Hidenori Shiotsuka

Direct Immobilization of Gold-Binding Antibody Fragments for Immunosensor Applications

A novel method that enables antibody fragments to be immobilized on a sensor substrate with a high binding capability using molecular recognition has been developed. Using genetic engineering, we fabricated bispecific recombinant antibody fragments, which consist of two kinds of antibody fragments: a gold antibody fragment and a target molecule antibody fragment. Surface plasmon resonance (SPR) analysis indicated that these gold-binding bispecific antibody fragments bind directly to the gold substrate with high affinity (K(D) approximately 10(-9) M). About 70% of the bispecific antibody fragments immobilized on the gold substrate retained their target protein-binding efficiency. The Sips isotherm was used to assess the heterogeneity in antibody affinity for the bispecific antibody fragments. The results showed that the immobilized bispecific antibody fragments exhibited an increased homogeneity of affinity (K(D)) to target molecules when compared with monospecific antibody fragments immobilized by conventional methods. The use of bispecific antibody fragments to directly immobilize antibody fragments on a solid-phase substrate offers a useful platform for immunosensor applications.

Biomimetic engineering of modular bispecific antibodies for biomolecule immobilization.

Modular bispecific antibodies (BsAbs) that interact directly with a gold surface were engineered for immobilization on biosensing devices. The BsAbs consist of the variable fragments of antigold and antilysozyme antibodies connected via one of three linkers derived from naturally occurring proteins. The BsAbs were bound tightly to both the gold surface and to lysozyme, thus functioning as interface molecules between lysozyme and the gold surface without a substantial loss of antigen-binding activity. The antigen-binding capacity (the ratio of the amount of immobilized lysozyme to the amount of immobilized BsAb) on the gold surface reached 82%. An analysis of the correlation between binding capacity and linker characteristics indicated that the presence of a long, rigid linker sequence derived from a cellulase resulted in a higher antigen-binding capacity than did the presence of a long but relatively flexible glycine-rich linker. This result suggests a strategy for designing linkers suitable for BsAb-based biomolecular immobilization.