Production of a common epitope specific anti-ankyrin monoclonal antibody

Abstract

Background: Ankyrin (Ank) is a protein family with crucial roles in retaining normal cellular physiology. In addition, ankyrin offers the potential as a non-antibody binder against various biomolecules. The designed ankyrin repeat protein (DARPin) selected from phage display libraries is useful for molecular detection and therapy. Monoclonal antibodies (mAbs) specific to the common epitope of DARPin are required to detect protein-protein interaction. Objectives: This study aimed to establish mAbs against common antigenic determinant of ankyrins for further application in immunological techniques. Materials and methods: Ank1D4 monomer and dimer were generated in the Escherichia coli expression system for immunogen preparation and validation of established mAbs. The binding activity of anti-Ank mAb obtained from different hybridoma clones was characterized using Ank1D4 by indirect ELISA. Candidate anti-Ank mAbs were validated for their cross-reactivity against irrelevant ankyrin (Ank2D3). The binding kinetic of mAbs from three candidate hybridoma clones (Ank-54, Ank-59, and Ank-94) was evaluated using bio-layer interferometry (BLI). The highest affinity clone (Ank-94 mAb) was further validated for its specificity against Ank1D4 and dimeric Ank1D4 using indirect ELISA. The interaction of three anti-Ank mAbs and ankyrins was compared by western immunoblotting analysis. The specificity of Ank-94 mAb was determined using a closely related scaffold, i.e., alpha-helicoidal HEAT-like repeat protein scaffold (αRep) by indirect ELISA. Ankyrins were detected by sandwich ELISA using Ank-94 mAb. Results: The culture supernatant from hybridoma clones were characterized for their anti-ankyrin binding properties. Using indirect ELISA, three clones exhibited positive reactivity against the immunized ankyrin antigen (Ank1D4). The interactive epitope was found to rely on common antigenic determinants found in Ank1D4, dimeric Ank1D4, and an irrelevant ankyrin, Ank2D3. The immunoblotting results suggest that all mAbs interact with the sequential epitope of ankyrins. The cross-reactivity of Ank-94 mAb was not observed with αRep. Ank-94 mAb was selected for further purification and evaluation of binding properties due to its highest degree of binding affinity against Ank1D4. Conclusion: The establishment of a novel Ank-94 mAb could be a valuable research tool in tracing the target of DARPins or developing immunoassays. Ank-94 mAb is superior over formerly produced Ank mAbs since it recognizes a common epitope on DARPins and relies on sequential epitope. Ank-94 mAb has no cross-reactivity with another scaffold, αRep.