Quality-controlled characterization of a monoclonal antibody specific to an EC5-domain of human desmoglein 3 for pemphigus research

Abstract

Background: Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease caused mainly by IgG autoantibodies (auto-abs) against the cadherin-type adhesion molecules desmoglein (Dsg) 1 and 3. Pathogenic anti-Dsg3 auto-abs 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 antigen-specific 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.

Keywords: 2G4; PV; antibody; autoimmunity; desmoglein (Dsg); pemphigus vulgaris; quality control.

Figure 1

Hybridoma characterization and antibody size and structural verification. (A) Representative flow cytometry of the 2G4 hybridoma cell line. Cells were gated as life singlet CD138+ IgG+, and Dsg3–specific cells were identified as dual stained for Dsg3-AF647 and Dsg3-PE. (B) Exemplified reducing SDS-page for purity analysis. Lane 1=marker, 2/3=medium before and after agarose binding, 4/5=flow trough, 6=elution (>90% IgG purity). (C) Dsg3 enzyme-linked immunosorbent assay applying a dilution series of 2G4 batches #1-6 (optical density [OD] at 405 nm). (D) Mass spectrometric analysis confirms the presence of a monoclonal antibody. Reduction with dithiothreitol leads to separation of heavy and light chain. (E) Zoom into the heavy chain reveals 4 glycosylation sites indicated by arrows.

Figure 2

Ex vivo binding verification using monkey esophagus or human skin sections. Characteristic intercellular epithelial staining on monkey esophagus visible with 2G4 as primary antibody at dilutions of (A) 1:1000, (B) 1:5000, (C) 1:10.000 indicated by arrow. (D) Control sample remains negative. Dsg3 characteristic basal and immediate suprabasal layers of skin visible in human skin on (E) cryosections (IgG green, DAPI blue) or (F) paraffin-embedded sections. Scale bar = 100µm.

Figure 3

Batch dependent 2G4 analysis confirms similar pathogenicity based upon disruption of desmosomes. Monolayer dissociation assay using human hTert-immortalized keratinocytes treated with 2G4, AK23, or human control IgG (all at 75 µg/ml for 24 h), n=triplicates/batch. *P <0.05, ns not significant.