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Xin He, Chang Fei Duan, Yong Hua Qi, Jun Dong, Geng Nan Wang, Guo Xian Zhao,
Jian Ping Wang, Jing Liu
PII: S0003-2697(16)30356-6
DOI: 10.1016/j.ab.2016.10.020
Reference: YABIO 12538
Please cite this article as: X. He, C.F. Duan, Y.H. Qi, J. Dong, G.N. Wang, G.X. Zhao, J.P. Wang, J. Liu,
Virtual mutation and directional evolution of anti-amoxicillin ScFv antibody for immunoassay of penicillins
in milk, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.10.020.
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4 Xin He 1 , Chang Fei Duan 1 , Yong Hua Qi 2, Jun Dong 1, Geng Nan Wang1, Guo Xian Zhao1,
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5 Jian Ping Wang 1, Jing Liu 1
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6 College of Veterinary Medicine, Agricultural University of Hebei, Baoding Hebei,
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7 China. 071000
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8 College of Animal Science, Henan Institute of Science and Technology, Xinxiang Henan,
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China. 453003
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Address correspondence to Jing Liu, College of Veterinary Medicine, Agricultural University of Hebei,
Baoding Hebei, China; E-mail: 444937594@qq.com.
The two authors contributed equally to this study.
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11 Abstract
12 In this study, an anti-amoxicillin single chain variable fragment (ScFv) antibody was
13 evolved by directional mutagenesis of a contact amino acid residue based on the analysis of
14 virtual mutation. Comparison with its parental ScFv, the mutant showed highly improved
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15 affinity for 11 penicillins with up to 6-folds increased sensitivity. Then, its recognition
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16 mechanisms for the 11 drugs were studied by using molecular docking. Results showed that
17 the mutant-penicillins intermolecular forces increased and the total binding energies decreased
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18 dramatically, which were responsible for the improvement of antibody sensitivity. The ScFv
19 mutant was used to develop an indirect competitive enzyme linked immunosorbent assay for
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determination of the 11 drugs in milk. The limits of detection were in the range of 0.2-3.0
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21 ng/mL, the crossreactivities were in the range of 31%-132%, and the recoveries from standards
fortified blank milk were in the range of 65.7%-92.4%. This is the first study reporting the
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23 directional evolution of a ScFv antibody based on virtual mutation and the use of ScFv
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26 Introduction
27 As a class of traditional antibiotics, penicillin drugs (PCs) have been broadly used to treat
28 various bacterial infections in humans and animals for over 70 years. However, the residues of
29 PCs in foods of animal origin are dangerous to the consumers because they can cause allergic
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30 reactions and accelerate the spreading of antimicrobial resistance [1]. Therefore, the European
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31 Union and China have set the maximum residue limits (MRLs) for different PCs in foods of
32 animal origin, e.g. penicillin, ampicillin and amoxicillin, 4 ng/mL in milk; oxacillin, cloxacillin,
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33 and nafcillin, 30 ng/mL in milk.
34 Up to now, there have been many methods reported to detect PCs residues in foods of
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animal origin, such as high performance liquid chromatography [2], liquid chromatography
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36 mass spectrometry [3], bioassay [4] and immunoassay (lateral flow assay [5] and enzyme
linked immunosorbent assay (ELISA) [6-9]). Among these methods, immunoassay is usually
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38 used as the screening tool due to its rapidity, simplicity and sensitivity. For an immunoassay,
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39 the core reagent is the used antibody, and the commonly used detection reagents are the
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40 conventional polyclonal and monoclonal antibodies. Polyclonal antibodies are the easiest and
41 quickest to produce, but they are lack of uniformity. Monoclonal antibodies are uniform and
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42 many clones can be obtained, but the preparation processes are complex and the expensive cell
43 culturing facilities are required. Furthermore, their intrinsic properties (e.g. affinity and
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46 various gene recombinant antibodies that can be used as alternative reagents for the
47 development of ELISA method. One of the remarkable recombinant antibodies is single chain
48 variable fragment (ScFv). In the past few years, the ScFv antibodies for some veterinary drugs
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49 (sulfonamides [10-13], clenbuterol [14, 15], fluoroquinolones [16], and ampicillin [17]) have
50 been produced by using ribosome display technology or phage display technology. Results
51 showed that their recognition abilities were similar to or better than their parental monoclonal
52 antibodies. Recently, the authors of the present study cloned the variable heavy chain (VH) and
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53 variable light chain (VL) from a hybridoma cell strain excreting the monoclonal antibody for
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54 amoxicillin, and transformed the ScFv gene into E. coli directly to express the ScFv antibody
55 [18]. Results showed that the obtained ScFv antibody showed similar recognition ability to the
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56 parental monoclonal antibody.
57 ScFv antibody has several advantages, and the most remarkable merit is that its intrinsic
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property such as affinity and specificity can be improved by gene mutagenesis technology. For
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59 mutagenesis of a ScFv antibody, the first thing is to determine which site(s) in the ScFv entity
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61 intermolecular interactions, e.g. finding the binding site, the key contact amino acids, and the
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62 intermolecular forces. In recent years, homology modeling and molecular docking are usually
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63 used to study the intermolecular interactions of different proteins (ScFv antibody, enzyme, and
64 receptor) with their respective ligands [10-12, 16, 18-24]. When the intermolecular interactions
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65 of ScFv-analyte are obtained, the ScFv antibody can be evolved in vitro by using different gene
66 mutagenesis methods.
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67 For example, a research team produced a ScFv antibody that showed similar recognition
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68 property to its parental monoclonal antibody: recognizing 9 sulfonamides. After its recognition
69 mechanisms were studied by using molecular docking, the ScFv antibody was evolved by
70 using random mutagenesis, site directed mutagenesis and DNA shuffling techniques [10-12].
71 Results showed that the ScFv mutant was able to recognize 18 sulfonamides simultaneously. In
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72 another report, an anti-norfloxacin ScFv antibody was produced and its recognition
73 mechanisms were studied by molecular docking, and then it was evolved by site directed
74 mutagenesis [16]. Results showed that the ScFv mutant showed constant sensitivity to 17
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76 to now, there has been only one paper reporting the evolution of ScFv antibody for PCs [17].
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77 In the report, an anti-ampicillin ScFv antibody was generated that was then evolved by
78 screening a mutated antibody library with several other PCs as the probes. Results showed that
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79 the ScFv mutant was broad specific for PCs (the computational method was not showed in
80 detail). As described above, the previous methods for evolution of ScFv antibodies required
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different mutated display libraries and several cycles of screening/enrichment experiments to
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82 obtain the ScFv mutants [10-12, 16,17, 19, 20, 25-28], so these methods were tedious, time
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84 In our recent study, the recognition mechanisms of an anti-amoxicillin ScFv antibody for 11
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85 PCs were studied by using homology modeling and molecular docking methods [18]. The
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86 ScFv binding site, key contact amino acid residues, main intermolecular forces and total
87 binding energies were obtained (Table 1). For improving its recognition performance, the ScFv
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88 antibody was evolved by directional mutagenesis of the specific contact amino acid residue
89 based on the results of virtual mutation and molecular docking. After characterization of its
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90 recognition ability, the obtained ScFv mutant was used to develop an ELISA method for
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94 The standards of amoxicillin anhydrous (AOC), ampicillin trihydrate sodium salt (APC),
95 oxacillin sodium salt monohydrate (OXC), penicillin G potassium salt (PCG), cloxacillin
96 sodium salt monohydrate (CLC), penicillin V potassium salt (PCV), methicillin (MTC),
97 dicloxacillin sodium salt monohydrate (DCLC), and nafcillin sodium salt monohydrate (NFC)
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98 were purchased from Sigma (St. Louis, Mo, USA). Carbencillin disodium salt (CBC) was
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99 obtained from Sangon Biotech Co., Ltd (Shanghai, China). Sulbenicillin (SBC) was obtained
100 from China Institute of Veterinary Drug Control (Beijing, China). Other chemical reagents
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101 were of analytical grade or better. The PCs standard stock solutions were prepared with
102 methanol respectively (10.0 g/mL), and their working solutions with series concentrations
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(0.1-100 ng/mL) were diluted from the stock solutions with water immediately before used.
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104 All the enzymes were of molecular biology grade. PMDTM 19-T Vector Cloning kit,
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105 restriction enzymes (Ecor I, Hind ), T4 DNA Ligase, X-Gal, IPTG (isopropyi--D-
107 from Takara Company (Dalian, China). EasyPure Quick Gel Extraction Kit, express vector
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108 pET-32a, pEASY-E2 Expression Kit, EasyPure Plasmid MiniPrep Kit, Luria-Bertani culture
109 medium (liquid and solid), Chemically Competent Cell BL21 (DE3), and Fast MultiSite
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110 Mutagenesis System were from TransGen Biotech (Beijing, China). DNA Purification Kit and
111 SDS-PAGE gel preparation kit were from Beijing ComWin Biotech Co. Ltd (Beijing, China).
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112 All the primers were synthesized by Beijing Liuhe Huada Gene Technology Company (Beijing,
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113 China).
115 The mutagenesis sites for the parental ScFv gene were selected according to the molecular
116 docking results obtained in our recent report [18]. Based on the parental ScFv-AOC docking
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117 model, the amino acid residue in the ScFv binding site that interfered with ScFv-AOC binding
118 was substituted with other amino acid directly by using the virtual mutation function of
119 YASARA 15.3.8 (YASARA Biosciences GmbH, Austria). The virtually mutated ScFv genes
120 were used to construct and optimize the 3D ScFv models for molecular docking with AOC
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121 respectively. When the substitution of a specific amino acid residue was beneficial to ScFv-
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122 AOC binding, then this amino acid residue was selected as the mutagenesis site. In this study,
123 VL CDR3 Ser 95 was mutated to Glu to produce ScFv mutant1, and two amino acid residues
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124 were mutated simultaneously to produce ScFv mutant2 (VL CDR3 Ser 95 Glu and VH
125 CDR1 Tyr 160 Arg). During the experiments, the parental ScFv gene in express vector
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ScFv-pET-32a was mutated directly to obtain the two mutated express vectors respectively by
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127 using Fast MultiSite Mutagenesis System following the producer recommended protocol
(TransGen Biotech, Beijing, China). The primers used for mutagenesis of VL CDR3 Ser 95
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133 purified and analyzed via agarose gel electrophoresis, and the gene sequences were determined
136 The procedures for expression of the two ScFv mutants were fully according to our recent
137 report [18]. Briefly, the mutated express vector was transformed into BL21 (DE3) competent
138 cell, and the bacteria solution was cultured in Luria-Bertani (LB) liquid culture media for
139 vibration culturation (200 rpm, 37 oC, 1 h). Then, 200 L of the bacteria solution was cultured
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140 on LB solid culture media at 37 oC overnight. The single bacteria colony was transferred into
141 LB liquid culture media for culturation (37 oC, 250 rpm). When the OD600 reached 0.5, 0.05
142 mM IPTG was added to induce ScFv expression (16 oC, 16 h). The bacteria solution was
143 centrifuged at 12,000 rpm for 5 min, and then the supernatant was discarded and the left
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144 precipitating protein (inclusion body) was resuspended with PBS. The bacteria solution was
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145 subjected to ultrosonication and centrifugation (12,000 rpm, 4 oC, 5 min), and the sediment
146 was treated with denature solution on ice for 1 h (8 mol/L urea, 50 mmol/L Tris-HCl, pH 8.0).
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147 The obtained mixture was purified with nickel-doped agarose gel FF column, and the eluent
148 was put into a dialysis bag for renature with renature solution for 20 h (50 mmol/L Tris-HCl, 2
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mol/L urea, 2 mmol/L reduced glutathione, 1 mmol/L oxidized glutathione). Finally, the
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150 obtained protein was dialyzed in 50 mmol/L Tris-HCl (pH 8.0) at 4 oC for 72 h, and the
dialyzed solution was concentrated with PEG10000 to obtain the soluble ScFv mutant.
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153 The ScFv mutants were characterized by the following three methods. Firstly, a volume of
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154 40 L ScFv mutant and 10 L protein loading buffer were added into a tube for boiling bath
155 for 10 min, and 10 L of the obtained solution was analyzed by SDS-PAGE (10 L, 200 V, 45
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156 min) with coomassie brilliant blue as the coloring agent and acetic acid-ethanol as the
157 decolorant. Secondly, a volume of ScFv mutant was added onto a nitrocellulose membrane that
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158 was immersed in blocking buffer for 1 h (4 % BSA in PBS (w/v), pH 7.4). Then, a volume of
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159 horseradish peroxidase labeled anti-His-tag antibody (1:2000) was added onto the block point,
160 and the membrane was incubated for 2 h at room temperature. Finally, a volume of substrate
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162 Thirdly, the specificity and sensitivity for AOC and other PCs were determined by using the
163 indirect competitive ELISA. The coating antigen amoxicillin-ovalbumin was prepared in our
164 previous report [18]. The ELISA procedures were as follows. Briefly, a microtiter plate was
165 coated with amoxicillin-ovalbumin overnight at 4 oC (100 L/well), and then blocked with 1%
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166 fetal calf serum. After the plate was washed with PBST for three times, 50 L of ScFv dilution
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167 and 50 L of PCs standard were added into the wells for competition (37 oC, 1 h). After
168 washes, horseradish peroxidase-labeled goat anti-His-tag antibody was added for incubation
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169 (100 L/well, 37 oC, 1 h). After washes, the TMB substrate solution was added to visualize the
170 result (100 L/well, 37 oC, 15 min). Finally, the reaction was stopped by adding 50 L of 2 M
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H2SO4, and the plate was read on an ELISA reader at 450 nm to obtain the OD values. The
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172 competitive inhibitory curves were developed by plotting the concentrations (Log C) versus the
B/B0 values (mean OD value of the standard divided by that of the zero standard). The
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174 concentrations showing 50% and 10% of inhibition were defined as the half of inhibition
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175 concentration (IC50) and the limit of detection (LOD) respectively. The crossreactivity (CR)
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176 was calculated as: CR (%) = IC50 AOC/ IC50 competitor 100.
178 The homology modeling and the molecular docking were carried out according to our recent
179 report [18]. The gene sequences of the two mutants were translated to amino acid sequences
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180 with DNAman software (Lynnon Biosoft, USA). The CDRs were analyzed on
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181 http://www.vbase2.org and the IGBLAST tool on NCBI. The homology modeling was
182 performed by using YASARA 15.3.8 (YASARA Biosciences GmbH, Austria), and the
183 parameters were: searching UniRef90 database; retrieving the templates from Protein Data
184 Bank; number of PSI-BLAST iterations, 3; maximum allowed (PSI-) BLAST E-value, 0.5;
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186 template, 5; maximum number of conformations per loop, 50. Then the 3D models of the two
187 ScFv mutants and the 11 PCs were constructed and optimized with YASARA. Finally, the two
188 mutants were docked with the 11 PCs respectively to study the mutants-PCs intermolecular
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189 interactions with Auto Dock 4.2.6 (Scripps Research Institute, USA). The docking parameters
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190 were set as: gird computing coordinate, (-16.354, 27.316, 10.181); number of points, 60; dock
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192 Sample Preparation and ELISA analysis
193 The extraction of PCs from milk sample was as follows. Briefly, 2 mL milk and 5 mL
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acetonitrile were added into a centrifuge tube that was vortexed vigorously for 5 min and
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195 centrifuged at 5000 rpm for 5 min. The supernatant was transferred into a clean tube and
evaporated to dryness. Finally, the dry residue was reconstituted in 2 mL of PBS and filtered
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197 with a 0.22 m Millipore filter for ELISA analysis. Some blank raw milk samples were
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198 obtained from the controlled farms that were used to assess the method. The 11 PCs were
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199 fortified into the blank milk sample respectively at different levels for analysis. Furthermore,
200 thirty real milk samples (15 raw milk samples and 15 packaged milk samples) from several
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201 farms and supermarkets in China were analyzed by the developed method.
204 In our recent study, the intermolecular interactions of parental ScFv-PCs were studied [18].
205 Results showed that the ScFv binding site was a small region surrounded by VL CDR1, VL
206 CDR3 and VH CDR3. The main intermolecular force hydrogen bonds were formed between
207 different contact amino acids and the oxygen(s) on PCs core structure (6-aminopenicilanic acid,
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208 APA) in 9 ScFv-PCs models (except CLC and DCLC). Furthermore, the different total ScFv-
209 PCs binding energies were showed to be responsible for the different IC50 for PCs. The main
210 docking results are shown in Table 1. In the docking model of ScFv-AOC, it was found that
211 the positively charged AOC side chain repelled with the corresponding positively charged VL
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212 CDR1 region, and the hydrophilic C=O bond in APA part repelled with the corresponding
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213 hydrophobic VH CDR1 region, which all interfered with ScFv-AOC binding. For evolution of
214 the ScFv antibody, the immune hapten AOC was used as the original ligand to find the optimal
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215 mutagenesis site. In the present study, several amino acids around the binding site (VL CDR3
216 Ser 95, Lys 96; VH CDR1 Tyr 160) were substituted with other amino acids respectively by
As shown in Figure 3A and Table 1, the binding site was surrounded by VL CDR1, VL CDR3,
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220 VH CDR1, VH CDR2 and VH CDR3, and the number of contact amino acids involving in
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221 mutant1-AOC binding increased, indicating the intermolecular interaction was improved. As
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222 shown in Figure 3B, the positive charges of the binding site increased, which improved the
223 interaction with the negatively charged APA part, but the interaction with AOC side chain was
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224 intact. As shown in Figure 3C, the interaction between the hydrophilic APA part and the
225 corresponding hydrophilic region in the binding site was improved, which was also beneficial
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226 to the binding, but the interaction with AOC side chain weakened. As a result, the binding
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227 energy of mutant1-AOC decreased from -5.56 to -6.15 kcal/mol, indicating a more stable
228 binding.
229 When two amino acids were substituted simultaneously (mutant2, VL CDR3 Ser 95 Glu
230 and VH CDR1 Tyr 160 Arg), the number of contact amino acids involving in mutant2-AOC
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231 binding also increased (Table 1), and the binding site was surrounded by VL CDR3, VH CDR1,
232 VH CDR2 and VH CDR3 (Figure 3D), indicating the intermolecular interaction was also
233 improved. As shown in Figure 3E, the negatively charged region in mutant2 increased, which
234 weakened the interaction with the negatively charged APA part, but largely improved the
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235 interaction with AOC side chain. As shown in Figure 3F, the hydrophilic region in mutant2
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236 broadened, which improved the interaction for both APA part and AOC side chain. As a result,
237 the binding energy decreased from -5.56 to -7.24 kcal/mol, also indicating a more stable
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238 binding.
239 The results described above meant that the more stable ScFv-AOC binding was obtained
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after mutagenesis of VL CDR3 Ser 95 and VL CDR3 Ser 95 + VH CDR1 Tyr 160. In
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241 comparison, the intermolecular interactions showed limit differences before and after virtual
mutation when VL CDR3 Lys 96 or VH CDR1 Tyr 160 were substituted with other amino acids.
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243 In this study, only three specific amino acids were selected for virtual mutation, and the
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244 selected mutagenesis sites maybe were not the really optimal sites. However, the mutagenesis
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245 of the one or two amino acids achieved the expected results, so the parental ScFv gene was
246 mutated twice to generate the two mutated ScFv genes respectively.
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248 In the previous reports for evolution of ScFv antibodies, the genes of ScFv mutants were
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249 usually obtained from the site-directed and randomly mutated display libraries [10-12,
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250 16,17,19, 20, 25-28]. Under these circumstances, it was not clear that which specific amino
251 acid was mutated and which the amino acid was mutated to until the gene sequences of ScFv
252 mutants were determined. In the present study, the specific amino acid was directly substituted
253 with target amino acid, which was simpler and more directional than the previous methods.
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254 The gene sequences of the parent ScFv and the two mutants were same except the one or two
255 codons. As shown in Table 2, the genes of both of the two ScFv mutants were 729 bp, and their
256 amino acid sequences were same as the parental ScFv antibody except the mutated sites (total
257 243 amino acids; VL chains, 112 amino acids; VH chains, 116 amino acids). These results were
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258 consistent with the original anticipations.
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259 In the previous reports, most of the ScFv antibodies were produced by using display
260 technologies [10-16,19, 20, 25-28], and only several ScFv antibodies were produced by direct
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261 transforming the genes into hosts for expression [29-31]. In comparison, the latter method is
262 simpler and more directional than the former methods. In our recent study, the ScFv gene was
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directly transformed into E. coli to express the parental ScFv antibody [18]. In the present
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264 study, the two mutated express vectors were also transformed into BL21 (DE3) directly to
express the two ScFv mutants. Results showed that the two mutants were expressed as the
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266 formation of inclusion body, so the target proteins were subjected to denature and renature to
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267 obtain the soluble mutants. The results of SDS-PAGE analysis showed that their molecular
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268 weights were all 46 kD, same as the parental ScFv antibody. Their expression concentrations
271 In the previous reports, the ScFv mutants showed higher sensitivity, higher affinity, or
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272 broader recognition spectrum than the parental ScFv antibodies [10-12, 16, 17, 19, 20, 25-28].
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273 In our recent study, the parental ScFv antibody simultaneously recognized 11 PCs, and the IC50
274 (8.9-53.3 ng/mL) and CRs (18%-107%) were similar to the parental monoclonal antibody
275 (Table 3). In the present study, the recognition abilities of the two mutants to the 11 PCs were
276 also determined. The IC50 and CRs are shown in Table 3.
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277 As shown in Table 3, the two mutants also recognized the 11 PCs simultaneously with IC50
278 of 3.1-16.3 ng/mL and CRs of 19%-132%. Comparison with the parental ScFv, the sensitivities
279 of the two mutants to the 11 PCs were improved to different extents. As shown in Table 3, the
280 mutant2 showed limit sensitivity improvement to six PCs (CBC, PCG, SBC, NFC, PCV,
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281 MTC), and showed highly improved sensitivity to other five PCs, and the best result was for
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282 DCLC with IC50 decreasing from 53.3 to 10.6 ng/mL (Table 3). In comparison, the mutant1
283 showed highly improved sensitivity to all the 11 PCs with IC50 decreasing for 2-6 folds. For
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284 example, the IC50 for DCLC decreased from 53.3 to 8.7 ng/mL, and that for PCG decreased
285 from 10.1 to 5.1 ng/mL (Table 3). This was because the two mutants showed different
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recognition abilities to the 11 PCs, and their recognition mechanisms were described in the
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287 following section Molecular docking of mutants-PCs. Because the mutant1 showed higher
sensitivity than mutant2, the former was used as detection reagent for determination of PCs
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289 residues in milk. The representative standard competitive curves of AOC and APC are shown
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290 in Figure 4. The results described above meant that the parental ScFv antibody was evolved
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291 successfully and the analysis result of virtual mutation was correct. This is the first study
292 reporting the directional evolution of a ScFv antibody based on the analysis of virtual mutation.
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294 In order to find out why the two mutants showed different sensitivities to the 11 PCs, their
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295 recognition mechanisms were studied by molecular docking. In our recent report, YASARA
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296 software was used to retrieve top five templates showing the highest homology to the parental
297 ScFv antibody, and then the five templates were combined to construct an optimal hybrid 3D
298 ScFv model [18]. In the present study, the amino acid sequences of the two mutants were same
299 as that of parental ScFv except one or two residues, so the parental ScFv model was used as the
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300 initial model to optimize the 3D models of two mutants based on the side chains of mutated
302 The docking results of two mutants with PCs are shown in Table 1. The docking results of
303 two mutants with AOC were identical with that of virtual mutations as described above. As
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304 shown in Table 1, the numbers of contact amino acids in these mutants-PCs models were
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305 generally more than that in parental ScFv-PCs models, indicating the intermolecular forces
306 increased. As the result, the binding energy of each mutants-PCs model was lower than that of
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307 the corresponding parental ScFv-PCs model (Table 1), indicating the mutants-PCs bindings
308 were more stable than the parental ScFv-PCs bindings, so the IC50 to the 11 PCs decreased to
During virtual mutation, the mutant1 showed highly increased interaction for APA part, but
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312 showed negligible interaction for AOC side chain, whereas the mutant2 showed highly
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313 increased interaction for both APA part and AOC side chain. Therefore, the binding energy of
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314 mutant2-AOC (-7.24 kcal/mol) was lower than mutant1-AOC (-6.15 kcal/mol), consequently
315 obtaining lower IC50 (3.1 ng/mL versus 4.6 ng/mL). However, the side chains of other 10 PCs
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316 were different from AOC (Figure 1), and the mutant2 could not recognize these side chains
317 exactly as the AOC side chain, thus disturbed its binding for other 10 PCs. In comparison, the
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318 different side chains of other 10 PCs showed limit influence on the bindings of mutant1 for
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319 APA part, so mutant1 showed generally improved sensitivities for the 10 PCs. Furthermore,
320 hydrogen bonds were formed mainly with the carboxyl oxygen on APA part in mutant1-PCs
321 models (except SBC, Figure 1), indicating APA part was the mutant1s main recognition site.
322 In mutant2-PCs models, hydrogen bonds were formed with oxygen and nitrogen atoms in PCs
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323 molecules, thus weakened its binding for APA part. Therefore, the total binding energies of the
324 mutant1-PCs models were generally lower than that of mutant2-PCs models (Table 1), and the
325 mutant1 showed generally lower IC50 values to the 10 PCs than mutant2 (Table 3).
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327 Then, the mutant1 was used to develop ELISA method for determination of PCs residues
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328 in milk. During the experiments, the 11 PCs were prepared with the extracts of blank milk
329 respectively to develop the matrix matched competitive curves. As shown in Figure 4, the
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330 representative matrix matched competitive curves of AOC and APC were similar to that of
331 their standards, revealing the matrix influence was minor. Therefore, the LODs for the 11 PCs
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in milk were according to the LODs for PCs standards (0.2-3.0 ng/mL). Then, the 11 drugs
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333 were fortified into blank milk sample respectively for analysis. The fortification levels for
AOC, APC, PCG, PCV, CLC, DCLC, OXC and NFC were according to their respective MRLs
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335 (4-30 ng/mL), and that for SBC, MTC, and CBC (without official MRLs) were set at a medium
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336 level (20 ng/mL). As shown in Table 4, the intra-assay recoveries were in a range of 68.7%-
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337 91.2% with CVs lower than 12.6%, and the inter-assay recoveries were in a range of 65.7%-
339 Finally, the 30 real milk samples were analyzed by the method. One raw milk sample was
340 determined as positive sample, and the residue level calculated as AOC were 3.2 ng/mL, but
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341 the specific analyte could not be verified. Other real milk samples were negative. This meant
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342 that one milk sample containing any of the 11 PCs can be determined as positive, but the result
343 can only be expressed as AOC equivalent. Therefore, the ELISA-positive results needed to be
344 confirmed with an instrumental method. Although, the ScFv mutant based ELISA method
345 could still be used as a rapid screening tool to detect low level of PCs residues in milk.
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346 Conclusion
347 The evolution of a ScFv antibody by using gene mutagenesis technique is the main method
348 to improve its intrinsic property. In the present report, an anti-AOC ScFv antibody was evolved
349 by directional mutagenesis of one contact amino acid residue based on the analysis of virtual
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350 mutation. Results showed that the obtained ScFv mutant showed highly improved sensitivity
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351 for 11 tested PCs. The molecular docking analysis found that the decrease of total binding
352 energy was responsible for the improvement of antibody recognition property. From the
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353 analysis of standards fortified blank samples and real samples, the ScFv mutant based ELISA
354 could be used for rapid screening the residues of PCs in milk.
355 Ackownledgements
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356 This study was financed by National Natural Science Foundation of China (31271869),
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357 Hebei Natural Science Foundation (C2011204021, C2015204049), Scientific and Technology
358 Project of HeBei Education Department (QN20131087), and Scientific and Technological
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360 References
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368 Rhijn, Quantitative analysis of penicillins in porcine tissues, milk and animal feed using
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384 (9) S.P. Fitzgerald, N.OLoan, R.I. McConnell, E.O. Benchikh, N.E. Kane, Stable competitive
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388 Vehniinen, P. Saviranta, Improving broad specificity hapten recognition with protein
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397 Wu, J. Shen, Selection of anti-sulfadimidine specific scFvs from a hybridoma cell by
399 (14) K. Pan, H. Wang, H.B. Zhang, H.W. Liu, H.T. Lei, L. Huang, Y.M. Sun, Production and
400 characterization of single chain Fv directed against -agonist clenbuterol, J. Agric. Food
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402 (15) X. Liu, H. Wang, Y. Liang, J. Yang, H. Zhang, H. Lei, Y. Shen, Y. Sun, Production and
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410 Cambillau, A. Pluckthun, Selection, characterization and X-ray structure of anti-ampicillin
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411 single-chain Fv fragments from phage-displayed murine antibody libraries, J. Mol. Biol. 309
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413 (18) J. Liu, H.C. Zhang, C. F. Duan, J. Dong, G.X. Zhao, J.P. Wang, N. Li, J. Z. Liu, Y. W. Li,
414 Production of anti-amoxicillin ScFv antibody and simulation studying its molecular
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416 (19) H. Gram, L.A. Marconi, C.F. Barbas, T.A. Collet, R.A. Lerner, A.S. Kang, In vitro selection
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421 with testosterone. Improved affinity results from small structural changes within the variable
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423 (21) Y.H. Liu, Y.R. Guo, C.M. Wang, W.J. Gui, G.N. Zhu, Homology modeling of anti-parathion
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424 antibody and its interaction with organophosphorous pesticides and analogues, J. Environ.
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429 (23) C.H.T. de Paula da Silva, S.M. Sanches, C.A. Taft, A molecular modeling and QSAR study
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433 A. Lucacchini, G. Giannaccini, A. Rossello, Synthesis, molecular docking and binding
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435 (25) N. Kokayashi, Y. Kato, H. Oyama, S. Taga, T. Niwa, P. Sun, M. Ohtoyo, J. Goto, Anti-
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436 estradiol-17 single-chain Fv fragments: Generation, characterization, gene randomization,
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438 (26) N. Kokayashi, H. Oyama, Y. Kato, J. Goto, E. Sderlind, C.A.K. Borrekaeck, Two-Step in
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439 Vitro Antibody Affinity Maturation Enables Estradiol-17 Assays with More than 10-Fold
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441 (27) X. Zhang, C. Zhang, Y. Liu, X. Yu, X. Liu, Construction of scFv phage display library with
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442 hapten-specific repertories and characterization of anti-ivermectin fragment isolated from the
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444 (28) S.M. Nejad, S.L. Veldhuis, G. Richard, J.C. Hall, Selection of single chain variable fragment
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445 (scFv) antibodies from a hyperimmunized phage display library for the detection of the
447 (29) L. Toleikis, O. Broders, S. Dubel, Cloning single-chain antibody fragments (scFv) from
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449 (30) J.A. Aebig, H.H. Albert, B.L. Zhu, J.S. Hu, H.T. Hsu, Cloning and construction of single-
450 chain variable fragments (SCFV) to cucumber mosaic virus and production of transgenic
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452 (31) F. Sellrie, J.A. Schenk, O. Behrsing, O. Drechsel, B. Micheel, Cloning and characterization
453 of a single chain antibody to glucose oxidase from a murine hybridoma, J. Biochem. Mol.
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457 Table 2. Gene sequences and amino acid sequences of parental ScFv and two mutants.
458 Table 3. Recognition performances of parental ScFv antibody and two mutants for PCs.
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459 Table 4. Recoveries of the 11 PCs from standards fortified blank milk sample (n=6).
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Figure captions:
Figure 1. Molecular structures of the 11 PCs (Red atoms were the hydrogen bond binding
positions of mutant1).
Figure 2. SDS-PAGE results of whole-cell proteins of two ScFv mutants. M, protein marker;
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lanes 1 and 2, negative controls of mutant1 and mutant2 without IPTG induction; lanes 3 and 4,
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expressed mutant1 and mutant2.
Figure 3. Molecular docking of mutant1-AOC (A, B, C) and mutant2-AOC (D, E, F). In A and
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D, VL CDR 1, 2, 3 are shown in red, green and blue, and VH CDR 1, 2, 3 are shown in yellow,
cyan and purple. In B and E, positive and negative charges are shown in blue and red. In C and
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F, hydrophobic and hydrophiclic region are shown in orange and cyan.
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Figure 4. Competitive inhibitory curves of standard AOC, APC, and their matrix matched
standards.
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Binding Binding
Hydrogen Hydrophobic Hydrogen Hydrophobic Hydrogen Hydrophobic Binding
energy a energy
bond interaction bond interaction bond interaction energy
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Asn31
-5.92 Arg100 Phe36 Tyr160 Tyr226 Ser184 Glu100 Arg160 Trp177
OXC Ser95 Tyr32 Phe36 Tyr160 -8.9 -7.18
Arg228 Tyr227 Glu186 Tyr179 Ser184
Arg100
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Arg100 Phe36 Ser95 Tyr160 -5.91 Arg100 Phe36 Glu95 Tyr160 Asn31
CBC -7.88 Val98 Trp177 -6.12
Arg228 Tyr226 Tyr227 Tyr226 Tyr179 Tyr226 Tyr227 Glu95
Asn31 Glu100
Arg100 Asn31 Phe36 Glu95
Lys96 -5.56 Glu186 Glu95 Arg160 Trp177
AOC Phe36 Ser95 Tyr227 Tyr160 Lys96 Tyr160 Tyr179 -6.15 -7.24
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Tyr226 Tyr226 Tyr179 Ser184
Tyr226 Tyr226 Tyr227
Arg228
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Arg158
-5.5 Arg100 Phe36 Glu95 Tyr160 Arg160 Trp177 Tyr179
PCG Tyr179 Tyr160 Tyr159 -7.47 Arg160 -6.26
Arg228 Tyr179 Tyr226 Tyr227 Ser184
Arg228
Ser95 Asn31 Glu95
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Asn31 Lys96 Tyr160 -5.3 Phe36 Glu95 Glu97
SBC Arg100 Arg100 -7.54 Ser184 Trp179 Ser184 Glu186 -6.44
Tyr179 Tyr226 Tyr227 Val98
Arg228 Arg228 Glu186
Arg100
Asn31 Phe36 Lys96 -5.07 Arg100 Tyr160 Trp177 Tyr226 Ser184 Glu95 Arg160 Tyr177
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NFC Tyr226 -7.84 -5.84
Tyr160 Tyr179 Tyr226 Tyr227 Arg228 Glu186 Trp179 Tyr226
Arg228
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Asn31 Ile34 Phe36
Tyr32 Ser95 Trp177 -4.95 Ser184 Arg160 Trp174 Tyr177
CLC ---- Arg100 Glu95 Lys96 Tyr179 -7.93 -7.18
Tyr226 Tyr228 Glu186 Trp179
Tyr226 Tyr227
Tyr160 Trp177 Tyr179 -4.89 Arg100 Phe36 Glu95 Lys96 Ser184 Trp177 Ser184 Glu186
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PCV Tyr226 -7.53 -5.32
Tyr227 Arg228 Arg228 Tyr160 Tyr179 Tyr227 Glu186
Phe36 Glu95 Tyr160
Arg100 Asn31 Tyr32 Phe36 -4.8 Ser184 Glu95 Glu100 Arg160
MTC Arg100 Tyr179 Tyr226 Tyr227 -7.21 -6.15
Tyr226 Tyr179 Glu186 Tyr177 Trp179 Ser184
Arg228
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Arg100 Tyr170 Ala182 Glu186 -4.69 Phe36 Glu95 Tyr160 Glu100 Glu97 Val98 Arg160
APC Arg100 -7.63 -7.02
Tyr226 Tyr226 Tyr179 Tyr226 Tyr227 Glu186 Trp177 Tyr179 Ser184
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Table 2. Gene sequences and amino acid sequences of parental ScFv and two mutants.
Reagent Sequence
GACATCGAGCTCACTCAGTCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATC
GCCTGCAGAGCCGGCGAAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACA
GAAACCAGGACAGCCACCCAAACTCCTCATCTATTCTGCATCTAACCGAGGATCCGGGGTCC
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CTGCCAGGTTTAGTGGCAGCGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAG
GAGGATGATACTGCAATGTATTTCTGTCAGCAA111AAAGAGGTTCCTCGGACGTTCGGTGGA
ScFv gene GGCACCAAACTGGAAATGAAACGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTG
GCGGATCGGAGGTGAAACTGCAGGAGTCAGGACCTGAGCTGGTGAAGCCCGGGGCTTCAGT
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(VL-linker-VH) GAAGCTGTCCTGCAAGGCTTCTGGCGACACCTTCACAAGGTAC222ATACACTGGGTGAGGCA
GAGGCCTGGACAGGGACTTGAGTGGATTGGCTGGATTTATCCTGGAGCTGGTAGTCCTGAGT
ACAATGAGAAGTTCAAGGGCAAGACTACATTGACTGTAGACAAATCCTCCAACACAGCCTAC
ATGTTCCTCAGAAGCCTGACCTCTGAGGACTCTGCGATCTATTTCTGTGCAGCCTACTATAGG
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TACGTCGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCC
parental: 111=AGT, 222=TAT mutant1: 111=GAG mutant2: 111=GAG, 222=AGG
DIELTQSPASLAVSLGQRATIACRAGESVDNYGISFMNWFQQKPGQPPKLLIYSASNRGSGVPARF
amino acid SGSGSGTDFSLNIHPMEEDDTAMYFCQQ1KEVPRTFGGGTKLEMKRGGGGSGGGGSGGGGSEVK
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residues LQESGPELVKPGASVKLSCKASGDTFTRY2IHWVRQRPGQGLEWIGWIYPGAGSPEYNEKFKGKT
TLTVDKSSNTAYMFLRSLTSEDSAIYFCAAYYRYVDYWGQGTTLTVSS
parental: 1, S=Ser; 2, Y=Tyr mutant1: 1, E=Glu mutant2:1, E=Glu; 2, R=Arg
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Table 3. Recognition performances of parental ScFv antibody and two mutants for PCs.
OXC CBC AOC PCG SBC NFC CLC PCV MTC APC DCLC
ScFv antibody
IC50 8.9 9.6 9.6 10.1 13.1 16.0 22.3 19.6 20.8 38.4 53.3
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CR 107 100 100 95 73 60 43 49 46 25 18
ScFv mutant1
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IC50 3.5 4.4 4.6 5.1 6.2 6.9 10.1 8.9 9.4 15.0 8.7
CR 132 104 100 90 74 67 46 52 49 31 53
a
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LOD 0.2 0.3 0.3 0.5 0.8 0.8 1.0 1.0 1.0 3.0 1.0
ScFv mutant2
IC50 3.3 9.3 3.1 8.1 10.0 12.1 8.0 12.5 16.3 9.2 10.6
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CR 94 33 100 34 31 26 39 25 19 34 29
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a b
The unit of IC50 and LOD is ng/mL. CR is expressed as %.
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Table 4. Recoveries of the 11 PCs from standards fortified blank milk sample (n=6).
intra-assay inter-assay
analyte added
(ng/mL) Recovery CV recovery CV
(%) (%) (%) (%)
4 85.3 7.5 80.2 9.4
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AOC
30 80.6 8.3 87.6 12.8
4 87.3 6.4 91.5 7.4
APC
30 91.2 9.4 84.5 7.0
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PCG 4 90.5 9.0 81.2 13.7
30 84.5 7.3 89.6 8.4
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PCV 4 76.8 6.3 72.3 9.5
30 82.1 7.2 86.7 11.3
20 71.0 9.8 65.7 14.8
CBC
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50 79.0 10.4 72.5 14.2
20 82.4 7.6 88.3 8.4
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SBC
50 81.6 8.1 80.8 7.2
20 90.5 6.0 92.4 6.8
MTC
50 85.7 8.7 80.6 6.4
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NFC
60 84.0 8.9 91.3 7.0
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Figure 1. Molecular structures of the 11 PCs (Red atoms were the hydrogen bond binding
positions of mutant1).
HO
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NH2
O O
NH NH
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O O
N N
S S
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HOOC HOOC
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Cl
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N
OCH3 O
SO3H COOH O
O O H3CO O
NH NH HN NH
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O O O O
N N N N
S S S S
D
Cl
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N
O
Cl
O
NH
C
O
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N
S
HOOC
Figure 2. SDS-PAGE results of whole-cell proteins of two ScFv mutants. M, protein marker;
lanes 1 and 2, negative controls of mutant1 and mutant2 without IPTG induction; lanes 3 and 4,
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Figure 3. Molecular docking of mutant1-AOC (A, B, C) and mutant2-AOC (D, E, F). In A and
D, VL CDR 1, 2, 3 are shown in red, green and blue, and VH CDR 1, 2, 3 are shown in yellow,
cyan and purple. In B and E, positive and negative charges are shown in blue and red. In C and
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C F
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Figure 4. Competitive inhibitory curves of standard AOC, APC, and their matrix matched
standards.
100
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80
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60
B/B0 (%)
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40
AOC standard
matrix matched AOC
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20 APC standard
matrix matched APC
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0
0.1 1 10 100
Log C (ng/mL)
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