Clinical significance of enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid

The NC16A domain of the 180-kDa bullous pemphigoid antigen (BP180) is the most immunogenic and, probably, pathogenic region in bullous pemphigoid (BP). In the present study, in order to determine whether serum level of circulating anti-BP180 autoantibodies is a valuable serum marker in BP, the immunoreactivity of sera against the NC16A domain of BP180 was measured using enzyme-linked immunosorbent assay (ELISA) in ten patients with BP. Serum levels of anti-BP180 autoantibodies correlated with the clinical course in BP patients, who received various therapeutic agents. The result suggests that this NC16A-ELISA is a useful method for evaluating the clinical course and efficacy of the therapy in patients with BP.     

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.     

Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin

Bullous pemphigoid is a subepidermal autoimmune blistering disease associated with autoantibodies to the hemidesmosomal bullous pemphigoid antigens 180 and 230. Most sera from bullous pemphigoid patients recognize epitopes within the N-terminal NC16A portion of the bullous pemphigoid 180 ectodomain. Using cryosections of human skin, patients' sera were shown to generate dermal-epidermal separation when coincubated with leukocytes and complement from healthy volunteers; however, the specificity of pathogenic autoantibodies in bullous pemphigoid patients has not yet been elucidated. In this study, by the use of a modified version of the cryosection model, we show that sera from all of 13 bullous pemphigoid patients and from two rabbits, immunized against bullous pemphigoid 180 NC16A, induced dermal-epidermal separation. This finding was confirmed with the use of IgG purified from patients' sera, whereas sera and purified IgG from healthy controls were not pathogenic. The induction of subepidermal splits in this experimental model was shown to be dependent on the presence of neutrophils, but not complement. Interestingly, patients' autoantibodies affinity purified against a recombinant form of bullous pemphigoid 180 NC16A retained their blister-inducing capacity, whereas patients' IgG depleted of reactivity to NC16A lost this ability. F(ab')2 fragments of antibodies specific to NC16A, lacking the Fc portion, did not induce splits. In addition, patients' autoantibodies purified against a recombinant fragment of the C-terminus of bullous pemphigoid 180 as well as rabbit antibodies to the intracellular portion of bullous pemphigoid 180 and to bullous pemphigoid 230 did not cause dermal-epidermal separation. Our in vitro results support the idea that autoantibodies to bullous pemphigoid 180 from patients with bullous pemphigoid are of pathogenic relevance.     

The intracellular and extracellular domains of BP180 antigen comprise novel epitopes targeted by pemphigoid gestationis autoantibodies

Pemphigoid gestationis (PG) is an autoimmune sub-epidermal bullous dermatosis of pregnancy associated with circulating autoantibodies targeting the extracellular non-collagenous (NC) 16A domain of bullous pemphigoid (BP) 180 antigen. In order to determine whether BP180 regions other than NC16A are recognized by PG autoantibodies, we have analyzed the reactivity of 15 PG patient sera against several BP180 antigenic sites by sensitive methods such as immunological screening and ELISA. Most PG sera tested (13 of 15) reacted with an epitope (amino acid 508-541) mapped in the NC16A domain. Of note, nine of 15 PG patient sera reacted with at least one additional antigenic site other than NC16A. Specifically, two epitopes in the BP180 extracellular domain and five epitopes in the intracellular one were recognized by three and seven PG sera, respectively. In addition, a representative intracellular epitope was recognized by PG autoantibodies as a portion of BP180 antigen both in denaturating and native conditions. Finally, reactivity against epitopes additional to NC16A was also detected at an early stage of the disease. The identification and characterization of hitherto unrecognized epitopes targeted by PG patient autoantibodies provide novel insights into the pathophysiology of humoral immune response to BP180 in PG.     

Autoantibodies in lichen planus pemphigoides react with a novel epitope within the C-terminal NC16A domain of BP180.

Lichen planus pemphigoides is an autoimmune subepidermal blistering disease. The finding of immunoglobulin G antibodies directed against the basement membrane zone differentiates it from bullous lichen planus. The aim of this study was to identify the target antigen of lichen planus pemphigoides autoantibodies. Sera from lichen planus pemphigoides patients (n = 4) stained the epidermal side of NaCl-split human skin in a pattern indistinguishable from that produced by bullous pemphigoid sera. In bullous pemphigoid, the autoimmune response is directed against BP180, a hemidesmosomal transmembrane collagenous glycoprotein. We previously demonstrated that bullous pemphigoid sera predominantly react with a set of four epitopes (MCW-0 through MCW-3) clustered within a 45 amino acid stretch of the major noncollagenous extracellular domain (NC16A) of BP180. By immunoblotting and enzyme-linked immunosorbent assay, lichen planus pemphigoides sera were also strongly reactive with recombinant bullous pemphigoid 180 NC16A. The lichen planus pemphigoides epitopes were further mapped using a series of overlapping recombinant segments of the NC16A domain. All lichen planus pemphigoides sera reacted with amino acids 46-59 of domain NC16A, a protein segment that was previously shown to be unreactive with bullous pemphigoid sera. Two lichen planus pemphigoides sera, in addition, reacted with the immunodominant antigenic region associated with bullous pemphigoid. In conclusion, there are now five bullous diseases that are associated with an autoimmune response to BP180: bullous pemphigoid; pemphigoid/herpes gestationis; cicatricial pemphigoid; linear immunoglobulin A disease; and lichen planus pemphigoides. In addition, we have identified a novel epitope within the BP180 NC16A domain, designated MCW-4, that appears to be uniquely recognized by sera from patients with lichen planus pemphigoides.     

Memory B cells specific for the NC16A domain of the 180 kDa bullous pemphigoid autoantigen can be detected in peripheral blood of bullous pemphigoid patients and induced in vitro to synthesize autoantibodies

Bullous pemphigoid is a subepidermal blistering disease characterized by the synthesis of autoantibodies against the 180 kDa and the 230 kDa bullous pemphigoid antigens. Whether autoimmunity is also reflected by the presence of circulating autoantigen-specific memory B cells is still a matter of debate. We used a new assay combining two-step immunomagnetic enrichment with multiparameter flow cytometry to detect and characterize bullous pemphigoid 180 kDa-specific IgG+ B cells in blood of bullous pemphigoid patients. In a first magnetic separation, B cells were isolated from peripheral blood mononuclear cells using releasable microbeads conjugated to a CD19 antibody. From pre-enriched B cells, bullous pemphigoid 180 kDa-specific cells were then positively selected using microbeads directly conjugated with a recombinant N-terminal fragment of the bullous pemphigoid 180 kDa ectodomain, containing the noncollagenous 16A domain, which was recently shown to harbor major epitopes of autoantibodies in bullous pemphigoid sera. Noncollagenous 16A domain-specific IgG+ B cells were detectable in blood of most, if not all patients with serum autoantibodies against the noncollagenous 16A domain. The specificity of the cells was confirmed by in vitro differentiation into antibody-forming cells and analysis of the culture supernatant for the presence of noncollagenous 16A domain-specific IgG antibodies. All noncollagenous 16A domain-specific IgG+ B cells showed a clear memory immunophenotype. Noncollagenous 16A domain-specific IgG+ memory B cells may be crucial for continuous noncollagenous 16A domain-specific autoantibody production and/or play a part as antigen-presenting cells for priming and restimulation of bullous pemphigoid 180 kDa-specific T helper cells.     

Autoantibodies to basement membrane proteins BP180 and BP230 are commonly detected in normal subjects by immunoblotting

Autoantibodies to basement membrane proteins BP180 and BP230 are characteristic of bullous pemphigoid and other subepidermal immunobullous disorders. These antibodies are, however, reported in other pruritic dermatoses, non-bullous disorders and non-cutaneous disease. Few studies have assessed basement membrane antibodies in normal subjects; antibody prevalence in this population is not clear. This study aims to examine basement membrane zone antibodies in normal middle-aged to elderly subjects. Sera from 61 healthy subjects (majority age 50–70 years) were assessed by immunoblot, indirect immunofluorescence and enzyme-linked immunosorbent assay. Ninety-one bullous pemphigoid patients acted as positive controls. Antigenic target, antibody class and titre were examined; sera binding BP180 were assessed for reactivity to the non-collagenous 16A (NC16A) domain. Thirty-six normal subjects (59%) had antibodies to either BP180 or BP230 on immunoblot analysis. BP180 was the commonest target antigen, detected in 35 subjects; binding to the immunodominant NC16A domain was not detected. Immunofluorescence was positive in three subjects. Of the bullous pemphigoid sera, 88% were positive on immunoblot or immunofluorescence; a higher frequency had antibodies against BP230. In conclusion, significant numbers of normal healthy subjects have circulating autoantibodies to basement membrane proteins, chiefly BP180 detectable by immunoblot, but these do not bind the NC16A domain.     

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

Background The 180‐kDa transmembrane hemidesmosomal protein (BPAG2) has been identified as an important autoantigen in bullous pemphigoid (BP). Using the NC16A domain as the antigenic target, a highly sensitive and specific enzyme‐linked immunosorbent assay (ELISA) for the detection of autoantibodies to BP180 was developed. Aim To investigate the correlation of clinical severity and ELISA indices in BP. Methods Antibody titers in the sera from 20 patients were measured using BP180NC16a‐ELISA, and an analysis of the correlation of ELISA indices with disease activity was performed. Serum was obtained from each patient with BP at least three times: before the initiation of treatment, during complete disease control just before the decrease in corticosteroid, and when the dosage of corticosteroid was successfully decreased to half the initial dose. Of the 20 patients, three showed recurrence at a later stage, caused by their decision to stop treatment; serum was obtained at the early stage of recurrence. Results ELISA indices were significantly decreased after successful therapy, although indirect immunofluorescence titers did not always show apparent correlations. Indices measured using BP180NC16a‐ELISA were well correlated with disease activity. Three patients decided to stop taking their medication; subsequently (within 1–2 weeks), blisters recurred, and the levels of antibodies to BP180 increased to close to those before the initiation of treatment. Conclusion BP180 antibody titers showed a closer correlation than indirect immunofluorescence titers to disease activity. The titer of BP180 antibody may be a useful tool for the evaluation of disease activity and for the assessment of the effectiveness of treatments in BP.     

Characterization of the anti-BP180 autoantibody reactivity profile and epitope mapping in bullous pemphigoid patients

Bullous pemphigoid is a subepidermal bullous disease of skin and mucosae associated with autoantibodies to BP180. To characterize the humoral response to BP180, we generated a random BP180 epitope library displayed on lambda bacteriophage. After validation of the library by epitope mapping of three BP180-specific monoclonal antibodies, 15 novel or known BP180 epitopes were identified using 10 bullous pemphigoid serum samples. Fifty-seven bullous pemphigoid and 81 control sera were then assayed against the selected epitopes. Thirty-one out of 57 (54%) bullous pemphigoid sera reacted with at least an additional antigenic site other than the NC16A, within the extracellular (37%) and intracellular (28%) domains of BP180. In addition, the reactivity with extracellular epitopes of BP180 contained within the residue stretches 508-541 and 1331-1404 appeared to be related to the presence of both skin and mucosal involvement. Finally, a preliminary analysis of the epitope pattern in the disease course indicated that bullous pemphigoid patients exhibit a specific reactivity pattern, and that binding to intracellular epitopes of BP180, in addition to NC16A, may be detectable at an early clinical stage. Our findings provide novel insights into the pathophysiology of bullous pemphigoid and show the potential of the utilized approach as a tool for a rapid diagnosis of bullous pemphigoid patients and their management.     

Autoantibodies in a subgroup of patients with linear IgA disease react with the NC16A domain of BP1801

Linear IgA disease is an autoimmune subepidermal blistering disease characterized by IgA deposits at the cutaneous basement membrane zone. IgA antibodies from linear IgA disease sera react with antigens of 97 kDa (LABD97) and 120 kDa (LAD-1), both of which appear to be fragments of the extracellular domain of bullous pemphigoid 180 (type XVII collagen). The aim of this study was to determine whether linear IgA disease sera react with the immunodominant region of BP180 (NC16A domain), which is a major target of IgG autoantibodies produced by patients with bullous pemphigoid. Indeed, 11 of 50 linear IgA disease sera were found to contain IgA autoantibodies that recognized a recombinant form of NC16A by immunoblotting. The same sera also reacted with NC16A by enzyme-linked immunosorbent assay. An epitope mapping analysis uncovered four linear IgA disease-associated epitopes located within the 45 amino acid N-terminal stretch of NC16A, all of which were previously identified as antigenic sites targeted by bullous pemphigoid autoantibodies. Eight of the linear IgA disease sera that were reactive with NC16A also recognized LAD-1 secreted by the SCC-25 cell line, and five sera recognized BP180 extracted from keratinocytes. Linear IgA disease sera depleted of reactivity to NC16A by immunoadsorption continued to react with both the LAD-1 antigen and BP180 by immunoblotting and with the basement membrane zone by indirect immunofluorescence microscopy. Our results demonstrate that IgA autoantibodies from a subset of linear IgA disease patients react with the same sites on BP180 that are targeted by IgG autoantibodies in bullous pemphigoid.     

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.     

Detection of laminin 5-specific auto-antibodies in mucous membrane and bullous pemphigoid sera by ELISA

Mucous membrane pemphigoid (MMP) is an autoimmune bullous disease that primarily affects mucous membranes leading to a scarring phenotype. MMP patients produce auto-antibodies (auto-ab) that preferentially recognize two components of the dermoepidermal basement membrane zone (BMZ): bullous pemphigoid (BP)180 and laminin 5 (LN5). Since detection of disease-specific auto-ab may be critical for diagnosis of MMP, we developed an ELISA with affinity-purified native human LN5. A total of 24 MMP, 72 BP, and 51 control sera were analyzed for LN5-specific auto-ab: 18/24 (75.0%) MMP and 29/72 (40.3%) BP sera were LN5 reactive. Sensitivity and specificity of the LN5 ELISA for MMP were 75% and 84.3%, respectively, and 40.3% and 88.2% for BP, respectively. The LN5 ELISA was more sensitive than a dot blot assay with native LN5, which detected LN5-reactive IgG in 14/24 (58.3%) MMP and 16/72 (22.2%) BP sera. In MMP, but not BP, levels of LN5-reactive IgG correlated with disease severity. Furthermore, IgG reactivity to LN5 of the MMP and BP sera was not significantly associated with IgG reactivity against other autoantigens of the BMZ, such as BP180 or BP230. Thus, the established LN5 ELISA holds great promise as a novel diagnostic and prognostic parameter for MMP.     

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.     

Immunoreactivity of bullous pemphigoid (BP) autoantibodies against the NC16A and C-terminal domains of the 180 kDa BP antigen (BP180): immunoblot analysis and enzyme-linked immunosorbent assay using BP180 recombinant proteins

The 180 kDa bullous pemphigoid (BP) antigen (BP180) is known to be recognized by sera from patients with BP, herpes gestationis (HG) and cicatricial pemphigoid (CP). A series of previous studies using BP180 recombinant proteins has shown that most sera from patients with BP and HG react with the NC16A domain of BP180, an extracellular non-collagenous region just adjacent to the plasma membrane. In contrast, the C-terminal region of BP180 has been reported to be one of the epitopes of CP. In the present study, we examined the immunoreactivity of 110 BP sera against the NC16A and C-terminal domains of BP180 using immunoblot analysis and enzyme-linked immunosorbent assay (ELISA). Immunoblot analysis revealed that 100 (91%) and 26 (23.5%) of the 110 BP sera recognized the NC16A and C-terminal domains, respectively. The results of the ELISA were correlated with those of immunoblotting. There were no specific or significant clinical features such as severe involvement of mucous membranes and scarring in BP patients whose sera reacted with the C-terminal region. These findings suggest that some BP sera react with the C-terminal region of BP180 without any association with the characteristic clinical features of CP.     

Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain

Bullous pemphigoid is a blistering skin disease characterized by autoantibodies directed against the NC16A domain of bullous pemphigoid 180 (collagen XVII), a transmembrane protein of epidermal basal cells. Passive transfer studies in mice have shown that antibodies that bind to this immunodominant region of bullous pemphigoid 180 are capable of inducing a skin disease that closely mimics bullous pemphigoid, supporting the hypothesis that epitopes within NC16A are involved in the pathogenesis of bullous pemphigoid. In this study, we examined the autoimmune T cell response in bullous pemphigoid patients. T cells from eight of 12 bullous pemphigoid patients, all of whom had circulating anti-bullous pemphigoid 180 autoantibodies, showed a specific proliferative response to recombinant forms of NC16A. T cell lines and clones developed from four of these patients recognize the same NC16A peptides as those targeted by autoantibodies from the corresponding individuals. These NC16A-responding T lymphocytes express alpha/beta T cell receptors and CD4 memory T cell surface markers and exhibited a Th1/Th2 mixed cytokine profile that may support the production of antibodies. This new information will aid in defining the key steps involved in the development of the autoimmune response in bullous pemphigoid.     

Cicatricial pemphigoid: IgA and IgG autoantibodies target epitopes on both intra- and extracellular domains of bullous pemphigoid antigen 180

BACKGROUND: Cicatricial pemphigoid (CP) is an autoimmune subepidermal blistering disease where autoantibodies target various components of the dermal-epidermal junction, including the bullous pemphigoid antigen 180 (BP180). OBJECTIVE: We determined the exact specificity of circulating IgG and IgA autoantibodies to BP180 in a large number of CP patients. METHODS: Twenty-six consecutive CP sera were analysed by Western blotting using a panel of cell-derived and recombinant proteins covering the entire BP180 molecule. RESULTS: Circulating autoantibodies were detected in all CP sera. Seven sera reacting with laminin-5 were excluded from further analyses; the remaining 19 sera recognized BP180, including six sera (32%) that showed only IgA reactivity to this protein. With the combined use of the soluble BP180 ectodomain (LAD-1) and recombinant BP180 NC16A, 16 of these 19 CP sera (84%) targeted BP180. IgG reactivity was preferentially found against NC16A, whereas IgA antibodies predominantly recognized LAD-1. Thirty-two per cent of the BP180-reative sera revealed reactivity with the intracellular domain of this protein. CONCLUSIONS: Our findings demonstrate that autoantibodies in CP target epitopes on both extra- and intracellular domains of BP180 and highlight the importance of testing for both IgG and IgA reactivity in these patients' sera.     

A Case of Chronic Bullous Disease of Childhood That Was Reactive to the Antigen of 120 kDa (LAD-1)

Chronic bullous disease of childhood (CBDC) is an autoimmune blistering disease that is characterized by Immunoglobulin A (IgA) deposits at the basement membrane zone. IgA autoantibodies (aAbs) from the serum of patients with CBDC react with antigens of 97 kDa (LABD97) and 120 kDa (LAD-1), and both of which are fragments of the extracellular domain of bullous pemphigoid 180 (BP180, type XVII collagen). The CBDC sera reacts with the immunodominant NC16a domain of BP180, which is the major region recognized by IgG aAbs in patients with bullous pemphigoid. A five-year-old boy presented with multiple pruritic tense blisters on the umbilical and inguinal areas for six weeks. The direct immunofluorescence of the perilesional area demonstrated linear deposits of IgA at the basement membrane zone. Using immunoblotting and an enzyme linked immunosorbent assay (ELISA), we identified the IgA aAbs reactive to antigens with a molecular weight of 120 kDa (LAD-1), which is a fragment of the extracellular domain of 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.     

Binding of bullous pemphigoid and pemphigus vulgaris sera to SCaBER cell line in an ELISA

Specific antibodies present in the sera of patients with bullous pemphigoid or pemphigus vulgaris were detected in an enzyme-linked immunosorbent assay (ELISA) employing a squamous carcinoma cell line, SCaBER, as substrate. Bullous pemphigoid sera bound preferentially to permeabilized cells, suggesting that the antigens are largely intracellular. The assay may prove to be a useful addition to current methods of detecting circulating antibodies in these patients.     

Cicatricial pemphigoid differs from bullous pemphigoid and pemphigoid gestationis regarding the fine specificity of autoantibodies to the BP180 NC16A domain

Bullous pemphigoid (BP), pemphigoid (herpes) gestationis (PG), cicatricial pemphigoid (CP), and lichen planus pemphigoides (LPP) are autoimmune subepidermal bullous diseases that are characterized by circulating autoantibodies to the transmembrane hemidesmosomal protein BP180/type XVII collagen. Previous studies demonstrated that the majority of patients with BP, PG, and LPP show antibodies to an immunodominant, membrane-proximal non-collagenous domain (NC16A) on the extracellular portion of BP180. By the use of non-overlapping peptides of the NC16A domain, we previously demonstrated that autoantibodies from BP and PG patients mainly react with epitopes clustered within the N-terminus of this immunodominant site of BP180; antibodies from patients with LPP also recognized the C-terminal portion of NC16A. However, some of these results had been obtained indirectly by preadsorption studies. The aim of the present study was to analyze the fine specificity of IgG autoantibodies to NC16A in sera from patients with CP and to compare their reactivity with antibodies from BP, PG, and LPP patients using a series of new overlapping fragments covering the entire NC16A domain. We confirm that BP and PG sera mainly react with N-terminal epitopes of NC16A, whereas sera from patients with LPP also bind to C-terminal portions, of this domain. Interestingly, out of ten patients with CP, the sera of seven reacted with NC16A; within NC16A, these sera bound to both C-terminal fragments and an N-terminal epitope right next to the cell membrane. Our data demonstrate a heterogeneous binding pattern of autoantibodies to BP180 NC16A in patients with CP.