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.
Shirasu
Naoto
N
Department of Electronics, Graduate School of Information Science and Electrical Engineering, Kyushu University, 744 Motooka, Nishi, Fukuoka 819-0935, Japan. shirasu@fukuoka-u.ac.jp
Onodera
Takeshi
T
Nagatomo
Kazutaka
K
Shimohigashi
Yasuyuki
Y
Toko
Kiyoshi
K
Matsumoto
Kiyoshi
K
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D001547:Benzaldehydes / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D006825:Hybridomas; D055767:Immobilized Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001547:Benzaldehydes; D055767:Immobilized Proteins; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; C032175:benzaldehyde