Quality-controlled characterization of a monoclonal antibody specific to an EC5-domain of human desmoglein 3 for pemphigus research
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Philipps-Universität Marburg
Abstract
Background: Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering
disease caused mainly by IgG autoantibodies (auto-abs) against the cadherintype
adhesion molecules desmoglein (Dsg) 1 and 3. Pathogenic anti-Dsg3 autoabs
bind to different Dsg3 epitopes, leading, among others, to signalling that is
involved in pathogenic events, such as Dsg3 depletion. As central tools in
research on PV, a limited number of antibodies such as AK23 are frequently
used by the autoimmune bullous disease community.
Methods: Previously, we have introduced a novel Dsg3 EC5-binding antibody
termed 2G4 that may potentially serve as a superior tool for numerous PV related
analysis. The purpose of this study was to develop a quality-controlled production
and verification process that allows I) a continuous quality improvement, and II) a
verified and comprehensible overall quality with regard to pathogenic antigenspecific
binding in a variety of pemphigus assays for each batch production.
Results: Thus, a workflow based on a standardized operating procedure was
established. This includes the verification of purity and in-vitro binding capacity
(SDS-page, direct and indirect immunofluorescence) as primary parameters, and
size analysis by mass-spectrometry and ex-vivo pathogenicity by monolayer
dissociation assay.
Conclusion: We here present an extensive point-by-point quality controlled IgG
production protocol, which will serve as a basis for a standardized antibody
assessment in PV research.