PMC8791857_01
Detection of IgG autoantibodies by various methods. (A) The results of indirect immunofluorescence (IIF) for the seven patient sera collected at indicated days. (B) The results of indirect immunofluorescence using salt-split skin (ssIIF) for the seven patient sera collected at indicated days. The IIF and ssIIF results of day 270 were similar to those of day 279 and therefore not shown. In both IIF and ssIIF, staining to cytoplasm but not to cell surfaces in the lower epidermis was seen at days 105 and 120, while clear cell surface staining in the lower epidermis was found only at days 270 and 279. Reactivity with BMZ was also negative at days 105 and 120 in both IIF and ssIIF. The upper, middle and lower images (from left to right) are days 0 and 7, 55 and 105, and 120 and 279, respectively. (C) The results of immunoblotting of normal human epidermal extract for the seven patient sera. The sera of pemphigus foliaceus and bullous pemphigoid patients were used as positive controls to indicate the protein bands for BP230, BP180, Dsg1, and the 97-kDa LAD-1, a truncated form of BP180. None of the seven patient sera reacted with intact BP180 and Dsg3. (D) The results of ELISAs using various recombinant proteins and peptides. Dot line indicates cutoff value. (E) Summary of the reactivity with major peptides and recombinant protein of the patient sera during the disease course.
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BMZ was found in sera at days 0, 7, 55, 270, and 279 and that with keratinocyte surfaces at the lowermost epidermis at days 270 and 279 ( Figure 1A ). By indirect immunofluorescence using salt-split skin (ssIIF), positive IgG reactivities with the epidermal side of the split skin and with cell surfaces in the lowermost epidermis were found in a similar manner to those found in IIF ( Figure 1B ). The findings in IIF and ssIIF at days 270 and 279 were almost the same, and therefore, the results at day 270 are not shown in  Figures 1A, B . The changes of titers of autoantibodies to BP230 RP, BP180 NC16a domain RP, and Dsg1 peptide LQ144 (intracellular domain, ICD) matched well to the disease course of this patient, while the changes of titers of autoantibodies to BP180 peptide LQ66 (ICD) and Dsg3 peptide LQ123 (extracellular domain, ECD) did not correlate with the disease course ( Figure 1D ). The changes of titers of autoantibodies to Dsg1 RP (ECD), Dsg3 RP (ECD), and Dsg3 peptide LQ133 (ECD) reflected the disease relapse ( Figure 1D ). Autoantibodies against Dsg3 peptide LQ133 (ECD) became positive after relapse probably via epitope spreading ( Figure 1E ).