Magnetic-assisted biotinylated single-chain variable fragment antibody-based immunoassay for amantadine detection in chicken
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A sensitive competitive immunoassay with simple operation was developed for the detection of the anti-virus drug amantadine (AMD). The single-chain variable fragment (scFv) antibody against AMD was site-specific biotinylated and overexpressed as a secreted body in Escherichia coli AVB101. Horseradish peroxidase-labeled streptavidin-biotinylated scFv antibody (HRP-SA-BIO-scFv) could specifically bind to AMD-functionalized magnetic beads (MBs) and then the immune complexes were separated from the matrix solution by magnet. The concentration of the AMD could be known by the measurement of the signal produced by the horseradish peroxidase. The newly established assay provides a significant improvement in comparison to the conventional ELISA without SA-BIO signal amplification and MBs separation. The limit of detection and assay time was 0.64 vs. 8.4 ng/mL and 50 vs. 150 min, respectively. The recoveries ranged from 77.8 to 112% with the coefficient of variation less than 13%. The immunoassay exhibited an obvious cross-reactivity to rimantadine (84%), 1-(1-adamantyl)ethylamine (72%), and somantadine (63%). These results demonstrated that the developed immunoassay provided a sensitive, rapid, and accurate approach for the detection of AMD in chicken by employing MBs as solid phase and SA-BIO as signal amplification. When applied in natural chicken samples, the newly established method provided results consistent with those from UPLC-MS/MS, suggesting that the proposed method could be used for rapid screening of the target of interest; the new immunoassay could also be extended to other small molecular contaminants and thus represents a universal strategy for food safety analysis.
KeywordsImmunoassay Biotinylated scFv Magnetic beads Amantadine Chicken
This work was supported by grants from the National Natural Science Foundation of China (No. 31472236 and 31672600).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Human and/or animal participation
This article does not contain any studies with human participants or animals performed by any of the authors.
- 2.Belshe RB, Smith MH, Hall CB, Betts R, Hay AJ. Genetic-basis of resistance to Rimantadine emerging during treatment of influenza-virus infection. J Virol. 1988;62:1508–12.Google Scholar
- 3.Gasparini R, Amicizia D, Lai PL, Bragazzi NL, Panatto D. Compounds with anti-influenza activity: present and future of strategies for the optimal treatment and management of influenza. Part I: influenza life-cycle and currently available drugs. J Prev Med Hyg. 2014;55:69–85.Google Scholar
- 4.Tsuruoka Y, Nakajima T, Kanda M, Hayashi H, Matsushima Y, Yoshikawa S, et al. Simultaneous determination of amantadine, rimantadine, and memantine in processed products, chicken tissues, and eggs by liquid chromatography with tandem mass spectrometry. J Chromatogr B. 2017;1044:142–8. https://doi.org/10.1016/j.jchromb.2017.01.014.CrossRefGoogle Scholar
- 7.Stumph MJ, Noall MW, Knight V. Gas-chromatographic determination of amantadine in human-urine. Clin Chem. 1980;26:295–6.Google Scholar
- 8.Peng D, Wei W, Pan Y, Wang Y, Chen D, Liu Z, et al. Preparation of a monoclonal antibody against amantadine and rimantadine and development of an indirect competitive enzyme-linked immunosorbent assay for detecting the same in chicken muscle and liver. J Pharmaceut Biomed. 2017;133:56–63. https://doi.org/10.1016/j.jpba.2016.11.009.CrossRefGoogle Scholar
- 12.Xu Z, Dong J, Wang H, Li Z, Beier RC, Jiang Y, et al. Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay. J Agric Food Chem. 2012;60:5076–83. https://doi.org/10.1021/jf300570q.CrossRefGoogle Scholar
- 20.Dong J, Li Z, Lei H, Sun Y, Ducancel F, Xu Z, et al. Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork. Anal Chim Acta. 2012;736:85–91. https://doi.org/10.1016/j.aca.2012.05.033.CrossRefGoogle Scholar
- 21.Wang Z, Wen K, Zhang X, Li X, Wang Z, Shen J, et al. New Hapten synthesis, antibody production, and indirect competitive enzyme-linked immunosorbent assay for amantadine in chicken muscle. Food Anal Method. 2017, 2017–07-23; https://doi.org/10.1007/s12161-017-1000-5.
- 22.Froger A, Hall JE. Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp : JoVE. 2007:253. doi: 10.3791/253. PubMed ID:18997900.Google Scholar
- 29.Wu H, Wang J, Yang H, Li G, Zeng Y, Xia W, et al. Development and application of an in-cell cleanup pressurized liquid extraction with ultra-high-performance liquid chromatography-tandem mass spectrometry to detect prohibited antiviral agents sensitively in livestock and poultry feces. J Chromatogr A. 2017;1488:10–6. https://doi.org/10.1016/j.chroma.2017.01.070.CrossRefGoogle Scholar