Noncompetitive immunodetection of benzaldehyde by open sandwich ELISA

Benzaldehyde (Bz) is a typical fragrant compound for peach-flavored beverages. In the food and beverage industries there is great demand for a sensitive and easy detection system of Bz in order to ensure product quality control and to avoid contamination. For the noncompetitive detection of Bz, we applied an open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) utilizing an antigen-dependent reassociation of antibody variable region fragments, VH and VL. We cloned the VH and VL genes of an anti-Bz monoclonal antibody, and the fragments were individually expressed and purified as a bacterial alkaline phosphatase (BAP)-conjugated form for VH and as a thioredoxine (Trx)-fused form for VL, respectively. Using these VH and VL fragments, we successfully constructed the OS-ELISA system for Bz detection. The Bz-induced formation of a trimolecular complex composed of VH-BAP/Bz/Trx-VL was readily detected by a dose-dependent increase in the BAP activity of the VH-fusion protein.