title: Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay.
abstract: A single-chain variable fragment (scFv) linked alkaline phosphatase (AP) fusion protein for detection of O,O-diethyl organophosphorus pesticides (O,O-diethyl OPs) was produced and characterized. The scFv gene was prepared by cloning V(L) and V(H) genes from hybridoma cells secreting monoclonal antibody with broad specificity for O,O-diethyl OPs. The amplified V(L) and V(H) regions were assembled using a linker (Gly(4)Ser)(3) by means of splicing overlap extension polymerase chain reaction to obtain the scFv gene, which was cloned into the expression vector pLIP6/GN containing an AP gene to produce the scFv-AP fusion protein in  Escherichia coli  strain BL21. The protein was purified by antigen-conjugated immunoaffinity chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and competitive direct enzyme-linked immunosorbent assay (cdELISA). The fusion protein is bifunctional, retaining both antigen binding specificity and AP enzymatic activity. Analysis of spiked and blind river water and Chinese cabbage samples demonstrated that the fusion protein based cdELISA(FP) exhibited good sensitivity and reproducibility.
authors.affiliation: Guangdong Provincial Key Laboratory of Food Quality and Safety,  South China Agricultural University , Guangzhou 510642, China.
authors.forename: Zhen-Lin
authors.initials: ZL
authors.lastname: Xu
authors.forename: Jie-Xian
authors.initials: JX
authors.lastname: Dong
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Zhen-Feng
authors.initials: ZF
authors.lastname: Li
authors.forename: Ross C
authors.initials: RC
authors.lastname: Beier
authors.forename: Yue-Ming
authors.initials: YM
authors.lastname: Jiang
authors.forename: Hong-Tao
authors.initials: HT
authors.lastname: Lei
authors.forename: Yu-Dong
authors.initials: YD
authors.lastname: Shen
authors.forename: Jin-Yi
authors.initials: JY
authors.lastname: Yang
authors.forename: Yuan-Ming
authors.initials: YM
authors.lastname: Sun
mesh_terms: D000469:Alkaline Phosphatase; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001483:Base Sequence; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005506:Food Contamination / Q000032:analysis; D006825:Hybridomas / Q000276:immunology; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000032:analysis*; D010575:Pesticides / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D009943:Organophosphorus Compounds; D010575:Pesticides; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D000469:Alkaline Phosphatase