Bullous Pemphigoid Sera that Contain Antibodies to BPAg2 also Contain Antibodies to LABD97 that Recognize Epitopes Distal to the NC16A Domain Conleth.

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Bullous Pemphigoid Sera that Contain Antibodies to BPAg2 also Contain Antibodies to LABD97 that Recognize Epitopes Distal to the NC16A Domain Conleth A. Egan, Ted B. Taylor, Marta J. Petersen Journal of Investigative Dermatology Volume 112, Issue 2, Pages 148-152 (February 1999) DOI: 10.1046/j.1523-1747.1999.00490.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Schematic representation of BPAg2. As described byGiudice et al. (1992), BPAg2 is a transmembrane protein. The NC16A domain is 73 amino acids long and has been divided into five regions (Zillikens et al. 1997). The previously described epitopes including MCW-1 are contained within the N-terminal 45 amino acids located in regions 1–3. LABD97’s N-terminus resides in the distal portion of region 3, distal to these epitopes. (Used with permission from G. Giudice, personal communication.) Journal of Investigative Dermatology 1999 112, 148-152DOI: (10.1046/j.1523-1747.1999.00490.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Western blot of epidermal extract incubated with BP sera. Twelve of 33 sera screened reacted with the 180 kDa BPAg2, extracted from human epidermis on western immunoblot. Five of these 12 sera also reacted with the 230 kDa BPAg1. There was no reactivity with either of these antigens seen with normal control sera. Lanes 1–12, BP sera;lanes 13–14, normal controls. Journal of Investigative Dermatology 1999 112, 148-152DOI: (10.1046/j.1523-1747.1999.00490.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Western blot of epidermal extract containing LABD97 incubated with BP sera. The 12 sera that were reactive with BPAg2 were reacted with an epidermal extract containing LABD97. Nine of these 12 sera reacted with LABD97. Three of the BPAg2 sera and two normal control sera showed no reactivity with LABD97. Lanes 1–12, BP sera;lanes 13–14, normal controls;lane 15, positive LABD97 control incubated with LABD serum and developed for IgA. Journal of Investigative Dermatology 1999 112, 148-152DOI: (10.1046/j.1523-1747.1999.00490.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Western blot of NC16A incubated with BP sera. The 12 sera with reactivity to BPAg2 were reacted with a recombinant form of the NC16A domain of BPAg2. All of the previously described major BPAg2 epitopes with which IgG antibodies react in BP are contained in this domain. Ten of the 12 BPAg2 reactive sera reacted with the recombinant NC16A domain. Lane 3 shows weak reactivity and lane 6 shows no reactivity, as do normal control sera. Lanes 1–12, BP sera;lane 13, positive control rabbit serum containing polyclonal antibodies to NC16A;lanes 14–15, normal controls. Journal of Investigative Dermatology 1999 112, 148-152DOI: (10.1046/j.1523-1747.1999.00490.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Patient serum stains the epidermal side of BMZ-split human skin. Normal human skin was split at the BMZ with 20 mM ethylenediamine tetraacetic acid and was used as a substrate for IIF. Both BP sera and the affinity purified IgG antibodies reacting with LABD97 demonstrate stain the epidermal side of split skin in an identical pattern. (A) BP serum dilution 1:320. (B) Affinity purified IgG antibodies eluted from LABD97 stain the epidermal side of BMZ-split human skin. (C) Affinity purified antibodies from a normal control serum do not bind to BMZ-split human skin. Scale bar: 100 μm. Journal of Investigative Dermatology 1999 112, 148-152DOI: (10.1046/j.1523-1747.1999.00490.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Western blot of epidermal extract incubated with affinity-purified IgG antibodies eluted from LABD97. IgG antibodies reactive with LABD97 were affinity purified from the 12 sera reactive with BPAg2. These eluted IgG antibodies were then reacted with an epidermal extract containing BPAg2. Nine of 12 purified antibodies reacted with BPAg2. Lanes 10–11 demonstrate weak reactivity with BPAg2 despite negative IIF. Lanes 1–12, eluted LABD97 IgG antibodies;lane 13, positive control BPAg2 reactive BP serum;lanes 14–15, normal controls. Journal of Investigative Dermatology 1999 112, 148-152DOI: (10.1046/j.1523-1747.1999.00490.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Western blot of NC16A incubated with affinity-purified IgG antibodies eluted from LABD97. The affinity-purified IgG antibodies reactive with LABD97 were reacted with recombinant NC16A. None reacted with NC16A in the presence of positive and negative controls. Lanes 1–12, eluted LABD97 IgG antibodies;lane 13, positive control rabbit serum containing polyclonal antibodies to NC16A;lanes 14–15, normal controls. Journal of Investigative Dermatology 1999 112, 148-152DOI: (10.1046/j.1523-1747.1999.00490.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions