用重组丙酮酸脱氢酶复合物E2亚单位检测M2抗体及其临床意义

目的用重组表达的丙酮酸脱氢酶复合物E2亚单位（PDC-E2）检测M2抗体,以利于原发性胆汁性肝硬化（PBC）的早期诊断。方法采用重组表达的PDC—E2建立了免疫印迹法（IBT）和酶联免疫吸附试验（ELISA）。检测40例PBC患者血清的M2抗体,以其他肝病患者、自身免疫病患者、健康体检者作对照。结果40例PBC患者血清中检测出抗PDC—E2抗体阳性37例,阴性3例,阳性率为92．5％,而疾病对照组和健康体检者血清中该抗体检测均为阴性。结论用重组表达的人PDC-E2检测抗体有较好的敏感性和特异性,有助于PBC的临床诊断。     

利用重组人源靶抗原二联体OP检测M2抗体及临床意义

目的 利用重组抗原检测自身免疫病患者血清中抗OP自身抗体并探讨其临床意义。方法 经Ni-NTA柱纯化表达重组OP融合蛋白作为抗原，分别用免疫印迹法（IBT）和酶链免疫吸附试验（ELISA）检测70份PBC患者血清，80份其它肝病患者血清，80份自身免疫病患者血清和100份正常人血清。结果 70份PBC患者血清中检测出阳性患者62例，阴性8例，阳性率为87．6％。其它肝病患者血清、自身免疫病患者血清和正常人血清均为阴性。重组抗原检测M2抗体有一定的敏感性。结论 利用重组抗原OP检测M2抗体，有较好的敏感性及特异性，有助于PBC的临床诊断。     

用ELISA检测抗丙酮酸脱氢酶复合物E2和E3结合蛋白自身抗体

目的　用重组丙酮酸脱氢酶复合物E2亚单位和E3结合蛋白 (PDC E2 ,PDC E3BP)建立筛查原发性胆汁性肝硬化 (PBC)患者相应自身抗体的ELISA。方法　用本室制备的重组PDC E2和PDC E3BP蛋白包被酶联反应板 ,检测PBC患者、正常人及其他肝病患者血清。结果　组建成筛查PBC患者血清中自身抗体的试剂盒 ,能够准确地检测PBC患者血清中的自身抗体。结论　建立的试剂盒能用于PBC患者血清中丙酮酸脱氢酶复合物自身抗体的检测。     

利用丙酮酸脱氢酶复合物原核表达产物检测原发性胆汁性肝硬变患者血清自身抗体

目的 建立一种利用重组表达的丙酮酸脱氢酶复合物E2亚单位(PDC-E2)和E3结合蛋白(PDC-E3BP)检测原发性胆汁肝硬变(PBC)患者血清的方法。方法 以适当的条件诱导PDC-E2和PDC-E3BP表达质粒pExSecI/PDC-E2和pET28/E3BP,利用表达产物通过酶联免疫吸附试验检测PBC、正常人及其他原因肝硬变患者血清,并与欧盟的检测试剂盒比较。结果 显示PDC-E2和PDC-E3BP表达产物检测PBC患者血清中的自身抗体阳性率为93.3％,与欧盟试剂盒比较,总符合率为98.03％。结论 建立了利用重组表达丙酮酸脱氢酶复合物E2亚单位和E3结合蛋白检测PBC患者血清中自身抗体的方法,且获得PDC-E2和PDC-E3BP的高效表达产物。     

M2抗体重组人源靶抗原二联体在原发性胆汁性肝硬化患者中的检测及应用

目的 利用重组抗原BCOADA-E2和PDC-E2的二联体（BP），检测PBC患者血清中的M2抗体并探讨其临床意义。方法 经Ni—NTA亲和柱纯化重组表达的BP融合蛋白，分别建立免疫印迹法（IBT）和酶链免疫吸附（ELISA）法检测60份PBC患者血清，以60份其他肝病患者、60份自身免疫病患者、80例正常人血清为对照组。结果 经常规试剂盒检测为M2（＋）的60份患者血清，利用重组抗原检测阳性53例，阴性7例，阳性率为88．3％；经常规试剂盒检测为M2（-）的60份自身免疫病患者血清、60份其他肝病患者和80例正常人血清，利用重组抗原检测为M2（-）。结论 利用重组抗原BP检测M2抗体敏感性较高。对临床辅助诊断PBC提供一定的手段。     

原发性胆汁性肝硬化患者清抗线粒体抗体M2亚型定量检测的临床价值

目的 研究抗线粒体抗体M2亚型(AMA-M2)定量检测对原发性胆汁性肝硬化(PBC)诊断的临床价值.方法 采用酶联免疫吸附试验(ELISA)检测20例健康人、21例PBC患者及56例其他患者的血清AMA-M2水平,应用矩阵决策法分析AMA-M2检测对PBC的诊断效率,并分析比较其与传统免疫印迹法检测结果的一致性.结果 ELISA法检测血清AMA-M2的线性回归方程为LogY=1.3517ΔA+1.1419,r=0.998,线性可达800RU/ml.与传统免疫印迹法相比较,Kappa值为0.87.健康人血清AMA-M2水平为(17.10±1.80)RU/ml;其他疾病患者中病毒性肝炎为(20.40±5.17)RU/ml,胆道疾病(23.00±11.54)RU/ml,SLE(18.24±3.36)RU/ml,其他自身免疫性疾病(19.80±9.39)RU/ml,分别与健康人群相比较差异均无统计学意义(P>0.05).PBC患者血清AMA-M2水平为(207.40±174.90)RU/ml,显著高于健康人群(P<0.01).AMA-M2检测对PBC诊断的敏感度为95.2%,特异度96.1%,诊断符合率95.9%,阳性预测值87.0%,阴性预测值98.6%.结论 ELISA法定量检测血清AMA-M2操作简便,结果准确可靠,诊断效率高,可作为PBC诊断的重要免疫学指标.     

利用重组抗原检测抗SSA/Ro-52kD自身抗体及其临床意义

目的重组表达SSA/Ro-52kD融合蛋白,并用于检测自身抗体. 方法应用DNA重组技术构建SSA/Ro-52kD工程菌,并表达SSA/Ro-52kD融合蛋白.经谷胱甘肽巯基转移酶(GST)柱层析纯化后,作为抗原,分别用免疫印迹法(IBT)和酶联免疫吸附试验(ELISA)检测20份SS患者血清、60份SLE患者血清和30份正常人血清. 结果重组抗原检测抗SSA/Ro-52kD自身抗体敏感性较高,而且抗体滴度与病程相关. 结论利用重组抗原对自身抗体进行定性、定量分析有助于自身免疫病的诊断和监控.     

细胞角蛋白19血清片段211、丙酮酸脱氢酶复合物E1α亚单位在非小细胞肺癌患者中的表达及意义

目的 分析非小细胞肺癌(NSCLC)患者细胞角蛋白19血清片段211(Cyfra211)、丙酮酸脱氢酶复合物E1α亚单位(PDHA1)的表达及临床意义。方法 收集2017年3月至2020年3月我院收治的139例NSCLC患者癌组织标本(癌组织组)及远离癌组织3 cm的癌旁正常组织(对照组)。分析不同特征NSCLC患者Cyfra211、PDHA1表达及患者预后情况。结果 癌组织组Cyfra211阳性表达率及PDHA1阴性表达率高于对照组(P<0.05);低分化、Ⅲ～Ⅳ期、淋巴结转移NSCLC患者Cyfra211阳性率及PDHA1阴性率更高(P<0.05);139例患者中死亡84例,生存55例,死亡NSCLC患者Cyfra211阳性率、PDHA1阴性率均高于生存患者(P<0.05);Cyfra211、PDHA1联合检测AUC、灵敏度、特异度均显著高于单一检测(P<0.05)。结论 Cyfra211、PDHA1C联合检测在NSCLC病情、预后评估中的作用更加可靠有效,可为NSCLC患者临床治疗指导及预后评估提供更好的参考依据。     

免疫印迹法检测1型糖尿病自身抗体的临床应用

目的探讨免疫印迹法检测1型糖尿病自身抗体的临床应用价值。方法用免疫印迹法和ELISA法分别测定43例1型糖尿病以及50例对照组血清中胰岛细胞抗体（ICA）、谷氨酸脱羧酶抗体（GADA）和胰岛素抗体（IAA）,评价试验的敏感性和特异性。结果免疫印迹法检测1型糖尿病患者血清中ICA、GADA和IAA的阳性率分别为69．7％、67．4％和23．3％,3种抗体联合检测的敏感性和特异性分别是93．0％和98．0％;ELISA法检测ICA、GADA和IAA的阳性率分别为72．1％、81．4％和41．9％,3种抗体联合检测的敏感性和特异性分别是97．7％和88．0％。结论免疫印迹法检测糖尿病自身抗体的敏感性低于ELISA法（P〈0．05）;而免疫印迹法的特异性明显高于ELISA法（P〈0.05）,免疫印迹法对DM的分型、指导临床用药以及判断预后具有重要的临床应用价值。     

血清抗线粒体抗体M_2亚型检测在原发性胆汁性肝硬化临床诊断中的价值

目的:探讨抗线粒体抗体(AMA)-M2亚型检测在原发性胆汁性肝硬化(primary billiary cirrhosis,PBC)诊断中的价值。方法:应用酶联免疫吸附试验,检测20名健康对照者、139例其他非PBC疾病患者和42例PBC患者血清中的AMA-M2水平,并采用矩阵图决策法分析血清AMA-M2检测对PBC的诊断效率。结果:健康对照者平均血清AMA-M2水平为(16.51±1.38)RU/mL,非PBC患者的平均血清AMA-M2水平为(24.33±7.29)RU/mL,两者比较差异无统计学意义(P>0.05)。PBC患者的平均AMA-M2水平为(211.51±126.68)RU/mL,与健康对照者比较,差异有统计学意义(P<0.01)。AMA-M2检测对PBC诊断的灵敏度为95.2%,特异度为97.4%,准确率达95.6%,阴性预测值为98.7%,阳性预测值为90.9%。结论:血清AMA-M2检测有较高的诊断效率,可作为PBC诊断的重要血清免疫学指标。     

Evaluation of newly developed ELISA using "MESACUP-2 test mitochondrial M2" kit for the diagnosis of primary biliary cirrhosis

OBJECTIVES: An enzyme-linked immunosorbent assay (ELISA) using MESACUP-2 Test Mitochondria M2 kit (new-M2 ELISA) has recently become commercially available. The aim of this study was to evaluate the clinical utility of this newly developed ELISA for the diagnosis of primary biliary cirrhosis (PBC). DESIGN AND METHODS: We tested the immunoreactivity of sera from 82 Japanese PBC patients to the 2-oxo-acid dehydrogenase complex (2-OADC) enzymes by indirect immunofluorescence, enzyme inhibition assay using commercially available TRACE Enzymatic Mitochondrial Antibody (M2) Assay (EMA) kit, commercial ELISAs using MESACUP Mitochondria M2 kit (old-M2 ELISA) and new-M2 ELISA, and immunoblotting on bovine heart mitochondria. RESULTS: Each test gave the following positive results; antimitochondrial antibodies (AMA) by immunofluorescence in 71 (87%) out of the 82 sera, enzymatic inhibitory antibody to pyruvate dehydrogenase complex (PDC) by EMA in 61 (74%), immunoglobulin (Ig) G class anti-PDC antibody by old-M2 ELISA in 55 (67%), IgG/M/A class anti-E2 subunit of PDC (PDC-E2)/anti-E2 subunit of branched chain oxo-acid dehydrogenase complex (BCOADC-E2)/anti-E2 subunit of 2-oxoglutarate dehydrogenase complex (OGDC-E2) antibodies by new-M2 ELISA in 73 (89%), and IgG, IgM, or IgA class antibodies against at least one of the 2-OADC enzymes by immunoblotting in 82 (100%). Fifty-three of the 82 sera (65%) were all positive by these five assays. Of the 18 sera that were positive by new-M2 ELISA but negative by old-M2 ELISA, 12 were theoretically interpretable. Of the 11 sera that were negative for AMA by immunofluorescence but positive for at least one of anti-2-OADC enzymes by immunoblotting, four (36%) were positive by new-M2 ELISA, whereas only two and one sera were positive by EMA and old-M2 ELISA, respectively. CONCLUSIONS: Our results indicated that the sensitivity of the newly developed new-M2 ELISA was higher than that of EMA and old-M2 ELISA, and comparable with that of immunofluorescence. However, it is still unclear whether the new-M2 ELISA could replace the conventional immunofluorescence testing for routine assay requests because six (7%) sera showed discrepant results between these two assays.     

Enzyme-linked immunosorbent assays for the determination of IgG,IgA, and IgM autoantibodies to pyruvate dehydrogenase in primary biliary cirrhosis

Summary Autoantibodies to the recently described mitochondrial autoantigen, pyruvate dehydrogenase, have been shown to be specific for primary biliary cirrhosis. In the present study we describe enzyme-linked immunosorbent assays to detect antibodies of IgG, IgA, and IgM classes reactive with pyruvate dehydrogenase. These assays showed high sensitivity (95%) and specificity (100%) for primary biliary cirrhosis when evaluated in 28 patients with primary biliary cirrhosis, 59 disease controls, and 214 healthy persons. Quantitation of these autoantibodies by calculating the areas under the sera titration curves of 10 primary biliary cirrhosis patients indicated that an increase in IgA antibodies to pyruvate dehydrogenase is related to more rapid disease progression.     

利用重组抗原检测抗SSA＼Ro—52kD自身抗体及其临床意义

目的 重组表达ＳＳＡ／Ｒｏ－５２ｋＤ融合蛋白,并用于检测自身抗体。方法 应用ＤＮＡ重组技术构建ＳＳＡ／Ｒｏ－５２ｋＤ工程菌,并表达ＳＳＡ／Ｒｏ－５２ｋＤ融合蛋白,经谷胱基肽巯基转移酶（ＧＳＴ）柱层析纯化后,作为抗原,分别用免疫印迹法（ＩＢＴ）和酶联免疫吸附试验（ＥＬＩＳＡ）检测２０份ＳＳ患者血清、６０份ＳＬＥ患者血清和３０份正常人血清。结果 重组抗原检测抗 ＳＳＡ／Ｒｏ－５２ｋＤ自身抗体敏感性较高     

脑梗死患者抗磷脂抗体的测定与临床意义

目的探讨抗磷脂抗体(APA)与脑梗死之间的关系及其临床意义。方法对157例脑梗死(C I)患者和82例正常对照组(NC)采用酶联免疫吸附试验(ELISA)检测ACA-IgG、IgM、IgA,血浆部分凝血活酶时间-狼疮抗凝物法(PTT-LA)筛选狼疮抗凝物(LA)。结果C I组血清中ACAIgG、IgM、IgA以及LA的阳性率均明显高于NC组(P<0.05);C I组总APA阳性率(25.5%)显著高于NC组(4.9%),P<0.01;多灶性C I组APA的阳性率(36.5%)明显高于单灶性C I组(15.7%),P<0.05;在40例APA阳性脑梗死的病因分析中,≤50岁C I组单纯APA阳性率(58.3%)显著高于>50岁C I组单纯APA阳性率(14.3%),P<0.05;女性C I组与男性C I组之间APA阳性率差异无统计学意义。结论抗磷脂抗体与脑梗死密切相关,且以多灶性脑梗死多见,尤其是原因不明的脑梗死患者重要的致病危险因素。     

2型糖尿病患者尿nephrin排泄量的变化及其临床意义

目的探讨2型糖尿病患者尿nephrin排泄量的变化及其与尿白蛋白排泄量的关系。方法87例2型糖尿病患者根据尿白蛋白、肌酐比值分为:正常白蛋白尿组,微量白蛋白尿组,临床白蛋白尿组。检测所有入选者尿nephrin(酶联免疫吸附法)及其相关因素。结果健康对照组患者尿中未检测到nephrin排泄,糖尿病组尿液中可检测到nephrin。正常白蛋白尿患者可检测到微量nephrin排泄,大量白蛋白患者尿nephrin/尿肌酐比值显著高于微量白蛋白尿组(P<0.01),尿nephrin/尿肌酐比值与尿白蛋白/尿肌酐呈显著性正相关。结论2型糖尿病患者尿nephrin排泄增加,并与糖尿病肾病的病情程度有关。     

Peripheral blood T-cell responses to pyruvate dehydrogenase complex in primary biliary cirrhosis: role of antigen-presenting dendritic cells

Patients with primary biliary cirrhosis (PBC) are usually characterized by the presence of antibody to pyruvate dehydrogenase complex (PDC) in the sera and PDC-specific T cells in the liver. However, most of the patients with PBC do not show peripheral blood T cells response to PDC. In this study, we re-evaluated the peripheral blood T cell responses to PDC in PBC using antigen-presenting dendritic cells (DCs). Twenty-four patients with PBC (AMA-positive: 16; AMA-negative: 8) and 13 normal controls were enrolled in the study. Peripheral blood mononuclear cells (PBMC) and highly enriched populations of T cells were stimulated with either only PDC or DCs plus PDC or PDC-pulsed DC plus PDC. Antibodies to different components of PDC were estimated by an immunoblotting technique. PBMC from only one out of ten AMA-positive PBC patients proliferated when cultured with only PDC. However, peripheral blood T cells from ten out of ten AMA-positive PBC patients and three out of ten AMA-negative PBC patients, but none of the five normal controls showed PDC-specific proliferation when cultured with PDC-pulsed DCs. Two of these three AMA-negative PBC patients, although negative for AMA, were positive for antibodies to other components of PDC. PDC-specific T cells are present in the peripheral blood from most of the patients with PBC. This is the first report on the effectiveness of antigen-pulsed DCs for the elucidation of autoantigen-specific immune response in human autoimmune diseases.