A Cysteine-Specific Cationization Strategy for Versatile Antibody Production against Intrinsically Disordered Proteins

Ryui Sakaguchi, Ai Miyamoto, Rikako Kutsuma, Takeru Mori, Daichi Nakashima, Mirei Masui, Tomoko Honjo, Midori Futami, Mariko Morii, Toshiyuki Oshiki, Junichiro Futami

Several autoantigens relevant to the immune system, especially those targeted by autoantibodies induced by antitumor responses, tend to be rich in disordered regions and are prone to aggregation. This inherent instability presents significant challenges for the production, purification, and analysis of autoantigens in laboratory settings. Cysteine-specific cationization can effectively solubilize and purify these challenging proteins, allowing the isolation of full-length water-soluble antigens in their denatured state. The purified antigens enable accurate multiplex autoantibody assays using a suspension Luminex bead array platform. However, well-validated positive control antibodies are essential to ensuring precise clinical diagnosis. In this study, we prepared and characterized a panel of control antibodies by immunizing rabbits with cysteine-specific S-cationized antigens. The resulting antibodies predominantly recognized linear epitopes and were highly effective as quality control reagents in autoantibody array assays. Additionally, these antibodies maintained their ability to bind to their native, unmodified intracellular counterparts, highlighting the usefulness of this approach for producing antibodies against intrinsically disordered proteins. Although a modest immune response against the S-cationized modification site was observed, it remained minimal and did not affect the usefulness of the antibodies for assay validation. We propose this versatile cysteine-specific cationization platform for managing unstable proteins rich in disordered regions, supporting antigen production for diagnostics, and antibody development for research and validation purposes.

Bioconjugate Chemistry

ACS

37

3

580

589

10.1021/acs.bioconjchem.6c00001

41777153