title: Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae.
abstract: A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.
authors.affiliation: Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Sakamoto
authors.forename: Benyakan
authors.initials: B
authors.lastname: Pongkitwitoon
authors.forename: Seiko
authors.initials: S
authors.lastname: Nakamura
authors.forename: Kaori
authors.initials: K
authors.lastname: Sasaki-Tabata
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Tanizaki
authors.forename: Katsumi
authors.initials: K
authors.lastname: Maenaka
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Satoshi
authors.initials: S
authors.lastname: Morimoto
mesh_terms: D015084:2,4-Dichlorophenoxyacetic Acid / Q000032:analysis / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000821:virology; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006458:Hemolymph / Q000737:chemistry / Q000378:metabolism / Q000821:virology; D006540:Herbicides / Q000032:analysis / Q000276:immunology*; D007814:Larva / Q000235:genetics / Q000378:metabolism / Q000821:virology; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D006540:Herbicides; D057127:Single-Chain Antibodies; D015084:2,4-Dichlorophenoxyacetic Acid