Engineering of a broad-specificity antibody: Detection of eight fluoroquinolone antibiotics simultaneously Janne Leivo ⇑ , Clement Chappuis, Urpo Lamminmäki, Timo Lövgren, Markus Vehniäinen Department of Biotechnology, University of Turku, FIN-20520 Turku, Finland article info Article history: Received 31 March 2010 Received in revised form 18 August 2010 Accepted 27 September 2010 Available online 4 November 2010 Keywords: Fluoroquinolone Recombinant antibody Broad-specificity immunoassay Phage display abstract Recombinant sarafloxacin-recognizing antibody was engineered with the use of novel fluoroquinolone (FQ) derivatives. A monoclonal FQ antibody, 6H7, was targeted to random mutagenesis to broaden the specificity of the antibody in development of a generic assay for FQ antibiotics. Engineering involved the synthesis of different small-sized FQ molecules to immobilize and detect the mutant antibodies. Selections with labeled FQs resulted in several mutant antibodies with increased affinity or wider spec- ificity toward different FQs. The best characterized mutant antibody was capable of recognizing seven of eight targeted FQs below maximum residue limits set by the European Union. The results are promising in regard to the development of a multiresidue immunoassay for FQs based on a single antibody. Ó 2010 Elsevier Inc. All rights reserved. Fluoroquinolones (FQs) 1 are a synthetic group of antibiotics de- rived from nalidixic acid. A number of different FQ derivatives have been synthesized for therapeutic use both in humans and in animals. Although the structure varies, the therapeutic mechanism is similar in that all FQs inhibit the activity of DNA replication in bacteria through the inactivation of DNA gyrase or topoisomerase IV enzyme [1]. In veterinary use, FQs, among other antibiotic substances, have also been adapted as antibiotic growth promoters. It is illegal to use FQs in the European Union (EU) because antibiotic residues in food can cause allergic reactions in humans and emergence of resis- tant bacteria strains in animal-borne foodstuffs [2]. Maximum residue limits (MRLs) set by the European Agency for the Evaluation of Medicinal Products (EMEA) have been estab- lished in EU region to control the use of FQs and other antibiotics in livestock and aquaculture. Screening of FQ antibiotics from food samples includes the conventional immunological [3–9], microbio- logical [10,11], and chromatographic [12–15] methods, and mass spectrometry (MS)-based assays have also been described [16– 18]. The varying structure between different FQs proposes an issue in the development of diagnostic techniques. An assay based on a single antibody that is capable of screening all different FQ derivatives would be optimal. Because all FQs have a similar core structure (Fig. 1), it should be possible to develop a generic immu- noassay for simultaneous recognition of multiple FQs. Such immunoassays have been developed with both monoclo- nal and polyclonal antibodies that can detect several FQs in a single assay. Polyclonal FQ assays capable of detecting 7–15 FQs simulta- neously in various matrices below MRLs have been described re- cently by Huet and coworkers [6] and Li and coworkers [9]. Monoclonal FQ assays capable of detecting 12 of the structurally similar FQs have been developed, as described by Wang and coworkers [7] and Kato and coworkers [8]. Ideally, the assay would contain only one type of antibody; however, a monoclonal anti- body with a wide enough specificity has yet to be described. Devel- opment of such a generic antibody using immunization involves conjugation of the FQ structure to carrier protein, which proposes a problem in that monoclonal antibodies tend to recognize all substructures of the immunogen, thereby leading to a narrow specificity of the antibody. Small molecule hapten antibodies also have the tendency to bind regions surrounding the conjugated antigen, mainly the carrier protein and the linker, because the binding surface of an antibody is usually significantly larger than the hapten [19]. Antibody engineering has been used previously for enhancing affinity and/or stability of the antibodies. Most of the studies in the protein engineering field are related in the mod- ification of antibody affinity or stability. Studies concentrated 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.09.041 ⇑ Corresponding author. Fax: +358 2 333 8050. E-mail address: jpleiv@utu.fi (J. Leivo). 1 Abbreviations used: FQ, fluoroquinolone; EU, European Union; MRL, maximum residue limit; EMEA, European Agency for the Evaluation of Medicinal Products; MS, mass spectrometry; DELFIA, dissociation-enhanced lanthanide fluoroimmunoassay; RAM, rabbit anti-mouse; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; MALDI–TOF, matrix-assisted laser desorption/ionization time-of-flight; BITC, biotin isothiocyanate; SAR, sarafloxacin; cHSA, cationized human serum albumin; DMF, dimethylformamide; RT, room temperature; DMSO, dimethyl sulfoxide; NOR, norfloxacin; CIP, ciprofloxacin; ENR, enrofloxacin; MAR, marboflox- acin; FLU, flumequine; IPTG, isopropyl b-d-thiogalactopyranoside; EP–PCR, error-prone polymerase chain reaction; BSA, bovine serum albumin; EDTA, ethyl- enediaminetetraacetic acid; Eu–cFQ, europium-labeled core FQ; scFv, single-chain variable fragment; IgG, immunoglobulin G; CDR, complementarity-determining region. Analytical Biochemistry 409 (2011) 14–21 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio