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    Home > Food News > Food Articles > Professor Peng Dapeng of Huazhong Agricultural University: ic-ELISA detection method and molecular recognition mechanism based on kitatamycin monoclonal antibody in animal tissues

    Professor Peng Dapeng of Huazhong Agricultural University: ic-ELISA detection method and molecular recognition mechanism based on kitatamycin monoclonal antibody in animal tissues

    • Last Update: 2021-04-16
    • Source: Internet
    • Author: User
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    Original title: Professor Peng Dapeng of Huazhong Agricultural University: ic-ELISA detection method and molecular recognition mechanism based on kitatamycin monoclonal antibody in animal tissues

    Recently, the team of Professor Peng Dapeng of Huazhong Agricultural University prepared KIT-specific antigens and developed monoclonal antibodies, and established ic-ELISA methods and sample preparation methods in animal tissues to monitor KIT residues.
    The research result "Development of a monoclonal-based ic-ELISA for the determination of kitasamycin in animal tissues and simulation studying its molecular recognition mechanism" was officially published in Food Chemistry (IF=6.
    301).
    Professor Peng Dapeng is the corresponding author of the paper, and doctoral student Li Long is the first author of the paper.

    Abstract

    In order to monitor the residue of kitasamycin (KIT), an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on the KIT-specific monoclonal antibody KA/2A9 was established.
    After system optimization of Ic-ELISA, the IC50 for KIT is 5.
    7±1.
    4 μg/L, the LOD range in different animal tissues is 22.
    47 μg/kg ~ 29.
    32 μg/kg, the recovery rate of addition is 70% ~ 120%, and The coefficient of variation is less than 20%.
    Then, the KIT-specific single chain antibody (scFv) named KA/2A9/3 was prepared for the first time.
    The results of homology modeling and molecular docking show that the key amino acids for KA /2A9/3 scFv to bind to KIT are TYR-92 (CDRL3), SER-93 (CDRL3), ASP-155 (CDRH1) and GLY-226 (CDRH3) , Where the hydrogen bond is the main force.
    Subsequently, virtual mutation provides a way to evolve the KA/2A9/3 scFv antibody.
    These results help to understand the antigen-antibody binding mechanism and provide theoretical support for the in vitro affinity maturation of KIT-specific scFv.

    Introduction

    Kitasamycin (KIT) is a macrolide antibiotic with a 16-membered ring (Figure S1) (Arsic, Barber, Čikoš, Mladenovic, Stankovic, & Novak, 2018).
    Because KIT has broad-spectrum antibacterial activity against mycoplasma, gram-positive bacteria, certain gram-negative bacteria, Leptospira and Rickettsia, it has been widely used in livestock and poultry breeding to control the respiratory tract and Intestinal diseases, promote growth and improve immunity (Leclercq, 2002).
    Similar to other antibiotics, KIT residues in animal tissues can induce drug resistance in bacteria present in the human body and eventually lead to the emergence of super bacteria (Alban, Nielsen, & Dahl, 2008; Ferri, Ranucci, Romagnoli, & Giaccone, 2017).
    Considering the harm of KIT, many countries set the maximum residue limit of 200 μg/kg in the edible tissues of pigs and poultry.
    Therefore, sensitive and accurate detection methods are essential for monitoring KIT residues in animal-derived foods.
    So far, instrumental methods are considered to be the most commonly used method for the detection of KIT residues in animal-derived foods, such as ultra high performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) (Tang, Lu, Lin, Shin, & Hwang, 2012) And liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Lan et al.
    , 2019), etc.
    However, it is well known that the instrument method is expensive and time-consuming, and is not suitable for rapid detection of large numbers of samples on site.
    In contrast, immunoassay methods such as indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and gold immunochromatographic assay (GICA) are more convenient, sensitive, have low hardware requirements, and are suitable for screening a large number of samples.
    However, immunoassay methods for KIT residues in animal-derived foods are rarely reported.
    Guo and colleagues prepared a monoclonal antibody against KIT.
    Based on this antibody, they developed a GICA method for detecting KIT residues.
    The IC50 of this method is 1.
    51 μg/L (Guo, Liu, Cui, Ma, Wu , & Kuang,
    2019).
    However, this GICA method can only be used to detect KIT residues in eggs and milk samples.
    Currently, there is no research on the KIT immunoassay in other animal products.
    As we all know, antibodies are the core reagents of immunoassays.
    At present, some specific antibodies for macrolide antibiotics have been developed (Z.
    Wang, Beier, & Shen, 2017), such as Tylosin (Lai, Lv, Zhang, Xiong, Lai, & Peng, 2020), Tilmicosin (Le, Zhu, & Yang, 2015), avermectin (Ni et al.
    , 2019; C.
    Wang, Wang, Jiang, Mi, & Shen, 2012) and ivermectin (Xie, Kong, Liu, Song, & Kuang, 2017).
    Unexpectedly, even if these macrolide antibiotics have similar chemical structures to KIT, these antibodies that can recognize other macrolide antibiotics are not sensitive to KIT.
    In recent years, with the widespread application of phage display technology, single-chain variable fragments (scFvs) composed of only heavy and light chain variable regions have become emerging materials for the development of immunoassays (Hammers & Stanley, 2014).
    A large number of studies have shown that the recognition ability of scFv antibody is not lower than that of its parental monoclonal antibody (Li, He, Ren, Zhang, Du, & Li, 2016; Lee, Syed A, Leow, Tan, & Leow, 2018) .
    In addition, scFv antibodies provide an excellent material for exploring the mechanism of antigen-antibody interaction, which helps to analyze why there is no cross-reaction between the specific antibodies of two drugs with similar structures.
    Antibody affinity represents the binding strength between an antibody and its corresponding antigen.
    Another advantage of ScFv antibodies is that they can explore the binding mechanism of scFv and antigen through molecular docking technology, thereby guiding the modification of scFv and further improving the affinity of the antibody (Tao et al.
    , 2020; Xu et al.
    , 2009).
    In previous reports, molecular docking technology was used to study the binding mechanism of different scFv antibodies to corresponding drugs, such as phenothiazine (Shi, Zhang, Xia, Liu, Zhang, & Wang, 2017), amoxicillin (He et al.
    , 2017), monensin (Makvandi-Nejad, Sheedy, Veldhuis, Richard, & Hall, 2010) and fluoroquinolone (Leivo, Lamminmäki, Lövgren, & Vehniäinen , 2013), and then applied different gene mutagenesis methods to scFv evolution.
    The results showed that compared with its parental scFv antibody, the evolved scFv showed increased affinity for each analyte.
    Therefore, exploring the interaction mechanism between scFv antibodies and corresponding antigens is of great significance for obtaining high-performance antibodies.
    In this study, we prepared KIT-specific antigens and developed monoclonal antibodies, and established ic-ELISA methods and sample preparation methods in animal tissues to monitor KIT residues.
    Subsequently, in order to explore the interaction mechanism between the antibody and the drug target, the scFv antibody against KIT was screened for the first time using phage display technology.
    Finally, virtual mutations were performed on the KIT-specific scFv antibody, providing a better evolutionary method for KIT-specific scFv.
    2013), and then applied different gene mutagenesis methods to scFv evolution.
    The results showed that compared with its parental scFv antibody, the evolved scFv showed increased affinity for each analyte.
    Therefore, exploring the interaction mechanism between scFv antibodies and corresponding antigens is of great significance for obtaining high-performance antibodies.
    In this study, we prepared KIT-specific antigens and developed monoclonal antibodies, and established ic-ELISA methods and sample preparation methods in animal tissues to monitor KIT residues.
    Subsequently, in order to explore the interaction mechanism between the antibody and the drug target, the scFv antibody against KIT was screened for the first time using phage display technology.
    Finally, virtual mutations were performed on the KIT-specific scFv antibody, providing a better evolutionary method for KIT-specific scFv.
    2013), and then applied different gene mutagenesis methods to scFv evolution.
    The results showed that compared with its parental scFv antibody, the evolved scFv showed increased affinity for each analyte.
    Therefore, exploring the interaction mechanism between scFv antibodies and corresponding antigens is of great significance for obtaining high-performance antibodies.
    In this study, we prepared KIT-specific antigens and developed monoclonal antibodies, and established ic-ELISA methods and sample preparation methods in animal tissues to monitor KIT residues.
    Subsequently, in order to explore the interaction mechanism between the antibody and the drug target, the scFv antibody against KIT was screened for the first time using phage display technology.
    Finally, virtual mutations were performed on the KIT-specific scFv antibody, providing a better evolutionary method for KIT-specific scFv.

    Materials and methods

    1.
    Reagents and materials The chemicals used in this study are all analytical reagent grades.
    The kitasamycin, tylosin, tilmicosin, spiramycin, erythromycin, zosamycin, avermectin and ivermectin used in ELISA were all purchased from the China Institute for Veterinary Drug Control.
    Hypoxanthine/Thymine/Aminopterin (HAT), Hypoxanthine/Thymine (HT), PEG 1450, Bovine Serum Albumin (BSA) and Ovalbumin (OVA), Carboxymethoxyamine Hemihydrochloride Salt (CMO), dimethyl sulfoxide (DMSO) and Freund's adjuvant were purchased from Sigma, USA.
    Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were provided by Sinopharm Chemical Reagent Co.
    , Ltd.
    DNA polymerase, DNA restriction endonuclease and T4 ligase were purchased from NEB, USA.
    HRP-anti E-tag antibody, HRP-anti M13 antibody, phagemid pCANTAB5E, Escherichia coli TG1 and Escherichia coli HB2151 were obtained from Amersham Biosciences, USA.
    All the solutions and buffers described herein are prepared using standard methods.
    2.
    The synthetic antigen KIT-CMO-BSA/OVAKIT-CMO-BSA/OVA conjugate was prepared by referring to the method of Kolosova et al.
    (Kolosova, Shim, Yang, Eremin, & Chung, 2006).
    A total of 77.
    10 mg (100 μmol) of KIT was dissolved in 5 mL of methanol, and 12 mg (110 μmol) of carboxymethoxyamine hemihydrochloride and 8.
    40 mg of NaHCO3 (100 μmol) were dissolved in 1 mL of ddH2O.
    Slowly add KIT methanol solution while stirring.
    After incubating for 2 h at room temperature and rotary evaporation at 60 °C, the product was dissolved in 5 mL CHCl3, and the organic phase was removed.
    The yellow product was dissolved in 2 mL of N,N-dimethylformamide solution containing 12 mg of N-hydroxysuccinimide and 20 mg of N,N-dicyclohexylcarbodiimide, and incubated overnight in the dark with stirring .
    BSA (32.
    5 mg, 0.
    5 μmol) was added to 20 mL of phosphate buffered saline (PBS, pH 7.
    4) and cooled at 4°C for 2 h to prepare a protein solution.
    The overnight mixed solution (2 mL) was slowly added dropwise to the protein solution and kept at 4 °C overnight.
    Subsequently, at 10, Centrifuge at a speed of 000 r/min for 10 min, and dialyze the supernatant for 3 days at 4 °C, and change the dialysate every 12 hours.
    KIT-CMO-OVA was prepared in the same way, except that 22.
    5 mg of OVA was used instead of BSA.
    3.
    Preparation of monoclonal antibodies All animal experiments described in this study follow the guidelines of the Animal Experiment Center of Huazhong Agricultural University and have been approved by the Animal Ethics Committee.
    Using KIT-CMO-BSA as the immunogen, KIT-specific monoclonal antibodies were prepared according to standard hybridoma production procedures.
    Dissolve 50 μg KIT-CMO-BSA in sterile H2O (0.
    25 mL), then emulsify it with an equal volume of Freund's complete adjuvant, and inject it into the neck and back by three-point or four-point subcutaneous injection once every two weeks Inject 8-week-old female Balb/C mice.
    Then, the same amount of immunogen was emulsified with an equal volume of Freund's incomplete adjuvant and injected in the same manner.
    After 8 days, serum was collected from the tail vein of the mice, and the titer of the serum was measured by ic-ELISA.
    72 to 84 hours before cell fusion, mice with high titers were injected intraperitoneally with 200 μg/ml immunogen without adjuvant.
    The total spleen cell suspension of immunized mice was fused with 5×107 SP2/0 myeloma cells in 0.
    8 mL of 50% PEG1450.
    After HAT selection and culture, ic-ELISA was used to evaluate the quality of the supernatant of hybridoma cells.
    The limiting dilution method was used to clone highly sensitive ELISA-positive hybridoma cells until all the hybridoma cells living in the supernatant were 3-fold positive.
    The cloned hybridoma cells (1.
    0×106 in RPMI-1640) were injected into Balb/C mice, and then the mice were treated by intraperitoneal injection of 0.
    5 mL of Freund's incomplete adjuvant.
    Ten days later, ascites was collected and used for ELISA.
    4.
    ELISA procedure According to the method described by the predecessors, the ELISA square matrix is ​​used to obtain the optimal concentration of coating antigen and monoclonal antibody (Peng et al.
    , 2016).
    Dilute KIT-CMO-OVA in coating buffer to concentrations of 1, 2, 4, 8, 16, 32, 64, and 128 mg/L, and incubate overnight at 4°C.
    The ELISA plate was washed 3 times with PBST, and the plate was blocked with 1% OVA (250 μL/well) at 37°C for 1 hour.
    After washing 3 times, add 100 μL/well of PBS serial dilution antibody (1:10 000-1:128, 000), and incubate at 37 ℃ for 1h.
    Then wash 3 times with PBST, add 100 μL/well of diluted (1:5000) goat anti-mouse IgG-HRP, and incubate at 37 °C for 1 h.
    After the final washing step, colorimetric detection is performed by adding TMB substrate (100 μL/well).
    Add 50 μL/well of 2 M H2SO4, incubate for 15 min to stop the reaction, and read the absorbance at 450 nm.
    From the results, we obtained the best ELISA coating antigen concentration and monoclonal antibody concentration.
    Using the above ELISA procedure, the cross-reaction rate (CR) was evaluated by comparing the IC50 of KIT and the macrolide analog.
    The macrolide analogues (including kitasamycin, midenomycin, josamycin, spiramycin, acetylspiramycin, erythromycin, tylosin, tilmicosin, abamectin, Ivermectin) was formulated to an appropriate concentration to determine CR.
    The concentration of the analyte standard solution ranges from 1 to 100,000 μg/L, and the calculation formula for CR is CR%=(KIT IC50/Analyte IC50)×100%.
    5 Sample preparation and ELISA analysis The tissue sample processing method is as follows: Weigh 2.
    00 g of homogeneous pig muscle, pig liver, chicken muscle, and chicken liver samples into a 50 mL centrifuge tube, and add 10 mL of 0.
    3% of 40% (v / v) methanol The metaphosphoric acid solution was vortexed for 1 min and centrifuged at 4000 r/min for 10 min.
    Take the supernatant 0.
    Put 5 mL in a 10 mL centrifuge tube, extract with 4 mL of CHCl3, vortex and mix for 1 minute.
    Centrifuge at 4000 r/min for 5 min.
    Remove the lower layer of CHCl3 solution, evaporate and dissolve in 2mL PBS.
    The dilution factor of the sample is 20.
    The minimum detection limit (LOD) of tissues was determined as follows: 20 different blank samples collected were used for ELISA verification.
    The above-mentioned ELISA method is used to determine the range of the blank matrix of the sample and determine the LOD.
    The LOD calculation method is the average of 20 blank samples plus 3 times the standard deviation.
    The recovery rate and coefficient of variation (CV) of this method on different samples: add KIT standard solutions of different concentrations (100, 200, 400 μg/kg) to the samples, mix and incubate at room temperature for 30 min.
    The samples were processed according to the above method and tested by ELISA.
    For each sample concentration, 5 parallel samples were measured and repeated 3 times at different time points.
    Use the following formula to calculate the recovery rate: recovery rate (%) = (measured concentration / spiked concentration) × 100.
    Cut-off value determination: accurately weigh 20 samples and add KIT standard solution with a maximum residue limit of 200 μg/kg.
    The established ELISA method was used to determine the concentration of the drug in the added sample and calculate the standard deviation.
    The cut-off value is calculated by subtracting 1.
    64 times the standard deviation from the measured drug concentration.
    6.
    Research on the recognition mechanism of antigen and antibody molecules

    Single chain antibody phage library preparation

    The collected KA/2A9 hybridoma cells (1×107) were centrifuged at 1300 r/min for 10 min at room temperature.
    According to the instruction manual, the cells were lysed with TRIzol reagent to extract RNA.
    Use Qiagen mRNA purification kit to purify mRNA, and use SuperScriptTM III First-Strand Synthesis System kit to perform RT-PCR to synthesize all mRNA into cDNA.
    Table S1 lists the primer sequences and ratios used for VH and VL gene amplification.
    SfiI and NotI restriction enzyme sites are included in the 5'primer of VL and the 3'primer of VH.
    Taq polymerase was used to amplify the VH and VL genes, and the 50 µL reaction mixture contained 2 µL cDNA and 1 µL VH or VL primer mixture.
    The PCR program is 95 ℃ 2 min; 94 ℃ 1 min, 50 ℃ 1 min, 72 ℃ 1 min 30 cycles.
    The VH and VL products amplified by PCR were mixed in equal amounts, and then SOE-PCR was used to assemble the full-length scFv gene.
    Use QIAquick PCR purification kit for sample purification.
    The amplified scFv and pCANTAB5E phage vectors were simultaneously digested with NotI and SfiI endonucleases, and the scFv fragments were inserted into the phage vector pCANTAB5E with T4 ligase at 16°C.
    The phagemid inserted with scFv was transformed into E.
    coli TG1 cells by electric shock transformation.
    The cells transformed by electroporation were cultured on SOBAG medium and incubated at 37 °C for 14 h.
    The colonies were then collected, mixed with glycerol and stored at -80°C.

    Panning and soluble expression of KIT-specific phage single-chain antibody

    Using M13K07 helper phage, the constructed phage library was used to prepare phage antibodies.
    The library stock solution was cultured in 2×YT-AG medium to form a logarithmic phase, rescued with M13KO7 helper phage, and amplified in 2×YT-AK medium at 30°C overnight.
    The phage was precipitated with 4% PEG/0.
    5 M NaCl solution and dissolved in sterile PBS.
    Before biopanning, the phage scFv and MPBS were incubated at room temperature at a ratio of 3:2 for 30 min, and then the phage scFv was added to the immunotube coated with 1% OVA and incubated at room temperature for 30 min.
    The immune tube is coated with KIT-CMO-OVA (40 ppm for the first round, 20 ppm and 10 ppm for the remaining two rounds), and blocked with a 2% MPBS solution.
    Add the phage antibody buffer to the immune tube and incubate at 37 °C for 1 h.
    Wash the immune tube 20 times with PBS and PBST respectively.
    The bound phage was eluted with 0.
    1 M glycine-hydrochloric acid, and then neutralized by adding 2 M Tris.
    Subsequently, the mixture was incubated with 10 mL of E.
    coli TG1 cells in log phase at 37 ℃ with shaking at 200 r/min for 50 min.
    Finally, the solution was spread on SOBAG plates at different dilution rates and incubated overnight at 30°C.
    The monoclonal bacteria were picked and cultured in 96 deep-well cell culture plates to screen for specific antibodies.
    Use KIT-CMO-OVA coated ELISA plate to screen phage antibodies.
    This method is similar to the established monoclonal antibody ELISA method and uses mouse HRP-anti M13 for screening.
    Add phage antibody to two wells containing 50 μL PBS and 50 μL 10 mg/L KIT standard solution.
    The positive phage antibody was applied to infect E.
    coli HB2151 cells in log phase and cultured on SOBAG-N medium.
    The growing clones were picked into 2×YT-AG medium, and incubated overnight at 30°C with shaking at 250 r/min.
    Subsequently, the overnight culture was divided into 1: 100% was added to 2×YT-AG medium, and incubated at 30 °C and 250 r/min for 1 h.
    Centrifuge the culture at 1500 g for 20 min to remove the supernatant, then resuspend the pellet in 50 mL of 2×YT-AI medium and incubate at 30°C with shaking at 250 r/min for 8 h.
    The recombinant phage antibody system was used to prepare soluble scFv antibodies.
    The ic-ELISA method was used to determine the titer and sensitivity of scFv.
    The coating antigen concentration is 8 mg/L, and the ic-ELISA method uses HRP-anti E-tag antibody.
    The FR (framework region) and CDR (complementarity determining region) of single-chain antibodies were analyzed using the IMGT database.

    Single-chain antibody sequence analysis and three-dimensional structure prediction

    The ScFv nucleotide sequence was sequenced by Shenggong Biotech (Shanghai, China).
    In the International ImmunoGeneTics gene technology information system database (
    cines.
    fr/%EF%BC%89%EF%BC%88Lefranc%EF%BC%8C2007%EF%BC%89%E4%B8%AD%E5%88%86%E6%9E%90%E4%BA%86%E5%8F%AF%E5%8F%98%E5%8C%BA%E5%BA%8F%E5%88%97%E5%8F%8AVH%EF%BC%8CFRL%EF%BC%88%E8%BD%BB%E9%93%BE%E6%A1%86%E6%9E%B6%E5%8C%BA%EF%BC%89%E5%92%8CCDR%E5%8C%BA%E7%9A%84%E5%91%BD%E5%90%8D%E5%8E%9F%E7%90%86%E3%80%82%E4%BD%BF%E7%94%A8%E7%BD%91%E7%AB%99%E4%B8%8A%E7%9A%84%E6%95%B0%E6%8D%AE%E5%BA%93%E5%88%86%E6%9E%90%E4%BA%86scFv%E5%BA%8F%E5%88%97%E5%90%8C%E6%BA%90%E6%80%A7%E7%82%B9%EF%BC%8C%E5%B9%B6%E5%9C%A8http://imgt.
    cines.
    fr/IMGT_vquest/share/textes/%E4%B8%8A%E9%80%89%E6%8B%A9%E4%BA%86%E5%B0%8F%E9%BC%A0%E5%AD%90%E5%BA%93%E8%BF%9B%E8%A1%8C%E5%88%86%E6%9E%90%E3%80%82ScFv">http://imgt.
    cines.
    fr/)
    ( cines.
    fr/%EF%BC%89%EF%BC%88Lefranc%EF%BC%8C2007%EF%BC%89%E4%B8%AD%E5%88%86%E6%9E%90%E4%BA%86%E5%8F%AF%E5%8F%98%E5%8C%BA%E5%BA%8F%E5%88%97%E5%8F%8AVH%EF%BC%8CFRL%EF%BC%88%E8%BD%BB%E9%93%BE%E6%A1%86%E6%9E%B6%E5%8C%BA%EF%BC%89%E5%92%8CCDR%E5%8C%BA%E7%9A%84%E5%91%BD%E5%90%8D%E5%8E%9F%E7%90%86%E3%80%82%E4%BD%BF%E7%94%A8%E7%BD%91%E7%AB%99%E4%B8%8A%E7%9A%84%E6%95%B0%E6%8D%AE%E5%BA%93%E5%88%86%E6%9E%90%E4%BA%86scFv%E5%BA%8F%E5%88%97%E5%90%8C%E6%BA%90%E6%80%A7%E7%82%B9%EF%BC%8C%E5%B9%B6%E5%9C%A8http://imgt.
    cines.
    fr/IMGT_vquest/share/textes/%E4%B8%8A%E9%80%89%E6%8B%A9%E4%BA%86%E5%B0%8F%E9%BC%A0%E5%AD%90%E5%BA%93%E8%BF%9B%E8%A1%8C%E5%88%86%E6%9E%90%E3%80%82ScFv">Lefranc, 2007), the variable region sequence and the VH, F.
    .
    .
    VH and VL sequences cines.
    fr/%EF%BC%89%EF%BC%88Lefranc%EF%BC%8C2007%EF%BC%89%E4%B8%AD%E5%88%86%E6%9E%90%E4%BA%86%E5%8F%AF%E5%8F%98%E5%8C%BA%E5%BA%8F%E5%88%97%E5%8F%8AVH%EF%BC%8CFRL%EF%BC%88%E8%BD%BB%E9%93%BE%E6%A1%86%E6%9E%B6%E5%8C%BA%EF%BC%89%E5%92%8CCDR%E5%8C%BA%E7%9A%84%E5%91%BD%E5%90%8D%E5%8E%9F%E7%90%86%E3%80%82%E4%BD%BF%E7%94%A8%E7%BD%91%E7%AB%99%E4%B8%8A%E7%9A%84%E6%95%B0%E6%8D%AE%E5%BA%93%E5%88%86%E6%9E%90%E4%BA%86scFv%E5%BA%8F%E5%88%97%E5%90%8C%E6%BA%90%E6%80%A7%E7%82%B9%EF%BC%8C%E5%B9%B6%E5%9C%A8http://imgt.
    cines.
    fr/IMGT_vquest/share/textes/%E4%B8%8A%E9%80%89%E6%8B%A9%E4%BA%86%E5%B0%8F%E9%BC%A0%E5%AD%90%E5%BA%93%E8%BF%9B%E8%A1%8C%E5%88%86%E6%9E%90%E3%80%82ScFv">were analyzed and submitted to the WAM-Web antibody model Database (
    bath.
    ac.
    uk/index.
    html%EF%BC%89%EF%BC%88Whitelegg%EF%BC%86Rees%EF%BC%8C2000%EF%BC%89%E8%BF%9B%E8%A1%8C%E5%90%8C%E6%BA%90%E5%BB%BA%E6%A8%A1%E3%80%82%E6%A0%B9%E6%8D%AE%E6%89%80%E6%9E%84%E5%BB%BA%E7%9A%84scFv%E4%B8%89%E7%BB%B4%E6%A8%A1%E5%9E%8B%EF%BC%8C%E4%BD%BF%E7%94%A8Autodock">http://antibody.
    bath.
    ac.
    uk/index.
    html) (Whitelegg&R.
    .
    .
    4.
    2.
    6 software for structure prediction.
    The best KIT and scFv antibody binding conformations are analyzed (Sotriffer, Flader, Winger, Rode) , Liedl and Varga, 2000).

    Results and Discussion

    1.
    Preparation of Monoclonal Antibodies

    According to reports, carboxymethoxy amine hemihydrochloride (CMO) is often used as a spacer to design haptens, and is used to develop specific monoclonal antibodies for erythromycin, tylosin and other small molecule drugs ( Lai et al.
    , 2020; Z.
    Wang et al.
    , 2015).
    Therefore, in this study, the hapten KIT was combined with bovine serum albumin (BSA) through the spacer CMO to synthesize the immunogen.
    After three injections of Balb/C mice with the immunogen KIT-CMO-BSA, the mouse serum was taken for checkerboard titration and ic-ELISA experiments to determine the serum antibody titer and affinity, respectively.
    The experimental results show that the mouse serum antibody titer is 1:64,000 and the IC50 is 242 μg/L, which meets the requirements for cell fusion.
    After 9 days of cell fusion, the culture supernatant of the 96-well plate was analyzed by ic-ELISA to screen out two stable hybridoma cells with high titer.
    After 5 times of subcloning, two best monoclonal hybridoma cell lines were successfully established, called KA /1H8 and KA/2A9, and monoclonal antibodies (mAbs) were obtained.
    The antibody subtype identification results showed that the antibody subtypes secreted by the KA/1H8 and KA/2A9 cell lines are all of the IgG1 isotype.

    2.
    Identification of monoclonal antibodies

    The monoclonal hybridoma cell lines KA/1H8 and KA/2A9 were selected for the preparation of mouse ascites.
    After 7~12 days, ascites were collected for detection of titer and sensitivity.
    As shown in Table S2, the optimal concentration of the coating agent is 8 mg/L, and the titers of the monoclonal antibodies extracted from the ascites of KA/1H8 and KA/2A9 are 1:640,000 and 1:8000, respectively.
    The IC50 values ​​of monoclonal antibodies KA/1H8 and KA/2A9 were 13.
    9 μg/L and 5.
    7 μg/L, respectively.
    Therefore, the highly sensitive monoclonal antibody KA/2A9 was selected for the development and optimization of ic-ELISA.

    3.
    Optimization of ic-ELISA conditions

    Figure 1.
    The standard curve of the established ELISA method was determined by the square matrix experiment.
    The optimal concentration of the coating source was 8 mg/L, and the optimal dilution concentration of the KA/2A9 monoclonal antibody was 1:40,000.
    According to the drug concentration of multiple dilutions, a standard curve was established, and the regression equation was y = -0.
    610x + 0.
    995, R2 = 0.
    995 (Figure 1).
    The range of the standard curve is 1.
    25 ~ 20 μg/L, the IC50 value of the standard curve is 5.
    7 ± 1.
    4 μg/L (n = 5), and the LOD is 1.
    49 μg/L, indicating that the established ic-ELISA method is highly sensitive (Table S3 ).
    The cross-reaction (CR) of the KA/2A9 monoclonal antibody with macrolide antibiotics is shown in Table 1.
    This antibody can recognize midemycin and josamycin, but has resistance to other macrolide antibiotics (such as Tai Lamycin and Tilmicosin) have no recognition ability.

    4.
    Validation of ic-ELISA method

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