Simultaneous occurrence of anti‐BP180 mucous membrane pemphigoid and mucosal‐dominant pemphigus vulgaris

We describe the simultaneous initial coexistence of mucous membrane pemphigoid (MMP) with blepharosynechia and anti‐bullous pemphigoid (BP)180 antibodies and pemphigus vulgaris (PV) with lesions limited to the oral mucosa, with the presence of anti‐desmoglein (Dsg)3 antibodies. A 75‐year‐old woman had severe oropharyngeal erosions and ulcers with blisters, and blepharoconjunctivitis and blepharosynechia of the left eye. Histopathological examination of the oral mucosa found acantholysis. Indirect immunofluorescence revealed IgG antikeratinocyte cell surface antibodies, and ELISA disclosed anti‐Dsg3 antibodies. Immunoblotting found positive reactivity with recombinant proteins of both the BP180‐NC16a domain and the BP180 C‐terminal domain. To our knowledge, the simultaneous initial occurrence of MMP and PV has never been reported previously. We present a rare case of concurrent PV and anti‐BP180 MMP, the diagnosis of which was confirmed by ELISA and immunoblotting assays.     

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.     

Refractory bullous pemphigoid leaving numerous milia during recovery

Recovery with milia may occur in bullous pemphigoid (BP), mucous membrane pemphigoid (MMP) and epidermolysis bullosa acquisita (EBA). Scarring commonly occurs in MMP and EBA. Here, we report a 62‐year‐old man patient with BP, who was left with numerous milia during recovery. The patient had immunoglobulin (Ig)G autoantibodies to the recombinant protein of the BP180‐NC16a domain and the soluble 120‐kDa ectodomain of BP180 (linear IgA bullous dermatosis [LAD]‐1). There are cases of BP with IgG autoantibodies to LAD‐1 and/or the recombinant protein of BP180 C‐terminal domain. Extensive milia formation during recovery may be associated with immunological predisposition and/or improper interaction between hemidesmosomes and the extracellular matrix components.     

Paraneoplastic pemphigus associated with fatal bronchiolitis obliterans and appearance of anti‐BP180 antibodies in the late stage of the disease

Paraneoplastic pemphigus (PNP) is an autoimmune bullous disease, which associates mainly with lymphoproliferative neoplasms. Bronchiolitis obliterans (BO) with progressive respiratory failure is a significant cause of death in PNP. We report a case of PNP associated with follicular lymphoma and BO, which showed findings suggesting coexistence of bullous pemphigoid (BP). The patient showed bullous and ulcerative lesions on the lips and oral cavity, and flaccid blisters on the trunk and thighs associated with anti‐desmoglein (Dsg)3 antibodies. At later disease stage after commencement of treatment, anti‐BP180 antibodies and tense blister formation were observed. It was proposed that persistent interface dermatitis is the first event in PNP, and subsequently induce the production of autoantibodies to Dsg and components of the basement membrane zone, resulting in both intraepidermal and subepidermal blisters. We speculate that interface dermatitis caused by autoreactive T cells induced autoantibody production against Dsg3, and subsequently against BP180.     

NaCl分离皮肤为底物的IIF法鉴别诊断表皮下大疱病

对1989~1991年间,在本所就诊的100例表皮下大疱病,通过临床、组织病理、直接免疫荧光(DIF)、间接免疫荧光(IIF)法以及1M NaCl分离皮肤为底物的IIF法进行了诊断和评价.结果发现DIF检查的100例中有76例皮肤BMZ有免疫反应物沉积,另24例为阴性,但有典型的自身免疫性大疱病的临床表现;根据临床、DIF和分离皮IIF法修正诊断出大疱性类天疱疮(BP)54例、获得性大疱性表皮松解症(EBA)8例、线状IgA大疱性皮病(LABD)7例(成人4、儿童3),瘢痕性类天疱疮(CP)7例、水疱性红斑狼疮(BSLE)3例、迟发性皮肤卟啉症(PCT)2例、BP和寻常型天疱疮(PV)并发1例,BP和疱疹样皮炎(DH)并发1例,有17例未定.研究结果发现分离皮IIF法对提高表皮下大疱病的诊断水平有较重要的应用价值.但由于CP的抗BMZ抗体可分别出现在表皮侧或真皮侧,所以分离皮IIF法不能单独鉴别CP和BP、CP和EBA.应该结合临床、免疫印迹和免疫电镜等方法进一步诊断,从而也表明分离皮IIF法有一定的局限性.     

Serum levels of anti‐type VII collagen antibodies detected by enzyme‐linked immunosorbent assay in patients with epidermolysis bullosa acquisita are correlated with the severity of skin lesions

Background Epidermolysis bullosa acquisita (EBA) is a chronic autoimmune subepidermal bullous disease characterized by circulating autoantibodies against type VII collagen. Detecting these autoantibodies is crucial for the diagnosis of this disease, and is also useful for measuring disease activity. Enzyme‐linked immunosorbent assay (ELISA), a quantitative method to measure anti‐type VII collagen antibody levels, is currently available to diagnose EBA. Objective The aim of this study was to investigate the relationship of ELISA with overall clinical severity. Methods Sera from patients with EBA (n = 30), bullous pemphigoid (n = 20), anti‐laminin γ1 pemphigoid (n = 9) and healthy donors (n = 24) were tested using ELISA, using the recombinant non‐collagenous 1 (NC1) and 2 (NC2) domains of type VII collagen. Relationships between clinical characteristics, indirect immunofluoroscence (IIF) titres and ELISA values were investigated. Results The sensitivity and specificity of the EBA ELISA were 96.7% and 98.1%, respectively. There was no significant difference between ELISA results for classic and inflammatory types. The severity of skin involvement was positively correlated with both ELISA value (r = 0.87, P < 0.01) and IIF titre (r = 0.59, P < 0.01). Time sequence analysis in four patients with EBA showed that ELISA values reflect disease activity better than IIF titres. Conclusions  Type VII collagen ELISA using the NC1 and NC2 domains is useful for diagnosing EBA and monitoring disease severity.     

Antidesmoplakin antibodies in pemphigus vulgaris

Background Besides being present in paraneoplastic pemphigus (PNP), circulating antidesmoplakin (DP) antibodies have been found anecdotally in other bullous diseases, including pemphigus foliaceus and pemphigus vulgaris. Objectives To verify how frequent anti‐DP antibodies are in pemphigus vulgaris. Methods We studied 48 sera from patients with proven pemphigus vulgaris (29 mucosal dominant pemphigus and 19 mucocutaneous pemphigus) by indirect immunofluorescence (IIF) with rat bladder epithelium (RBE) as a substrate and by immunoblotting (IB) on human keratinocyte cultures enriched in DP. Results Ten sera (21%) were positive in IIF on RBE. By IB, eight sera proved to have antibodies to both DP I (250 kDa) and DP II (210 kDa), one serum had antibodies directed to DP I only, and two sera to DP II only. Conclusions Our data confirm that RBE is not a specific IIF substrate for the serological diagnosis of PNP. It remains a sensitive and specific substrate for the detection of anti‐DP antibodies, which, in patients with pemphigus vulgaris, are probably caused by an epitope‐spreading phenomenon.     

The subclass distribution of IgG autoantibodies in cicatricial pemphigoid and epidermolysis bullosa acquisita

To study the subclass distribution of autoantibodies and their complement-fixing capacity in cicatricial pemphigoid (CP) and epidermolysis bullosa acquisita (EBA) we studied the sera from 23 patients by both indirect immunofluorescence (IIF) on 4-microns cryostat sections of normal human skin and immunoblotting of epidermal or dermal extracts. Monoclonal antibodies of strict specificity for human IgG subclasses were used. Sera from 20 patients with BP served as controls. In addition, total IgG subclass levels were determined by indirect competitive ELISA in all sera. Complement binding capacity was studied by IIF using antibodies to C3 after incubation of skin section with autoantibodies and source of fresh complement. CP autoantibodies reacting with the 230-240 kD and/or the 180-kD epidermal bands showed an IgG4/IgG1 subclass restriction, with a predominance of IgG4 in 10 cases, of IgG1 in four. In BP sera, IgG4 and IgG1 autoantibodies were detected with a similar frequency (100% and 83%, respectively). In EBA sera, autoantibodies reacting with the 290 kD and 145 kD dermal bands also showed an IgG1/IgG4 restriction. Concordant results were obtained by IIF. However, the IIF method had a lower sensitivity for the detection of IgG4 CP antibodies and IgG1 EBA antibodies than immunoblotting. Finally, when CP antibodies were analyzed for their complement-binding activity, it was found that sera containing IgG4 autoantibodies alone never fixed complement whereas all complement-fixing CP sera had IgG1 autoantibodies, suggesting that only this subclass of antibodies is capable of fixing complement.     

Case of bullous pemphigoid coexisting with anti‐desmoglein autoantibodies

A 79‐year‐old Japanese woman had clinical and histopathological features of bullous pemphigoid, while direct immunofluorescence test revealed C3 and immunoglobulin G depositions in the lower cell surfaces of the epidermis in addition to those in the dermoepidermal junction. Chemiluminescent enzyme immunoassays were positive for desmoglein‐1 and ‐3 antibodies in addition to anti‐BP180 antibodies. In an immunoblotting study, antibodies against both 180‐kDa bullous pemphigoid antigen and 130‐kDa pemphigus vulgaris antigen were detected. Based on these results, bullous pemphigoid coexisting with anti‐desmoglein autoantibodies was diagnosed in this case.     

Correlation of antibodies against desmogleins 1 and 3 with indirect immunofluorescence and disease status in a Greek population with pemphigus vulgaris

Background The use of ELISA testing of antibodies to desmogleins 1 and 3 (anti‐Dsg1 and anti‐Dsg3) and indirect immunofluorescence (IIF) has been strongly supported for the serologic diagnosis of pemphigus. The purpose of this study was to correlate anti‐Dsg1 and anti‐Dsg3 with IIF values, disease localization, treatment and clinical course in Greek patients with pemphigus vulgaris (PV). Methods A total of 54 patients with PV had ELISA serum testing for the presence and titers of anti‐Dsg1, anti‐Dsg3 and IIF. Anti‐Dsg1, anti‐Dsg3 and IIF were correlated with treatment and disease localization. For 40 patients, titers of anti‐Dsg1 and anti‐Dsg3 were assessed in relation to treatment and clinical course after 12 months. Results Anti‐Dsg3 and anti‐Dsg1 positivity in patients with negative IIF was 70.6% and 58.8%, respectively. Anti‐Dsg1 and anti‐Dsg3 were positive in 89.3% and 100% of patients with mucocutaneous disease, respectively, 88.9% and 66.7% of patients with skin limited disease, respectively and 52.9% and 76.5% of patients with mucosal limited disease, respectively. Both antibody titers showed significant correlation with IIF and treatment status. Improvement of clinical status was associated with significant decrease of both anti‐Dsg1 and anti‐Dsg3 after 12 months. Conclusions Serum testing of anti‐Dsg1 and anti‐Dsg3 in PV patients not only provides significant correlations with IIF, treatment and disease type, but may serve as a monitoring tool for clinical course and treatment guidance.     

Two Brazilian cases of IgA pemphigus

IgA pemphigus is a rare, neutrophilic, acantholytic skin disorder with approximately 70 cases described in the literature. We report two patients with the subcorneal pustular dermatosis (SPD) type of IgA pemphigus. Initially, both patients were misdiagnosed as subcorneal pustular dermatosis of Sneddon and Wilkinson. The correct diagnosis was only made after detecting intercellular IgA depositions in the epidermis by direct immunofluorescence. Immunoblotting (IB) of normal human epidermal extracts, performed on both sera, was negative for Dsg 1, Dsg 3, BP 230, BP 180, 210 kDa envoplakin, and 190 kDa periplakin. ELISA for desmogleins (Dsg 1 and Dsg 3) showed that neither of the cases had IgA antibodies to Dsg. The c-DNA transfection test for desmocollins (Dsc) revealed that the IgA antibodies of both patients reacted with desmocollin 1. This result supports the hypothesis that the autoantigen in SPD type IgA pemphigus is desmocollin 1.     

IgA autoantibodies in the pemphigoids and linear IgA bullous dermatosis

Please cite this paper as: IgA autoantibodies in the pemphigoids and linear IgA bullous dermatosis. Experimental Dermatology 2010; 19: 648–653. Background: Patients with bullous pemphigoid (BP), mucous membrane pemphigoid (MMP) and pemphigoid gestationis (PG) have IgG antibodies against BP180 and BP230, components of the hemidesmosomes. Patients with linear IgA bullous dermatosis (LABD) have IgA autoantibodies against a 97/120‐kDa protein which is highly homologous to a shedded fragment of the BP180‐ectodomain. Objectives: The aim of our study was to determine the incidence of IgA autoantibodies directed against BP180/BP230 in the pemphigoids and LABD and to determine the antigenic regions that are targeted by IgA autoantibodies. Methods: Utilizing baculovirus‐expressed recombinant BP180 and BP230 proteins, we performed immunoblot analyses for IgA reactivity of sera from patients with BP (n = 30), MMP (n = 10), PG (n = 6), LABD (n = 6) and from control patients with non‐related pruritic dermatoses (n = 8). Results: IgA reactivity against BP180 and/or BP230 was detected in 19/30 of the BP, in 7/10 of the MMP, in 6/6 of the LABD and in 3/6 of the PG sera, respectively, but not in the control group. In all subgroups, the major antigenic site recognized by IgA antibodies was located within the NH₂‐terminus of the BP180‐ectodomain, but only a minority of the sera showed also IgA reactivity against the BP180‐NC16a‐domain. IgA reactivity against the central domain of BP180 was more frequently seen than against its COOH‐terminus. IgA against the COOH‐ and NH₂‐terminus of BP230, respectively, was detected in 6/30 of the BP, 1/10 of the MMP, 1/6 of the LABD and 0/8 control sera. Conclusion: IgA reactivity against BP180 and/or BP230 is a common finding in the pemphigoids.     

大疱性系统性红斑狼疮的皮肤基底膜带相关抗原

免疫印迹和盐裂皮肤间接免疫荧光检测 ５例大疱性系统红斑狼疮（ＢＳＬＥ）血清，对照为 ５例获得性大疤性表皮松解症（ＥＢＡ）、２０例类天疱疮（ＢＰ）、２０例ＳＬＥ和１０例正常人血清。结果表明，３例（３／５）ＢＳＬＥ血清结合盐裂皮肤真皮侧和真皮提取物中２９０ ０００抗原，其中２例ＢＳＬＥ血清也结合表皮提取物中 １６５ ０００抗原，结果与 ＥＢＡ和部分ＢＰ血清相同。ＳＬＥ血清未结合 ２９０ ０００和 １６５ ０００抗原。提示ＢＳＬＥ血清中存在ＥＢＡ和ＢＰ抗体，推测ＥＢＡ和ＢＰ抗原可能是ＢＳＬＥ的皮肤基底膜带相关抗原。     

A sensitive and restricted enzyme-linked immunosorbent assay for detecting a heterogeneous antibody population in serum from people suffering from a new variant of endemic pemphigus

We recently described a new variant of endemic pemphigus foliaceus (EPF) in El Bagre, Colombia, that resembles Senear-Usher syndrome and identified autoantibodies to desmoglein 1 (Dsg1), as well as to multiple known and unknown antigens including plectins, in the serum of these patients. Here, we developed a cost-effective ELISA assay capable of detecting the heterogeneous antibody population observed in these EPF patients, and useful for serum epidemiological studies. A protein extract obtained from trypsin-digested fresh bovine skin and further purified on a concanavalin A matrix was used as antigen. This extract contains an important conformational epitope (a 45 kDa tryptic fragment of the Dsg1 ectodomain), which is recognized by antibodies in serum from patients with all varieties of pemphigus foliaceus (PF), and from half of those with pemphigus vulgaris with active clinical disease. The cut-off and threshold values were normalized using human serum obtained from both endemic and non-endemic areas for PF. The efficiency of this ELISA was tested using 600 serum samples from controls and patients diagnosed with EPF, non-endemic PF and other bullous diseases. The overall sensitivity and specificity of the assay were determined to be 95% and 72%, respectively, with reproducibilities of 98% (intraassay) and 95% (interassay). Comparing the ELISA with other tests to detect EPF autoantibodies, this ELISA was the most sensitive, followed by direct immunofluorescence (DIF), indirect immunofluorescence using anti-IgG4 monoclonal antibodies and immunoprecipitation (IP), respectively. The most specific assay was IP, followed by DIF. Immunoblotting to Dsg1 exhibited both poor sensitivity and poor specificity, although plectins were well visualized. We conclude that this ELISA is an excellent tool for field serological studies, allowing testing of multiple serum samples simultaneously and for detecting, with appropriate restriction and sensitivity, the heterogeneous antibody population seen in patients with this variant of EPF. Finally, autoantibody serum levels obtained with this ELISA correlated well with the clinical activity and extent of disease in patients with El Bagre EPF.Abbreviations BMZ Basement membrane zone - BP Bullous pemphigoid - BP180 Bullous pemphigoid 180 kDa antigen - ConA Concanavalin A - CPF Cazanaves pemphigus foliaceus - DIF Direct immunofluorescence - ELISA Enzyme-linked immunosorbent assay - EPF Endemic pemphigus foliaceus - IB Immunoblotting - IIF Indirect immunofluorescence - IP Immunoprecipitation - mAb Monoclonal antibody - PBS Phosphate-buffered saline - PBS^– Phosphate-buffered saline lacking divalent cations - PF Pemphigus foliaceus - PV Pemphigus vulgaris - SLE Systemic lupus erythematosus This paper is part of the doctoral (PhD) thesis of Ana María Abréu Vélez, MD, while at the University of Antioquia.     

Atypical pemphigus with immunoglobulin G autoantibodies against desmoglein 3 and desmocollin 3

In patients with pemphigus vulgaris (PV), pathogenic immunoglobulin (Ig)G antibodies are most commonly directed against desmoglein 3 (Dsg3). It has recently been reported, however, that IgG anti‐desmocollin 3 (Dsc3) antibodies are detected in some cases of pemphigus with or without IgG anti‐Dsg3 antibodies. We present a case of pemphigus with IgG antibodies against Dsg3 and Dsc3. Subsequent studies showed that the cell surface distribution pattern of Dsc3 but not Dsg3 was altered, suggesting that suprabasal acantholytic blisters were induced by IgG anti‐Dsc3 antibodies rather than IgG anti‐Dsg3 antibodies. Our case suggests that anti‐Dsc3 antibodies may be pathogenic in cases positive for the dual cadherin autoantibodies.     

Acquired bullous diseases of childhood: re-evaluation of diagnosis by indirect immunofluorescence examination on 1 M NaCl split skin and immunoblotting

Summary Acquired autoimmune bullous diseases of childhood are rare, and can be difficult to distinguish clinically. We have studied 12 children, with an initial diagnosis of bullous pemphigoid (BP) in eight patients, cicatricial pemphigoid (CP) in one, chronic bullous disease of childhood (CBDC) in one, and epidermolysis bullosa acquisita (EBA) in two.
All patients had positive indirect immunofluorescence (HF) of the BMZ with IgG. Using 1 M NaCl split skin, six patients showed epidermal binding of IgG, with additional IgA in three cases, and in five patients IgG antibodies bound a dermal protein. Immunoblotting studies revealed an antibody to type VII collagen (EBA antigen) in three patients who had a dermal pattern on IIF. Six sera reacted with an epidermal protein of 180 and/or 220 kDa, characteristic of BP and CP. One of the three IgA-positive sera detected 220-and 180-kDa epidermal proteins using anti-IgA antibody. Following these studies the diagnosis was changed in three of the children. The diagnosis of CBDC was changed to either BP or EBA because of the presence of circulating IgG autoantibodies. In two children with an initial diagnosis of BP the diagnosis was changed to EBA.
We conclude that the clinical picture in bullous disorders of childhood shows considerable overlap, and is often misleading. Additional circulating IgA autoantibodies seem to be more common in BP than has been recognized previously. Indirect immunofluorescence investigation on 1 M NaCl split skin may be helpful in differentiating between BP and EBA, but does not replace immunoblotting studies. EBA is apparently more common in children than in adults. No difference was found between the children with BP and EBA with regard to the duration of disease. The long-term outlook is good, although the course may be protracted.     

The predominance of IgG4 in prodromal bullous pemphigoid

Background Prodromal bullous pemphigoid (PBP) and bullous pemphigoid (BP) demonstrate immunoglobulin G (IgG) and/or C3 deposition at the basement membrane zone (BMZ) on direct immunofluorescence. BP‐180‐specific IgG1, IgG4, and IgE antibodies have been detected in BP. However, the distribution of IgG subclasses is unknown in PBP. Objectives We will describe the role of anti‐BMZ IgG subclasses in PBP and we will correlate these findings to better understand the pathogenesis of PBP. Methods Skin biopsies and serum samples were obtained from 45 patients who had PBP. The skin tissue was processed for direct immunofluorescence studies. Sera were analyzed by indirect immunofluorescence for the presence of circulating anti‐BMZ IgG antibodies (by standard IIF) and IgG subclasses antibodies (by sandwich double antibody immunofluorescence [SDAI]). Sera were also analyzed for antibodies against BP‐180 and BP‐230 antigens by enzyme‐linked immunosorbent assay (ELISA). Results Thirty‐two patients (71%) had IgG and C3 staining at the BMZ, while 13 patients (29%) had isolated C3 staining at the BMZ on direct immunofluorescence. All patients demonstrated staining on the epidermal side of the salt‐split skin. Of the seven skin specimens that were available for C5‐9 SDAI testing, all were found to be positive along BMZ area. Standard IIF studies demonstrated the presence of circulating BMZ antibodies in 11 of the 30 patients (36.6%). When SDAI for IgG subclass differentiation was utilized, 17 of 30 (56.6%) patients were found to have circulating anti‐BMZ antibodies. All of these 17 patients had IgG4 subclass antibodies. Thirteen patients did not have detectable IgG subclass anti‐BMZ antibody on SDAI. Sixteen of 30 patients had detectable anti‐BP‐180 or anti‐BP‐230 antibodies, while 12 (40%) did not have detectable antibody against BP antigens on ELISA. Conclusions IgG4 is the initial and predominant anti‐BMZ antibody subclass detected in PBP. Demonstration of linear C5‐9 at the BMZ enhances the early diagnosis of PBP. Predominance of IgG4 and the initial presence of IgG4 on skin lesions as well as the presence of only IgG4 subclass anti‐BMZ antibody suggest that IgG4 subclass antibody could be the initial immunologic event encountered in patients with PBP.     

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.     

Epitope mapping of BP230 leading to a novel enzyme-linked immunosorbent assay for autoantibodies in bullous pemphigoid

Background Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by circulating autoantibodies against BP180 and BP230. For BP180, the NC16A domain has previously been identified as the main antigenic target in BP, while data about the diagnostic value of epitopes on BP230 were inconclusive. Objectives To identify the most appropriate epitopes on BP230 to be applied in a simple, sensitive, and highly specific enzyme‐linked immunosorbent assay (ELISA) for routine detection of serum autoantibodies. Methods Ten overlapping linear fragments covering the whole length of BP230 were expressed in Escherichia coli. Based on Western blot analysis with sera from patients with BP (n = 49) and healthy controls (n = 94), the diagnostic performance of the fragments was compared by receiver operating characteristics curve analysis. The BP230‐C3 fragment comprising the C‐terminal portion (amino acids 2326–2649) was subsequently applied in a novel ELISA. The operating characteristics of this ELISA were analysed by probing sera from patients with BP (n = 118), pemphigus vulgaris (n = 50), rheumatoid arthritis and other inflammatory arthritides (n = 170), and systemic lupus erythematosus (n = 56), and from healthy blood donors (n = 483). Results Among all the fragments, BP230‐C3 provided the best efficiency in serologically diagnosing BP by Western blot. An ELISA employing BP230‐C3 revealed a diagnostic sensitivity of 56·8% and specificity of 97·6%. Its diagnostic added value amounted to 4·2% compared with the anti‐BP180‐NC16A‐4X ELISA alone. Conclusions Recombinant BP230‐C3 is a suitable target antigen for the detection of serum autoantibodies against BP230.     

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.