title: Cloning, expression, purification and characterization of a bispecific single-chain diabody against fluoroquinolones and sulfonamides in Escherichia coli.
abstract: A recombinant bispecific single-chain diabody (scDb), recognizing fluoroquinolones (FQs) and sulfonamides (SAs), was successfully constructed with two single-chain variable fragment antibodies (scFvs). The scDb gene was cloned into the expression vector pJB33, and 6×His-tagged scDb was expressed as soluble bodies in Escherichia coli RV308 host, then purified by one step affinity chromatography of immobilized metal ion affinity chromatography (IMAC). SDS-PAGE and Western blotting analysis of the purified scDb indicated that the prepared scDb was successfully expressed as a ∼60 kDa and the final purity of the scDb protein was up to 95% with yields of approximately 6 mg/L of bacterial culture. The scDb was further characterized by indirect competitive enzyme linked immunosorbent assay (icELISA), showing that the affinity and specificity of scDb were fully retained from the two parental scFvs, capable of simultaneously binding FQs and SAs. The 50% inhibition concentration (IC50) values of the optimized immunoassay were 0.45 ng mL(-1) for FQs and 0.75 ng mL(-1) for SAs, respectively. The scDb exhibited high affinity to 20 FQs and 14 SAs. Taken together, these findings suggested that the prepared scDb could be used to develop future novel immunoassay for simultaneous determination of 20 FQs and 14 SAs.
authors.affiliation: College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
authors.forename: Min
authors.initials: M
authors.lastname: Chen
authors.affiliation: College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Wen
authors.affiliation: College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
authors.forename: Xiaoqi
authors.initials: X
authors.lastname: Tao
authors.affiliation: College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Xie
authors.affiliation: College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
authors.forename: Limiao
authors.initials: L
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
authors.forename: Shuangyang
authors.initials: S
authors.lastname: Ding
authors.affiliation: College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. Electronic address: haiyang@cau.edu.cn.
authors.forename: Haiyang
authors.initials: H
authors.lastname: Jiang
mesh_terms: D000900:Anti-Bacterial Agents / Q000276:immunology*; D018033:Antibodies, Bispecific / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D024841:Fluoroquinolones / Q000276:immunology*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D013449:Sulfonamides / Q000276:immunology*
chemical_list: D000900:Anti-Bacterial Agents; D018033:Antibodies, Bispecific; D024841:Fluoroquinolones; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D013449:Sulfonamides
keywords: Enzyme-linked immunosorbent assay; Fluoroquinolones; Single-chain diabody; Sulfonamides