Production and Directional Evolution of Antisarafloxacin ScFv

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Production and directional evolution of anti-sarafloxacin ScFv antibody for immunoassay of fluoroquinolones in milk Jian ping Wang, Jun Dong, Chang Fei Duan, Hui Cai Zhang, Xin He, Geng Nan Wang, Guo Xian Zhao, and Jing Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03356 • Publication Date (Web): 10 Oct 2016 Downloaded from http://pubs.acs.org on October 10, 2016

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Journal of Agricultural and Food Chemistry

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Production and directional evolution of anti-sarafloxacin ScFv antibody for

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immunoassay of fluoroquinolones in milk

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Jian Ping Wang1, Jun Dong1, Chang Fei Duan1, Hui Cai Zhang2, Xin He1, Geng Nan Wang1,

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Guo Xian Zhao2, Jing Liu1 ∗ 1

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071000

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College of Veterinary Medicine, Agricultural University of Hebei, Baoding Hebei, China.

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College of Animal Science and Technology, Agricultural University of Hebei, Baoding Hebei, China. 071000

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∗

Address correspondence to Jing Liu, College of Veterinary Medicine, Agricultural University of Hebei, Baoding Hebei, China; Tel: +86 312 7528477, Fax: +86 312 7528369; E-mail: [email protected].

ACS Paragon Plus Environment

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ABSTRACT A recombinant anti-sarafloxacin ScFv antibody was produced by direct transformation of its

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gene into Rosetta-gami(DE3) for expression, and then its recognition mechanisms for 12

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fluoroquinolones were studied by using molecular docking method. Based on the results of

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virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino

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acid residue Tyr99 to His. The ScFv mutant showed highly increased affinity for the 12 drugs

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with up to 7-folds improved sensitivity. Finally, the mutant was used to develop an indirect

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competitive enzyme linked immunosorbent assay for determination of the 12 drugs in milk.

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The limits of detection were in the range of 0.3-8.0 ng/mL, the crossreactivities were in the

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range of 5%-106%, and the recoveries from the standards fortified blank milk were in the

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range of 62.0%-89.3%. This is the first study reporting the evolution of a ScFv antibody by

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using directional mutagenesis strategy based on virtual mutation.

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Keywords: sarafloxacin; ScFv; fluoroquinolones; recognition mechanism; directional

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mutagenesis; milk

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INTRODUCTION

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Fluoroquinolone drugs (FQs) are the third generation quinolone drugs that are widely used

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to treat bacterial infections in human, animal, and aquaculture. Twelve representative FQs are

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shown in Figure 1. The broad use of FQs in foods producing animals may produce their

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residues in animal derived foods that are dangerous to the consumers. Therefore, China, the

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European Union and the United States have established different maximum residue levels

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(MRLs) for various FQs in different animal derived foods, e.g. 30 ng/g of sarafloxacin in fish

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and 100 ng/g of ciprofloxacin in meat and milk. By now, there have been many instrumental

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methods reported to determine the residues of FQs in foods of animal origin. However, these

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methods require well-equipped laboratories, trained personals, and time-consuming sample

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preparation steps. In comparison, enzyme linked immunosorbent assay (ELISA) is a simple,

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rapid and sensitive screening method. By now, many ELISA methods have been reported to

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detect FQs residues.1-11 The core reagent of an ELISA method is the used antibody. In the

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previous methods, the conventional polyclonal and monoclonal antibodies were usually used as

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the detection reagents. However, the intrinsic properties of the two antibodies are fixed once

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they are generated (e.g. affinity and specificity).

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As the development of gene engineering technique, it is possible to produce various gene

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recombinant antibodies, and single chain variable fragment (ScFv) is a remarkable

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recombinant antibody that is produced by connecting variable heavy chain (VH) and variable

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light chain (VL). ScFv antibody can be produced in bacteria and yeast,12, 13 and its intrinsic

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property can be improved by various mutagenesis technologies. 14, 15 In the past decade, the

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ScFv antibodies for sulfonamides, 16 clenbuterol 17 and penicillins 18 have been produced. By

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now, there has been only one article reporting the production of ScFv antibody for FQs. 19 In


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the report, an anti-norfloxacin ScFv antibody was produced by using phage display technology,

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and its binding properties were consistent with its parental monoclonal antibody: capable of

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recognizing 17 FQs below MRLs, except for other 3 FQs.

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As mentioned above, the recognition property of a ScFv antibody can be evolved in vitro.

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For evolution of a ScFv antibody, the first thing is to study its recognition mechanism, i.e.

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finding the binding site, the contact amino acids and the intermolecular forces. In recent years,

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molecular docking has been usually used to study the intermolecular interactions of different

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ScFv antibodies with the respective competitors, and then the ScFv mutants are obtained by

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using random mutagenesis, site directed mutagenesis or DNA shuffling technique. 14-16, 19-23

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For example, the recognition mechanisms of the anti-norfloxacin ScFv antibody for FQs were

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studied by molecular docking, and then it was evolved by screening a mutated phage display

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library. 19 The obtained ScFv mutant showed constant sensitivity to the 17 FQs while showed

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up to 10-folds improved sensitivity to other 3 analogs, and then the ScFv mutant was used as

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detection reagent for determination of FQs residues in animal derived foods. 24 Besides the

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evolution of ScFv antibodies, a research team engineered two recombinant antibodies for FQs

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from the randomly mutated phage display libraries, and the binding properties of the two

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mutants were improved. 25, 26

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As discussed above, all the previously reported recombinant antibodies were generated by

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using ribosome display technology and phage display technology. 14-17, 19-26 The two display

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technologies can be used to obtain many clones, but the selection of target clone among large

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number of clones is tedious and inconvenient. Besides the two strategies, the cloning VH and

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VL from a hybridoma cell strain and the direct transformation of ScFv gene into a host for

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expression can also generate a ScFv antibody, so this method is simpler than the two display


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technologies. By now, there have been several articles reporting the production of ScFv

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antibodies for some macromolecules by using the third method. 27-29 In our previous study, the

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anti-amoxicillin ScFv antibody was generated by using this method, and the ScFv antibody

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showed similar recognition ability to its parental monoclonal antibody. 18 In the previous

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reports about antibody evolution, a mutated display library was required that was screened and

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enriched for several cycles to obtain the mutants, 16, 19-23, 25,26 which were time consuming and

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lack of directive property.

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To the best knowledge of the authors, there has been no article reporting the evolution of a

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ScFv antibody by directional mutagenesis of the contact amino acid residue. In our previous

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report, a monoclonal antibody against sarafloxacin was produced that showed high

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crossreactivities and high sensitivities to 9 FQs, but showed low crossreactivities and low

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sensitivities to other 3 FQs (Table 1). 11 In order to study its recognition mechanism and verify

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if its recognition property (crossreactivity and sensitivity) can be improved by directional

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mutagenesis method, the relative ScFv antibody was produced and evolved in the present study.

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Then, the obtained ScFv mutant was used to develop an ELISA method for determination of

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FQs residues in milk.

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MATERIALS AND METHODS

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Reagents and Chemicals. Sarafloxacin (SAR), difloxacin (DIF), ciprofloxacin (CIP),

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enrofloxacin (ENR), norfloxacin (NOR) and pefloxacin (PEF) were obtained from the China

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Institute of Veterinary Drug Control (Beijing, China). Lomefloxacin (LOM), enofloxacin

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(ENO), amifloxacin (AMI), danofloxacin (DAN), ofloxacin (OFL) and marbofloxacin (MAR)

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were purchased from Sigma (St. Louis, MO, USA). All the chemical reagents used in this

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study were of analytical grade or better. The standard stock solutions of these FQs were


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prepared with methanol (10 µg/mL), and their working solutions with series concentrations

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(0.1-200 ng/mL) were diluted from the stock solutions with PBS. All the standard solutions

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were stored at 4 oC to be stable for 8 weeks. PBS (pH 7.2) was prepared by dissolving 0.2 g

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KH2PO4, 0.2 g KCl, 1.15 g Na2HPO4, and 8.0 g NaCl in 1000 mL deionized water. Washing

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buffer (PBST) was PBS buffer containing 0.05% Tween. Coating buffer was carbonate buffer

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(0.1 M, pH 9.6). Substrate buffer was 0.1 M citrate (pH 5.5). The substrate system was

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prepared by adding 200 µL 1% (w/v) TMB in DMSO and 64 µL 0.75% (w/v) H2O2 into 20 mL

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substrate buffer.

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All the restriction enzymes and DNA modification enzymes were molecular biology grade.

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RNase prep pure Cell /Bacteria Kit was from Tiangen Biotech Co. Ltd (Beijing, China). Prime

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script RT-PCR Kit, IPTG (isopropyi-β-D-thirgalactopyranoside), X-Gal, PMDTM 19-T Vector

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Cloning kit, horseradish peroxidase-labeled goat anti-His-tag antibody, restriction enzymes

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(Ecor I, Hind Ⅲ) and T4 DNA Ligase were from Takara Company (Dalian, China). EasyPure

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Quick Gel Extraction Kit, EasyPure Plasmid MiniPrep Kit, pEASY-E2 Expression Kit, express

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vector pMALTM-C5X, competent cell Rosetta-gami(DE3), Fast MultiSite Mutagenesis System

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and Luria-Bertani culture medium (liquid and solid) were from TransGen Biotech (Beijing,

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China). DNA Purification Kit and SDS-PAGE gel preparation kit were from Beijing ComWin

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Biotech Co. Ltd (Beijing, China). The synthesis of primers and the analysis of gene sequence

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were performed at Beijing Liuhe Huada Gene Technology Company (Beijing, China).

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Construction of ScFv gene. The hybridoma cell strain R8M8 excreting anti-SAR

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monoclonal antibody was prepared in our lab. 11 The VH and VL gene fragments were extracted

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and the ScFv gene was constructed according to our previous report. 18 Briefly, the total RNA

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was obtained by extraction of 1×107 hybridoma cells with RNase Prep Pure Cell/Bacteria Kit,


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and the cDNA was generated by using PrimeScript RT-PCR Kit. The VH and VL gene

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fragments were obtained by PCR amplification. After the PCR products were analyzed via

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agarose gel electrophoresis, the bands corresponding to 330 bp were recovered by using

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EasyPure Quick Gel Extraction Kit. The obtained VH and VL fragments were linked with T-

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vector (pEASY-E2) respectively for gene sequence analysis. Then the two gene fragments were

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assembled to construct the ScFv gene by splicing overlap extension PCR. After analysis and

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purification via agarose gel electrophoresis, the ScFv gene was linked with T-vector (pEASY-

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E2) for gene sequence analysis. Finally, the ScFv gene and the express vector pMALTM-C5X

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were digested respectively (EcoR I, Hind III), and the two digested products were linked to

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construct the recombinant express vector ScFv-pMALTM-C5X. All the primers used in this

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study are shown in Table 2.

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Expression of ScFv antibody. The express vector was added into a tube containing 50 µL

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of E. coli Rosetta-gami(DE3) solution. The mixture was heat shocked for 90 seconds at 42 oC,

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kept in ice water for 3 min, and cultured on Luria-Bertani (LB) solid culture media at 37 oC

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overnight. The single bacterial colony was transferred into a tube containing 2.5 mL LB liquid

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culture media for vibration culturation overnight (37 oC, 220 rpm). Then, a volume of bacteria

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solution was diluted for 100 folds and cultured in 2.5 mL LB liquid culture media for 3 h.

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When the OD600 value reached 0.6, 0.5 mM IPTG was added to induce ScFv expression (20 oC

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14 h or 37 oC 4 h). The obtained bacteria solution was centrifuged at 4,000 rpm for 10 min.

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The supernatant was discarded, and the left sediment was resuspended in 500 µL breakage

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buffer (50 mM Tris, 300 mM NaCl, 0.2%Triton X-114, 0.5 mM EDTA, 2 mM DTT, pH 8.0).

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After the bacteria solution was sonicated for 6 min and centrifuged for 5 min (4 oC, 12,000


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rpm), the supernatant was purified with nickel-doped agarose gel FF column to obtain the ScFv

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antibody.

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Characterization of ScFv antibody. Western blot. A volume of ScFv solution was added

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onto a nitrocellulose membrane that was immersed in the blocking buffer (4 % BSA in PBS

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(w/v)) for 1 h. Then, a volume of horseradish peroxidase labeled anti-His-tag antibody (1:2000)

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was added onto the block point, and the membrane was incubated for 2 h at room temperature.

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Finally, a volume of substrate solution (4-chloro-1-naphthol) was added to visualize the result.

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Indirect competitive ELISA. The procedures of indirect competitive ELISA were according

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to our previous reports. 11, 18 The optimal concentrations of coating antigen and antibody were

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determined by the checkerboard procedure. A microtiter plate was coated with SAR-OA

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overnight at 4 oC (100 µL/well), and then blocked with 1% fetal calf serum. After the plate was

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washed with PBST for three times, 50 µL of ScFv dilution and 50 µL of FQs standard were

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added into the wells for incubation (37 oC, 1 h). After washes, horseradish peroxidase-labeled

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goat anti-His-tag antibody was added for incubation (100 µL/well, 37 oC, 1 h). After washes,

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the TMB solution was added to visualize the result (100 µL/well, 37 oC, 15 min). Finally, 2 M

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H2SO4 was added to stop the reaction (50 µL/well), and the OD value of each well was read on

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an ELISA reader at 450 nm. The analyte concentrations (Log C) and the B/B0 values (OD of

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the standard divided by that of the zero standard) were used to develop the competitive

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inhibitory curve. The concentrations showing 50% and 10% of inhibition were defined as the

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half of inhibition concentrations (IC50) and the limits of detection (LOD) respectively. The

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crossreactivity (CR) was calculated as: CR (%) = 100 × IC50 SAR / IC50 FQs.

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Homology modeling and molecular docking. Homology modeling and molecular docking were all carried out according to our recent study. 18 The amino acid sequence was translated


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from the ScFv gene sequence with DNAman software (Lynnon Biosoft, USA), and was

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analyzed with the online ExPASy software. The complementary determining regions (CDRs)

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were analyzed with the online tool on http://www.vbase2.org and the IGBLAST tool on NCBI.

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The homology modeling was performed by using YASARA 15.3.8 (YASARA Biosciences

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GmbH, Austria), and the parameters were: searching UniRef90 database; retrieving the

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templates from Protein Data Bank; number of PSI-BLAST iterations, 3; maximum allowed

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(PSI-) BLAST E-value, 0.5; maximum templates, 5; maximum oligomerization, 4; maximum

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alignment variations per template, 5; maximum number of conformations per loop, 50. Then

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the 3D models of the ScFv antibody and the 12 FQs was constructed and optimized with

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YASARA. Finally, the 3D model of ScFv antibody was docked with the 12 FQs respectively

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to study their intermolecular interactions by using Auto Dock 4.2.6 (Scripps Research Institute,

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USA). The parameters for molecular docking were set as follows: gird computing coordinate,

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(125.128, 10.263, -4.898); number of points, 60; dock GA Runs, 100; other parameters, default.

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Directional mutagenesis of ScFv antibody. The ScFv gene was mutated based on the

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molecular docking results of ScFv-SAR. The contact amino acid residue interfering with ScFv-

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SAR bind was substituted with other amino acid directly by using the virtual mutation function

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of YASARA software. During virtual mutation, if an amino acid was substituted with a

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specific amino acid and the ScFv-SAR binding energy largely decreased, then this amino acid

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residue was selected as the mutagenesis site. The virtual mutated ScFv model was optimized

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and docked with SAR again. In this study, the VH CDR3 Tyr99 was substituted with His to

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produce the ScFv mutant. During the experiments, the ScFv gene in express vector ScFv-

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pMALTM-C5X was mutated directly to obtain the mutated express vector by using Fast

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MultiSite Mutagenesis System following the producer recommended protocol. Then, the


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mutated express vector was expressed to obtain the ScFv mutant as the procedures described

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above. The amino acid sequence of ScFv mutant was used for homology modeling and

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molecular docking with FQs as described above.

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Sample Preparation and ELISA analysis. The extraction of FQs from milk sample was as

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follows. Briefly, 2 mL milk and 10 mL 0.1 mol/L EDTA-Mcllvaine buffer were added into a

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centrifuge tube. The mixture was stirred vigorously on a vortex mixer for 3 min, and

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centrifuged at 10000 rpm for 5 min. A volume of supernatant was filtered with a 0.22 µm

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Millipore filter for ELISA analysis. Some raw milk samples obtained from several controlled

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farms were used as blank samples to assess the method. The 12 FQs were fortified into the

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blank milk sample respectively at levels of 10-100 ng/mL for analysis. Furthermore, 35 real

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milk samples (15 raw milk samples and 20 packaged milk samples) from several farms and

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supermarkets in China were analyzed by the method. For confirmation of the ELISA method,

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the 35 real samples were determined with our recently reported high performance liquid

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chromatography method (HPLC), 30 and one HPLC-positive milk sample was also analyzed by

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the ELISA method.

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RESULTS AND DISCUSSIONS

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ScFv antibody. In our previous report, the anti-SAR monoclonal antibody showed different

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IC50 and CRs to 12 FQs (Table 1). 18 For studying its recognition mechanism, the ScFv

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antibody was produced in this study. During the experiments, some universal methods were

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used to extract the total RNA, reverse transcribe the cDNA and clone the VH and VL genes

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according to the previous reports. 14-19 In this study, a group of degenerate primers was used to

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amplify VL and VH genes as many as possible (Table 2). The VL and VH genes were expected

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to about 330 bp and 350 bp respectively, and the ScFv gene (VH-(Gly4Ser)3-VL) was expected


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to about 720 bp. The final sequence analyses showed that the VL gene was 321 bp (encoding

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107 amino acids), the VH gene was 348 bp (encoding 116 amino acids), and the ScFv gene was

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714 bp (encoding 238 amino acids),.

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In many previous reports, the ScFv genes were transformed into different display vectors to

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construct the display libraries, and the target ScFv antibodies were screened from the libraries.

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14-17, 19-26

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transformed into E. coli Rosetta-gami(DE3) to express the ScFv antibody according to the

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previous reports. 18,27-29 As shown in Figure 2, the SDS-PAGE electrophorogram of whole-cell

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protein indicated that the ScFv antibody was expressed as fusion protein (77.2 KD), and its

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molecular weight was 25.67 kD. Furthermore, the ScFv expression amounts in supernatant

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were much more than that in precipitating protein under both induction conditions (16 oC 14 h

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and 37 oC 4 h), so the ScFv antibody was soluble formation (Figure 2).

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In the present study, the recombinant express vector ScFv-pMALTM-C5X was directly

Then, the IC50 and CRs of ScFv antibody for the 12 FQs were determined. As shown in

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Table 1, the ScFv showed high affinity to SAR and DIF (IC50 of 8.7-9.2 ng/mL and CRs of

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95%-100%), showed low affinity to DAN, OFL and MAR (IC50 of 86-143 ng/mL and CRs of

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6%-11%), and showed medium affinity to other 7 FQs (IC50 of 12.9-36 ng/mL and CRs of

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24%-67%). These results were consistent with that of the monoclonal antibody. This indicated

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that the ScFv antibody fully inherited the recognition property of the monoclonal antibody, so

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the ScFv antibody was used to study the recognition mechanisms for FQs.

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Intermolecular interactions of ScFv-FQs. Homology modeling and molecular docking are

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two commonly used methods to simulate the protein-ligand interaction. In the previous reports,

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the optimal homology templates of ScFv antibodies were all retrieved manually from NCBI,

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and their 3D models were optimized with different software. 14-16, 19-23 In the present study,


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YASARA software was used to search the protein databases automatically, retrieve top five

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templates with the highest homology to the ScFv antibody, and optimize the 3D models of

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ScFv and FQs according to our recent study. 18 The crystal structures of five templates were

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combined to obtain an optimal hybrid 3D ScFv model for molecular docking.

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It is well known that the binding pocket of an antibody is bulkier than the hapten molecule.

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Therefore, SAR, as the immunogen hapten, was docked into the ScFv model to find the

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specific binding site. As shown in Figure 3A, the binding site was a small region surrounded

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by VL CDR1 (Tyr162), VL CDR3 (Trp221, Ser222, Tyr226), VH CDR1 (Tyr33), VH CDR2

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(Trp50), and VH CDR3 (Tyr99, Tyr102). Among the eight contact amino acids, VH Tyr33, VH

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Trp50, VH Tyr99, VH Tyr102, VL Tyr162 and VL Ser222 constructed a hydrophobic region that

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maintained the conformation of binding site. This meant that hydrophobic interaction was the

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main intermolecular force of ScFv-SAR complex. Furthermore, two hydrogen bonds were

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formed between the C-3 carboxyl group in SAR and Trp221 and Tyr226 (Figure 3A),

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indicating hydrogen bond was the secondary intermolecular force. Besides, Cation-Pi and Pi-Pi

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bond also involved in ScFv-SAR binding, but their actions were minor.

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Then, the ScFv was docked with other 11 FQs respectively. The main results are shown in

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Table 3. DIF was docked into the binding site exactly because the contact amino acids in ScFv-

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DIF complex were same as that in ScFv-SAR (Table 3), indicating the intermolecular forces of

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the two docking complexes were equal, so the ScFv antibody showed the highest affinity to it.

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As shown in Table 3, the docking results with other 7 FQs (CIP, ENR, NOR, PEF, AMI, LOM

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and ENO) showed that only three or four amino acids constructing the hydrophobic region

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(Tyr99 and Tyr102 plus Tyr33 and/or Tyr162) involved in the 7 ScFv-FQs complexes, so the

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ScFv antibody showed medium affinity to them. The docking results with DAN, OFL and


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MAR showed that poor hydrophobic actions were formed in the 3 ScFv-FQs complexes (Table

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3), indicating the 3 drugs were not docked into the binding site properly, so the ScFv antibody

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showed low affinity to them. In the 12 ScFv-FQs complexes, the contact amino acids forming

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hydrogen bonds were different (Table 3), but they all formed hydrogen bond(s) with the A ring

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and B ring in FQs molecules, so the A ring and B ring in FQs molecules were the main ScFv

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binding positions.

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According to the theory of molecular docking, the low binding energy indicates the stable

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antibody-hapten interaction, i.e. high affinity. In the 12 ScFv-FQs complexes, the combined

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actions of intermolecular forces, contact amino acids and specific antibody binding positions

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led to different total binding energies. As shown in Table 1 and Table 3, the trend of ScFv-FQs

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binding energies was generally consistent with the CRs trend. For example, the binding

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energies of ScFv for DAN, OFL and MAR were the highest (-4.37, -4.14, -4.24 kcal/mol), so

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the affinity to them were the lowest, whereas the binding energy of ScFv-SAR was the lowest

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(-5.16 kcal/mol), so the affinity to SAR was the highest. Based on these results, it could be

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concluded that the total ScFv-FQs binding energies were responsible for the differences of IC50

269

and CRs.

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Directional mutagenesis. In the previous reports, the genes of ScFv mutants were usually

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obtained by screening the mutated display libraries. 14-16, 19-23 Under this circumstance, it was

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not clear that which amino acid was mutated and which the amino acid was mutated to until the

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full gene sequences of ScFv mutants were determined. Therefore, this strategy was somewhat

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blind and low directive. In the present study, the specific amino acid in ScFv binding site was

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mutated to certain amino acid directly, so this strategy was simpler and more directive than the

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previous methods. During the docking of ScFv-SAR, it was found that the negative charge on


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A ring and B ring repelled with the negatively charged region of VH CDR1 and CDR3 (Figure

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3B), which interfered with ScFv-SAR binding. Furthermore, the hydrophobic piperazinyl ring

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in SAR repelled with the corresponding hydrophilic region of VH CDR3 (Figure 3C), which

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was also an interference. Based on these results, the contact amino acids in VH CDR3 were

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substituted with other amino acids respectively for virtual mutation.

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There were nine amino acids in VH CDR3 region, but only two of them (Tyr99 and Tyr102)

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evolved in the ScFv binding, so the two amino acids was selected as virtual mutation sites and

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the mutation purpose was to increase the positive charge of VH CDR3 and/or improve its

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hydrophobicity. For the purpose, one method was to substitute the two contact amino acids

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with alkaline amino acid. For Tyr102, its next amino acid was an alkaline amino acid with

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positive charge (Arg101), and the isoelectric point of Arg101 was higher than that of other

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alkaline amino acids (Lys and His), so it was unnecessary to substitute Tyr102 with an alkaline

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amino acid here. During the experiments, Tyr99 was substituted with several other amino acids

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(Ser, Gln, Thr, Arg, Lys, Glu and Asp). Results showed that the binding energies for SAR after

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virtual mutation ranged from -4.37 to -6.63 kcal/mol, and these virtual mutations showed

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minor influences on the hydrophobic actions, so these mutations were omitted.

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When Tyr99 was substituted with His, the total binding energy decreased from -5.16 to -

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7.03 kcal/mol (Table 1), and the number of hydrogen bonds and the amino acids forming

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hydrophobic interaction all increased in the binding site (Figure 3A and 3D), indicating the

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intermolecular forces of ScFv-SAR increased. At the same time, the interference actions

297

mentioned above weakened. As shown in Figure 3E, the positively charged region in the

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binding site broadened, which well matched with the negatively charged A ring and B ring. As

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shown in Figure 3F, the hydrophilicity of binding site decreased, which improved binding with


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the hydrophobic piperazinyl ring. As described above, the substitution of Tyr99 with His

301

increased the affinity for A ring and B ring due to the improved electrostatic interaction (Figure

302

3E), and largely improved the affinity for piperazinyl ring due to the new hydrogen bond and

303

the increased hydrophobic action (Figure 3D, 3F). Therefore, VH CDR3 Tyr99 was substituted

304

with His for directional mutagenesis of the ScFv antibody in the present study.

305

Characterization of ScFv mutants. In the previous reports, the ScFv mutants showed

306

higher sensitivity or broader recognition spectrum than the parental ScFv antibodies. 14-16, 19-23

307

In the present study, the IC50 and CRs of ScFv mutant for the 12 FQs were also determined. As

308

shown in Table 1, the ScFv mutant recognized the 12 FQs simultaneously with IC50 of 3.2-63

309

ng/mL and CRs of 5%-106%. Comparison with the parental ScFv, the CRs changes were

310

minor (3%-11%), but the sensitivity for each analyte was largely improved. For example, the

311

IC50 for MAR and OFL decreased from 132 and 143 ng/mL to 22.1 and 20.0 ng/mL (improved

312

about 6 and 7 folds respectively), and the IC50 for DAN decreased from 86 ng/mL to 63 ng/mL

313

(improved about 1.4 folds). The representative standard competitive curves for SAR and OFL

314

are shown in Figure 4 with concentrations of 0.1-200 ng/mL. This meant that the parental ScFv

315

antibody was evolved successfully.

316

Then, the mutant was docked with the 12 FQs respectively to study their intermolecular

317

interactions again. The main results are shown in Table 3. The docking results of mutant-SAR

318

were same as that of virtual mutation. As shown in Table 3, the mutated His99 involved in all

319

the 12 mutant-FQs complexes, and the hydrogen bonds and hydrophobic interactions generally

320

increased, indicating the intermolecular forces were improved. As a result, the binding energy

321

of each mutant-FQs complex was lower than that of the corresponding ScFv-FQs complex

322

(Table 3), indicating the mutant-FQs bindings were more stable than the ScFv-FQs bindings.


15


Page 16 of 33

323

As discussed above, the parental ScFv interacted with FQs molecules mainly via the A ring

324

and B ring, and the interaction with the piperazinyl ring (except DAN) was weak. In

325

comparison, the ScFv mutant increased the interaction with A ring and B ring, and highly

326

increased the interaction with piperazinyl ring (Figure 3 D-F). Therefore, the IC50 to FQs

327

containing A ring, B ring and piperazinyl ring decreased for 2-7 folds, while the IC50 to DAN

328

decreased only about 1.4 folds (Table 1).

329

In the previous report, the IC50 of anti-NOR ScFv mutant to 17 FQs were constant with that

330

of its parental ScFv, while the IC50 to other 3 FQs were improved for up to 10 folds. 19 This

331

was because the mutant showed highly improved affinities for the piperazinyl rings in the

332

molecules of the 3 FQs, but showed unchanged affinities for the piperazinyl rings in the

333

molecules of other 17 FQs. In the present study, the ScFv mutant showed generally improved

334

sensitivities for the 12 FQs, which was better than the previous report. This study for the first

335

time reported the evolution of a ScFv antibody by using directional mutagenesis strategy,

336

which was simpler and more directive than the previous method. 19 Therefore, virtual mutation

337

could be used as an assistant tool for the directional mutagenesis of gene recombinant antibody.

338

ELISA of FQs in milk. The obtained ScFv mutant was used to develop an ELISA method

339

for determination of the residues of the 12 FQs in milk. During the experiments, the 12 FQs

340

were prepared with the extracts of blank milk to develop the matrix matched competitive

341

curves respectively. As shown in Figure 4, the matrix matched competitive curves of SAR and

342

OFL were similar to that of their standards, revealing the matrix influence was minimal.

343

Therefore, the LODs for the 12 FQs in milk were according to their standards (0.3-8.0 ng/mL,

344

Table 1). Then, the 12 FQs were fortified into blank milk respectively for analysis (10, 50 and

345

100 ng/mL). As shown in Table 4, the intra-assay recoveries ranged from 62.4% to 85.3% with


16

Page 17 of 33


346

CVs lower than 14.4%, and the inter-assay recoveries ranged from 62.0% to 89.3% with CVs

347

lower than 13.6%.

348

Furthermore, the 35 real milk samples were analyzed by the method. Results showed that

349

four raw milk samples (R3, R8, R11, R12) and one packaged milk sample (P17) were

350

determined as positive samples, and other milk samples were determined as negative. Due to

351

the antibody’s high CRs, the detection results could only be expressed as SAR equivalent. The

352

residue levels in the five samples (R3, R8, R11, R12 and P17) were 5, 106, 25, 23, and 41

353

ng/mL respectively. This meant that one milk sample containing any of the 12 FQs could be

354

determined as positive, but the specific analyte could not be identified.

355

In our recent report, a molecular imprinted polymer-HPLC method was developed to

356

determine four FQs in milk (NOR, CIP, LOM, ENR), and one positive sample was obtained

357

(ENR, 136 ng/mL). 30 In the present study, the HPLC-positive milk sample was analyzed by

358

the ELISA method, and the result calculated as SAR was 108 ng/mL. Furthermore, the 35 real

359

milk samples were also determined by the HPLC. Results showed that the 30 ELISA-negative

360

milk samples were determined as HPLC-negative samples, and the 4 ELISA-positive samples

361

(R3, R8, R11, R12) contained residues of ENR (8.5 ng/mL), ENR (264 ng/mL), CIP (43

362

ng/mL) and ENR (67 ng/mL) respectively. Taking into account the CRs and recoveries of the

363

ELISA method to ENR and CIP, the ELISA results were comparable to the HPLC results. The

364

ELISA-positive sample P17 could not be verified by the HPLC method, which maybe was

365

because P17 contained other FQs that was out of the HPLC detection range, or it was an

366

ELISA-false-positive result due to the non-specific recognition. Therefore, an instrumental

367

method capable of confirmatory determination of the 12 FQs or all FQs remained to be studied.

368

From analysis of the fortified blank samples, the real samples and the authentic positive sample,


17


369

the ELISA method could be used as a rapid screening tool to detect the trace levels of FQs

370

residues in milk.

371

ACKNOWLEDGEMENTS

372

Page 18 of 33

The authors are grateful for the financial supports from National Natural Science Foundation

373

of China (31271869), Hebei Natural Science Foundation (C2011204021, C2015204049), and

374

Scientific and Technology Project of Hebei Education Department (QN20131087).


18

Page 19 of 33


375

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Wan, Y.P.; Zhang, S.X.; Kai, Z.P.; Yang, X.L.; Shen, J.Z. Development of a monoclonal

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antibody based broad-specificity ELISA for fluoroquinolone antibiotics in foods and

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molecular modeling studies of cross-reactive compounds. Anal. Chem. 2007, 79, 4471-

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4478.

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[2] Adrian, J.; Pinacho, D.G.; Granier, B.; Diserens, J.M.; Sanchez-Baeza, F.; Marco, M.P. A

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multianalyte ELISA for immunochemical screening of sulfonamide, fluoroquinolone and

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beta-lactam antibiotics in milk samples using class-selective bioreceptors. Anal. Bioanal.

384

Chem. 2008, 391, 1703-1712.

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[3] Zhao, C. Preparation of anti-gatifloxacin antibody and development of an indirect

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competitive enzyme-linked immunosorbent assay for the detection of gatifloxacin residue

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in milk. J. Agric. Food. Chem. 2007, 55, 6879-6884.

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[4] Kato, M.; Ihara, Y.; Nakata, E.; Miyazawa, M.; Sasaki, M.; Kodaira, T.; Nakazawa, H.

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Development of enrofloxacin ELISA using a monoclonal antibody tolerating an organic

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solvent with broad cross-reactivity to other new quinolones. Food Agric. Immunol. 2007,

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18, 179-187.

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[5] Li, Y.; Ji, B.; Chen, W.; Liu, L.; Xu, C.; Peng, C.; Wang, L. Production of new class-

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specific polyclonal antibody for determination of fluoroquinolones antibiotics by indirect

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competitive ELISA. Food Agric. Immunol. 2008, 19, 251-264.

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[6] Hu, K.; Huang, X.Y.; Jiang, Y.S.; Fang, W.; Yang, X.L. Monoclonal antibody based

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enzyme-linked immunosorbent assay for the specific detection of ciprofloxacin and

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enrofloxacin residues in fishery products. Aquaculture. 2010, 310, 8-12.


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[7] Brás Gomes, F.B.M.; Riedstra, S.; Ferreira, J.P.M. Development of an immunoassay for

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ciprofloxacin based on phage-displayed antibody fragments. J. Immunol. Method. 2010,

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[8] Lu, S.X.; Zhang, Y.L.; Liu, J.T.; Zhao, C.B.; Liu, W.; Xi, R.M. Preparation of anti-

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pefloxacin antibody and development of an indirect competitive enzyme-linked

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immunosorbent assay for detection of pefloxacin residue in chicken liver. J. Agric. Food

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Chem. 2006, 54, 6995-7000.

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[9] Holtzapple, C. K.; Buckley, S.A.; Stanker, L.H. Production and characterization of

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monoclonal antibody against sarafloxacin and cross-reactivity studies of related

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fluoroquinolones. J. Agric. Food Chem. 1997, 45, 1984-1990.

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Huet, A.C.; Charlier, C.; Tittlemier, S.A.; Singh, G.; Benrejeb, S.; Delahaut, P.

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eggs, and muscle by enzyme-linked immunosorbent assay (ELISA). J. Agric. Food Chem.

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2006, 54, 2822-2827.

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Liu, Y.Z.; Zhao, G.X.; Wang, P.; Liu, J.; Zhang, H.C.; Wang, J.P. Production of the

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broad specific monoclonal antibody against sarafloxacin for rapid immunoscreening of 12

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fluoroquinolones in meat. J. Environ. Sci. Health B. 2013, 48, 139-146

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[12]

Miller, K.D.; Weaver-Feldhaus, J.; Gray, S.A.; Siegel, R.W.; Feldhaus, M.J. Production,

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purification, and characterization of human scFv antibodies expressed in Saccharomyces

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cerevisiae, Pichia pastoris, and Escherichia coli. Protein Expr. Purif. 2005, 42(2), 255-267.

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Cabezas, S.; Rojas, G.; Pavon, A.; Alvarez, M.; Pupo, M.; Guillen, G.; Guzman, M.G.

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Selection of phage-displayed human antibody fragments on Dengue virus particles

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captured by a monoclonal antibody: Application to the four serotypes. J. Virol. Methods.


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2008, 147(2), 235-243. [14]

Gram, H.; Marconi, L.A.; Barbas, C.F.; Collet ,T.A.; Lerner, R.A.; Kang, A.S. In vitro

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selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin

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library. Proc Natl Acad Sci USA. 1992, 89(7), 3576-3580.

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Valjakka, J.; Hemminki, A.; Niemi, S.; Soderlund, H.; Takkinen, K.; Rouvinen, J.

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Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in

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complex with testoster-one. Improved affinity results from small structural changes within

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the variable domains. J. Biol. Chem. 2002, 277(46), 44021-44027.

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Korpimaki, T.; Hagren, V.; Brockmann, E.C.; Tuomola, M. Generic lanthanide

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fluoroimmunoassay for the simultaneous screening of 18 sulfonamides using an engineered

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antibody. Anal. Chem. 2004, 76 (11), 3091-3098.

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Liu, X.; Wang, H; Liang, Y.; Yang, J.; Zhang, H.; Lei, H.; Shen, Y.; Sun, Y.

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Production and characterization of a single-chain Fv antibody–alkaline phosphatase fusion

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protein specific for clenbuterol. Mol. Biotechnol. 2010, 45, 56-64.

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Liu, J.; Zhang, H.C.; Duan, C.F.; Dong, J.; Zhao, G.X.; Wang, J.P.; Li, N.; Liu, J.Z.; Li,

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Y.W. Production of anti-amoxicillin ScFv antibody and simulation studying its molecular

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recognition mechanism for penicillins. J. Environ. Sci. Health B. 2016, 51(11), 742-750.

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Wen, K.; Nölke, G.; Schillberg, S.; Wang, Z.; Zhang, S.; Wu, C.; Jiang, H.; Meng, H.;

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Shen, J. Improved fluoroquinolone detection in ELISA through engineering of a broad-

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specific single-chain variable fragment binding simultaneously to 20 fluoroquinolones.

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Anal Bioanal Chem. 2012, 403, 2771-2783.

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Kokayashi, N.; Oyama, H.; Kato, Y.; Goto, J.; Söderlind, E.; Borrekaeck, C.A.K. Two-

Step in Vitro Antibody Affinity Maturation Enables Estradiol-17β Assays with More than


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10-Fold Higher Sensitivity. Anal. Chem. 2010, 82(3), 1027-1038. [21]

Zhang, X.; Zhang, C.; Liu, Y.; Yu, X.; Liu, X. Construction of scFv phage display

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library with hapten-specific repertories and characterization of anti-ivermectin fragment

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isolated from the library. Eur. Food Res. Technol. 2010, 231(3), 423-430

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Nejad, S.M.; Veldhuis, S.L.; Richard, G.; Hall, J.C. Selection of single chain variable

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fragment (scFv) antibodies from a hyperimmunized phage display library for the detection

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of the antibiotic monensin. J. Immunol. Methods. 2010, 360(1-2), 103-118.

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Kokayashi, N.; Kato, Y.; Oyama, H.; Taga, S.; Niwa, T.; Sun, P.; Ohtoyo, M.; Goto, J.

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Anti-estradiol-17β single-chain Fv fragments: Generation, characterization, gene

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randomization, and optimized phage display. Steroids, 2008, 73(14), 1485-1499.

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Tao, X.; Chen, M.; Jiang, H.; Shen, J.; Wang, Z.; Wang, X.; Wu, X.; Wen, K.

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Chemiluminescence competitive indirect enzyme immunoassay for

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20 fluoroquinolone residues in fish and shrimp based on a single-chain variable fragment.

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Anal. Bioanal. Chem. 2013, 405(23), 7477-7484.

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Leivo, J.; Chappuis, C.; Lamminmäki, U.; Lövgren, T.; Vehniäinen, M. Engineering of

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a broad-specificity antibody: detection of eight fluoroquinolone antibiotics simultaneously.

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Anal. Biochem. 2011, 409, 14-21.

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Leivo, J.; Lamminmäki, U.; Lövgren, T.; Vehniäinen, M. Multiresidue detection of

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fluoroquinolones: specificity engineering of a recombinant antibody with oligonucleotide-

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directed mutagenesis. J. Agric. Food Chem. 2013, 61(49), 11981-11985.

464 465 466

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Toleikis, L.; Broders, O.; Dubel, S. Cloning single-chain antibody fragments (scFv)

from hybridoma cells. Methods Mol. Med. 2004, 94, 447-458. [28]

Aebig, J.A.; Albert, H.H.; Zhu, B.L.; Hu, J.S.; Hsu, H.T. Cloning and construction of


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single-chain variable fragments (SCFV) to cucumber mosaic virus and production of

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transgenic plants. Acta Horticulture. 2006, 722, 129-136.

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Sellrie, F.; Schenk, J.A.; Behrsing, O.; Drechsel, O.; Micheel, B. Cloning and

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Characterization of a Single Chain Antibody to Glucose Oxidase from a Murine

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Hybridoma. J. Biochem. Mol. Biol. 2007, 40, 875-880.

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[30]

Wang, G.N.; Yang, K.; Liu, H.Z.; Feng, M.X.; Wang. J.P. Molecularly imprinted

473

polymer-based solid phase extraction combined high performance liquid chromatography

474

for determination of fluoroquinolones in milk. Anal. Methods. 2016, 8, 5511-5518.

475


23


Page 24 of 33

FIGURE CAPTIONS: Figure 1. Chemical structures of twelve fluoroquinolone drugs. Figure 2. SDS-PAGE results of expressed ScFv fusion protein. M, protein marker; lane 1, whole cell protein before induction; lane 2, supernatant at 16oC 14 h; lane 3, precipitating at 16oC 14 h; lane 4, supernatant at 37oC 4 h; lane 5, precipitating at 37oC 4 h. Figure 3. Molecular docking of ScFv-SAR (A, B, C) and mutant-SAR (D, E, F). A and D, Ligplus results; B and E, surface electrostatic potential maps; C and F, hydrophobic interaction maps. Positive and negative charges are shown with blue and red in B and E respectively, and hydrophobic and hydrophiclic region are shown with orange and cyan in C and F respectively. Figure 4. Representative competitive inhibitory curves of SAR and OFL standards, and their matrix matched curves.


24

Page 25 of 33


Table 1. Performances of the monoclonal antibody, ScFv antibody and ScFv mutant for FQs.

Monoclonal antibody Parental ScFv ScFv mutant a

IC50 a CR IC50 CR IC50 CR LOD a

SAR 9.0 100 8.7 100 3.4 100 0.3

DIF 9.3 97 9.2 95 3.2 106 0.3

CIP 13.2 68 12.9 67 4.6 74 0.5

ENR 15.3 59 14.8 58 6.2 55 1.0

NOR 20.9 43 21.2 41 9.6 35 1.5

PEF 25.0 36 23.0 38 8.7 39 1.4

AMI 23.1 39 24.6 35 9.3 37 1.5

ENO 25.7 35 25.2 35 9.2 37 1.5

LOM 33.3 27 36.0 24 11.0 30 2.2

DAN 90 10 86 11 63.0 5 8.0

OFL 150 6 143 6 20.5 17 4.0

MAR 128 7 132 7 22.1 15 4.0

The units of IC50 and LOD are ng/mL. b The CR is expressed as %.


25


Page 26 of 33

Table 2. Primers for production of the ScFv antibody and ScFv mutant. target

parental ScFv

ScFv mutant

primer

sequence

VL forward

CCGGAATTCGACATYGAGCTCACYCAGTCTCCA (EcoR I)

VL reverse

CCCAAGCTTGCGTTTBATYTCCAGYTTGG

VL-Linker reverse

GCCAGAGCCACCTCCGCCTGAACCGCCTCCACCGCGTTTCA TTTCCAGTTTGG a

VH forward

CATGCCATGGAGGTSMARCTGCAGSAGTCWGG

VH reverse

CCCAAGCTTGGAGGAGACTGTGAGAGT (Hind III)

VH-Linker forward

TCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGAGGTGA AACTGCAGGAGTCAGG a

VH forward

TATTTCTGTGCAGCCCACTATAGGTACGTCG

VH reverse

GGGCTGCACAGAAATAGATCGCAGAGTCCTC

a

This primer contains the overlapped linker part. B=C/T, D=A/G/T, H=A/C/T, V=A/C/G, K=G/T, M=A/C, R=A/G, S=C/G, W=A/T, X=C/T.


26

Page 27 of 33


Table 3. Molecular docking results of the parental ScFv and ScFv mutant with FQs. Parental ScFv-FQs Drug Hydrogen bond SAR

Trp221 Tyr226

Tyr33 Trp50 Tyr99 Tyr102 Tyr162 Ser222

DIF

Trp221 Tyr226

Tyr33 Trp50 Tyr99 Tyr102 Tyr162 Ser222

CIP

Trp221

Tyr33 His35 Trp50 Tyr99 Tyr162 Ser222 Tyr226

Ser222 ENR

NOR

Tyr33 His35 Trp50 Tyr99 Tyr102 Ser161 Tyr162 Asn224

Binding energy a

-5.16 -5.12

-4.83

-5.04

Arg31 Tyr162

Tyr33 Tyr99 Arg101 Tyr102 Tyr162 Tyr32

-5.09

Arg31

Tyr33 Tyr99 Tyr102 Tyr226

-4.92

PEF

AMI

Tyr33 Trp221

Trp50 Tyr99 Tyr102 Tyr162 Tyr226

-4.52

ENO

Trp221 Tyr226

Trp50 Tyr99 Tyr102 Tyr162 Ser222 Tyr226

-4.76

Arg31 Tyr162

Tyr33 Tyr99 Tyr100 Arg101 Tyr102 Tyr162

Arg31

LOM

OFL

Tyr33 Trp221

Tyr32 Tyr33 Tyr99 Tyr100 Arg101 Tyr33 Trp50 Tyr99 Tyr226

MAR

Tyr102 Tyr226

Trp50 Tyr99 Tyr102 Trp221

DAN

a

Hydrophobic interaction

ScFv mutant-FQs

-4.83

-4.37 -4.14

-4.24

Hydrogen bond Tyr100 Tyr102 Tyr226 Arg31 Tyr33 Tyr102 Trp221

Trp221

Tyr100 Tyr102 Tyr226 Tyr102 Tyr226 His35 His99 Tyr102 Tyr226 Val103 Trp221 Tyr100 Tyr102 Tyr226 Tyr100 Tyr102 Tyr100 Tyr102 Tyr226 Tyr33 Trp221

Hydrophobic interaction Arg31 Tyr32 Tyr33 His35 Trp50 Tyr52 His99 Arg101 Trp221 His99 Tyr100 Tyr102 Trp221 Tyr226 Tyr33 His35 Trp50 His99 Tyr100 Arg101 Tyr162 Tyr226 His35 His99 Tyr100 Arg101 Tyr102 Val103 Ser222 Ser223 Asn224 Tyr226 Arg31 Tyr32 Tyr33 His35 Trp50 His99 Arg101 Trp221 Arg31 Tyr32 Tyr33 Trp50 His99 Tyr100 Arg101 Trp221 Trp50 Tyr100 Arg101 Trp221 Tyr33 His35 His99 Tyr100 Arg101 Tyr102 Tyr162 Tyr226 Arg31 Tyr32 Tyr33 His35 Trp50 His99 Arg101

Binding energy

-7.03 -6.87

-6.93

-6.71

-6.89

-6.77

-6.68

-6.84

-6.26

Arg31 Tyr32 Tyr33 His35 Trp50 His99

-6.12

Trp50 His99 Arg101 Trp221

-6.51

Tyr32 His35 Trp50 His99 Tyr100 Tyr102 Tyr162 Ser222

-6.72

Unit of binding energy is kcal/mol.


27


Page 28 of 33

Table 4. Recoveries of FQs from standards fortified blank milk samples (n=6). Intra-assay analyte

SAR

DIF

CIP

ENR

NOR

PEF

added (ng/mL)

Inter-assay

Intra-assay

Recovery (%)

CV (%)

Recovery (%)

CV (%)

10

85.3

6.4

82.1

6.7

50

76.3

8.3

77.2

10.5

100

63.4

11.0

72.1

10

76.8

7.6

50

69.8

100

analyte

added (ng/mL)

Inter-assay

Recovery (%)

CV (%)

Recovery (%)

CV (%)

10

68.5

7.6

74.8

8.5

50

70.2

7.5

72.5

8.7

10.4

100

64.8

16.2

80.4

12.0

72.0

9.4

10

76.3

7.4

82.5

8.1

8.2

67.5

7.6

50

81.5

6.7

80.7

10.2

63.4

8.5

69.7

13.2

100

78.3

9.4

80.1

9.0

10

78.9

8.2

71.5

11.4

10

83.2

6.7

77.4

10.5

50

76.9

9.4

84.0

8.9

50

82.4

11.4

76.4

8.7

100

70.3

14.4

74.5

9.4

100

62.4

8.7

68.9

8.9

10

82.6

6.4

89.3

7.3

10

65.9

10.2

71.9

12.0

50

68.0

13.2

66.4

9.4

AMI

ENO

LOM

DAN

50

66.5

7.6

70.3

13.6

100

68.5

8.5

75.4

9.0

100

73.8

7.9

62.0

13.4

10

74.2

9.3

77.5

8.7

10

82.6

9.5

74.6

9.2

50

80.3

6.8

72.1

9.8

50

75.0

9.3

72.9

8.1

100

73.2

8.3

81.1

8.2

100

75.4

8.7

80.2

12.6

10

80.6

7.2

76.8

13.1

10

75.3

8.3

77.1

9.1

50

76.9

8.1

70.4

9.4

50

77.5

7.1

83.1

8.7

100

77.3

12.3

75.2

10.8

100

74.1

9.7

82.5

8.0

OFL

MAR


28

Page 29 of 33


Figure 1 O

O

F

COOH A

F

O

B

F

F

N

N

O

COOH N

N

COOH

COOH N

N NH

N

N

N H3C

N

F

Sarafloxacin

H3CH2C

NH

F

Difloxacin

Ciprofloxacin

Enrofloxacin O O

O

O

F

F

N

N

N

N

Norfloxacin

NH

F

COOH N

Enoxacin

H3C

N

COOH

N

N

CH3

H3C

H3C

Ofloxacin

F

N O

N

O

COOH

N

CH3

N

Lomefloxacin

O F

N O

N H3C

Amifloxacin

COOH

N CH2CH3

CH3

N CH3

O

O

N

CH3

H3C

Pefloxacin

F

F

N

H3C

NH

N

N

N

N

N

COOH

COOH

CH2CH3

CH2CH3

N

F

COOH

COOH

F

Marbofloxacin


N

Danofloxacin

29


Page 30 of 33

Figure 2


30

Page 31 of 33


Figure 3 A

D

B

C

E

F


31


Page 32 of 33

Figure 4

100

B/B0 (%)

80

60

40

SAR standard matrix matched SAR OFL standard matrix matched OFL

20

0 0.1

1

10

100

Log C (ng/mL)


32

Page 33 of 33


Table of contents (TOC)


33

Production and Directional Evolution of Antisarafloxacin ScFv

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