title: Production and characterization of a monoclonal antibody against enrofloxacin.
abstract: Enrofloxacin is a fluoroquinolone antibiotic approved for the treatment of infections in animals. Because of the side effects to consumers of animal products, the maximum residue limits (MRLs) of enrofloxacin in animal tissues for consumption are regulated. In this study, a monoclonal antibody (mAb) against enrofloxacin was prepared and characterized for the development of a direct competitive enzyme-linked immunosorbent assay (ELISA). The obtained mAb, Enro44, was highly specific for enrofloxacin and had a 50% inhibition concentration (IC(50)) of 1.99 ng/ml in a competitive ELISA, and the limit of detection (LOD) was 0.50 ng/ml. The cross-reactivity of the mAb with other quinolones and fluoroquinolones was lower than 0.01%. The subclass of the mAb Enro44 was identified as IgG1. The antigen (Ag)-captured direct competitive ELISA using the mAb Enro44 was tested on different spiked samples, including chicken muscle, cattle milk, and cattle urine, and the assay demonstrated recoveries of 82-112%, 80-125%, and 78-124%, respectively. Furthermore, the quantitation of enrofloxacin obtained from the ELISA and from high-performance liquid chromatography (HPLC) was in good agreement, with the linear regression coefficient between 0.933 and 1.056. The cDNAs encoding a heavy-chain Fd fragment (VH and CH1) and a light chain of the mAb Enro44 were cloned and sequenced. Taken together, the results obtained reveal a potential use of this mAb in an ELISA for the detection of enrofloxacin in food samples. The information of amino acid sequence of this mAb will be useful for further modification and production of the mAb in a bioreactor.
authors: Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors: Manaspong
authors: M
authors: Chusri
authors: Pitikarn
authors: P
authors: Wongphanit
authors: Tanapat
authors: T
authors: Palaga
authors: Songchan
authors: S
authors: Puthong
authors: Sarintip
authors: S
authors: Sooksai
authors: Kittinan
authors: K
authors: Komolpis
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002417:Cattle; D002623:Chemistry Techniques, Analytical; D002645:Chickens; D003001:Cloning, Molecular; D003429:Cross Reactions; D018076:DNA, Complementary / Q000235:genetics; D000077422:Enrofloxacin; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D024841:Fluoroquinolones / Q000032:analysis* / Q000276:immunology*; D005504:Food Analysis / Q000379:methods*; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D020128:Inhibitory Concentration 50; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D024841:Fluoroquinolones; D007074:Immunoglobulin G; D000077422:Enrofloxacin