应用免疫印迹技术检测天疱疮 抗体方法的建立

目的　建立应用免疫印迹技术检测天疱疮抗体方法。方法　环切包皮分离表皮后提取天疱疮抗原,进行印迹反应。对27份寻常型天疱疮（pemphigusvulgaris,PV）患者血清进行检测,并与间接免疫荧光（indirectimmunofluorescence，IIF）比较。结果　在26份血清中检出与表皮分离抗原结合的IgG抗体(96.3%)，其中24份与相对分子质量（Mr）为130×10     

Ro60/SSA基因转染HEp-2细胞作为间接免疫荧光检测底物及其应用

本研究拟改造间接免疫荧光技术(IIF)抗核抗体(ANA)检测法的底物HEp-2细胞,建立抗60kDRo60/SSA抗体免疫荧光检测法。采用PCR扩增人源Ro60 cDNA,克隆入真核表达载体pEGFP-C1,并转染HEp-2细胞。通过荧光显微镜、免疫印迹法(IBT)及IIF鉴定转染细胞(HEp-Ro60)中Ro60-绿色荧光蛋白(GFP)融合蛋白的表达和抗原性。分别以HEp-Ro60以及HEp-2为底物的IIF检测10份抗Ro/SSA对流免疫电泳(CIE)检测阳性、其他抗体阴性血清以及对照血清。获得的转染细胞传十几代后仍具有较强的Ro60-GFP表达,融合蛋白保持Ro60的抗原性。IIF检测中HEp-Ro60的滴度比HEp-2增加了6.7倍(P<0.01),而且2例HEp-2细胞上IIF检测为阴性的血清在HEp-Ro60上为阳性。10例阳性血清中有8例出现了特征性荧光模式。结论是HEp-Ro60可用于IIF检测抗Ro抗体,并增加了ANA检出的敏感性。     

五种方法检测抗dsDNA抗体性能比较与临床评价

用化学发光免疫分析法(CLIA)、免疫印迹法(WB)、间接免疫荧光法(IIF)、胶体金免疫层析法(CGCIA) 和ELISA检测dsDNA抗体,评价方法的分析性能、相关性及对系统性红斑狼疮(SLE)的诊断价值.用五种方法检测300例SLE组患者、300例疾病对照组患者和100名健康人对照组血清抗dsDNA抗体.经统计学...     

Ro60/SSA基因转染HEp-2细胞作为间接免疫荧光检测底物及其应用

本研究拟改造间接免疫荧光技术（IIF）抗核抗体（ANA）检测法的底物HEp-2细胞，建立抗60kDRo60／SSA抗体免疫荧光检测法。采用PCR扩增人源Ro60 cDNA，克隆入真核表达载体pEGFP-C1，并转染HEp-2细胞。通过荧光显微镜、免疫印迹法（IBT）及IIF鉴定转染细胞（HEp-Ro60）中Ro60-绿色荧光蛋白（GFP）融合蛋白的表达和抗原性。分别以HEp-Ro60以及HEp-2为底物的IIF检测10份抗Ro／SSA对流免疫电泳（CIE）检测阳性、其他抗体阴性血清以及对照血清。获得的转染细胞传十几代后仍具有较强的Ro60-GFP表达，融合蛋白保持Ro60的抗原性。IIF检测中HEp-Ro60的滴度比HEp-2增加了6．7倍（P〈0．01），而且2例HEp-2细胞上IIF检测为阴性的血清在HEI-Ro60上为阳性。10例阳性血清中有8例出现了特征性荧光模式。结论是HEp-Ro60可用于IIF检测抗Ro抗体，并增加了ANA检出的敏感性。     

抗核抗体谱检测的临床诊断意义

目的:分析抗核抗体线性免疫印迹分析法( ANA-LIA)和抗核抗体间接免疫荧光法在自身免疫性疾病临床的诊断价值.方法:ANA-LIA使用德国IMTEC公司IMTEC-Human GmbH抗核抗体谱试剂检测；抗核抗体用间接免疫荧光法检测.结果:用间接免疫荧光法检测抗核抗体300例,用免疫印迹法检测抗核抗体谱263例,ANA荧光法与ANA-LIA免疫印迹法检测阳性结果无显著性差异(P＞0.05)；30例同时进行ANA、ANA-LIA检测,结果一致率73.3％；ANA-LIA漏检率20％,错检率6.7％.结论:以间接免疫荧光法检测抗核抗体缺乏特异性,而抗核抗体谱免疫印迹法具有高特异性,但存在着较高的漏检率,因此在临床实际应用中将两种方法联合应用,检测效果更好.     

免疫荧光法检查自身抗体183例分析

本文利用免疫荧光法检查183例自身抗体,发现阳性者46例,占受检总例数的25.14%。镜下观察阳性者呈黄绿色荧光,即证明病人存在对该组织成分的自身抗体。对阳性病例可进一步作滴度检测。作者认为,利用免疫荧光法进行自身抗体检查对自身免疫疾病的临床诊断和治疗有较大的实用价值。     

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。     

抗核抗体谱检测在诊断系统性红斑狼疮中的意义

探讨抗核抗体(ANA)和抗核抗体谱(ANAs)检测对诊断系统性红斑狼疮(SLE)患者的临床意义。用间接免疫荧光法(IIF)和免疫印迹法检测106例SLE组和30名对照组血清ANA及ANAs中的12种抗体。结果表明:SLE组ANA阳性率为86.8%,ANAs中抗ds-DNA、抗Scl-70、抗Jo-1、抗nRNP、抗Sm、抗SS-A、抗SS-B、抗Ro-52、抗CENP-B、抗AnuA、抗AHA和抗核糖体P蛋白的阳性率分别为28.3%、0.9%、0.9%、35.9%、17.0%、39.6%、18.9%、41.5%、9.4%、29.3%、31.1%和9.4%。ANA和ANAs联合检测提高了诊断的敏感性,对SLE的诊断和治疗有重要意义。     

不同方法检测抗双链DNA抗体在系统性红斑狼疮诊断中的价值评估

目的 评估流式荧光法(FFIA)、间接免疫荧光法(IIF)及酶联免疫吸附法(ELISA)检测抗双链DNA(dsDNA)抗体用于系统性红斑狼疮(SLE)诊断的临床价值,为临床实践寻找适宜的检测策略。方法 选取我院就诊的SLE患者92例,非SLE自身免疫性疾病患者127例及50例健康体检者,分别采用FFIA、IIF和ELISA检测血清中抗dsDNA抗体,计算各方法检测灵敏度和特异性,采用Kappa检验评估结果一致性,采用受试者工作特征曲线(ROC曲线)比较各方法用于区分SLE和非SLE自身免疫性疾病的效能。结果 用FFIA、IIF、ELISA检测抗dsDNA抗体,SLE组阳性率分别为44.6%、50.0%和62.0%,均高于疾病对照组和健康对照组(P<0.0001)。IIF与FFIA、ELISA具有高度的一致性,Kappa值分别为0.64和0.72。FFIA与ELISA检测结果具有中等的一致性,Kappa值为0.56。以串联的方式FFIA-IIF或ELISA-IIF联合检测,可提高SLE诊断的灵敏度,分别达到59.8%和67.4%,其特异性分别为92.1%和89.3%,阴阳性与诊断符...     

肾脏疾病患者血清中ANCA的荧光模式及其意义

目的：进一步探讨肾脏疾病患者血清中抗中性粒细胞细胞质抗体(ANCA)的荧光模式及其意义。方法：应用间接免疫荧光(IIF)和ELISA方法检测了251例肾脏疾病患者血清中ANCA及其靶抗原。结果：在251例肾脏疾病患者中，ANCA阳性51例占20．3％，其中10例为cANCA阳性(10／251)；30例为cANCA阳性(30／251);11例为aANCA阳性(11／251)。结论：同时用IIF法和ELISA法检测ANCA，可以提高ANCA检出的阳性率；不同ANCA荧光模式与疾病的种类病情及预后密切相关；提高对不典型ANCA和pANCA伴ANA荧光模式的鉴别，有助于临床对血管炎和其它自身免疫性疾病的诊断。     

Diagnostic features of pemphigus vulgaris in patients with pemphigus foliaceus: detection of both autoantibodies, long-term follow-up and treatment responses

There are several studies that describe the simultaneous presence and conversion of pemphigus foliaceus into pemphigus vulgaris and vice versa. We describe eight patients with clinical, histological and immunopathological features of pemphigus foliaceus, at the time of the initial diagnosis. After a mean period of 2.5 years, additional serological features of pemphigus vulgaris were observed. During a long-term follow-up, systemic therapies, their durations and treatment outcomes were recorded. These patients did not respond to conventional systemic therapy and developed multiple side-effects from these drugs. Hence, they were treated with intravenous immunoglobulin therapy (IVIg). Prior to the initiation of IVIg therapy, different assays were performed to detect the presence of autoantibodies, including indirect immunofluorescence (IIF), immunoblot assay using bovine gingival lysate, and ELISA. Twenty-five healthy normal individuals, 12 patients with pemphigus vulgaris, and eight patients with pemphigus foliaceus served as controls for comparison of serological studies. At the time of initial diagnosis, the sera of all eight study patients also demonstrated binding on an immunoblot assay to a 160-kDa protein (desmoglein 1) only. This is typically observed in pemphigus foliaceus. Prior to staring IVIg therapy, binding was observed to both the 160 kDa and 130 kDa (desmoglein 3) proteins on an immunoblot assay which was characteristic of pemphigus vulgaris. The antidesmogleins, 1 and 3 autoantibodies, were predominantly of the IgG4 subclass in all eight patients studied. IVIg therapy induced remission in four patients and control in four of the eight patients. The total follow-up period ranged from 2.6 to 9.5 years (mean 5.3 years). It is difficult to determine the exact time at which these patients with pemphigus foliaceus developed pemphigus vulgaris. It is possible that the disease was nonresponsive to conventional immunosuppressive therapy owing to the simultaneous presence of two autoantibodies.     

Analysis of the reactivity of indirect immunofluorescence in patients with pemphigus foliaceus and pemphigus vulgaris using rat bladder epithelium as a substrate

OBJECTIVES:

To evaluate the reactivity of indirect immunofluorescence using rat bladder epithelium as a substrate in patients with pemphigus foliaceus and pemphigus vulgaris from the Department of Dermatology, University of São Paulo Medical School, Brazil.

METHODS:

Thirty-two patients (8 male and 24 female) from the Department of Dermatology, University of São Paulo Medical School, were selected. Three had mucosal pemphigus vulgaris, 20 had mucocutaneous pemphigus vulgaris, and 9 had pemphigus foliaceus. Patients'' sera were tested by indirect immunofluorescence performed on human foreskin and rat bladder epithelium and by ELISA assays utilizing baculovirus-expressed recombinant desmoglein 3 and desmoglein 1.

RESULTS:

No patients with mucosal pemphigus vulgaris, 5 of 20 patients with mucocutaneous pemphigus vulgaris (25%) and 4 of 9 patients with pemphigus foliaceus (44%) had positive indirect immunofluorescence using rat bladder epithelium as a substrate.

CONCLUSION:

Indirect immunofluorescence using rat bladder epithelium as a substrate is recommended whenever a diagnosis of paraneoplastic pemphigus is considered. The identification of a subset of pemphigus foliaceus and pemphigus vulgaris patients that recognizes desmoplakins by this laboratory tool is critical to avoid the misdiagnosis of paraneoplastic pemphigus.     

Simultaneous presence of mucous membrane pemphigoid and pemphigus vulgaris: molecular characterization of both autoantibodies

There are several reports in the literature describing the coexistence of features of pemphigus vulgaris and pemphigoid in the same patient. We describe 15 patients with clinical, histological, and immunopathological features of mucous membrane (cicatricial) pemphigoid at the time of initial diagnosis. All 15 patients failed to respond clinically to conventional systemic agents over a mean period of 7.2 years. Hence, IVIg therapy was used. Prior to initiating IVIg therapy, features of mucous membrane pemphigoid and pemphigus vulgaris were demonstrated by various serological tests. Different assays were performed to identify molecular characteristics of these two autoantibodies. Twenty-five healthy normal individuals, 22 patients with mucous membrane pemphigoid, 17 patients with pemphigus vulgaris, and 12 patients with pemphigus foliaceus served as controls for comparison of serological studies. On indirect immunofluorescence, using monkey esophagous as substrate, sera of all 15 patients had demonstrable levels of anti-intercellular cement substance (ICS) or anti-keratinocyte cell surface antibody. Sera of 14 patients on salt split skin bound to the epidermal side of the split, which was consistent with mucous membrane pemphigoid. Sera of all 15 patients demonstrated binding to a 205-kDa protein (human B4 integrin) and a 130-kDa protein (desmoglein 3) on immunoblot. In a sample of sera from each of the 6 patients with mucous membrane pemphigoid and pemphigus vulgaris, the anti-ICS antibody was of the IgG4 subclass. The IgG4 subclass is a characteristic feature associated with pathogenic autoantibodies in pemphigus vulgaris. Hence, in such patients, a dual diagnosis should be considered and confirmed by various serological assays. It is possible that the presence of two pathogenic autoantibodies in these patients could have contributed to the lack of response to conventional immunosuppressive therapy.     

寻常性天疱疮抗原EC1-2和EC3-4表位的分子克隆与蛋白表达

目的：克隆并表达寻常性天疱疮抗原（ＰＶＡ,即桥粒芯糖蛋白－３）的ＥＣ１－２和ＥＣ３－４表位,用于通过血清学方法特异性诊断天疱疮和了解ＰＶＡ的表位与抗ＰＶＡ抗体间的联系。方法：从角朊细胞抽提ＲＮＡ,逆转录合成ｃＤＮＡ,扩增目的基因ＥＣ１－２和ＥＣ３－４后与质粒载体ＰＧＥＸ－４Ｔ－１连接,电转导入大肠杆菌ＢＬ２１,经ＩＰＴＧ诱导后表达ＥＣ１－２和ＥＣ３－４融合蛋白,ＳＤＳ－ＰＳＧＥ电泳后转移至硝酸纤素     

Autoimmunity to desmocollin 3 in pemphigus vulgaris

Pemphigus vulgaris is a blistering disease associated with autoantibodies to the desmosomal adhesion protein, desmoglein 3. Genetic deficiency of desmoglein 3 in mice mimics autoimmunity to desmoglein 3 in pemphigus vulgaris, with mucosal-dominant blistering in the suprabasal layer of the epidermis. Mice with an epidermal-specific deletion of desmocollin 3, the other major desmosomal cadherin isoform expressed in the basal epidermis, develop suprabasal blisters in skin that are histologically identical to those observed in pemphigus vulgaris, suggesting that desmocollin 3 might be a target of autoantibodies in some pemphigus vulgaris patients. We now demonstrate that desmocollin 3 is an autoantigen in pemphigus vulgaris, illustrated in a patient with mucosal-dominant blistering. Six of 38 pemphigus vulgaris and one of 85 normal serum samples immunoprecipitate desmocollin 3 (P = 0.003). Incubation of patient IgG with human keratinocytes causes loss of intercellular adhesion, and adsorption with recombinant desmocollin 3 specifically prevents this pathogenic effect. Additionally, anti-desmocollin 3 sera cause loss of keratinocyte cell surface desmocollin 3, but not desmoglein 3 by immunofluorescence, indicating distinct cellular pathogenic effects in anti-desmocollin and anti-desmoglein pemphigus, despite their identical clinical presentations. These data demonstrate that desmocollin 3 is a pathogenic autoantigen in pemphigus vulgaris and suggest that pemphigus vulgaris is a histological reaction pattern that may result from autoimmunity to desmoglein 3, desmocollin 3, or both desmosomal cadherins.     

Subclass distribution of human IgG autoantibodies in pemphigus

The distribution of IgG subclasses in the intercellular substance (ICS) reactive autoantibodies in serum of 10 patients with pemphigus was analyzed by semiquantitative indirect immunofluorescence (IF) using the HP series of monoclonal antibodies specific for the four human IgG subclasses. IgG4 ICS specific autoantibody was present in all 10 sera at a titer of 10 to 320, while IgG1 antibodies were found in 9 of 10 sera at a seemingly lower level. IgG3 autoantibodies were detected in the serum of one patient, only after isolation of IgG using ion-exchange chromatography. Autoantibodies of IgG subclass 2 were not detectable in any of the 10 sera tested. One of the ten patients displayed circulating anti-ICS antibodies of only the IgG4 isotype.     

Isotypes and antigenic profiles of pemphigus foliaceus and pemphigus vulgaris autoantibodies

In this study we systematically characterized isotype profiles and antigenic and tissue specificity of antidesmoglein autoantibodies from patients with pemphigus foliaceus (PF) and pemphigus vulgaris (PV) using enzyme-linked immunoabsorbent assays (ELISA), indirect immunofluorescence (IIF) staining, and immunoblotting (IB). In PF, we found that IgG1 antidesmoglein-1 (Dsg1) reacts with a linear epitope(s) on the ectodomain of Dsg1, while its IgG4 counterpart recognizes a conformational epitope(s). These two subclasses of anti-Dsg1 are both capable of recognizing tissues from monkey esophagus and adult human skin, but IgG1 is not able to react with mouse skin, which may explain why this isotype of anti-Dsg1 failed to induce PF-like lesions in the passive transfer animal model. In mucosal PV patients, we found that both IgG1 and IgG4 only recognized monkey esophagus tissue by IIF, except in one patient, indicating that these antibodies react with a unique conformational epitope(s) that is present in mucosal but not skin tissue. In generalized PV, IgG1 anti-Dsg3 autoantibodies appeared to recognize a linear epitope(s) on the Dsg3 ectodomain. In contrast, IgG4 anti-Dsg3 antibodies recognized both linear and conformational epitopes on the Dsg3 molecule. Interestingly, the IgG1 anti-Dsg3 antibodies failed to react with human and mouse skin tissues, suggesting that this subclass of autoantibodies may not play an essential role in the development of PV suprabasilar lesions. In summary, we conclude that this study further elucidates the pathological mechanisms of PF and PV autoantibodies by revealing their distinct isotype and antigenic profiles. This information may help us to better understand the autoimmune mechanisms underlying the development of pemphigus.