Title: Severity and Phenotype of Bullous Pemphigoid Relate to Autoantibody Profile Against the NH2- and COOH-Terminal Regions of the BP180 Ectodomain
Abstract: Bullous pemphigoid, the most common autoimmune subepidermal bullous disorder, is associated with autoantibodies targeting antigenic sites clustered within the extracellular domain of BP180. To investigate epitope and subclass specificity of autoantibodies in bullous pemphigoid, we developed an enzyme-linked immunosorbent assay utilizing baculovirus-expressed recombinant forms of the NH2- and COOH-terminal regions of the extracellular domain of BP180 and examined sera obtained from patients with active bullous pemphigoid (n=116) and controls (n=100). Ninety-three (80%) and 54 (47%) of the 116 bullous pemphigoid sera recognized the NH2- and COOH-terminal regions, respectively, of the extracellular domain of BP180. Detailed analysis demonstrates that (i) this novel enzyme-linked immunosorbent assay is highly specific (98%) and sensitive (93%) as 108 of 116 bullous pemphigoid sera reacted with at least one of the baculovirus-derived recombinants, (ii) in active bullous pemphigoid, autoantibodies against the NH2-terminus of the extracellular domain of BP180 were predominantly of the IgG1 class, whereas a dual IgG1 and IgG4 response to this region was related to a more severe skin involvement, (iii) autoreactivity against both the NH2- and COOH-terminal regions was more frequently detected in patients with mucosal lesions, and (iv) levels of IgG (and IgG1) against the NH2-terminal, but not against the COOH-terminal portion of the extracellular domain of BP180, reflected disease severity indicating that autoantibodies against the NH2-terminus are critical in the pathogenesis of bullous pemphigoid. In conclusion, this novel enzyme-linked immunosorbent assay represents a highly sensitive and specific assay for rapid diagnosis of bullous pemphigoid and related disorders and may provide predictive parameters for the management of bullous pemphigoid patients. Bullous pemphigoid, the most common autoimmune subepidermal bullous disorder, is associated with autoantibodies targeting antigenic sites clustered within the extracellular domain of BP180. To investigate epitope and subclass specificity of autoantibodies in bullous pemphigoid, we developed an enzyme-linked immunosorbent assay utilizing baculovirus-expressed recombinant forms of the NH2- and COOH-terminal regions of the extracellular domain of BP180 and examined sera obtained from patients with active bullous pemphigoid (n=116) and controls (n=100). Ninety-three (80%) and 54 (47%) of the 116 bullous pemphigoid sera recognized the NH2- and COOH-terminal regions, respectively, of the extracellular domain of BP180. Detailed analysis demonstrates that (i) this novel enzyme-linked immunosorbent assay is highly specific (98%) and sensitive (93%) as 108 of 116 bullous pemphigoid sera reacted with at least one of the baculovirus-derived recombinants, (ii) in active bullous pemphigoid, autoantibodies against the NH2-terminus of the extracellular domain of BP180 were predominantly of the IgG1 class, whereas a dual IgG1 and IgG4 response to this region was related to a more severe skin involvement, (iii) autoreactivity against both the NH2- and COOH-terminal regions was more frequently detected in patients with mucosal lesions, and (iv) levels of IgG (and IgG1) against the NH2-terminal, but not against the COOH-terminal portion of the extracellular domain of BP180, reflected disease severity indicating that autoantibodies against the NH2-terminus are critical in the pathogenesis of bullous pemphigoid. In conclusion, this novel enzyme-linked immunosorbent assay represents a highly sensitive and specific assay for rapid diagnosis of bullous pemphigoid and related disorders and may provide predictive parameters for the management of bullous pemphigoid patients. bullous pemphigoid baculoprotein containing the NC16A domain and Col15 domain of BP180 baculoprotein containing the COOH-terminal domains NC3–NC1 of BP180 baculoprotein containing the extracellular domain of BP180 extracellular domain enzyme-linked immunosorbent assay optical density phosphate-buffered saline Bullous pemphigoid (BP) is an autoimmune blistering disorder of the skin that usually affects the elderly. The disease is associated with tissue-bound and circulating IgG autoantibodies directed against components of the epidermal basement membrane zone (Jordon et al., 1967Jordon R.E. Beutner E.H. Witebsky E. et al.Basement zone antibodies in bullous pemphigoid.JAMA. 1967; 200: 751-756Crossref PubMed Scopus (399) Google Scholar). Clinically, BP is characterized by generalized tense subepidermal blisters arising on apparently normal or erythematous skin with involvement of mucous membranes in 20%–30% of cases (Lever, 1953Lever W.F. Pemphigus.Medicine. 1953; 32: 1-123Crossref PubMed Scopus (358) Google Scholar). Previous studies have demonstrated that BP autoantibodies predominantly recognize BP230 (BPAG1) and BP180 (BPAG2 or type XVII collagen) (Labib et al., 1986Labib R.S. Anhalt G.J. Patel H.P. et al.Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting.J Immunol. 1986; 136: 1231-1235PubMed Google Scholar; Stanley et al., 1988Stanley J.R. Tanaka T. Müller S. et al.Isolation of complementary DNA for bullous pemphigoid antigen by use of patients' autoantibodies.J Clin Invest. 1988; 82: 1864-1870Crossref PubMed Scopus (311) Google Scholar; Giudice et al., 1992Giudice G.J. Emery D.J. Diaz L.A. Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180.J Invest Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar), two structural components of hemidesmosomes. Hemidesmosomes are multiprotein junctional complexes that promote attachment of basal keratinocytes to the underlying basement membrane zone (reviewed inBorradori and Sonnenberg, 1999Borradori L. Sonnenberg A. Structure and function of hemidesmosomesmore than simple adhesion complexes.J Invest Dermatol. 1999; 112: 411-418Crossref PubMed Scopus (474) Google Scholar). BP230, an entirely cytoplasmic protein belonging to the plakin family, is involved in the anchorage of intermediate filaments to the basal cell surface (reviewed inRuhrberg and Watt, 1997Ruhrberg C. Watt F.M. The plakin familyversatile organizers of cytoskeletal architecture.Curr Opin Genet Dev. 1997; 7: 392-397Crossref PubMed Scopus (184) Google Scholar). In contrast, BP180 is a transmembrane protein with a type II orientation. Its large extracellular portion consists of 15 interrupted collagenous subdomains (Giudice et al., 1992Giudice G.J. Emery D.J. Diaz L.A. Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180.J Invest Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar; Hopkinson et al., 1992Hopkinson S.B. Riddelle K.S. Jones J.C. Cytoplasmic domain of the 180-kD bullous pemphigoid antigen, a hemidesmosomal componentmolecular and cell biologic characterization.J Invest Dermatol. 1992; 99: 264-270Abstract Full Text PDF PubMed Scopus (125) Google Scholar). It serves as a cell surface receptor (Giudice et al., 1992Giudice G.J. Emery D.J. Diaz L.A. Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180.J Invest Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar; Hopkinson et al., 1992Hopkinson S.B. Riddelle K.S. Jones J.C. Cytoplasmic domain of the 180-kD bullous pemphigoid antigen, a hemidesmosomal componentmolecular and cell biologic characterization.J Invest Dermatol. 1992; 99: 264-270Abstract Full Text PDF PubMed Scopus (125) Google Scholar) and plays an important role in the maintenance of epidermal–stromal adhesion (reviewed inBorradori and Sonnenberg, 1999Borradori L. Sonnenberg A. Structure and function of hemidesmosomesmore than simple adhesion complexes.J Invest Dermatol. 1999; 112: 411-418Crossref PubMed Scopus (474) Google Scholar). This notion is supported by the observation that mutations in the BP180 gene (COL17A1) are the cause of a clinical variant of non-Herlitz junctional epidermolysis bullosa, a congenital disorder characterized by skin fragility and blistering (reviewed inPulkkinen and Uitto, 1998Pulkkinen L. Uitto J. Hemidesmosomal variants of epidermolysis bullosa. Mutations in the α6β4 integrin and the 180-kD bullous pemphigoid antigen/type XVII collagen genes.Exp Dermatol. 1998; 7: 46-64Crossref PubMed Scopus (96) Google Scholar). Recently, strong evidence has been provided that autoantibodies to BP180 play a key role in subepidermal blister formation in BP patients. In a passive transfer model, rabbit antibody raised against the murine homolog of the human immunodominant region of BP180, the NC16A domain, were able to induce sub-epidermal blister formation, reproducing all key features of BP (Liu et al., 1993Liu Z. Diaz L.A. Troy J.L. et al.A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180.J Clin Invest. 1993; 92: 2480-2488Crossref PubMed Scopus (543) Google Scholar). Furthermore, it has been found that the serum levels of IgG autoantibodies directed against the extracellular domain (ECD) of BP180 were related to the severity of BP (Haase et al., 1998Haase C. Büdinger L. Borradori L. et al.Detection of IgG autoantibodies in the sera of patients with bullous and gestational pemphigoidELISA studies utilizing a baculovirus- encoded form of bullous pemphigoid antigen 2.J Invest Dermatol. 1998; 110: 282-286Abstract Full Text Full Text PDF PubMed Google Scholar; Schmidt et al., 2000Schmidt E. Obe K. Bröcker E.B. Zillikens D. Serum levels of autoantibodies to BP180 correlate with disease activity in patients with bullous pemphigoid.Arch Dermatol. 2000; 136: 174-178Crossref PubMed Scopus (289) Google Scholar). Autoantibodies from patients with BP and some closely related autoimmune bullous disorders, such as gestational and cicatricial pemphigoid, have been found to predominantly recognize two major antigenic regions of the ECD of BP180. Specifically, the NC16A subdomain, a region adjacent to the membrane spanning domain of BP180, harbors major epitopes recognized by almost all sera from patients with BP as well as gestational pemphigoid, a blistering disorder associated with autoimmunity against BP180 that occurs during pregnancy (Giudice et al., 1993Giudice G.J. Emery D.J. Zelickson B.D. et al.Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain.J Immunol. 1993; 151: 742-5750Google Scholar; Matsumura et al., 1996Matsumura K. Amagai M. Nishikawa T. Hashimoto T. The majority of bullous pemphigoid and herpes gestationis serum samples react with the NC16a domain of the 180-kDa bullous pemphigoid antigen.Arch Dermatol Res. 1996; 288: 507-509Crossref PubMed Scopus (152) Google Scholar). In these patients, reactivity with the ECD of BP180 can be almost completely abolished by pre-adsorption of IgG with recombinant NC16A protein (Zillikens et al., 1997aZillikens D. Mascaro J.M. Rose P.A. et al.A highly sensitive enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid.J Invest Dermatol. 1997; 109: 679-683Crossref PubMed Scopus (206) Google Scholar; Hata et al., 2000Hata Y. Fujii Y. Tsunoda K. Amagai M. Production of the entire extracellular domain of BP180 (type XVII collagen) by baculovirus expression.J Dermatol Sci. 2000; 23: 183-190Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar; Lin et al., 2000Lin M.S. Fu C.L. Giudice G.J. et al.Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain.J Invest Dermatol. 2000; 115: 955-961Crossref PubMed Scopus (72) Google Scholar). Second, the COOH-terminal region of the ECD of BP180 has been shown to be recognized by approximately one-third of BP sera (Murakami et al., 1998Murakami H. Nishioka S. Setterfield J. et al.Analysis of antigens targeted by circulating IgG and IgA autoantibodies in 50 patients with cicatricial pemphigoid.J Dermatol Sci. 1998; 17: 39-44Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar; Nakatani et al., 1998Nakatani C. Muramatsu T. Shirai T. 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.Br J Dermatol. 1998; 139: 365-370Crossref PubMed Scopus (76) Google Scholar) as well as by sera from a subset of patients with cicatricial pemphigoid (Balding et al., 1996Balding S.D. Prost C. Diaz L.A. et al.Cicatricial pemphigoid autoantibodies react with multiple sites on the BP180 extracellular domain.J Invest Dermatol. 1996; 106: 141-146Crossref PubMed Scopus (216) Google Scholar; Bedane et al., 1997Bedane C. McMillan J.R. Balding S.D. et al.Bullous pemphigoid and cicatricial pemphigoid autoantibodies react with ultrastructurally separable epitopes on the BP180 ectodomainevidence that BP180 spans the lamina lucida.J Invest Dermatol. 1997; 108: 901-907Crossref PubMed Scopus (140) Google Scholar; Murakami et al., 1998Murakami H. Nishioka S. Setterfield J. et al.Analysis of antigens targeted by circulating IgG and IgA autoantibodies in 50 patients with cicatricial pemphigoid.J Dermatol Sci. 1998; 17: 39-44Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar; Nie and Hashimoto, 1999Nie Z. Hashimoto T. IgA antibodies of cicatricial pemphigoid sera specifically react with C- terminus of BP180.J Invest Dermatol. 1999; 112: 254-255Crossref PubMed Scopus (96) Google Scholar), a chronic autoimmune blistering disorder characterized by predominant involvement of mucosal surfaces with scarring. The purpose of this study was to develop a sensitive and highly specific in vitro assay for the detection of circulating autoantibodies in BP utilizing baculovirus-derived recombinants of the NH2- and COOH-terminal regions of the ECD of BP180. The baculovirus expression system has proved to be extremely useful for the production of recombinant proteins retaining many of the post-translational modifications of the native proteins (Kidd and Emery, 1993Kidd I.M. Emery V.C. The use of baculoviruses as expression vectors.Appl Biochem Biotechnol. 1993; 42: 137-159Crossref PubMed Scopus (74) Google Scholar). Because of the putative key role of BP180 in disease initiation, we assessed the reactivity against these two BP180 recombinant proteins in serum samples obtained from BP patients and from a control group and further characterized the epitope specificity as well as the isotype distribution of the autoantibody response to BP180. Furthermore, we investigated the potential relationship between the autoantibody profile of these BP sera and the clinical phenotype and severity of BP. Serum samples were obtained from patients with clinically active BP (n=116), BP patients in remission (n=6), and control donors; the latter included patients with pemphigus vulgaris (n=28) and healthy volunteers (normal human sera, n=72). All patients and volunteers gave written consent to participate in this study. In the group of patients with active BP, sera were taken before immunosuppressive treatment in 99 patients, but 17 patients with chronic BP had already received systemic immunosuppressive treatment. Diagnosis of BP was based on the following criteria: (i) typical clinical presentation with tense cutaneous blisters (including 16 cases with additional involvement of mucous membranes without scarring); (ii) histopathologic evidence of subepidermal blister formation; (iii) linear IgG and/or C3 deposits at the dermo-epidermal junction of perilesional skin by direct immunofluorescence; and (iv) IgG-positive indirect immunofluorescence on saline-separated human skin. Control sera were collected from randomly chosen healthy individuals who had no history of bullous skin disorder and from 28 pemphigus vulgaris patients whose sera showed intercellular IgG reactivity on monkey esophagus or human skin and also reacted with recombinant desmoglein 3 by enzyme-linked immunosorbent assay (ELISA). To investigate the relationship between autoantibody profiles of the BP sera and the clinical phenotype, sera from patients with active BP were compared to those of patients in remission; five of the latter patients had received systemic immunosuppressive treatment, and one patient received no therapy. A total of 114 BP patients (i.e., 108 patients with active BP and six patients in remission) were clinically well characterized with regard to disease severity and the duration of disease (Table III). Disease severity was classified according to the extent and number of blisters: 0, no skin lesions; 1, localized disease (few bullae or bullae at limited areas of the body); 2, generalized disease. The clinical stage of BP was defined as "acute" (duration ≤3 mo) or "chronic" (duration ≥3 mo with persisting bullae or erosions). "Remittent" was defined by the absence of skin lesions for more than 1 mo. Patient and control sera were stored at –20°C prior to analysis. Sera from 95 BP patients with IgG against the NH2-terminus of the BP180 ectodomain (Table IVTable V ) and 48 sera reactive to the COOH-terminus (Table IV) were analyzed with regard to the antibody levels and isotype distribution of BP180-reactive IgG, disease activity, duration, and mucosal involvement.Table IIISpecificity of autoantibodies against BP180 and clinical statusap-values were determined by Fisher's exact probability test.BP180-NbNH2-terminal domain of the ECD of BP180./CcCOOH-terminal domain of the ECD of BP180.Extent of disease (n=114)Disease duration (n=114)Mucosal involvement (n=114)GeneralizedLocalizedRemittentAcuteChronicRemittent+––/–4 (5.3)2 (6.1)0 (0)2 (2.6)4 (12.9)0 (0)2 (12.5)4 (4.1)+/–36 (48.0)18 (54.5)5 (83.3)38 (49.4)16 (51.6)5 (83.3)5 (31.3)54 (55.1)–/+9 (12.0)4 (12.1)0 (0)9 (11.7)4 (12.9)0 (0)0 (0)13 (13.3)+/+26 (34.7)9 (27.3)1 (16.7)28 (36.4)7 (22.6)1 (16.7)9 (56.2)27 (27.6)Total75 (100%)p=0.8733 (100%)6 (100%)77 (100%)p=0.2731 (100%)6 (100%)16 (100%)p=0.02198 (100%)a p-values were determined by Fisher's exact probability test.b NH2-terminal domain of the ECD of BP180.c COOH-terminal domain of the ECD of BP180. Open table in a new tab Table IVComparison of IgG levelsaGeometric mean±geometric standard error. against the NH2-terminal regions of the BP180 ectodomain (BP180-N) and clinical activity of BPIg classDisease activitypbSignificance of pairwise comparisons within analysis of covariance models with simultaneous adjustment for age and gender.Generalized, n=62Localized, n=27Remittent, n=6Generalized versus localizedGeneralized versus remittentIgG0.649 (0.605–0.696)0.413 (0.372–0.458)0.272 (0.221–0.335)0.001< 0.0001IgG10.768 (0.702–0.840)0.475 (0.425–0.529)0.235 (0.211–0.261)0.001<0.0001IgG40.322 (0.281–0.368)0.186 (0.163–0.211)0.159 (0.137–0.185)0.0070.042a Geometric mean±geometric standard error.b Significance of pairwise comparisons within analysis of covariance models with simultaneous adjustment for age and gender. Open table in a new tab Table VComparison of IgG isotype reactivity against the NH2-terminal region of the BP180 ectodomain (BP180-N) and the clinical statusap-values were determined by Fisher's exact probability test.IgG1/IgG4 reactivityExtent of disease (n=95)Disease duration (n=95)Mucosal involvement (n=95)GeneralizedLocalizedRemittentAcuteChronicRemittent+––/–3 (4.8)5 (18.5)1 (16.6)5 (7.6)3 (13.0)1 (16.7)0 (0)9 (11.1)+/–26 (41.9)14 (51.9)1 (16.6)32 (48.5)8 (34.8)1 (16.7)3 (21.4)38 (46.9)–/+5 (8.1)1 (3.7)3 (50.0)3 (4.5)3 (13.0)3 (50.0)3 (21.4)6 (7.4)+/+28 (45.2)7 (25.9)1 (16.6)26 (39.4)9 (39.2)1 (16.7)8 (57.2)28 (34.6)Total62 (100%)p=0.00927 (100%)6 (100%)66 (100%)p=0.03023 (100%)6 (100%)14 (100%)p=0.06381 (100%)a p-values were determined by Fisher's exact probability test. Open table in a new tab The recombinant proteins BP180-N (NC16A domain and the collagenous Col15 domain of BP180) and BP180-C (COOH-terminal domains NC3–NC1 of BP180) are linked to glutathione-S-transferase (GST) and 6xHis tag (Figure 1) and were produced in a baculovirus expression system as described below. The generation of cDNAs encoding for human BP180 has been described previously (Borradori et al., 1997Borradori L. Koch P.J. Niessen C.M. et al.The localization of bullous pemphigoid antigen 180 (BP180) in hemidesmosomes is mediated by its cytoplasmic domain and seems to be regulated by the beta4 integrin subunit.J Cell Biol. 1997; 136: 1333-1347Crossref PubMed Scopus (114) Google Scholar). These cDNAs were used as a template to amplify by polymerase chain reaction (PCR) truncated variants of BP180 using Pfu DNA polymerase following the manufacturer's instructions (Stratagene, La Jolla, CA). Forward primers contained an EcoR I site and reverse primers contained either an EcoR I or a Not I site adjacent to the sequence homologous to the desired BP180 sequence. The cDNA fragments were subcloned into the pAcGHLT-A baculovirus transfer vector (Pharmingen, San Diego, CA) by ligation of double digested PCR fragments into the correspondingly cut vectors. Correctness of both constructs was verified by nucleotide sequencing. pAcGHLT-A vectors were cotransfected with BaculoGold DNA (Pharmingen) into Sf21 insect cells and recombinant baculoviruses were amplified using a previously described protocol (Amagai et al., 1994Amagai M. Hashimoto T. Shimizu N. Nishikawa T. Absorption of pathogenic autoantibodies by the extracellular domain of pemphigus vulgaris antigen (Dsg3) produced by baculovirus.J Clin Invest. 1994; 94: 59-67Crossref PubMed Scopus (299) Google Scholar). The recombinant BP180 proteins and the GST/6xHis control protein (generated by cotransfection of an empty pAcGHLT-A vector) were expressed in Sf21 cells as previously described (Haase et al., 1998Haase C. Büdinger L. Borradori L. et al.Detection of IgG autoantibodies in the sera of patients with bullous and gestational pemphigoidELISA studies utilizing a baculovirus- encoded form of bullous pemphigoid antigen 2.J Invest Dermatol. 1998; 110: 282-286Abstract Full Text Full Text PDF PubMed Google Scholar). Recombinant BP180 proteins were purified as follows: 3 d after infection, 10×106 baculovirus-infected insect cells were suspended in 1 ml of icecold lysis buffer (Pharmingen), incubated for 1 h at 4°C, and then centrifuged (2000×g, 15 min). Solubilized proteins were purified by affinity chromatography over Nickel-NTA agarose (Quiagen, Hilden, Germany) using the batch method according to the manufacturer's protocol. The BP180 recombinant BP180-N and the GST/6xHis control protein were purified under native conditions, whereas purification of BP180-C was performed under denaturing conditions by adding 6 M guanidine hydrochloride to the lysis buffer and 8 M urea to washing and elution buffers. Purified proteins were finally dialyzed against phosphate-buffered saline (PBS) (pH 7.5) at 4°C for 48 h (BP180-N, GST/6xHis) or gradually dialyzed against PBS supplemented with 8 M to 3 M urea (BP180-C). Affinity-purified BP180 proteins were stored in aliquots at -80°C. Protein concentrations were determined with a commercial kit (DC Protein Assay, Bio-Rad, Munich, Germany) according to a modified protocol by Lowry. BP180-N and BP180-C were fractionated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualized by Coomassie staining and Western blot with a monoclonal mouse anti-GST antibody (1:2000, clone GST3-4C, Zymed, San Francisco, CA) to verify molecular weight and immunoreactivity of the recombinant BP180 proteins (Figure 1). Optimal conditions for the ELISA were established by various chessboard titrations following published guidelines (Kemeny, 1991Kemeny D.M. A Practical Guide to ELISA. Pergamon Press, Oxford1991Google Scholar). The affinity-purified recombinants of BP180 were immobilized on 96-well polystyrene plates (Maxisorb Immunoplate, Nunc, Wiesbaden, Germany) by coating each well with 0.5 μg of BP180-N, 0.5 μg of BP180-C, or a molar equivalent amount of the control protein GST/6xHis in 100 μl of 0.1 M bicarbonate buffer, pH 9.8, at 4°C for 5 h. An incubation time of 5 h was arbitrarily chosen and worked well in the laboratory routine; shorter incubation times led to a reduced sensitivity of the assay. ELISA plates were then washed five times with TBST (TBS pH 7.5 with 0.05% Tween 20; Merck, Nürnberg, Germany) and blocked for 2 h with 100 μl of 5% skimmed milk powder in TBST at room temperature. Patients' and control sera diluted 50-fold in blocking buffer were added to the wells in duplicate. After an incubation period of 2 h at room temperature and consecutive washes as above, wells were reacted with alkaline phosphatase (AP) labeled goat antihuman IgG (γ) (1:6000, Kirkegaard and Perry, Gaithersburg, MD), monoclonal biotinylated mouse antihuman IgG1 (1:800, clone HP6069) or monoclonal biotinylated mouse antihuman IgG4 (1:500, clone HP6025) (both Zymed, San Francisco, CA), biotinylated mouse antihuman IgE (1:500, Zymed) or peroxidase-conjugated sheep antihuman IgA (1:5000, The Binding Site, Birmingham, U.K.) for 1–2 h. After incubation of biotinylated antibody for 1 h, wells were washed as above and incubated with streptavidin–AP (ZyMAX, Zymed) diluted at 1:2000 in TBS for 1 h at room temperature. After another series of washes, 100 μl of p-nitrophenylphosphate (Kirkegaard and Perry) or the peroxidase substrate 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma, Deisenhofen, Germany) were added to the wells and optical density (OD) was measured at 405 nm when a defined patient serum (positive reference control) reached an OD of 1.0 as determined with a Wallac 1420 microplate reader (Wallac, Freiburg, Germany). All extinction data were standardized based on the positive and negative internal standards, which were set to 1 and 0 OD units, respectively. The background reactivity of IgG to the control protein GST/6xHis was subtracted from the mean OD values obtained with BP180-N and BP180-C proteins. For the detection of IgG1, IgG4, IgE, and IgA reactivity to BP180, the GST/6xHis control protein was not used routinely, because a relevant total IgG reactivity against GST/6xHis had not been detected in any of the sera by ELISA. To evaluate plate-to-plate variability, each plate included identical internal controls (two positive sera, one control serum). The specificity of serum reactivity to BP180-N and BP180-C was verified by immunoadsorption of sera with these BP180 recombinants prior to ELISA ("inhibition ELISA"), which led to a significant decrease of serum reactivity to these particular BP180 recombinants. A total of 4 μg of BP180-N, BP180-C, or GST/6xHis proteins was added to nitrocellulose membranes, followed by two washes with TBST for 10 min. Subsequently, membranes were blocked with TBST containing 5% skimmed milk powder for 1 h at room temperature and then incubated with sera diluted 50-fold in blocking buffer overnight at 4°C. Patient and control sera before and after pre-adsorption were then reacted with GST/6xHis and the BP180 recombinants on ELISA and IgG reactivity was detected as described above. Statistical analysis was performed using SPSS 10.0 (SPSS, Chicago, IL). Receiver operating characteristic (ROC) curves were used to evaluate the ability of the ELISA to detect autoantibodies against the NH2- and COOH-terminal domains of the ECD of BP180. The areas under the curves (AUC) are reported with their 95% confidence interval (95%-CI). The Youden index (J=sensitivity+specificity-1) was used to select the best cut-off values. To assess the reproducibility of the ELISA results, coefficients of variance were calculated for a BP reference serum with an intermediate OD reading. The interassay variability of the OD405 readings for the different ELISAs ranged between 6.9% and 12.6% depending on the investigated BP180 recombinant and the Ig subclass. Disease activity and disease duration were compared with ELISA values using multivariate analysis of covariance additionally consisting of age and sex. To enhance the assumption of normality, ELISA values were log-transformed. The independence of the distribution of epitope and Ig subtype specific autoantibody and the clinical phenotype was tested by Fisher's exact test. p<0.05 was considered statistically significant. Values are expressed as geometric mean±geometric standard error. Sera from patients with active BP (n=116), from normal volunteers (n=72), and from patients with pemphigus vulgaris (n=28) were tested against the BP180 recombinants, BP180-N and BP180-C, that were immobilized onto ELISA microtiter plates. These BP180 recombinants presumably possessed a conformation identical or similar to the native BP180 protein (Figure 2). Based on the maximization of the Youden index (sensitivity+specificity-1), the cut-off point for the ELISA with BP180-N protein was set at 0.149 OD units, which corresponds to a Youden index of 0.792. Reactivity to BP180-C was considered positive when the OD of the sera exceeded 0.065 units (Youden index=0.436). The diagnostic accuracy of each test, as determined by ROC curves, is represented by the area under the curve (AUC (95%-CI)), which was 0.925 (0.887–0.963) for the BP180-N and 0.796 (0.737–0.855) for the BP180-C ELISA (Figure 3). Using the selected cut-off values, 93 (80%) and 54 (47%) of 116 BP sera exhibited specific reactivity with BP180-N and BP180-C, respectively (Figure 3). One (specificity 99%) and three (specificity 97%) of the control sera yielded marginal OD readings against BP180-N and BP180-C, respectively.Figure 3Detection of IgG a