To compare the parameters in the COL17-depletion assay and COL17-ELISA, one-way ANOVA test was used. by Western blotting. By contrast, BP-IgG with IVIg was found to result in 70C90% increases in COL17 and to restore adhesion to the plate. Interestingly, IVIg significantly inhibited the binding of BP-IgG to the COL17-enzyme-linked immunosorbent assay plate, and this was due to anti-idiotypic antibodies against BP-IgG. When anti-idiotypic antibodies against BP-IgG in 0.02% of IVIg were depleted from IVIg, those antibodies did not exhibit inhibitory effects on COL17 depletion. When cryosections of human skin were incubated with BP-IgG in the presence of AT7867 2HCl leukocytes, dermalCepidermal separation was observed. BP-IgG treatment with IVIg or anti-idiotypic antibodies did not induce such separation. These findings strongly suggest the presence of anti-idiotypic antibodies against anti-COL17 IgG in IVIg. This mechanism of IVIg could be a target for therapies against BP. DermalCEpidermal Separation Assay (Cryosection Assay) autoantibody-induced, neutrophil-dependent dermalCepidermal separation was performed as described (28, 29). Briefly, 5-m cryosections from normal human skin were incubated with BP-IgG (2?mg/ml) in the presence or absence of 5?mg/ml IVIg at 37C for 1?h. After washing with PBS, AT7867 2HCl the slides were covered with a second slide that was taped at each end to form a chamber. Subsequently, 107?cells/ml of freshly isolated normal human leukocyte suspension was injected into the chamber and incubated at 37C for 5?h. Sections were subsequently stained with H&E. We tested the experiments using two different blood donors. Statistical Analyses Statistical calculations were performed using SigmaPlot (Version 12.0, Systat Software, Chicago, IL, USA). To compare the parameters in the COL17-depletion assay and COL17-ELISA, one-way ANOVA test was used. A comparison of the COL17-ELISA using patient sera with/without IVIg was performed using the Wilcoxon signed-rank test. A assay, demonstrating the dermalCepidermal separation induced by BP-IgG in the presence of leukocytes (28, 29). In case of BP-IgG with PBS, dermalCepidermal separation was observed 5?h after incubation with normal human leukocytes (Figure ?(Figure5,5, upper left). Next, we added IVIg, BSA and normal human IgG and incubated them with BP-IgG on the cryosections for 1?h, followed by incubation with normal human leukocytes for 5?h. IVIg depleted (5?mg/ml, upper right) and normal human IgG (5?mg/ml, lower middle) did not block the dermalCepidermal separations. By contrast, IVIg (5?mg/ml, upper middle) and the idiotype (2.6?g/ml, lower left) protected the dermalCepidermal separation. Open in a separate window Figure 5 Intravenous immunoglobulin (IVIg) prevents dermalCepidermal separation assay (Figure ?(Figure66B). Open in a separate window Figure 6 A different companys intravenous immunoglobulin (IVIg) reproduces the results. Using IVIg from a different company, the type XVII collagen (COL17)-depletion assay and assay were performed. (A) COL17-depletion assay using IVIg from a different company (pretreatment). (B) assay using IVIg from a different company. Discussion Intravenous immunoglobulin therapy is currently applied in autoimmune diseases, and various mechanisms of action AT7867 2HCl for IVIg are involved. Previous clinical studies concluded that IVIg therapy for AIBD is a safe, effective strategy (2, 3, 31C34). The modes of action of IVIg are divided into two major mechanisms (1). One mechanism involves the F(ab)2 fragment, which is responsible for antigen recognition. The other mechanism involves the Fc fragment, which contributes to effector cell activation. Anti-idiotypic antibodies work the F(ab)2 fragment, which neutralize the pathogenic antibodies in several autoimmune disorders (1, 12). However, until now, there have been no reports on anti-idiotypic antibodies in BP. According to our COL17-depletion results, we expected IVIg to contain anti-idiotypic antibodies against BP-IgG and to prevent BP-IgG binding to autoantigen COL17. We clearly demonstrated the presence of very small amounts of anti-idiotypic antibodies against anti-COL17 IgG (0.02%) in IVIg. Even very small amounts of anti-idiotypic antibodies can Rabbit polyclonal to Catenin alpha2 prevent COL17 depletion. In addition, after the depletion of anti-idiotypic antibodies from IVIg, the ability of IVIg to block COL17 depletion was not observed. IVIg is purified from serum pooled from healthy volunteers; therefore, the IVIg used in this study may have contained anti-idiotypic antibodies to BP-IgG by chance. However, we ruled this.