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, V(H) and V(L). We cloned the V(H) and V(L) genes of an anti-Bz monoclonal antibody, and the fragments were individually expressed and purified as a bacterial alkaline phosphatase (BAP)-conjugated form for V(H) and as a thioredoxine (Trx)-fused form for V(L), respectively. Using these V(H) and V(L) fragments, we successfully constructed the OS-ELISA system for Bz detection. The Bz-induced formation of a trimolecular complex composed of V(H)-BAP/Bz/Trx-V(L) was readily detected by a dose-dependent increase in the BAP activity of the V(H)-fusion protein.