Standardized Production of Anti‐Desmoglein 3 Antibody AK23 for Translational Pemphigus Vulgaris Research

Abstract

AbstractAntibody‐mediated receptor activation is successfully used to develop medical treatments. If the activation induces a pathological response, such antibodies are also excellent tools for defining molecular mechanisms of target receptor malfunction and designing rescue therapies. Prominent examples are naturally occurring autoantibodies inducing the severe blistering disease pemphigus vulgaris (PV). In the great majority of patients, the antibodies bind to the adhesion receptor desmoglein 3 (Dsg3) and interfere with cell signaling to provoke severe blistering in the mucous membranes and/or skin. The identification of a comprehensive causative signaling network downstream of antibody‐targeted Dsg3 receptors (e.g., shown by pharmacological activators or inhibitors) is currently being discussed as a basis to develop urgently needed first‐line treatments for PV patients. Although polyclonal PV IgG antibodies have been used as proof of principle for pathological signal activation, monospecific anti‐Dsg3 antibodies are necessary and have been developed to identify pathological Dsg3 receptor–mediated signal transduction. The experimental monospecific PV antibody AK23, produced from hybridoma cells, was extensively tested in our laboratory in both in vitro and in vivo models for PV and proved to recapitulate the clinicopathological features of PV when generated using the standardized production and purification protocols described herein. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Bovine IgG stripping from FBS and quality controlBasic Protocol 2: AK23 hybridoma expansion and IgG productionBasic Protocol 3: AK23 IgG purificationBasic Protocol 4: AK23 IgG quality controlSupport Protocol 1: Detection of endotoxin levelsSupport Protocol 2: Detection and removal of mycoplasma