title: Cloning, expression, purification, and characterization of a novel single-chain variable fragment antibody against the 2-nitrobenzaldehyde derivative of a furaltadone metabolite in Escherichia coli.
abstract: Furaltadone is an illicit veterinary drug that shows toxic, carcinogenic, and mutagenic effects, as does its metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ)(1). Recombinant antibodies with desirable affinity and specificity that can replace polyclonal or monoclonal antibodies are important factors for effective AMOZ immunoassays. In the present study, a novel single-chain variable fragment (scFv) antibody against the 2-nitrobenzaldehyde derivative of AMOZ (NPAMOZ) was prepared and characterized. The scFv gene was cloned into the pET-22b(+) expression vector, and 6His-tagged scFv antibodies expressed as inclusion bodies in Escherichia coli BL21 (DE3), which were then purified by nickel nitrilotriacetic acid column chromatography. Characterization of the target protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and a novel indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) showed that the scFv antibody was ∼27kDa and exhibited HRP-anti-His-tag antibody-recognized activity. The final purity, yield and mg of this scFv antibody after ultrafiltration concentration were 97%, 20% and 29.1mg, respectively. The icCLEIA indicated that the antibody competitively combined with NPAMOZ, exhibiting an IC(50) value of 1.46±0.01 ng/ml (n=6). Cross-reactivity studies revealed that the antibody showed desirable specificity to NPAMOZ and little reactivity to analogs except the parent furaltadone. In summary, these findings suggested that the prepared recombinant scFv antibody can be used for future immunoassay screening for AMOZ.
authors.affiliation: Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, China. yangwuyi2003@yahoo.com.cn
authors.forename: Wuying
authors.initials: W
authors.lastname: Yang
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Jiexian
authors.initials: J
authors.lastname: Dong
authors.forename: Zhenlin
authors.initials: Z
authors.lastname: Xu
authors.forename: Yudong
authors.initials: Y
authors.lastname: Shen
authors.forename: Yuanming
authors.initials: Y
authors.lastname: Sun
authors.forename: Xixia
authors.initials: X
authors.lastname: Liu
authors.forename: Zhenfeng
authors.initials: Z
authors.lastname: Li
authors.forename: Hongtao
authors.initials: H
authors.lastname: Lei
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Du
mesh_terms: D000818:Animals; D001547:Benzaldehydes / Q000737:chemistry* / Q000276:immunology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007124:Immunoenzyme Techniques / Q000379:methods; D051379:Mice; D009581:Nitrofurans / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D023303:Oxazolidinones / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D001547:Benzaldehydes; C471213:His-His-His-His-His-His; D009581:Nitrofurans; D009842:Oligopeptides; D023303:Oxazolidinones; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C043850:2-nitrobenzaldehyde; D006639:Histidine; C007795:furaltadon