Serum levels of autoantibodies to BP180 correlate with disease activity in patients with bullous pemphigoid

OBJECTIVE: To investigate the possible correlation of levels of circulating anti-BP180 autoantibodies with disease activity in bullous pemphigoid (BP). DESIGN: Diagnostic study. SETTING: Regional referral center at a university dermatology department. PATIENTS: Fifteen patients with typical clinical, histologic, and immunofluorescence findings of BP who had not received prior systemic treatment. INTERVENTIONS: Initially, 6 consecutive patients with BP were treated with oral doxycycline and niacinamide. Subsequently, 9 consecutive patients with BP received a combination of oral dapsone and prednisolone. MAIN OUTCOME MEASURES: Disease activity, serum levels of autoantibodies to BP180, and titers of antibasement membrane zone autoantibodies were assayed before initiation of treatment and 4 and 8 weeks later. Reactivity to BP180 was analyzed by enzyme-linked immunosorbent assay using a recombinant form of BP180 NC16A. Titers of anti-basement membrane zone autoantibodies were assayed by indirect immunofluorescence on 1-mol/L sodium chloride-split human skin. RESULTS: In both treatment groups, disease activity correlated with serum levels of autoantibodies to BP180 NC16A (P = .004 [dapsone-prednisolone] and .007 [doxycycline-niacinamide]). No correlation was seen between disease activity and indirect immunofluorescence reactivity (P = .18 and .16, respectively). In patients receiving dapsone plus prednisolone, the dose of corticosteroids necessary to suppress new blister formation correlated with anti-BP180 reactivity (P = .002). CONCLUSIONS: In contrast to indirect immunofluorescence reactivity that reflects reactivity to both BP 180 and BP230, serum levels of autoantibodies to BP180 correlate with disease activity in BP. Assaying reactivity to BP180 should be a helpful guide for the therapeutic management of patients with this disease. Our results underline the pathogenic relevance of autoantibodies to human BP180.     

Correlation of clinical severity and ELISA indices for the NC16A domain of BP180 measured using BP180 ELISA kit in bullous pemphigoid

BACKGROUND: Titres of circulating autoantibodies detected by indirect immunofluorescence (IIF) have been used for the diagnosis and evaluation of disease activity in bullous pemphigoid (BP). In BP, the major pathogenic epitope is known to be the non-collagenous extracellular domain (NC16A) of the 180-kDa transmembrane hemidesmosomal protein (BPAG2). Recently, an enzyme-linked immunosorbent assay (ELISA) kit using the NC16A domain recombinant protein (BP180 ELISA kit) has become commercially available to measure the quantities of pathogenic autoantibodies circulating in BP patients. OBJECTIVE: To investigate the correlation of clinical severity and ELISA indices in BP. METHODS: Fourteen patients with a typical form of BP and one refractory BP patient who died despite extensive treatment were included in this study. Antibody titres in sera from these patients were measured using BP180 ELISA kit and an analysis of ELISA indices with disease activity was performed. RESULTS: ELISA indices were significantly reduced after successful therapy, although IIF titres did not always show apparent correlations. In the patient with refractory BP, ELISA indices also showed a good correlation with disease course. ELISA indices measured using the BP180 ELISA kit were well correlated with the disease activity. CONCLUSION: This commercially available kit more closely followed disease activities than the IIF titres. The BP ELISA system may be a useful tool to evaluate the disease activity and to assess the effectiveness of the treatment of BP.     

BP180NC16a与大疱性类天疱疮病情变化的关系

目的探讨大疱性类天疱疮(BP)患者外周血血清IIF滴度、BP180NC16a-ELISA指数与病情变化的相关性。方法监测12例以系统糖皮质激素治疗为主的泛发性BP患者不同时期病情变化及其相应的IIF滴度和BP180NC16a-ELISA指数,并对结果进行分析。结果IIF滴度与病情变化没有平行关系,BP180NC16a-ELISA指数与病情变化较平行。结论BP180NC16a-ELISA指数可作为BP病情的监测和调整治疗方案的重要参考。     

BP180NC16a-ELISA对大疱性类天疱疮血清学诊断的评价

目的 评价BP180NC16a-ELISA对大疱性类天疱疮血清学诊断的效能.方法 BP患者42例,对照组42例(其中正常人对照24例,天疱疮18例),在患者用药前采血,比较BP180NC16a-ELISA和盐裂试验免疫荧光(IIF)检测的结果.结果 用BP180NC16a-ELISA检测时,BP患者中有1例呈阴性反应,其敏感性为97.62%;正常人对照组中有1例呈阳性反应,其特异性为97.62%,且BP180NC16a-ELISA法的A值与IIF滴度之间无相关性.结论 BP180NC16a-ELISA在疾病初始阶段是检测血清中抗BP180抗体的有效方法.     

Role of BP180NC16a-enzyme-linked immunosorbent assay (ELISA) in the diagnosis of bullous pemphigoid in China

Background Autoantibodies of bullous pemphigoid (BP) patients react with two components of the hemidesmosome of stratified epithelia: the BP antigen 230 (BP230) and the BP antigen 180 (BP180). Recently, strong evidence has been provided that autoantibodies to BP180 play a key role in subepidermal blister formation in BP patients, and NC16A contains an important antigen determinant of BP. Objective To study the role of BP180NC16a enzyme‐linked immunosorbent assay (BP180NC16a‐ELISA) in the diagnosis of BP in China. Methods Sera from BP patients (n = 42) and control subjects (normal controls, n = 24; pemphigus patients, n = 18) were measured by BP180NC16a‐ELISA. All BP sera were obtained at presentation from patients who had not received previous systemic treatment. The values of immunoglobulin G (IgG) antibody levels measured by ELISA were compared with those measured by indirect immunofluorescence (IIF) (gold standard for the diagnosis of BP) on salt‐split skin. Results Using BP180NC16a‐ELISA, 41 of the 42 BP sera were positive, whereas only one of the serum samples from 24 normal controls was positive and all the pemphigus sera showed a negative result. Thus, the sensitivity and specificity of BP180NC16a‐ELISA were both 97.62%. There was no correlation between the mean ELISA values and IIF titers. The ELISA and IIF results were further compared and analyzed using a 2 × 2 contingency table, which showed that they were not significantly different. Conclusions It is suggested that BP180NC16a‐ELISA is a useful tool for the diagnosis of BP.     

BP180 ELISA using bacterial recombinant NC16a protein as a diagnostic and monitoring tool for bullous pemphigoid

Bullous pemphigoid (BP) is an acquired autoimmune subepidermal blistering disease against hemidesmosomal cytoplasmic BP230 and transmembrane BP180 proteins. Epitope mapping studies have shown that the membrane-proximal noncollagenous (NC) 16a domain of BP180 harbors clusters of antigenic sites recognized by the vast majority of BP sera. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) using bacterial recombinant NC16a protein and evaluated its clinical benefit for diagnosis and monitoring disease activity. Fifty four (84.4%) of 64 sera from BP patients were positive, while only one (1.1%) of 91 sera from collagen disease patients and five (1.5%) of 336 sera from normal control barely exceeded the cut-off value. None of 69 pemphigus vulgaris sera and none of 42 pemphigus foliaceus sera exceeded the cut-off value. Thus, the sensitivity and specificity of NC16a ELISA were 84.4 and 98.9%, respectively. The correlation between ELISA scores and disease activity along the time course was examined using seven BP patients. NC16a ELISA scores tended to fluctuate in parallel with the disease activity along the time course and reflected the disease activity much better than indirect immunofluorescence. These findings indicate that NC16a ELISA will be a valuable tool not only for the diagnosis of patients with BP but also for the monitoring of the disease activity.     

Evaluation of a BP180-NC16a enzyme-linked immunosorbent assay in the initial diagnosis of bullous pemphigoid

BACKGROUND: Bullous pemphigoid (BP) is the most common subepidermal immunobullous disease, characterized by circulating IgG autoantibodies targeting BP180 and BP230 hemidesmosomal proteins. Several immunological studies have demonstrated that the membrane proximal noncollagenous domain NC16a of BP180 is the immunodominant region targeted by BP autoantibodies. Recently, a commercial BP180 NC16a-specific enzyme-linked immunosorbent assay (ELISA) has become available for detecting pathogenic anti-BP180 autoantibodies in BP sera. However, it remains unclear whether the diagnostic potential of the ELISA is equivalent to that of the 'gold-standard' diagnostic technique of immunofluorescence (IF). OBJECTIVES: To examine the usefulness of a commercially available BP180-NC16a ELISA in the initial serodiagnosis of BP. METHODS: Sera from a large cohort of patients with BP (n = 102) and control subjects (age- and sex-matched normal volunteers, n = 60; pemphigus foliaceus, n = 18; pemphigus vulgaris, n = 16) were assayed by BP180-NC16a ELISA. All BP sera were obtained at presentation before initiation of systemic immunosuppressive therapy. The values of IgG antibody levels measured by ELISA were compared with those measured by indirect IF on salt-split skin. Results Receiver operating characteristic analysis was used to calculate the cut-off value for the ELISA in the diagnosis of BP which maximizes both sensitivity and specificity, and to estimate the diagnostic accuracy of the ELISA as represented by the area under the curve (AUC = 0.965). A cut-off value of 9 was associated with a sensitivity of 89% (91 of 102 BP sera showed a positive result) and a specificity of 98%. Fifty-eight of 60 normal controls and all the pemphigus sera showed a negative result. There was a correlation between the mean ELISA values and indirect IF titres (Spearman rank correlation 0.286; P = 0.004). CONCLUSIONS: Our results suggest that the BP180-NC16a ELISA is a useful tool for the detection of pathogenic anti-BP180 IgG autoantibodies at the initial disease stage of BP. Because it is not only highly sensitive and specific, but is also easy to perform, is objective, and semiquantitative, the ELISA may provide valuable information for the accurate and reliable serodiagnosis of BP.     

Detection of anti-BP180NC16a and anti-BP230 autoantibodies in blister fluid of patients with bullous pemphigoid: the first survey in Greece

Bullous pemphigoid (BP) is an acquired bullous disease with an increasing prevalence among elderly people worldwide, including in Greece. Blister formation in most patients with BP is caused by autoantibodies against structural components of the basement membrane zone of the skin, predominantly BP180NC16a and BP230 antigens on the hemidesmosome adhesion complex. Routine diagnostic methods such as histological examination and direct and indirect immunofluorescence are combined to determine diagnosis. In this study, an ELISA was used to measure levels of both anti-BP180NC16A and anti-BP230 autoantibodies in the blister fluid of 13 patients with newly diagnosed BP, before starting treatment. The aim of the study was to evaluate this method as a diagnostic tool in BP. Our results indicate that blister-fluid examination by ELISA can be a useful tool to diagnose bullous pemphigoid, especially in elderly patients who refuse biopsy or have poor venous access.     

Subacute prurigo variant of bullous pemphigoid: autoantibodies show the same specificity compared with classic bullous pemphigoid

We describe a 76-year-old white woman with a 6-month history of intensive pruritus and excoriated papules resembling subacute prurigo. Histopathology showed signs of chronic dermatitis, whereas findings by direct and indirect immunofluorescence microscopy were compatible with bullous pemphigoid (BP). The patient's serum contained IgG autoantibodies that recognized epitopes on both BP180 and BP230 by Western blot analysis of epidermal extracts. In addition, we found strong reactivity with recombinant NC16A, an immunodominant region of BP180 targeted in the majority of BP sera, whereas no antibodies against the keratinocyte-derived soluble BP180 ectodomain (LAD-1) or the recombinant intracellular domain of BP180 were detected. The patient's disease responded well to oral methylprednisolone and mycophenolate mofetil. Disease activity correlated with enzyme-linked immunosorbent assay reactivity of antibodies to BP180 but not with titers of antibodies to the dermoepidermal junction as determined by indirect immunofluorescence on salt-split skin. Our findings suggest that the subacute prurigo form of BP is a true variant of BP.     

Absence of anti-BP180 antibodies in mothers of infants with bullous pemphigoid

BACKGROUND: It is not clear whether bullous pemphigoid (BP) of infancy is linked to maternal transmission of pathogenic autoantibodies. Objectives To search for anti-BP180 antibodies in the sera of infants with BP and their mothers, using sensitive and specific methods. METHODS: Four infants (<6 months) with BP and their mothers were tested for anti-BP180 antibodies by indirect immunofluorescence, immunoblotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: We found anti-BP180 antibodies in the sera of the four infants with all methods. These antibodies reacted with the extracellular domain NC16A. In the serum of their mothers we found 180 and 160 kDa proteins, each in one case, but indirect immunofluorescence and ELISA were negative, suggesting the absence of anti-BP180 autoantibodies reacting with the extracellular domain NC16A. CONCLUSIONS: BP of infants is not due to maternofetal transmission of pathogenic autoantibodies. Other hypotheses for the pathophysiology of BP are discussed.     

大疱性类天疱疮疾病严重程度与BP180抗体水平关系的研究

目的 研究大疱性类天疱疮患者临床疾病严重程度和BP180抗体滴度之间的关系.方法 用BP180NC16a-ELISA分别测定大疱性类天疱疮患者治疗前,病情缓解、糖皮质激素开始减量时和糖皮质激素减量至相当于泼尼松0.5 mg·kg^-1·d^-1时,体内BP180抗体滴度,观察其与患者病情严重程度的一致性.结果 在治疗前,19例大疱性类天疱疮患者的BP180 ELISA平均A值为0.520(0.832～0.372);在病情得到控制、糖皮质激素准备减量时,其BP180 ELISA平均A值为0.405(O.723～0.204);在病情缓解、糖皮质激素减至相当于泼尼松0.5 mg·kg^-1·d^-1时,其BP180 ELISA平均A值为0.215(0.412～0.093).结论 BP180抗体的滴度与大疱性类天疱疮患者疾病严重程度相关,是一种评估疾病病情的有用手段,也可对治疗的有效性作出评价.     

Immunpathologische Veränderungen bei 115 Patienten mit bullösem Pemphigoid

Bullous pemphigoid (BP) is an autoimmune bullous disease of the elderly. Patients' auto-antibodies are directed against BP180, a transmembrane hemidesmosomal glycoprotein of basal keratinocytes. Between 1989 and 1998, we identified 115 patients with BP in the Department of Dermatology at the University of Würzburg. This is one of the largest groups of patients with BP studied to date. In all patients, direct and/or indirect immunofluorescence microscopy studies were positive. The average age (+/- standard deviation) of the patients was 75 +/- 12 years, the median was 78 years. The youngest patient was 39 years of age; the oldest, 99 years. 54% of the patients were female and 46% male. In 24% of the patients, an involvement of oral, and in 7% of genital mucous membranes was seen. 98% of the patients complained about pruritus. By direct immunofluorescence microscopy, linear deposits of C3 and/or IgG were detected at the basement membrane zone of perilesional skin biopsies. By indirect immunofluorescence microscopy using NaCl-separated human skin, 87% of cases revealed circulating serum antibodies whereas on monkey esophagus, only 72% of sera were found positive. In 85% of the patients, we detected elevated serum levels of total IgE before the initiation of treatment. In those sera that were analyzed for reactivity with BP180, autoantibodies to the immunodominant NC16 A domain of BP180 were detected in 89% by immunoblot analysis and in 93% by ELISA. Therefore, in most patients, BP can be diagnosed serologically.     

盐裂皮肤间接免疫荧光技术在大疱性类天疱疮诊断中的价值评估

目的:评价盐裂皮肤间接免疫荧光（IIF-SSS）在大疱性类天疱疮（BP）诊断中的价值。方法:采用单中心临床回顾性研究。纳入2013年1月至2019年1月在中国医学科学院皮肤病医院就诊的初诊BP患者163例,对照组404例,包括天疱疮161例、湿疹67例、药疹26例、多形红斑23例、结节性痒疹18例等。于患者用药前采血,...     

Serum levels of immunoglobulins G1 and G4 targeting the non‐collagenous 16A domain of BP180 reflect bullous pemphigoid activity and predict bad prognosis

Bullous pemphigoid (BP) is an autoimmune subepidermal bullous disease, and different immunoglobulin (Ig)G autoantibody subclasses may play different roles in the pathogenesis of BP. This study aims to evaluate the relationship between specific IgG subclasses and BP. Enzyme‐linked immunoassays (ELISA) were developed to test the IgG subclasses targeting the non‐collagenous 16A (NC16A) domain of BP180. A statistical analysis was carried out to assess the relationship of BP and IgG subclasses as well as other factors. The correlation coefficients between the ELISA scores for four IgG subclasses and disease severity scores were 0.586 for IgG, 0.441 for IgG1, 0.594 for IgG2, 0.345 for IgG3, and 0.448 for IgG4 before treatment. After treatment, the correlation coefficient was 0.376 for IgG, 0.522 for IgG1, 0.314 for IgG2, 0.582 for IgG3 and 0.503 for IgG4. Spearman's rank correlation coefficient was 0.801 for IgG1, 0.66 for IgG2, 0.575 for IgG3 and 0.463 for IgG4 between the ELISA scores of IgG subclasses and the disease severity score variation. The ELISA scores of IgG subclasses in patients with mucosal involvement were higher than those without. Survival analysis showed that sex, IgG1 and IgG4 were the independent predictors for BP. In conclusion, the serum levels of IgG1 and IgG4 targeting BP180NC16A were paralleled with disease severity in BP patients. IgG1 and IgG4 and sex were the independent prognostic factors for an early prognosis of BP.     

大疱性类天疱疮和妊娠疱疹患者血清抗BP180 NC16A抗体的纯化和鉴定

目的 探讨大疱性类天疱疮（BP）和妊娠疱疹（HG）患者血清抗BPl80 NC16A 抗体的纯化和鉴定方法。方法 原核表达系统pGEX-2TBP180NC16A表达GST/NC16A融合蛋白,将融合蛋白与谷胱甘肽琼脂糖凝聚微珠进行共价偶联。微珠亲和层析法纯化BP和HG患者血清中抗BP180 NC16A抗体,并用ELISA、免疫荧光、及Western印迹进行鉴定。结果 原核表达系统pGEX-2TBP180NC16A表达37 000 GST/NC16A融合蛋白,微珠亲和层析法纯化后得单一抗BP180 NC16A抗体。经ELISA方法定量后确定其含量为2.4 mg/ml;该抗体能与人皮肤基底膜带结合,证明抗体活性;免疫印迹可见单一片段,显示抗体纯度。结论 微珠亲和层析法纯化的BP和HG患者血清中抗BP180 NC16A自身抗体活性高、特异性强。     

A case of bullous pemphigoid successfully treated by plasmapheresis: assessment of the change in titers of circulating antibodies by immunoblotting and enzyme-linked immunosorbent assay

We report a case of bullous pemphigoid successfully treated with double filtration plasmapheresis. The changes in titers of circulating autoantibodies were assessed by immunoblotting and enzyme-linked immunosorbent assay (ELISA) using a recombinant protein of the non-collagenous 16a (NC16a) domain of the 180 kDa bullous pemphigoid antigen (BP180). The ELISA was shown to be more sensitive in detecting disease-specific antoantibodies in the bullous pemphigoid sera. The reduction of titers of circulating autoantibodies in the sera correlated well with the decrease in the disease activity in both the first and second rounds of plasmapheresis treatment in this case.     

Enzyme‐linked immunosorbent assay (ELISA) and indirect immunofluorescence testing in a bullous pemphigoid and pemphigoid gestationis

Enzyme‐linked immunosorbent assay (ELISA) is an excellent tool for detection of circulating antibodies against the NC16A portion of BP180 antigen. We compared the sensitivity and specificity of a commercially available BP180‐NC16a domain ELISA with that of an indirect immunofluorescence (IIF) testing in the evaluation of bullous pemphigoid (BP) and pemphigoid gestationis (PG), and analyzed the relationship between ELISA results and the presence of IgG deposition, in an epidermal or combined pattern, on direct immunofluorescence (DIF) testing of salt‐split skin. ELISA was performed on serum from 28 patients (24 BP, 4 PG) and 50 controls. IIF testing was performed on serum from 27 patients and 98 controls. For the group of 28 patients with BP or PG, ELISA had a sensitivity of 93% and specificity of 96% (P < 0.001), while sensitivity was 74% and specificity 96% (P < 0.001) for IIF testing. In these patients, ELISA has a higher sensitivity than IIF testing, but similar specificity. Evaluation of controls who had IgG deposition on the dermal side of salt‐split skin on DIF testing showed specificity for the ELISA of 100% (all four cases negative) and 80% for IIF testing (one of five positive). Positive ELISA correlated with a diagnosis of BP or PG only in patients who had IgG at the basement membrane zone (BMZ) by DIF testing. Overall, ELISA appears to have greater sensitivity and specificity for BP or PG than does IIF testing.     

盐裂皮肤间接免疫荧光及BP180 NC16a酶联免疫吸附测定在大疱性类天疱疮诊断中的意义

目的 探讨盐裂皮肤间接免疫荧光及大疱性类天疱疮（BP）180 NC16a-酶联免疫吸附测定（ELISA）检测在BP诊断中的意义。方法 收集2015年1月至2017年8月在中国医学科学院皮肤病医院用盐裂皮肤间接免疫荧光（IIF?SSS）和BP180 NC16a?ELISA检测BP患者174例和对照组129例血清。其中25例BP患者用直接免疫荧光（DIF）进行检测并与IIF?SSS和BP180 NC16a?ELISA敏感性进行比较。结果 IIF?SSS、BP180 NC16a?ELISA的敏感性分别为93.67%、96.55%;特异性分别为100%、96.12%。IIF?SSS与BP180 NC16a?ELISA相关系数0.147,为弱相关。其中25例BP患者血清学诊断方法（IIF?SSS,BP180 NC16a?ELISA）和DIF敏感性比较差异无统计学意义。结论 BP血清学诊断方法特异性强、敏感性高,值得临床推广应用。     

Association between effective dose of prednisolone, alone or in conjunction with other immunosuppressants, and titre of anti-bullous pemphigoid 180 antibody: a retrospective study of 42 cases

Background. Corticosteroids, especially prednisolone or prednisone, are the most commonly used drugs for the treatment of bullous pemphigoid (BP). However, the appropriate initial effective prednisolone dose has not been established. Recently, a highly sensitive and specific ELISA for detection of autoantibodies to the non‐collagenous extracellular domain (NC16A) of the 180 kDa transmembrane hemidesmosome component [bullous pemphigoid (BP)180] was developed, and the titre of anti‐BP180 antibody was found to be closely related to disease activity. Aim. To investigate the relationship between anti‐BP180 antibody titre and effective prednisolone dose alone or in conjunction with other immunosuppressants. Methods. Anti‐BP180 antibody titres were measured by ELISA for the NC16A domain of BP180 in the sera of patients with BP (n = 42) at the start of treatment. The effective prednisolone dose was calculated from the patients’ records. Results. Higher anti‐BP180 antibody titres correlated with a higher effective prednisolone dose. In particular, patients with antibody titres > 200 required a significantly higher effective prednisolone dose than did those with antibody titres ≤ 200. Conclusions. A higher effective prednisolone dose may be necessary for patients who have both a high titre of anti‐BP180 antibody and severe clinical disease.     

BP180NC16a-ELISA检测大疱性类天疱疮多中心随机双盲对照试验

目的 探讨BP180抗体诊断试剂盒检测大疱性类天疱疮（BP）的灵敏度和特异度。方法 多中心随机双盲平行对照临床试验。使用BP180抗体诊断试剂盒（ELISA）检测106例临床已经确诊的活动期BP患者和106例对照人群（含非BP的其他大疱性疾病以及硬皮病、银屑病、SLE、妊娠晚期孕妇、健康献血员等）血清样本,并与间接免疫荧光（IIF）试验进行灵敏度和特异度的对比。结果 106份BP患者血清样本中81份BP180抗体诊断试剂盒为阳性,其灵敏度为76.4%;83份抗表皮抗原抗体检测试剂盒（IIF法）为阳性,其灵敏度为78.3%。106份对照组血清标本中95份BP180抗体诊断试剂盒为阴性,其特异度为89.6%;102份抗表皮抗原抗体检测试剂盒为阴性,其特异度为96.2%。BP180抗体诊断试剂盒（ELISA）与对照试剂盒的灵敏度和特异度相比较,差异无统计学意义（P > 0.05）。结论 BP180抗体诊断试剂盒可作为诊断BP的一种辅助手段。