Basic studies for the diagnosis of bullous diseases having pathologic changes at the basement membrane zone

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There are a number of skin diseases in which epidermal basement membrane zone (BMZ) is affected. Therefore, the research for epidermal BMZ is important for both diagnostic and therapeutic respects. First, we investigated a series of antibodies against various components of BMZ, including LH7.2 and GB3 monoclonal antibodies and human monoclonal antibody against 23OkD bullous pemphigoid (BP) antigen. One approach is the precise localization of the antigens for these antibodies. For this purpose we have established cryofixation and cryosubstitution for immunoelectron microscopy in collaboration with Professor Eady, England.Another approach is the determination of such antigens by using biochemical and molecular biological techniques. We discussed for these projects with Drs. Stanley and Amagai, U. S. A. and with Dr. Black, England, who also provided us with sera from their BP cases. Using immunoblot and immunoprecipitation method, we first showed that autoantigen profile is almost the same among Japanese, British and U. S. cases. We also found that although 230kD BP antigen is restricted to BMZ, 170kD BP antigen may exist in both BMZ and cell membrane of basal keratinocytes. Furthermore, although 170kD BP antigen was thought not to be detectable by immunoprecipitation, we showed 170kD BP antigen can be detectable by our improved immunoprecipitation method. In addition, we also established ELISA method using recombinant antigen protein produced by CDNA clone for 230kD BP antigen. This method can substitute the conventional immunofluorescence technique to detect anti-BMZ antibodies in BP sera.Another important disease is linear IgA bullous dermatosis (LAD). We have investigated for LAD autoantigen as collaboration work with Professor Zone, U. S. A. and Drs. Black and Wojnarowska, England. We confirmed that some LAD sera specifically react with 97kD protein. However, the real nature of this 97kD protein should be clarified by further confirmative studies.