BP230- and BP180-specific Auto-Antibodies in Bullous Pemphigoid

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BP230- and BP180-specific Auto-Antibodies in Bullous Pemphigoid Sybille Thoma-Uszynski, Wolfgang Uter, Susanne Schwietzke, Silke C. Hofmann, Thomas Hunziker, Philippe Bernard, Regina Treudler, Christos C. Zouboulis, Gerold Schuler, Luca Borradori, Michael Hertl Journal of Investigative Dermatology Volume 122, Issue 6, Pages 1413-1422 (June 2004) DOI: 10.1111/j.0022-202X.2004.22603.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Bullous pemphigoid (BP)230 recombinant proteins utilized in this ELISA study. (A) BP230 is an intracellular hemidesmosomal component which consists of a central rod flanked by globular end domains. (B) The recombinant BP230 fusion proteins are 6xHis- and glutathione-S-transferase (GST) tagged at their NH2 terminus. Two COOH-terminal constructs (BP230-C1, containing residues 1881–2649 and BP230-C2, residues 2077–2649) and one NH2-terminal construct (BP230-N, residues 1–1307) were expressed. The purified BP230 recombinant proteins were transferred to nitrocellulose membranes and Coomassie stained. (C) SF21 insect cells infected with recombinant BP230 baculovirus for 3 d were recognized by a representative BP serum. Journal of Investigative Dermatology 2004 122, 1413-1422DOI: (10.1111/j.0022-202X.2004.22603.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Bullous pemphigoid (BP)180 recombinant protein utilized in this ELISA study. (A) BP180 is a type II transmembrane protein containing an intracellular, a transmembrane, and an extracellular domain with 15 interrupted collagenous regions. (B) The recombinant fusion protein BP180ex is 6xHis- and glutathione-S-transferase (GST)-tagged at its NH2 end (residues 485–1497). The purified BP180ex protein was transferred to nitrocellulose membranes and Coomassie stained. (C) SF21 insect cells infected with BP180ex baculovirus for 3 d were recognized by a representative BP serum. Journal of Investigative Dermatology 2004 122, 1413-1422DOI: (10.1111/j.0022-202X.2004.22603.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 IgG reactivity against the extracellular domain of bullous pemphigoid (BP)180ex is highly indicative for BP. BP patient and control serum samples were tested by ELISA using recombinant BP180ex. Sera of patients with BP (n=127) and of 51 controls were incubated with 0.5 μg of immobilized BP180ex protein. IgG binding was detected with an alkaline-phosphatase-labeled anti-human IgG ab. Samples were run in duplicate and mean outer diameter reading at 405 nm of a serum sample with BP230 recombinants. Data are displayed as box plots: The box shows first and third quartiles, the median (as line), and the arithmetic mean (as dot), the whiskers represent values below the first quartile and above the third quartile within the 1.5-fold inter-quartile range, respectively, and outliers beyond the whiskers are shown as squares. The dotted lines indicate the cut-off values as evaluated by maximization of Youden index. The inset visualizes the corresponding receiver operating characteristic curve. There was a highly significant difference between clinical variants and controls as well as between “generalized” and “focal” BP. Journal of Investigative Dermatology 2004 122, 1413-1422DOI: (10.1111/j.0022-202X.2004.22603.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Cutpoint determination by receiver operating characteristic (ROC) curves for bullous pemphigoid (BP)230-reactive total IgG. Detection of serum IgG against the NH2-terminus (BP230-N) and COOH-terminus (BP230-C1 and BP230-C2) of BP230 is shown as ROC curves. Sera of patients with BP (n=127) and 51 controls were incubated with 0.5 μg of immobilized BP230-N, BP230-C-1, BP230-C-2 proteins. IgG binding was detected with an alkaline phosphatase (AP)-labeled anti-human IgG ab. Samples were run in duplicate and mean OD reading at 405 nm of a serum sample with BP230-like recombinant proteins. The solid lines indicate the cut-off values as evaluated by maximization of Youden index. Journal of Investigative Dermatology 2004 122, 1413-1422DOI: (10.1111/j.0022-202X.2004.22603.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Bullous pemphigoid (BP)230 IgG levels in clinical variants. Total IgG responses against the BP230 recombinant proteins were compared between serum samples from patients with “generalized”versus“focal” BP. Box plot presentation depicting median, arithmetic mean, and range from first to third quartile as in Figure 3. BP230-specific IgG reactivity against all three BP230 recombinants in both generalized and focal BP. Overall, higher, significantly different IgG levels were observed in localized BP. Thus, BP230 IgG-reactivity was significantly associated with the outcome “focal” BP. Journal of Investigative Dermatology 2004 122, 1413-1422DOI: (10.1111/j.0022-202X.2004.22603.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 (A—D) IgG subclass analysis (IgG1 and IgG4) of bullous pemphigoid (BP)230 and BP180 reactive auto-antibodies (auto-ab). IgG1 and IgG4 auto-ab against BP230 (A–C) and BP180 (D) were compared between the sera from patients with “generalized”versus“focal BP”. Box plot presentation depicting median, arithmetic mean, and range from first to third quartiles as in Figure 3. There was neither a significant correlation for IgG1 or IgG4 reactivity against BP230 (BP230-N; BP230-C1/C2) nor to BP180 (BP180ex) with the outcome “generalized”versus“focal” BP. Journal of Investigative Dermatology 2004 122, 1413-1422DOI: (10.1111/j.0022-202X.2004.22603.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions