Abstract

We tested a mouse monoclonal along with an "enhanced validated" rabbit polyclonal antibody directed against human TGM2 by a new antibody validation workflow: The three-step approach was based on a protein array screening for protein hit discovery, a multiplexed epitope mapping for hit validation and epitope identification, and a final epitope substitution scan for an in-depth analysis of conserved and variable amino acid positions. The goal was to compare validation methods and to analyze of off-target binding of two different types of research antibodies. While the rabbit polyclonal antibody showed a strong cross-reactivity on the protein microarray and no target-binding, it turned out to be highly target specific and less cross-reactive in the multiplexed epitope mapping on the peptide microarray, in accordance with the expectations. A higher target specificity and less off-target binding was found for the mouse monoclonal antibody. Multiplexed epitope mapping showed only a single response corresponding to the epitope of the antibody, and the final epitope substitution scan highlighted 7 well conserved amino acid positions---a prerequisite for a low cross-reactivity and no off-target binding