purified M9-fraction was prepared by subjecting a 100,000 g supernatant from rat liver mitochondria to ion exchange chromatography. This fraction was devoid of the previously described M1-M8 antigens except for M4. Trypsin treatment of the fraction enabled a distinction to be made between M4 which was protease resistant, and M9 which was trypsin sensitive. Applying this M9-fraction in Western blotting anti-M9 positive sera recognized two proteins at a molecular weight of 98 kD and 59 kD. Anti-M9 antibodies were detected in 37% of 156 anti-M2 positive as well as in 82% of 22 anti-M2 negative patients with histologically proven PBC. It is concluded that anti-M9 is a new AMA type in PBC which may be helpful especially for the early diagnosis of PBC in patients who are still anti-M2 negative. As one of the earliest immunological signs in PBC further characterization of M9 could provide new insights into the etiopathogenesis of the disease.     

Positive rate of anti-mitochondrial antibody in Japanese corporate workers

Anti-mitochondrial antibody(AMA) has been reported to be detectable in approximately 85% of patients with primary biliary cirrhosis(PBC). Therefore, a test for AMA is acceptable to be essential for diagnosing PBC. However, the positive rate in Japanese general population has not yet been determined. We tested sera from 1,145 corporate workers who took an annual health check and evaluated the liver of AMA-positive subjects. An indirect immunofluorescence method was used for screening AMA. ELISA and immunoblotting method were used for detecting anti-M2 in AMA-positive cases. AMA was detected in 5 of 1,145(0.44%) corporate workers. AMA positive rate was higher in females than in males(0.91% and 0%, respectively) and the AMA-positive people are all females over age 40. All of the AMA-positive sera are also positive for Anti-M2. Liver biopsy was performed in two AMA-positive cases and the histology was compatible with PBC in both cases.     

Anti-M4 antibodies in primary biliary cirrhosis react with sulphite oxidase, an enzyme of the mitochondrial inter-membrane space.

Testing three anti-M2/anti-M4-positive and three anti-M2 positive/anti-M4-negative primary biliary cirrhosis (PBC) marker sera against different mitochondrial enzymes by ELISA it could be shown that only the anti-M4-positive sera reacted with pyruvate dehydrogenase (M2) and sulphite oxidase (SO), an enzyme of the mitochondrial inter-membrane space in parallel. Absorption of these sera with SO abolished completely the anti-M4 antibodies but had no effect on the anti-M2 activity. The specificity of this reaction was also documented by examining 30 anti-M2/anti-M4-positive sera showing that 28 of them were positive with SO. Among ten anti-M2/anti-M8-positive but anti-M4-negative PBC sera, four became positive when tested against SO, indicating a higher sensitivity of SO for the demonstration of anti-M4. Retesting sera from 76 PBC patients with defined anti-mitochondrial antibody (AMA) profiles who had been followed for up to 18 years against SO by ELISA and complement fixation test (CFT), none of 32 patients with profile A B (positive for anti-M2 and/or anti-M9 by ELISA; benign course) but 33 of 44 patients with profile C/D (anti-M2/anti-M4 and or anti-M8 positive by CFT and or ELISA; progressive course) were positive. These data indicate that sulphite oxidase can be used in the ELISA for the detection of anti-M4 antibodies which may be of prognostic relevance.     

Use of ATPase-associated antigen (M2) for detection of antimitochondrial antibodies in primary biliary cirrhosis by fluorometric immunoassay

An indirect binding assay, the fluorometric immunoassay (FIAX), was established for the detection of anti-M2 antibodies which are specific markers for primary biliary cirrhosis (PBC). Submitochondrial particles (SMP) from beef heart and rat liver and the ATPase-associated antigen (M2) were used. The antigens were fixed to a cellulose acetate surface, SMP at a concentration of 2 mg/ml, ATPase at a concentration of 0.2 mg/ml. Sera were used at 1:60 and 1:120 and bound antimitochondrial antibodies (AMA) were demonstrated by fluorescent isothiocyanate labelled monospecific anti-human IgG, IgM and IgA antibodies. The fluorescent signals were proportional to the AMA titre in the serum samples and were measured in a fluorometer (FIAX 100). Of 94 patients with PBC, 92 had AMA against SMP from beef heart compared with 76 in the complement fixation test (CFT) and 84 in the immunofluorescence test (IFL). Ninety reacted with the ATPase-associated M2 antigen. Sera from patients known to have AMA of different specificities (anti-M1, anti-M3, anti-M5, anti-M6) reacted with SMP from beef heart and/or rat liver but not with M2.     

Diagnostic Relevance and Clinical Significance of the New Enhanced Performance M2 (MIT3) ELISA for the Detection of IgA and IgG Antimitochondrial Antibodies in Primary Biliary Cirrhosis

Background/aims: Antimitochondrial antibodies (AMAs) are the serological hallmark of primary biliary cirrhosis (PBC). We evaluated the sensitivity and specificity of a new M2 enhanced performance enzyme-linked immunosorbent assay (ELISA) (MIT3) for the detection of IgG- and IgA-specific isotypes of AMA in PBC patients including a number of PBC patients negative for AMA by indirect immunofluorescence (IIF) as well as in patients with diverse, non-PBC disorders. We also investigated the clinical significance of IgG and IgA AMA in PBC. Methods: One hundred and three Greek PBC patients including 27 with AMA IIF-negative at the time of the investigation, 29 with autoimmune hepatitis-1 (AIH-1), 12 with primary sclerosing cholangitis (PSC), 26 with hepatitis C virus (HCV), 15 with hepatitis B virus (HBV), and 29 healthy were investigated for AMA (IgG and IgA) using the MIT3-based ELISAs (INOVA Diagnostics, San Diego, CA). The samples were also tested by conventional anti-M2 ELISA (INOVA Diagnostics, Inc.). Results: The IgG MIT3-based ELISA significantly increased AMA detection in the cohort of PBC patients, over 26% of whom were AMA IIF-negative, from 63.1% by the conventional anti-M2, and 73.7% by IIF to 79.6% by MIT3-based ELISA (p<0.001). IgA AMAs were detected in 47.6% patients. Overall, IgG/IgA AMAs were detected in 84/103 (81.6%). IgG MIT3-based ELISA detected 12/27 IIF AMA-negative samples (44.4%), while IgG/IgA MIT3-based ELISAs detected 13/27 IIF AMA-negative patients (48.1%). The specificities of MIT3-based ELISAs (IgG and IgA) were 82.8% and 89.7%, respectively, in AIH-1, 100% and 93.3%, respectively, in HBV, 100% in PSC, and 96% and 93.3%, respectively, in HCV. Patients positive for IgG AMA had significantly more severe disease as shown by worse histology and elevated biochemical markers; IgG and IgA AMA titers were associated positively with the Mayo risk score but none of the isotypes were able to predict disease outcome. Conclusions: The new IgG and IgA MIT3-based ELISAs seem to have higher specificity and sensitivity for AMA detection than IIF and the conventional anti-M2. Interestingly, these assays were able to unmask AMA presence in almost half of the AMA-negative samples by IIF. These findings may suggest the use of MIT3-based ELISAs as first-line investigation for AMA detection, particularly, when the laboratories are unfamiliar with the use and interpretation of the IIF patterns of AMA. The presence of IgG AMA seems to characterize PBC patients with more severe disease, but both IgG and IgA isotypes of AMAs were not predictive markers of disease outcome.