Development of tetravalent antibody–enzyme complexes employing a lactate oxidase and the application to electrochemical immunosensors

Antibody-enzyme complexes (AECs) are ideal for immunosensing. Although AECs using antibody fragments can be produced by bacterial hosts, their low affinity limits their sensing applications. We have improved the affinity of AECs by combining two antibodies using Catcher/Tag systems; however, it requires multiple antibodies and an enzyme production process. In this study, to realize the production of AECs harboring multiple antibody fragments in a single production process, we report a versatile development method of unique AECs based on a multimeric enzyme structure. Using the homotetrameric enzyme, lactate oxidase (LOx), as a labeling enzyme, tetravalent AECs were developed as an electrochemical immunosensor. Homogeneous tetravalent AECs were successfully fabricated by fusing the anti-epidermal growth factor receptor (EGFR) variable domain of a heavy chain of heavy chain antibody to the N-terminus of LOx. The prepared AECs bound to EGFR, maintain their enzyme activity, and worked well as sensing elements in electrochemical sandwich enzyme-linked immunosorbent assay. Moreover, tetravalent AECs exhibited higher sensitivity than monovalent AECs because of their avidity. The fabricated AECs were successfully used in a wash-free homogeneous electrochemical detection system combined with magnetic separation. Our findings offer new insights into the applications of the LOx tetrameric enzyme for the fabrication of AECs with tetravalent antibodies, which may serve as scaffolds for immunosensors.