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Clinical significance of enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid
Journal of Dermatological Science 26 (2001) 14 – 18 www.elsevier.com/locate/jdermsci
Clinical significance of enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid Yasuyuki Amo a,*, Tsukasa Ohkawa a, Minako Tatsuta a, Yuhko Hamada a, Takao Fujimura a, Kensei Katsuoka a, Takashi Hashimoto b a
Department of Dermatology, Kitasato Uni6ersity School of Medicine, 1 -15 -1 Kitasato, Sagamihara, Kanagawa 228 -8555, Japan b Department of Dermatology, Kurume Uni6ersity School of Medicine, 67 Asahimachi, Kurume, Fukuoka 830 -0011, Japan Received 28 June 2000; received in revised form 16 August 2000; accepted 16 August 2000
Abstract 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. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Autoantibody; Bullous pemphigoid; BP180; Recombinant protein
1. Introduction Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease caused by autoantibodies to the basement membrane zone (BMZ). Two different BP antigens of 230-kDa (BP230) and 180-kDa (bullous pemphigoid antigen (BP180)) have been identified. BP230 localizes in the intracellular hemidesmosomal plaques, * Corresponding author. Tel.: +81-427-788111; fax: + 81427-788628. E-mail address: [email protected] (Y. Amo).
whereas BP180 is a transmembrane glycoprotein, whose extracellular domain consists of 15 interrupted collagenous domains. Epitope mapping studies of human BP180 showed that the NC16A domain of this protein harbors all of the major extracellular antigenic sites recognized by BP sera [1]. Furthermore, the study using a newborn mouse animal model by Liu et al. [2] indicated that the antibodies to BP180 play a crucial role in blister formation. In the present study, to study whether serum level of anti-BP180 autoantibodies is a valuable marker for BP, the immunoreactivity of BP sera was measured by enzyme-linked im-
0923-1811/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 0 0 ) 0 0 1 4 9 - 3
Y. Amo et al. / Journal of Dermatological Science 26 (2001) 14–18
munosorbent assay (ELISA) using a recombinant protein of the NC16A domain of BP180.
2. Materials and methods
15
sodium chloride-separated human skin, all the BP sera showed IgG antibodies reactive with the epidermal side of the split. Normal control sera were obtained from 12 healthy volunteers, eight males and four females (mean age, 32.9 years; range, 25–53 years). All the serum samples were stored at − 80°C until use.
2.1. Patients and sera We investigated ten patients with well-characterized BP, five males and five females (mean age, 66.9 years; range, 44 – 84 years) (Table 1). All patients had widespread bullous disease, and no systemic treatment had been initiated. Four patients (cases 6, 8–10) showed a recurrence, 1 – 14 months after the successful treatment of corticosteroids with or without adjuvant therapy. Disease activity was evaluated according to the number of blisters and erosions on skin and/or mucous membrane. Serum samples were taken before initial treatment and after complete remission (skin or mucous membrane lesions disappeared completely). Sera were also taken in patients with recurrence, before additional treatment and after the second remission. Direct immunofluoresence (IF) revealed linear deposits of IgG and C3 on the BMZ in the biopsied perilesional skin of all the ten BP patients. By indirect IF using 1 mol/l
2.2. ELISA The bacterial recombinant protein of BP180 NC16A domain fused with glutathione-S-transferase (GST) was prepared and purified as described previously [3,4]. The optimized ELISA was run under the following conditions. The entire assay was performed at room temperature on 96-well microtiter plates. Each well was coated overnight with 100 ml of 1 mg/ml purified recombinant GST-NC16A protein in 0.05 M bicarbonate buffer, pH 9.6. The wells were then washed once with phosphate-buffered saline (PBS) containing 0.05% Tween-20, pH 7.2, and blocked for 2 h with 200 ml of PBS containing 1% skim milk. Subsequently, the wells were incubated for 1 h with 50 ml of 1600-fold diluted sera in PBS containing 1% skim milk. After washing three times as above, each well was incubated for 1 h with
Table 1 Serum levels of anti-BP180 autoantibodies to BP180 NC16A domain as determined by ELISA before and after treatmenta Case number
1 2 3 4 5 6 7 8 9 10
Age (years)
62 77 65 84 57 44 84 72 67 57
Sex
M F F F M M F M M F
Therapy
S S S S, S, S, S, S, S, S,
Mino DDS DDS, C C, CyA Mino, DDS, C DDS, C, CyA Mino, DDS, C, CyA, P
NC16A ELISA (OD) Initial treatment
Additional treatment (recurrence cases)
Before
After
Before
After
0.257 0.339 1.896 1.848 0.314 0.765 1.455 0.531 0.297 1.638
0.038 0.117 1.253 0.269 0.238 0.008 0.097 0.390 0.181 0.165
0.916
0.060
1.505 0.764 1.784
0.529 0.172 0.165
a Cases 6, 8–10 showed recurrence within 1–14 months. S, corticosteroids; Mino, minocycline hydrochloride; DDS, diaphenylsulfone; C, cyclophosphamide; CyA, cyclosporin A; P, plasmapheresis
Y. Amo et al. / Journal of Dermatological Science 26 (2001) 14–18
16
Fig. 1. The immunoreactivities of normal and BP sera against the NC16A domain of BP180 as determined by ELISA. The immunoreactivities of the ten BP sera before treatment were significantly higher than those of the 12 normal control sera (PB0.001).
100 ml of horseradish peroxidase-labeled goat anti-human IgG antiserum (TAGO Inc, Burlingame, CA, USA) diluted 4000-fold in PBS containing 1% skim milk. Following three washings, 100 ml of substrate solution (o-phenylenediamine in 0.1% H2O2) was added to each well. After 30 min incubation, the reaction was stopped by the addition of 20 ml of 2.5N H2SO4, and the optical density (OD) at 492 nm, with the correlation wavelength set at 620 nm, was measured using a microplate reader. Each serum was assayed in duplicate.
3. Results
3.1. ELISA titer of anti-BP180 autoantibodies correlated with the clinical course of BP patients The immunoreactivity of 12 normal control
Fig. 2. The results of ELISA before and after the treatment for the ten BP patients. (A) The results of the initial treatment. The immunoreactivities of ten BP sera were significantly higher before treatment than after treatment (P B0.05). (B) The results of the second treatment. A significant decline in the immunoreactivity was noted in all the four patients with recurrence after additional treatment (PB 0.01).
Y. Amo et al. / Journal of Dermatological Science 26 (2001) 14–18
17
the NC16A domain of BP180 for ten BP sera taken before treatment (0.9349 0.693, range 0.297 –1.896) were significantly higher than that of normal control sera (PB 0.001) (Fig. 1). A significant decline in immunoreactivity (0.2769 0.362, range 0.008 –1.253, PB 0.05) was noted in all the BP patients, when they were in complete remission after treatment (Fig. 2A). After treatment, only one patient (case 3) still had a markedly elevated level of anti-BP180 antibodies. This patient initially had extensive and rapidly progressive skin lesions, and was treated with high dosage of betamethasone (5 mg daily). The dose of betamethasone tapered gradually, and the patient was given 2 mg betamethasone daily when she reached complete remission. The immunoreactivity (1.2429 0.482, range 0.764 –1.784) of four patients showing recurrence of skin lesions (cases 6, 8–10) before additional treatment was significantly higher than that of normal control sera (PB 0.001). A significant decline in immunoreactivity was again noted in all the four patients with recurrence after additional treatment (0.2329 0.205, range 0.060 –0.529, PB 0.01) (Fig. 2B). The immunoreactivity of cases 4 and 6, who received adjuvant therapy in addition to corticosteroids, correlated clearly with the clinical course (Fig. 3). Immunoreactivity increased when BP patients did not respond to initial treatment consisting of systemic corticosteroids and adjuvant therapy (case 6 and others). In contrast, the immunoreactivity had declined in all the patients, when they became in complete remission.
4. Discussion Fig. 3. The relationship between ELISA results and clinical course in two representative BP patients. Disease activity was evaluated according to the number of blisters and erosions on skin and/or mucous membrane. The immunoreactivities of both cases 4 and 6 correlated well with the clinical course.
sera against the NC16A domain of BP180 using ELISA (mean 9 S.D.) was 0.0699 0.037, with a range of 0.013 –0.132. Values\3 S.D. above the mean control OD value (0.181) were considered as the cut-off point. The immunoreactivities against
The majority of BP patients react with BP180 NC16A domain, which indicates that the recombinant protein of this region is more suitable than epidermal extracts as an antigen source for detecting circulating anti-BP180 autoantibodies [3–6]. Furthermore, autoantibodies against the NC16A domain of BP180 have been shown to play a crucial role in blister formation in a passive transfer animal model [2]. Blister formation in this animal model depends upon complement activation and neutrophil recruitment [2]. It is also
18
Y. Amo et al. / Journal of Dermatological Science 26 (2001) 14–18
indicated that neutrophil elastase is essential for the dermal-epidermal cleavage in the same experimental BP model [7]. Previous studies reported the absence of correlation between disease activity and BP antibody levels as detected by indirect IF [8,9]. It has also been suggested that assessment of the titer of the antibodies to BP180 may not be important in treating BP [10]. In this study, we demonstrated by ELISA using the recombinant protein of BP180 (NC16AELISA) that the immunoreactivity to the NC16A domain of BP180 was significantly higher in BP sera than in normal control sera, and was clearly correlated with the clinical course of the BP patients. Furthermore, immunoreactivity increased in BP patients who did not respond to treatment, while the immunoreactivity was declined in patients in complete remission. The results in the present study suggest that the NC16A-ELISA is a highly sensitive and specific test that allows the objective and semi-quantitative detection of pathogenic anti-BP180 autoantibodies. Recently, Schmidt et al. [10] also reported that serum levels of anti-BP180 autoantibodies detected by similar NC16A-ELISA correlate with disease activity in BP. Therefore, the NC16A-ELISA for the detection of circulating anti-BP180 autoantibodies is useful for evaluating, not only disease activity in patients with BP, but also for evaluating the disease’s clinical course and the effect of therapy.
References [1] Zillikens D, Mascaro JM, Rose PA, Liu Z, Ewing SM, Caux F, Hoffmann RG, Diaz LA, Giudice GJ. A
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
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– 83. Liu Z, Diaz LA, Troy JL, Taylor AF, Emery DJ, Fairley JA, Giudice GJ. 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– 8. Matsumura 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– 9. Giudice GJ, Wilske KC, Anhalt GJ, Fairley JA, Taylor AF, Emery DJ, Hoffmann RG, Diaz LA. Development of ELISA to detect anti-BP180 autoantibodies in bullous pemphigoid and herpes gestationis. J Invest Dermatol 1994;102:878– 81. Nakatani 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 enzymelinked immunosorbent assay using BP180 recombinant proteins. Br J Dermatol 1998;139:365– 70. Giudice GJ, Emery DJ, Zelickson BD, Anhalt GJ, Diaz LA. Bullous pemphigoid and herpes gestationis autoantibodies recognized a common non-collagenous site on the BP180 ectodomain. J Immunol 1993;151:5724– 50. Liu Z, Shapiro SD, Zhou X, Twining SS, Senior RM, Giudice GJ, Fairley JA, Diaz LA. A critical role for neutrophil elastase in experimental bullous pemphigoid. J Clin Invest 2000;105:113– 23. Ahmed AR, Maize JC, Provost TT. Bullous pemphigoid: clinical and immunologic follow-up after successful therapy. Arch Dermatol 1977;113:1043– 6. Zillikens D, Ambach A, Schuessler M, Dummer R, Hartmann AA, Burg G. The interleukin-2 receptor in lesions and serum of bullous pemphigoid. Arch Dermatol Res 1992;284:141– 5. Schmidt E, Obe K, Bro¨cker EB, Zillikens D. Serum levels of autoantibodies to BP180 correlate with disease activity in patients with bullous pemphigoid. Arch Dermatol 2000;136:174– 8.
Clinical significance of enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid Yasuyuki Amo a,*, Tsukasa Ohkawa a, Minako Tatsuta a, Yuhko Hamada a, Takao Fujimura a, Kensei Katsuoka a, Takashi Hashimoto b a
Department of Dermatology, Kitasato Uni6ersity School of Medicine, 1 -15 -1 Kitasato, Sagamihara, Kanagawa 228 -8555, Japan b Department of Dermatology, Kurume Uni6ersity School of Medicine, 67 Asahimachi, Kurume, Fukuoka 830 -0011, Japan Received 28 June 2000; received in revised form 16 August 2000; accepted 16 August 2000
Abstract 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. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Autoantibody; Bullous pemphigoid; BP180; Recombinant protein
1. Introduction Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease caused by autoantibodies to the basement membrane zone (BMZ). Two different BP antigens of 230-kDa (BP230) and 180-kDa (bullous pemphigoid antigen (BP180)) have been identified. BP230 localizes in the intracellular hemidesmosomal plaques, * Corresponding author. Tel.: +81-427-788111; fax: + 81427-788628. E-mail address: [email protected] (Y. Amo).
whereas BP180 is a transmembrane glycoprotein, whose extracellular domain consists of 15 interrupted collagenous domains. Epitope mapping studies of human BP180 showed that the NC16A domain of this protein harbors all of the major extracellular antigenic sites recognized by BP sera [1]. Furthermore, the study using a newborn mouse animal model by Liu et al. [2] indicated that the antibodies to BP180 play a crucial role in blister formation. In the present study, to study whether serum level of anti-BP180 autoantibodies is a valuable marker for BP, the immunoreactivity of BP sera was measured by enzyme-linked im-
0923-1811/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 0 0 ) 0 0 1 4 9 - 3
Y. Amo et al. / Journal of Dermatological Science 26 (2001) 14–18
munosorbent assay (ELISA) using a recombinant protein of the NC16A domain of BP180.
2. Materials and methods
15
sodium chloride-separated human skin, all the BP sera showed IgG antibodies reactive with the epidermal side of the split. Normal control sera were obtained from 12 healthy volunteers, eight males and four females (mean age, 32.9 years; range, 25–53 years). All the serum samples were stored at − 80°C until use.
2.1. Patients and sera We investigated ten patients with well-characterized BP, five males and five females (mean age, 66.9 years; range, 44 – 84 years) (Table 1). All patients had widespread bullous disease, and no systemic treatment had been initiated. Four patients (cases 6, 8–10) showed a recurrence, 1 – 14 months after the successful treatment of corticosteroids with or without adjuvant therapy. Disease activity was evaluated according to the number of blisters and erosions on skin and/or mucous membrane. Serum samples were taken before initial treatment and after complete remission (skin or mucous membrane lesions disappeared completely). Sera were also taken in patients with recurrence, before additional treatment and after the second remission. Direct immunofluoresence (IF) revealed linear deposits of IgG and C3 on the BMZ in the biopsied perilesional skin of all the ten BP patients. By indirect IF using 1 mol/l
2.2. ELISA The bacterial recombinant protein of BP180 NC16A domain fused with glutathione-S-transferase (GST) was prepared and purified as described previously [3,4]. The optimized ELISA was run under the following conditions. The entire assay was performed at room temperature on 96-well microtiter plates. Each well was coated overnight with 100 ml of 1 mg/ml purified recombinant GST-NC16A protein in 0.05 M bicarbonate buffer, pH 9.6. The wells were then washed once with phosphate-buffered saline (PBS) containing 0.05% Tween-20, pH 7.2, and blocked for 2 h with 200 ml of PBS containing 1% skim milk. Subsequently, the wells were incubated for 1 h with 50 ml of 1600-fold diluted sera in PBS containing 1% skim milk. After washing three times as above, each well was incubated for 1 h with
Table 1 Serum levels of anti-BP180 autoantibodies to BP180 NC16A domain as determined by ELISA before and after treatmenta Case number
1 2 3 4 5 6 7 8 9 10
Age (years)
62 77 65 84 57 44 84 72 67 57
Sex
M F F F M M F M M F
Therapy
S S S S, S, S, S, S, S, S,
Mino DDS DDS, C C, CyA Mino, DDS, C DDS, C, CyA Mino, DDS, C, CyA, P
NC16A ELISA (OD) Initial treatment
Additional treatment (recurrence cases)
Before
After
Before
After
0.257 0.339 1.896 1.848 0.314 0.765 1.455 0.531 0.297 1.638
0.038 0.117 1.253 0.269 0.238 0.008 0.097 0.390 0.181 0.165
0.916
0.060
1.505 0.764 1.784
0.529 0.172 0.165
a Cases 6, 8–10 showed recurrence within 1–14 months. S, corticosteroids; Mino, minocycline hydrochloride; DDS, diaphenylsulfone; C, cyclophosphamide; CyA, cyclosporin A; P, plasmapheresis
Y. Amo et al. / Journal of Dermatological Science 26 (2001) 14–18
16
Fig. 1. The immunoreactivities of normal and BP sera against the NC16A domain of BP180 as determined by ELISA. The immunoreactivities of the ten BP sera before treatment were significantly higher than those of the 12 normal control sera (PB0.001).
100 ml of horseradish peroxidase-labeled goat anti-human IgG antiserum (TAGO Inc, Burlingame, CA, USA) diluted 4000-fold in PBS containing 1% skim milk. Following three washings, 100 ml of substrate solution (o-phenylenediamine in 0.1% H2O2) was added to each well. After 30 min incubation, the reaction was stopped by the addition of 20 ml of 2.5N H2SO4, and the optical density (OD) at 492 nm, with the correlation wavelength set at 620 nm, was measured using a microplate reader. Each serum was assayed in duplicate.
3. Results
3.1. ELISA titer of anti-BP180 autoantibodies correlated with the clinical course of BP patients The immunoreactivity of 12 normal control
Fig. 2. The results of ELISA before and after the treatment for the ten BP patients. (A) The results of the initial treatment. The immunoreactivities of ten BP sera were significantly higher before treatment than after treatment (P B0.05). (B) The results of the second treatment. A significant decline in the immunoreactivity was noted in all the four patients with recurrence after additional treatment (PB 0.01).
Y. Amo et al. / Journal of Dermatological Science 26 (2001) 14–18
17
the NC16A domain of BP180 for ten BP sera taken before treatment (0.9349 0.693, range 0.297 –1.896) were significantly higher than that of normal control sera (PB 0.001) (Fig. 1). A significant decline in immunoreactivity (0.2769 0.362, range 0.008 –1.253, PB 0.05) was noted in all the BP patients, when they were in complete remission after treatment (Fig. 2A). After treatment, only one patient (case 3) still had a markedly elevated level of anti-BP180 antibodies. This patient initially had extensive and rapidly progressive skin lesions, and was treated with high dosage of betamethasone (5 mg daily). The dose of betamethasone tapered gradually, and the patient was given 2 mg betamethasone daily when she reached complete remission. The immunoreactivity (1.2429 0.482, range 0.764 –1.784) of four patients showing recurrence of skin lesions (cases 6, 8–10) before additional treatment was significantly higher than that of normal control sera (PB 0.001). A significant decline in immunoreactivity was again noted in all the four patients with recurrence after additional treatment (0.2329 0.205, range 0.060 –0.529, PB 0.01) (Fig. 2B). The immunoreactivity of cases 4 and 6, who received adjuvant therapy in addition to corticosteroids, correlated clearly with the clinical course (Fig. 3). Immunoreactivity increased when BP patients did not respond to initial treatment consisting of systemic corticosteroids and adjuvant therapy (case 6 and others). In contrast, the immunoreactivity had declined in all the patients, when they became in complete remission.
4. Discussion Fig. 3. The relationship between ELISA results and clinical course in two representative BP patients. Disease activity was evaluated according to the number of blisters and erosions on skin and/or mucous membrane. The immunoreactivities of both cases 4 and 6 correlated well with the clinical course.
sera against the NC16A domain of BP180 using ELISA (mean 9 S.D.) was 0.0699 0.037, with a range of 0.013 –0.132. Values\3 S.D. above the mean control OD value (0.181) were considered as the cut-off point. The immunoreactivities against
The majority of BP patients react with BP180 NC16A domain, which indicates that the recombinant protein of this region is more suitable than epidermal extracts as an antigen source for detecting circulating anti-BP180 autoantibodies [3–6]. Furthermore, autoantibodies against the NC16A domain of BP180 have been shown to play a crucial role in blister formation in a passive transfer animal model [2]. Blister formation in this animal model depends upon complement activation and neutrophil recruitment [2]. It is also
18
Y. Amo et al. / Journal of Dermatological Science 26 (2001) 14–18
indicated that neutrophil elastase is essential for the dermal-epidermal cleavage in the same experimental BP model [7]. Previous studies reported the absence of correlation between disease activity and BP antibody levels as detected by indirect IF [8,9]. It has also been suggested that assessment of the titer of the antibodies to BP180 may not be important in treating BP [10]. In this study, we demonstrated by ELISA using the recombinant protein of BP180 (NC16AELISA) that the immunoreactivity to the NC16A domain of BP180 was significantly higher in BP sera than in normal control sera, and was clearly correlated with the clinical course of the BP patients. Furthermore, immunoreactivity increased in BP patients who did not respond to treatment, while the immunoreactivity was declined in patients in complete remission. The results in the present study suggest that the NC16A-ELISA is a highly sensitive and specific test that allows the objective and semi-quantitative detection of pathogenic anti-BP180 autoantibodies. Recently, Schmidt et al. [10] also reported that serum levels of anti-BP180 autoantibodies detected by similar NC16A-ELISA correlate with disease activity in BP. Therefore, the NC16A-ELISA for the detection of circulating anti-BP180 autoantibodies is useful for evaluating, not only disease activity in patients with BP, but also for evaluating the disease’s clinical course and the effect of therapy.
References [1] Zillikens D, Mascaro JM, Rose PA, Liu Z, Ewing SM, Caux F, Hoffmann RG, Diaz LA, Giudice GJ. A
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
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– 83. Liu Z, Diaz LA, Troy JL, Taylor AF, Emery DJ, Fairley JA, Giudice GJ. 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– 8. Matsumura 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– 9. Giudice GJ, Wilske KC, Anhalt GJ, Fairley JA, Taylor AF, Emery DJ, Hoffmann RG, Diaz LA. Development of ELISA to detect anti-BP180 autoantibodies in bullous pemphigoid and herpes gestationis. J Invest Dermatol 1994;102:878– 81. Nakatani 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 enzymelinked immunosorbent assay using BP180 recombinant proteins. Br J Dermatol 1998;139:365– 70. Giudice GJ, Emery DJ, Zelickson BD, Anhalt GJ, Diaz LA. Bullous pemphigoid and herpes gestationis autoantibodies recognized a common non-collagenous site on the BP180 ectodomain. J Immunol 1993;151:5724– 50. Liu Z, Shapiro SD, Zhou X, Twining SS, Senior RM, Giudice GJ, Fairley JA, Diaz LA. A critical role for neutrophil elastase in experimental bullous pemphigoid. J Clin Invest 2000;105:113– 23. Ahmed AR, Maize JC, Provost TT. Bullous pemphigoid: clinical and immunologic follow-up after successful therapy. Arch Dermatol 1977;113:1043– 6. Zillikens D, Ambach A, Schuessler M, Dummer R, Hartmann AA, Burg G. The interleukin-2 receptor in lesions and serum of bullous pemphigoid. Arch Dermatol Res 1992;284:141– 5. Schmidt E, Obe K, Bro¨cker EB, Zillikens D. Serum levels of autoantibodies to BP180 correlate with disease activity in patients with bullous pemphigoid. Arch Dermatol 2000;136:174– 8.