Importance of serological tests in diagnosis of autoimmune blistering diseases

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1 doi: / Journal of Dermatology 2015; 42: 3 10 REVIEW ARTICLE Importance of serological tests in diagnosis of autoimmune blistering diseases Ken ISHII Department of Dermatology, Toho University Omori Medical Center, Tokyo, Japan ABSTRACT Autoimmune blistering diseases are organ-specific autoimmune diseases characterized by autoantibodies against structural proteins that maintain cell cell (pemphigus diseases) and cell matrix adhesions (pemphigoid diseases) in the skin and mucous membranes. Over the last few decades, identification of autoantigens and extensive characterization of autoantibodies have improved understanding of the pathogenesis of these diseases. In addition, the development of new sensitive and specific immunoassays enabled accurate diagnosis and proper evaluation of disease activity in autoimmune blistering diseases. In this review, we describe practical updates for molecular diagnostic tests for autoimmune blistering diseases and the basis for interpreting the results of the assays. Key words: autoantibodies, autoimmunity, bullous pemphigoid, immunoassays, pemphigus. INTRODUCTION Autoimmune blistering diseases are organ-specific autoimmune diseases characterized by autoantibodies against structural proteins that maintain cell cell and cell matrix adhesions in the skin and mucous membranes. They can be categorized into two major groups: (i) pemphigus group which shows intraepidermal blisters; and (ii) pemphigoid group which shows subepidermal blisters. For the diagnosis of autoimmune blistering diseases, in addition to clinical observation and histopathological examination, it is necessary to demonstrate immunologically the presence of tissue-bound and/or circulatory autoantibodies. Tissue-bound autoantibodies can be detected by a direct immunofluorescence test using perilesional skin. This is the most reliable method for the diagnosis of autoimmune blistering diseases. The detection of circulating autoantibodies can be made by different immunoassays such as indirect immunofluorescence, immunoblot, immunoprecipitation and enzymelinked immunosorbent assay (ELISA). Identification of autoantigens in autoimmune blistering diseases in the last few decades has allowed the development of specific laboratory diagnostic tests using recombinant proteins which enable more accurate and rapid diagnosis of autoimmune blistering diseases. In Japan, the diagnostic tests, desmoglein (Dsg)1 and 3 ELISA are available for the diagnosis of pemphigus, BP180 and BP230 ELISA for bullous pemphigoid (BP), and type VII collagen ELISA for epidermolysis bullosa acquisita (EBA). In this review, we describe the practical updates of the molecular diagnostic tests for autoimmune blistering diseases and the basis for interpretation of the results of the assays. PEMPHIGUS Pemphigus is defined as a group of autoantibody-mediated blistering diseases of the skin and mucous membranes characterized by the loss of cell cell adhesion due to the binding of autoantibodies against keratinocyte cell surfaces. 1 Pemphigus has two major subtypes: pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Autoantigens associated with these diseases are Dsg3 and Dsg1, respectively, which are members of the cadherin type of cell adhesion molecules. Pemphigus vegetans is a variant of PV, while pemphigus erythematosus and fogo selvagem represent localized and endemic variants of PF, respectively. More recently, paraneoplastic pemphigus (PNP) was recognized as a distinct subtype of pemphigus. PNP has immunoglobulin (Ig)G class autoantibodies against Dsg3 and the plakin family. Pemphigus disease variants and the associated autoantigens are summarized in Table 1. Histology demonstrated that intraepidermal blisters in the skin occur at different levels in PV and PF. In PV, blisters occur at the suprabasal layer, while PF blisters occurs at superficial layers. The difference in histology is related to the difference in the pattern of expression of Dsg1 and Dsg3 in the skin and mucous membrane. Desmoglein: autoimmune target of pemphigus Desmogleins (Dsg) are cadherin type cell adhesive molecules localized at desmosomes where keratin intermediate filaments are tethered. Dsg consist of four isoforms, Dsg1 4, which have a different expression pattern in different tissues. In the skin, Dsg1 is expressed throughout the epidermis with a gradual increase in upper layers. Dsg3 is expressed in an inverse Correspondence: Ken Ishii, M.D., Ph.D., Department of Dermatology, Toho University Omori Medical Center, Omori-Nishi, Ota-Ku, Tokyo , Japan. ken.ishii@med.toho-u.ac.jp Received 7 October 2014; accepted 8 October Japanese Dermatological Association 3

2 K. Ishii Table 1. Subtype of pemphigus and target antigen Type Immunoglobulin class Target antigen Pemphigus vulgaris (mucosal type) IgG Dsg3 Pemphigus vulgaris (mucocutaneous IgG Dsg3, Dsg1 type) Pemphigus foliaceus IgG Dsg1 Paraneoplastic pemphigus IgG Dsg3, Dsg1 Plakin family (plectin, epiplakin, desmoplakin, 210-kD envoplakin, 190-kD periplakin, BP230) a2-macroglobulin-like 1 (A2ML1) IgA pemphigus (SPD type) IgA Dsc1 IgA pemphigus (IEN type) IgA Unidentified Pemphigus herpetiformis IgG Dsg1, Dsg3, Dsc Dsc, desmocollins; Dsg, desmoglein; IEN, intraepidermal neutrophilic; Ig, immunoglobulin; SPD, subcorneal pustular dermatosis. pattern with the strongest expression in the basal layer. In the mucous membrane, Dsg3 is expressed throughout all layers, while Dsg1 is weakly expressed in the superficial layers. The clinical phenotype and the histological localization of blisters of pemphigus can be explained by the different distribution of Dsg1 and Dsg3 and the ability of different Dsg isoforms to compensate for each other with respect to their cell adhesive function (Dsg compensation theory). 2 Many studies demonstrated that the autoantibodies against Dsg in pemphigus cause the disease. For example, IgG-purified sera from PV patients cause blisters when injected into neonatal mice. 3 In addition, adsorption of pemphigus sera with recombinant Dsg resulted in loss of pathogenic activity in blister formation of those sera. Monoclonal anti-dsg antibodies cause disease when injected into neonatal mice or human skin organ culture. 4 7 Recent studies using monoclonal antibodies from patients or pemphigus model mice demonstrated that there are pathogenic and non-pathogenic anti-dsg antibodies in blister formation. 4 6 Epitope mapping of the pathogenic antibodies showed that these pathogenic antibodies tend to bind the amino-terminal domain of Dsg. This finding is consistent with previous reports using patient sera in which immunoadsorption with the amino-terminal domain of recombinant Dsg eliminated the ability of PF sera to induce cutaneous blisters in neonatal mice. 8,9 Diagnosis of pemphigus For the diagnosis of pemphigus, in addition to clinical and histopathological findings, it is necessary to demonstrate the in vivo deposition of IgG type Ig on the cell surface of the epidermis and show circulating autoantibodies against the cell surface of the epidermis. Since the autoantigens for PV and PF were identified as Dsg3 and Dsg1, respectively, ELISA using recombinant Dsg1 and Dsg3 have been developed and are widely used to easily detect autoantibodies in patient sera. 10,11 In the original ELISA system, recombinant ectodomains of Dsg1 and Dsg3 that properly reflect their 3-D structures were produced by baculovirus expression system and were coated Table 2. Anti-Dsg autoantibody profile and subtype of pemphigus Anti-Dsg1 Ab Anti-Dsg3 Ab Diagnosis + Pemphigus vulgaris (mucosal) + + Pemphigus vulgaris (mucocutaneous) + Pemphigus foliaceus Normal or other diseases Ab, antibody; Dsg, desmoglein. on the ELISA plate because most, if not all, pathogenic antibodies in patient sera recognize conformational epitopes of Dsg1 and Dsg3. Recently, by analysis of monoclonal Dsg antibodies isolated from patients, it has been shown that pathogenic anti-dsg antibodies preferentially bind the mature form of Dsg but not the proprotein form. 12,13 An ELISA using recombinant ectodomains of Dsg1 and Dsg3 produced by a mammalian expression system has been developed because the baculovirus system technically comprises a proprotein form in addition to a mature form and it is easier to purify the mature form of Dsg only with the mammalian expression system. These ELISA systems demonstrated high sensitivity and specificity (97.9% and 98.9% for anti-dsg1 ELISA; 97.5% and 97.8% for anti-dsg3 ELISA, respectively). Subtype of pemphigus and anti-dsg antibody profile The serological differentiation of PV and PF by the Dsg antibody profile measured by ELISA provides an advantage in most cases (Table 2). When a serum demonstrates a positive reaction for Dsg1 but a negative one for Dsg3, it suggests a diagnosis of PF. When anti-dsg3 is positive but anti-dsg1 antibody is negative, it suggests a diagnosis of the mucosal-dominant type of PV. Presence of both anti-dsg1 and Dsg3 antibodies support a diagnosis of the mucocutaneous type of PV. As this is a serological diagnosis, the final diagnosis should be made in combination with clinical pictures and histological findings Japanese Dermatological Association

3 Serology in autoimmune blistering diseases Monitoring Comparing titers measured by these ELISA with those from standard serum enable the calculation of an ELISA score (index value). It has been shown that ELISA scores fluctuate with disease activity in the most cases (Fig. 1). It is useful to evaluate disease activity and determine schedules for tapering corticosteroids. 14 After blisters and erosions are no longer clinically evident, ELISA scores are useful for the assessment of disease activity. A decrease in ELISA value can be a useful guide for steroid tapering. If the ELISA index value rises, the patient s clinical course should be monitored very closely. Changes in the ELISA value alone are not generally sufficient to require the addition of a new treatment. If new development of blisters or erosions is observed, the treatment regimen should be reconsidered. As ELISA is a measurement by enzyme reaction, the ELISA value is saturated when antibody titer is high. 14 ELISA index should be measured after appropriate dilutions when the index is over 150 to get a true index value. The titers do not necessarily reflect the disease activity when compared among different patients, as patients who have same disease activities may have different ELISA values. This discrepancy may be explained by the fact that patients sera are polyclonal with heterogenous antibodies which contain both pathogenic and non-pathogenic antibodies in blister formation. This was proved by isolation of monoclonal antibodies from mice and humans. 4 7 Anti-Dsg antibody measurement by CLEIA New immunoassays for detection of anti-dsg1 and Dsg3 autoantibodies have been developed using chemiluminescence enzyme immunoassay (CLEIA) (MBL, Aichi, Japan). CLEIA is based on enzyme antibody conjugates using a chemiluminescent substrate, and luminometer for measurements. Magnetic microparticles coated with Dsg are applied in CLEIA as the solid phase separating agent. The assay has several advantages over ELISA. First, it is a more sensitive assay than ELISA. Second, the assay is a fully automatic system with more rapid assay time than ELISA. In addition, because it has a larger detection linear range, it is not necessary to dilute samples more, even if the titer of samples is high. Functional assay to assess the pathogenic activity of patients sera ELISA or the immunofluorescence tests described above are serological assays to detect the binding capacity of autoantibodies against Dsg. In contrast, functional biological assays have been developed to measure the ability of autoantibodies to induce the loss of cell cell adhesion. Passive transfer of PV sera to neonatal mice is a well-established in vivo functional assay to assess the pathogenic ability to induce bullous formation of pemphigus. 3 However, this assay requires a large volume of sera to induce blister formation in the skin of mice. In addition, some autoantibodies do not cross-react with mouse Dsgs. PSL (mg) ELISA (Index) PDAI PDAI (pemphigus disease acvitity index) Dsg1 ELISA Dsg3 ELISA Time course (week) Figure 1. Time course of anti-desmoglein (Dsg) antibodies titers and clinical disease activity in a pemphigus vulgaris patient. Dsg1 and Dsg3 enzyme-linked immunoassay (ELISA) scores fluctuate in parallel with disease activity. Clinical disease activity is shown by pemphigus disease index (PDAI). PSL, prednisolone Japanese Dermatological Association 5

4 K. Ishii To address these problems, an in vitro dissociation assay using primary human epidermal keratinocytes has been developed. 15 After incubation with pemphigus sera, cell sheets of confluent keratinocytes were released by dispase treatment and subjected to mechanical stress such as pipetting. The number of fragmented cell sheets is used as an indicator of pathogenic activity in inducing the loss of cell cell adhesion (Fig. 2). Another assay is an ex vivo pathogenicity assay using organ-cultured human skin. In this assay, autoantibodies are injected into human skin specimens and the blister formation is confirmed by histology after 24-h incubation. 16 These functional assays are widely used for evaluating the pathogenicity of patients sera for research purpose. Paraneoplastic pemphigus Paraneoplastic pemphigus (PNP) is a unique subtype of pemphigus associated with underlying neoplasms. PNP is characterized by refractory stomatitis and pseudomembranous conjunctivitis. Skin lesions are variable, including erythema, blisters, erosion, erythema multiforme-like lesions and lichen planus-like lesions. PNP patients are known to possess characteristic autoantibodies against several members of the plakin family (250-kD desmoplakin I, 230-kD BPAG1, 210-kD envoplakin, 190-kD periplakin and 500-kD plectin) and the proteinase inhibitor a2-macroglobulin-like 1 (170 kd) (Table 1). 17 In addition, PNP has antibodies against Dsg3, which have been shown to be pathogenic. 7,18 Diagnosis of PNP can be made by positive reaction of IgG by indirect immunofluorescence using rat bladder section, which is rich in plakins. In contrast, PV and PF do not react to rat bladder section because the transitional epithelium does not express Dsg3 and Dsg1. This assay is useful to differentiate PNP from PV. Most PNP patients show reactivity to 210-kD envoplakin and 190-kD periplakin by immunoblotting using human epidermal extract. 19 ELISA employing recombinant envoplakin has been developed. 20 PV serum Normal Figure 2. In vitro keratinocyte dissociation assay. Keratinocytes sheets are incubated with pemphigus vulgaris (PV) serum or control serum for 6 h in combination with exfoliative toxin A to digest desmoglein 1. After dispase treatment to release monolayers from the bottom of wells, the keratinocyte sheets were subjected to mechanical stress by pipetting. Note that cell sheets broke into numerous small fragments when treated with PV serum whereas the cell sheets were minimally dissociated when treated with normal control. IgA pemphigus Immunoglobulin A pemphigus is a subtype of pemphigus characterized by the presence of IgA against the keratinocyte surface. IgA pemphigus is subdivided into the subcorneal pustular dermatosis (SPD) type and intraepidermal neutrophilic (IEN) IgA dermatosis type. The SPD type of IgA pemphigus targets desmocollin Autoantigen in IEN IgA dermatosis appears to be heterogenous. Direct immunofluorescence shows IgA deposition against the keratinocyte cell surface. Immunofluorescence using desmocollin 1 transfected Cos7 cells has been developed to detect circulating IgA autoantibodies. 21 ELISA using recombinant desmocollin has also been developed although the sensitivity is not high. 22 Pemphigus herpetiformis Pemphigus herpetiformis is a rare clinical variant of pemphigus with the clinical features of dermatitis herpetiformis and the immunological characteristics of pemphigus. 23 Skin lesions manifest urticarial plaques and vesicles arranged in a herpetiform or annular pattern. The target autoantigen is usually Dsg1 or less frequently Dsg 3, suggesting that pemphigus herpetiformis is a clinical variant of PF or PV. 24 It has been recently reported that some pemphigus herpetiformis cases also demonstrate autoantibodies against desmocollin. 25 PEMPHIGOID DISEASES The pemphigoid diseases are a heterogeneous group of subepidermal autoimmune blistering diseases induced by autoantibodies against components that maintain epidermal dermal adhesive structures. The disease group includes bullous pemphigoid, mucous membrane pemphigoid, linear IgA bullous dermatosis, anti-laminin-c1 pemphigoid, epidermolysis bullosa acquisita and dermatitis herpetiformis. The target autoantigens are shown in Table 3. Bullous pemphigoid Bullous pemphigoid (BP) is the most common autoimmune blistering disease. BP occurs most frequently in elderly patients. Clinically, it is characterized by large, tense bullae on the trunk, proximal extremities, groin and axillae. Approximately 20% of BP cases involve oral membrane lesions. The histological characteristic is subepidermal separation at the dermoepidermal junctions. Inflammatory cell infiltration with eosinophils can be seen. Autoantibodies in BP target two hemidesmosomal proteins, BP antigen 180 (BP180, BPAG2, type XVII collagen) and BP antigen 230 (BP230, BPAG1). BP180 is a transmembrane protein with a large collagenous extracellular domain. BP230 is a cytoplasmic protein belonging to the plakin family. They are localized at the hemidesmosomes, which is the adhesive complex structure promoting epithelial extracellular matrix adhesion. Direct immunofluorescence shows IgG and complement deposition along the basement membrane zone (BMZ) in a linear pattern. In some cases, complement deposition can be seen even when IgG deposition is not detected. This is a valuable finding for the diagnosis for BP Japanese Dermatological Association

5 Serology in autoimmune blistering diseases Table 3. Subepidermal blistering diseases and their target autoantigens Type Ig class Target antigen Bullous pemphigoid IgG BP180, BP230 Herpes gestationis IgG BP180, BP230 Mucous membrane pemphigoid Anti-BP180 type IgG, BP180 IgA Anti-laminin-332 type IgG Laminin-332 Ocular type IgG a6b4-integrin Linear IgA bullous dermatosis Lamina lucida type IgA 120-kD/97-kD LAD-1 Sub-lamina densa type IgA Unidentified (type VII collagen) Anti-laminin-c1 IgG Laminin-c1 pemphigoid Epidermolysis bullosa IgG Type VII collagen acquisita Dermatitis herpetiformis IgA Tissue/epidermal transglutaminase Ig, immunoglobulin. Immunoblot and immunoprecipitation using keratinocyte extract reveal that % of patients sera react to 180- or 230-kD protein. Immunoblot using the recombinant BP180NC16a domain, the proximal region of the extracellular domain of BP180, demonstrates that 90% of BP sera react to NC16a domain of BP On the other hand, in MMP with anti-bp180 antibody, sera tend to react to C-terminal extracellular domains of BP180. BP180 and BP230 ELISA ELISAs for BP180 and BP230 have been developed. BP180 ELISA uses recombinant NC16a domain that is the proximal region of extracellular domain of BP As major epitopes in BP180 reside within this NC16a domain, 82.2% of sera that showed BMZ staining by indirect immunofluorescence demonstrate a positive reaction in the ELISA. In addition, it has been shown that the ELISA score correlates well with disease activity. BP230 is another autoimmune target of BP. It is a member of the plakin family and a cytoplasmic molecule localized at the adhesive plaque of hemidesmosomes. BP230 ELISA uses recombinant domain of the N- and C-terminus. It does not contain the middle part of BP230. However, 60% of BP sera show positive reaction with this assay. 28 When the combination of BP180 and BP230 ELISA were used, 97% of BP sera react either to BP180 ELISA or BP230 ELISA. In contrast to BP 180 ELISA, ELISA scores measured with BP230 do not always correlate well with disease activity over time. Epidermolysis bullosa aquisita Epidermolysis bullosa acquisita (EBA) is an acquired mechanobullous disease characterized by the presence of autoantibodies against type VII collagen. The diagnostic clinical criteria for EBA are trauma-induced blisters that heal with milia and scarring in the absence of family history of epidermolysis bullosa. The immunopathological criteria are subepidermal blisters and linear deposits of IgG in the BMZ. The demonstration of IgG autoantibodies labeling the dermal side of salt-split skin is the most specific diagnostic assay. Immunoblot using dermal extract shows 290-kD type VII collagen. Collagen VII, the main structural constituent of anchoring fibrils, is a homotrimer of three identical a-chains. Each a-chain is flanked by an N-terminal 145-kD non-collagenous domain (NC1) and a 34-kD NC2 domain. The NC1 domain has been identified as the immunodominant region of collagen VII. 29,30 ELISA using a combination of NC1 and NC2 domains of type VII collagen were developed and are commercially available in Japan. 31 The assay showed sensitivity of 93.8% and specificity of 98.1%. The assay is useful for differentiating EBA from BP and to monitor the disease activity. In addition, an ELISA with NC1 domain only has been developed and is commercially available. 32,33 Mucous membrane pemphigoid Mucous membrane pemphigoid (MMP) is a group of subepidermal blistering diseases which are characterized by the predominant involvement of mucous membrane. As scarring of the mucous membranes in MMP is common, it was previously called cicatricial pemphigoid. Several target autoantigens of MMP have been identified. The two major target autoantigens are the C-terminal of BP180 and laminin-332. Of MMP, 70 80% target the C-terminal of BP180. Approximately 10% of MMP target laminin-332. Diagnosis is confirmed by demonstration of IgG and/or IgA and C3 deposits at the basement membrane by direct immunofluorescence. Indirect immunofluorescence is often negative when the titer is low. For BP180 type MMP, IgG and IgA antibodies react with BP180 by immunoblot using epidermal extracts and recombinant protein BP180 C-terminal domain. IgG autoantibodies in sera of laminin-332 type MMP react with laminin-332 by immunoprecipitation of cultured keratinocytes, immunoblot with extracellular matrix of cultured human keratinocytes or purified laminin Autoantibodies to a6b4-integrin are reported to be detected in ocular type MMP. 35 Linear IgA bullous dermatosis Linear IgA bullous dermatosis exhibits linear IgA deposit at the BMZ by direct and indirect immunofluorescence. Clinically, linear IgA bullous dermatosis develops pruritic small vesicles at the periphery of erythema similar to dermatitis herpetiformis Duhring. 36 Most patients demonstrated IgA binding to the epidermal side of salt-split skin by immunofluorescence, while some patients show IgA binding to the dermal side of salt-split skin. The target autoantigen of the lamina lucida type of LAD is 120-kD/97-kD LAD-1, which is a proteolytic cleavage product of BP ,38 IgA autoantibodies to LAD-1 can be detected by immunoblot using concentrated HaCaT cell culture media. Autoantigen of sub-lamina densa-type is undermined, however, in some patients, IgA bind to type VII collagen of the anchoring fibrils Japanese Dermatological Association 7

6 K. Ishii Blisters of skin and mucous membranes Histopathology Intraepidermal blisters subepidermal blisters DIF Intercellular pa ern In epidermis BMZ pa ern IgG IgA IgG IgA Linear Granular/fibrillar Pemphigus IgA pemphigus IIF (Split skin) LAD DH Dsg1/3 ELISA Dsc1-3 Epidermal Dermal PV or PF PNP? BP MMP(BP180) MMP(laminin 332) EBA Anti-laminin γ1pemphigoid IIF with rat bladder Immunoblot Plakins BP180 ELISA BP230 ELISA Immunoblot Type Vll collagen ELISA Immunoblot Figure 3. Flow charts of serodiagnosis for autoimmune blistering diseases. BMZ, basement membrane zone; DIF, direct immunofluorescence; Dsc, desmocollin; Dsg, desmoglein; EBA, epidermolysis bullosa acquisita; ELISA, enzyme-linked immunoassay; Ig, immunoglobulin; IIF, indirect immunofluorescence; MMP, mucous membrane pemphigoid; PF, pemphigus foliaceus; PNP, paraneoplastic pemphigus; PV, pemphigus vulgaris. Anti-laminin-c1 pemphigoid Anti-laminin-c1 pemphigoid is a subepidermal blistering disease which is characterized by autoantibodies against a 200-kD protein of the epidermal basement membrane, recently identified as laminin-c1. 40 IgG autoantibodies react with the dermal side of salt-split skin by immunofluorescence. Immunoblot using dermal extracts shows IgG binding to 200-kD protein. ELISA using a recombinant form of the C-terminus of laminin-c1 was developed with 70% sensitivity and 98.7% specificity. 41 Dermatitis herpetiformis Dermatitis herpetiformis is a chronic and recurrent subepidermal blistering disease which clinically exhibits pruritic papules and vesicles on the knee, elbows and buttocks. It is associated with distinct human leukocyte antigen haplotype (DQ8, DQ2) in Caucasian but not in Japanese subjects. 42,43 In addition, it is associated with celiac disease, a gluten-sensitive enteropathy, although no association has been reported in Japanese patients. Direct immunofluorescence shows granular or fibrillar deposit of IgA and C3 in the papillary dermis. The target autoantigen has been identified as epidermal transglutaminase (TG3). 44 Sensitive and specific ELISA systems for IgA detection against epidermal and tissue transglutaminase are commercially available. 45 CONCLUSION Flow charts for the diagnosis for autoimmune blistering diseases are shown in Figure 3. Accurate diagnosis and proper evaluation of the disease activity using immunoassays are necessary for providing appropriate treatment to patients. In the future, diagnostic immunoassays for all autoantigens should be available for clinical use. In addition, functional assays to evaluate disease activity in autoimmune blistering diseases should be introduced for practical use. CONFLICT OF INTEREST: None declared. REFERENCES 1 Stanley JR, Amagai M. Pemphigus, bullous impetigo, and the staphylococcal scalded-skin syndrome. N Engl J Med 2006; 355: Japanese Dermatological Association

7 Serology in autoimmune blistering diseases 2 Amagai M. Desmoglein as a target in autoimmunity and infection. J Am Acad Dermatol 2003; 48: Anhalt GJ, Labib RS, Voorhees JJ, Beals TF, Diaz LA. Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease. N Engl J Med 1982; 306: Tsunoda K, Ota T, Aoki M et al. Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3. J Immunol 2003; 170: Payne AS, Ishii K, Kacir S et al. Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display. J Clin Invest 2005; 115: Ishii K, Lin C, Siegel DL, Stanley JR. Isolation of pathogenic monoclonal anti-desmoglein 1 human antibodies by phage display of pemphigus foliaceus autoantibodies. J Invest Dermatol 2008; 128: Saleh MA, Ishii K, Yamagami J, Shirakata Y, Hashimoto K, Amagai M. 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Methods Mol Biol 2013; 961: Schepens I, Jaunin F, Begre N et al. The protease inhibitor alpha- 2-macroglobulin-like-1 is the p170 antigen recognized by paraneoplastic pemphigus autoantibodies in human. PLoS ONE 2010; 5: e Amagai M, Nishikawa T, Nousari HC, Anhalt GJ, Hashimoto T. Antibodies against desmoglein 3 (pemphigus vulgaris antigen) are present in sera from patients with paraneoplastic pemphigus and cause acantholysis in vivo in neonatal mice. J Clin Invest 1998; 102: Nagata Y, Karashima T, Watt FM, Salmhofer W, Kanzaki T, Hashimoto T. Paraneoplastic pemphigus sera react strongly with multiple epitopes on the various regions of envoplakin and periplakin, except for the c-terminal homologous domain of periplakin. J Invest Dermatol 2001; 116: Probst C, Schlumberger W, Stocker W et al. Development of ELISA for the specific determination of autoantibodies against envoplakin and periplakin in paraneoplastic pemphigus. Clin Chim Acta 2009; 410: Hashimoto T, Kiyokawa C, Mori O et al. Human desmocollin 1 (Dsc1) is an autoantigen for the subcorneal pustular dermatosis type of IgA pemphigus. J Invest Dermatol 1997; 109: Hashimoto T, Komai A, Futei Y, Nishikawa T, Amagai M. Detection of IgA autoantibodies to desmogleins by an enzyme-linked immunosorbent assay: the presence of new minor subtypes of IgA pemphigus. Arch Dermatol 2001; 137: Kasperkiewicz M, Kowalewski C, Jablonska S. Pemphigus herpetiformis: from first description until now. J Am Acad Dermatol 2014; 70: Ishii K, Amagai M, Komai A et al. Desmoglein 1 and desmoglein 3 are the target autoantigens in herpetiform pemphigus. Arch Dermatol 1999; 135: Kozlowska A, Hashimoto T, Jarzabek-Chorzelska M et al. Pemphigus herpetiformis with IgA and IgG antibodies to desmoglein 1 and IgG antibodies to desmocollin 3. J Am Acad Dermatol 2003; 48: Matsumura K, Hashimoto T, Amagai M, Nishikawa T. The majority of bullous pemphigoid and herpes gestationis serum samples react with NC16a domain of the 180 kd bullous pemphigoid antigen. Arch Dermatol Res 1996; 288: Kobayashi M, Amagai M, Kuroda-Kinoshita K et al. BP180 ELISA using bacterial recombinant NC16a protein as a diagnostic and monitoring tool for bullous pemphigoid. J Dermatol Sci 2002; 30: Yoshida M, Hamada T, Amagai M et al. Enzyme-linked immunosorbent assay using bacterial recombinant proteins of human BP230 as a diagnostic tool for bullous pemphigoid. J Dermatol Sci 2006; 41: Lapiere JC, Woodley DT, Parente MG et al. Epitope mapping of type VII collagen. Identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa. J Clin Invest 1993; 92: Gammon WR, Murrell DF, Jenison MW et al. Autoantibodies to type VII collagen recognize epitopes in a fibronectin-like region of the noncollagenous (NC1) domain. J Invest Dermatol 1993; 100: Saleh MA, Ishii K, Kim YJ et al. 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