Topic review

Anti-Ciprofloxacin ScFv in Animal-Derived Food

Submitted by: Yali Zhao

Definition

Ciprofloxacin (CIP) is a synthetic third-generation fluoroquinolone (FQ) antibiotic that has been developed and is widely used to treat bacterial infections in humans and animals. This antibiotic exerts effects by inhibiting DNA gyrase or topoisomerase II in susceptible bacteria and exhibits high activity against a broad spectrum of Gram-negative and Gram-positive bacteria. However, the unreasonable and extensive use of antibiotics has resulted in the potential for residual antibiotics in food of animal origin, which can damage multiple systems in the body and cause bacterial resistance. Therefore, the European Union, the Joint FAO/WHO Expert Committee on Food Additives (JECFA, Rome, Italy) and China established maximum residue limits of CIP in animal-derived food to prevent the accumulation of antimicrobial residues, e.g., 100 µg/kg in milk and meat.

1. Overview

An immunized mouse phage display scFv library with a capacity of 3.34 × 109 CFU/mL was constructed and used for screening of recombinant anti-ciprofloxacin single-chain antibody for the detection of ciprofloxacin (CIP) in animal-derived food. After four rounds of bio-panning, 25 positives were isolated and identified successfully. The highest positive scFv-22 was expressed in E. coli BL21. Then, its recognition mechanisms were studied using the molecular docking method. The result showed the amino acid residue Val160 was the key residue for the binding of scFv to CIP. Based on the results of virtual mutation, the scFv antibody was evolved by directional mutagenesis of contact amino acid residue Val160 to Ser. After the expression and purification, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on the parental and mutant scFv was established for CIP, respectively. The IC50 value of the assay established with the ScFv mutant was 1.58 ng/mL, while the parental scFv was 26.23 ng/mL; this result showed highly increased affinity, with up to 16.6-fold improved sensitivity. The mean recovery for CIP ranged from 73.80% to 123.35%, with 10.46% relative standard deviation between the intra-assay and the inter-assay. The RSD values ranged between 1.49% and 9.81%. The results indicate that we obtained a highly sensitive anti-CIP scFv by the phage library construction and directional evolution, and the scFv-based IC-ELISA is suitable for the detection of CIP residue in animal-derived edible tissues.

2. Phage Display Technology

Ciprofloxacin (CIP) is a synthetic third-generation fluoroquinolone (FQ) antibiotic that has been developed and is widely used to treat bacterial infections in humans and animals. This antibiotic exerts effects by inhibiting DNA gyrase or topoisomerase II in susceptible bacteria and exhibits high activity against a broad spectrum of Gram-negative and Gram-positive bacteria [1]. However, the unreasonable and extensive use of antibiotics has resulted in the potential for residual antibiotics in food of animal origin, which can damage multiple systems in the body [2][3] and cause bacterial resistance [4][5]. Therefore, the European Union, the Joint FAO/WHO Expert Committee on Food Additives (JECFA, Rome, Italy) and China established maximum residue limits of CIP in animal-derived food to prevent the accumulation of antimicrobial residues, e.g., 100 µg/kg in milk and meat.
By now, many physicochemical methods have been reported for the detection of residues of FQs in foods of animal origin. These analytical methods are highly sensitive and dependable; however, such methods require specialized instrumentation, trained personals, and are time consuming. They are unsuitable for the rapid evaluation of large numbers of samples. Immunoassays, especially the indirect competitive enzyme-linked immunosorbent assay (IC-ELISA), which is based on the principle that antibodies specifically bind to antigens, are considered the most reliable method for detecting antibodies [6][7]. In previous studies, researchers have developed IC-ELISA based on monoclonal antibodies (MAbs) to determine fluoroquinolone in food of animal origin [8][9][10]. Although ELISA is a mature and widely used method, it has many rigorous programs for preparing traditional antibodies (PAbs and MAbs) from antigen-immunized animals [11]. Hence, a simple, rapid, and effective technology for preparing novel antibodies must be developed.
The development of gene engineering techniques facilitated the production of various gene recombinant antibodies, and single-chain variable fragment (scFv) is the most popular format of recombinant antibody that has been successfully constructed by assembling the variable-heavy (VH) region and light chain (VL) domain of an antibody with a flexible linker [12]. The intrinsic properties of scFv antibodies can be improved by various mutagenesis techniques [13]. The recognition property of an scFv antibody can be evolved in vitro [14]. For the evolution of the scFv antibody, its recognition mechanism should be studied first, and binding sites, contact amino acids, and intermolecular forces should be determined [15]. In recent years, molecular docking has been used in analyzing the interactions between ligands and scFv antibodies, and random mutagenesis and site-directed mutagenesis have been used in obtaining scFv mutants [16][17].
Phage display technology (PDT) is the integration of foreign genes into specific coat protein genes of phage and fusion, with coat protein to promote ligand recognition and binding [18][19]. It is considered to be the most suitable technology for the production of single-chain antibodies. The phage antibody library uses genetic engineering methods to amplify VH and VL genes. After random combination, it is inserted into the phage coat protein gene and fused and expressed on the surface of the phage [20]. Specific single-chain antibodies are obtained through specific panning, which is extensively used for preparing antigen-specific artificial antibodies in biomedicine, environmental pollutants analysis, and food safety detection fields. For example, Xu et al. [21] and Zhao et al. [22] obtained the broad-specificity domain antibodies for Bt Cry toxins and pyrethroid pesticides by rounds of specific phage library biopanning, respectively, which are all based on phage antibody library technology.

3. Conclusions

A highly sensitive anti-CIP single-chain antibody was obtained through phage display and directional evolution, and a rapid and highly sensitive IC-ELISA method for detecting CIP residues in products of animal origin was developed. The method showed good stability, reproducibility, and accuracy for detecting CIP, indicating a wide application prospect for the rapid and sensitive detection of antibiotic residues in animal-derived food.

References

  1. Dalhoff, A. Antiviral, antifungal, and antiparasitic activities of fluoroquinolones optimized for treatment of bacterial infections: A puzzling paradox or a logical consequence of their mode of action? Eur. J. Clin. Microbiol. Infect. Dis. 2015, 34, 661–668.
  2. Bird, S.T.; Etminan, M. Risk of acute kidney injury associated with the use of fluoroquinolones. CMAJ 2013, 185, E475–E482.
  3. Patel, K.; Goldman, J.L. Safety Concerns Surrounding Quinolone Use in Children. J. Clin. Pharmacol. 2016, 56, 1060–1075.
  4. Li, J.; Hao, H. The effects of different enrofloxacin dosages on clinical efficacy and resistance development in chickens experimentally infected with Salmonella Typhimurium. Sci. Rep. 2017, 7, 11676.
  5. Xu, L.; Wang, H. Integrated pharmacokinetics/pharmacodynamics parameters-based dosing guidelines of enrofloxacin in grass carp Ctenopharyngodon idella to minimize selection of drug resistance. BMC Vet. Res. 2013, 9, 126.
  6. Cui, L.; Jinxin, H. Preparation of a Chicken scFv to Analyze Gentamicin Residue in Animal Derived Food Products. Anal. Chem. 2016, 88, 4092–4098.
  7. Abdelwahab, M.; Loa, C.C. Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus. J. Virol. Methods 2015, 217, 36–41.
  8. Huang, B.; Yin, Y. Preparation of high-affinity rabbit monoclonal antibodies for ciprofloxacin and development of an indirect competitive ELISA for residues in milk. J. Zhejiang Univ. Sci. B 2010, 11, 812–818.
  9. Fan, G.-Y.; Yang, R.-S. Development of a class-specific polyclonal antibody-based indirect competitive ELISA for detecting fluoroquinolone residues in milk. J. Zhejiang Univ. Sci. B 2012, 13, 545–554.
  10. Zhang, H.-T.; Jiang, J.-Q. Development of an indirect competitive ELISA for simultaneous detection of enrofloxacin and ciprofloxacin. J. Zhejiang Univ. Sci. B 2011, 12, 884–891.
  11. Li, C.; Luo, X. A Class-Selective Immunoassay for Sulfonamides Residue Detection in Milk Using a Superior Polyclonal Antibody with Broad Specificity and Highly Uniform Affinity. Molecules 2019, 24, 443.
  12. Makvandi-Nejad, S.; Sheedy, C. Selection of single chain variable fragment (scFv) antibodies from a hyperimmunized phage display library for the detection of the antibiotic monensin. J. Immunol. Methods 2010, 360, 103–118.
  13. Norihiro, K. Anti-estradiol-17beta single-chain Fv fragments: Generation, characterization, gene randomization, and optimized phage display. Steroids 2008, 73, 1485–1499.
  14. Kobayashi, N.; Oyama, H.; Kato, Y.; Goto, J.; Söderlind, E.; Borrebaeck, C.A. Two-step in vitro antibody affinity maturation enables estradiol-17beta assays with more than 10-fold higher sensitivity. Anal. Chem. 2010, 82, 1027–1038.
  15. Liu, J.; Zhang, H.C. Production of anti-amoxicillin ScFv antibody and simulation studying its molecular recognition mechanism for penicillins. J. Environ. Sci. Health Part B Pestic. Food Contam. Agric. Wastes 2016, 51, 742–750.
  16. Wen, K.; Nolke, G. Improved fluoroquinolone detection in ELISA through engineering of a broad-specific single-chain variable fragment binding simultaneously to 20 fluoroquinolones. Anal. Bioanal. Chem. 2012, 403, 2771–2783.
  17. Tao, X.; Chen, M. Chemiluminescence competitive indirect enzyme immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single-chain variable fragment. Anal. Bioanal. Chem. 2013, 405, 7477–7484.
  18. Kumar, R.; Parray, H.A. Phage display antibody libraries: A robust approach for generation of recombinant human monoclonal antibodies. Int. J. Biol. Macromol. 2019, 135, 907–918.
  19. Zhao, A.; Tohidkia, M.R.; Siegel, D.L.; Coukos, G.; Omidi, Y. Phage antibody display libraries: A powerful antibody discovery platform for immunotherapy. Crit. Rev. Biotechnol. 2016, 36, 276–289.
  20. Arap, M.A. Phage display technology: Applications and innovations. Genet. Mol. Biol. 2005, 28, 1–9.
  21. Xu, C.; Miao, W. Construction of an immunized rabbit phage display antibody library for screening microcystin-LR high sensitive single-chain antibody. Int. J. Biol. Macromol. 2019, 123, 369–378.
  22. Zhao, Y.; Liang, Y. Isolation of broad-specificity domain antibody from phage library for development of pyrethroid immunoassay. Anal. Biochem. 2016, 502, 1–7.

Keywords

scFv ciprofloxacin recognition mechanism directional mutagenesis IC-ELISA