Development of monoclonal antibodies and quantitative ELISAs targeting insulin-degrading enzyme

Abstract

Background: Insulin-degrading enzyme (IDE) is a widely studied zinc-metalloprotease implicated in the pathogenesis of type 2 diabetes mellitus, Alzheimer disease (AD) and varicella zoster virus infection. Despite more than six decades of research on IDE, progress has been hampered by the lack of well-characterized reagents targeting this biomedically important protease. To address this important need, we generated and characterized new mouse monoclonal antibodies (mAbs) targeting natively folded human and rodent IDE. Results: Eight monoclonal hybridoma cell lines were derived in house from mice immunized with full-length, natively folded, recombinant human IDE. The mAbs derived from these lines were shown to detect IDE selectively and sensitively by a wide range of methods. Two mAbs in particular - designated 6A1 and 6H9 - proved especially selective for IDE in immunocytochemical and immunohistochemical applications. Using a variety of methods, we show that 6A1 selectively detects both human and rodent IDE, while 6H9 selectively detects human, but not rodent, IDE, with both mAbs showing essentially no cross reactivity with other proteins in these applications. Using these novel anti-IDE mAbs, we also developed sensitive and quantitative sandwich ELISAs capable of quantifying IDE levels present in human brain extracts. Conclusion: We succeeded in developing novel mAbs that selectively detect rodent and/or human IDE, which we have shown to be suitable for a wide range of applications, including western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, and quantitative sandwich ELISAs. These novel anti-IDE mAbs and the assays derived from them constitute important new tools for addressing many unresolved questions about the basic biology of IDE and its role in multiple highly prevalent human diseases.