Detection of Laminin 5-Specific Auto-antibodies in Mucous Membrane and Bullous Pemphigoid Sera by ELISA Vassiliki Bekou, Sybille Thoma-Uszynski, Olaf.

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Detection of Laminin 5-Specific Auto-antibodies in Mucous Membrane and Bullous Pemphigoid Sera by ELISA Vassiliki Bekou, Sybille Thoma-Uszynski, Olaf Wendler, Wolfgang Uter, Susanne Schwietzke, Thomas Hunziker, Christos C. Zouboulis, Gerold Schuler, Lydia Sorokin, Michael Hertl Journal of Investigative Dermatology Volume 124, Issue 4, Pages 732-740 (April 2005) DOI: 10.1111/j.0022-202X.2005.23646.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 (A, B) Affinity-purified laminin 5 (LN5). (A) Silver stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels run under reducing conditions of LN5 from SCC-25 cell culture media, before and after affinity purification from culture supernatants. (B) Mouse anti-LN α3, goat anti-LN β3, and mouse anti-LN γ2, all showed reactivity with the purified LN5 preparation. The bands shown represent the normal unprocessed and processed forms of the LN5 chains. α3 chain, processed at 165 kDa; β3 chain, unprocessed at 140 kDa; and γ2 chain, unprocessed at 155 kDa and processed at 105 kDa. The content of residual fibronectin (FN) in the LN5 preparation was visualized by a goat anti-FN ab. Numbers on the left of the gels indicate migration positions of molecular weight marker. Journal of Investigative Dermatology 2005 124, 732-740DOI: (10.1111/j.0022-202X.2005.23646.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 (A–C) Sensitivity and specificity of the laminin 5 (LN5) ELISA. The diagnostic properties of the LN5 ELISA are depicted as receiver operating characteristic curves for (A) mucous membrane pemphigoid (MMP) and (B) bullous pemphigoid (BP) sera. Sera of patients with active MMP (n=24), BP (n=72), and controls (n=51) were screened for LN5 IgG reactivity by ELISA. (C) Comparing the IgG reactivity against LN5 in the MMP, BP, and control sera, a significant difference in the LN5 IgG autoantibody status was found between all three groups in pairwise tests. Journal of Investigative Dermatology 2005 124, 732-740DOI: (10.1111/j.0022-202X.2005.23646.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 (A, B) Anti-laminin 5 (LN5) reactivity of the mucous membrane pemphigoid (MMP) and bullous pemphigoid (BP) sera by dot blot analysis. (A) A total of 24 MMP, 72 BP, and 51 control sera were analyzed for LN5 IgG reactivity by dot blot. 14/24 (58.3%) MMP and 16/72 (22.2%) BP sera reacted with LN5 by dot blot analysis. Representative dots of the LN5-reactive patients' and control sera are shown. Fibronectin (FN) was used as a control protein. (B) The clinical phenotype of the LN5-reactive MMP and BP patients is depicted. Journal of Investigative Dermatology 2005 124, 732-740DOI: (10.1111/j.0022-202X.2005.23646.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunoserological profile of mucous membrane pemphigoid (MMP) and bullous pemphigoid (BP) patients. The IgG reactivity pattern of MMP and BP sera with saline-split human skin (SSS) compared with IgG reactivity to laminin 5 (LN5) by ELISA and dot blot analysis are shown. A significant portion of the 13 SSS-negative MMP sera and eight SSS-negative BP sera were reactive for LN5 by ELISA or dot blot analysis. Journal of Investigative Dermatology 2005 124, 732-740DOI: (10.1111/j.0022-202X.2005.23646.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 (A, B) IgG titers against laminin 5 (LN5) correlate with the clinical severity of mucous membrane pemphigoid (MMP) but not of bullous pemphigoid (BP). (A) MMP patients with extensive skin involvement had higher LN5-specific IgG autoantibody levels than MMP patients with limited or exclusive mucous membrane involvement. (B) In contrast, anti-LN5 IgG reactivity of the BP sera did not show such a relationship with the clinical status (ns, not significant). Journal of Investigative Dermatology 2005 124, 732-740DOI: (10.1111/j.0022-202X.2005.23646.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 (A–C) Anti-laminin 5 (LN5) IgG titers correlate with disease activity in a patient with mucous membrane pemphigoid (MMP). The clinical phenotype of a MMP patient (A) over a course of 120 wk and corresponding serum IgG reactivity to LN5 by ELISA (B) and by dot blot analysis (C) are shown. Remarkably, upon clinical remission the titers of LN5-reactive IgG declined to threshold levels. Journal of Investigative Dermatology 2005 124, 732-740DOI: (10.1111/j.0022-202X.2005.23646.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 (A, B) Relationship of IgG reactivity for laminin 5 (LN5) and bullous pemphigoid (BP)180 or BP230 of mucous membrane (MMP) and BP sera. A total of 6/24 (25.0%) of the MMP sera were reactive to BP180, four of which (16.7%) showed IgG reactivity to LN5 (A). Among the BP sera, 55/72 (76.4%) were positive for BP180 and 34/72 (47.2%) for BP230. A total of 22/72 (30.6%) of the BP180-reactive BP sera were also LN5 reactive and 12/72 (16.7%) of the BP230-positive BP sera showed IgG reactivity to LN5 (B) (ns, not significant). Journal of Investigative Dermatology 2005 124, 732-740DOI: (10.1111/j.0022-202X.2005.23646.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions