Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Determining the specificity of an anti-PD-1 antibody using a new bead array format covering about 80% of the human proteome (#558)

Matthias Harbers 1 , Shusei Sugiyama 1 , Miwako Denda 1 , Satoshi Ozawa 1 , Ryo Morishita 2
  1. CellFree Sciences Co., Ltd., Yokohama, Kanagawa , Japan
  2. CellFree Sciences Co., Ltd., Matsuyama, Ehime, Japan

Anti-programmed cell death protein 1 (PD-1/PDCD1/CD279) is a suppressor of the immune system playing an important role in preventing autoimmunity. Anti-PD-1 antibodies are therefore used to enable immune responses during tumor therapy. We used a commercial monoclonal anti-PD-1 antibody against the human PD-1 protein as an example to establish routines for proteome-wide antibody specificity testing applying our new bead array format representing ~80% of the human proteome. This format allows parallel in-solution testing of many more proteins than possible in any individual cell line experiment where proteins on the array are presented in a similar concentration range. For testing the specificity of a given antibody, our process comprises three steps: First, we confirm the binding conditions and detection method for the antibody using a set of positive and negative controls. Second, the specificity of the antibody is analyzed on the bead array in a special 1536-well plate format. In each well 14 different proteins are pooled allowing for quick overviews on the specificity of the antibody. This bead array format may also be used for further monitoring the specificity of different antibody batches or testing different secondary antibodies and detection methods. Third, to identify the actual targets that possibly cross-react with the antibody, we individually test each protein from the wells found positive in the initial screen on the 1536-well plate. This process led to the identification of three unrelated proteins that cross-reacted with the anti-PD-1 antibody. Alignments of the amino acid sequences from those cross-reacting proteins revealed a common epitope that is shared with human PD-1 protein thus demonstrating that our process and the new bead array platform are providing very accurate conditions for testing antibody specificity on a proteome-wide scale. This is an essential process when further characterizing antibodies for use in research, diagnostics or even therapy.