Native Autoantigen Complex Detects Pemphigoid Autoantibodies

Abstract

Pemphigoid diseases are a group of autoimmune disorders characterized by subepidermal blistering in the skin and mucosa. Among them, mucous membrane pemphigoid (MMP) autoantibodies are characterized by targeting multiple molecules in the hemidesmosomes, including collagen XVII, laminin-332, and integrin a6/β4. Traditionally, recombinant proteins of the autoantigens have been employed to identify circulating autoantibodies by immune assays. However, developing an efficient detection system for MMP autoantibodies has been challenging because the autoantibodies have heterogeneous profiles and the antibody titers are typically low. In this study, we introduce an ELISA that takes advantage of a native autoantigen complex rather than simple recombinant proteins. We generated HaCaT keratinocytes with a DDDDK-tag knocked in at the COL17A1 locus by CRISPR/Cas9-mediated gene editing. Immunoprecipitation using the DDDDK-tag isolated a native complex that contained full-length and processed collagen XVII and integrin α6/β4. Then, we used the complex proteins to prepare an ELISA system and enrolled 55 MMP cases to validate its diagnostic performance. The sensitivity and specificity of the ELISA for detecting MMP autoantibodies were 70.9% and 86.7%, respectively, far superior to those of conventional assays. In autoimmune diseases such as MMP, in which autoantibodies target various molecules, isolating the antigen-protein complexes can help establish a diagnostic system.