Magnetic-assisted biotinylated single-chain variable fragment antibody-based immunoassay for amantadine detection in chicken

Abstract

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. Graphical abstract ᅟ.

Development of Fluorescence Polarization Immunoassay With scFv to Detect Fumonisin Bs in Maize and Simultaneous Study of Their Molecular Recognition Mechanism

“…It is often difficult to obtain scFvs that can maintain the same specificity as the maternal mAbs. Some previous articles reported that scFv antibodies showed similar or better performances than that of the parental mAbs ( Wen et al, 2012 ), but other articles reported that scFv antibodies performed worse than the parental mAbs ( Xie et al, 2018 ). Min et al amplified the anti-FB 1 single-stranded antibody gene on the basis of anti-FB 1 hybridoma cells and converted it to E. coli to obtain anti-FB 1 single-stranded antibody with the an IC 50 reducing 12 times approximately 2000 ng/ml compared with the original mAb ( Min et al, 2010 ).…”

Preparation of Ractopamine Single-Chain Variable Fragment and Development of icELISA Based on Immunomagnetic Beads

“…IMBs can quickly separate samples from the matrix, preserve the biological activity and integrity of samples, and reduce the complex pretreatment steps in the instrument detection method, thereby improving the detection efficiency. In 2018, Xie et al 42 established a highly sensitive ELISA assay based on MB and BAS based on biotinylated scFv and measured an IC 50 of 1.93 ng/mL for amantadine for actual chicken meat sample detection. Enzyme-catalyzed biotin-labeled scFv can only be linked by one biotin to one scFv and cannot amplify the signal well; therefore, in this study, chemobiotin-labeled RAC-scFv was used and directly conjugated with streptavidin beads, and the selected bead size was 1 μm, which has good dispersion stability and facilitates the detection of small molecules.…”

Single‐chain fragment variable produced by phage display technology: Construction, selection, mutation, expression, and recent applications in food safety

“…Wang et al, 2010), fluoroquinolones (Tao et al, 2013;Wang, Dong, et al, 2016;Wen et al, 2012), β-lactams (X. He et al, 2017;He, Wei, et al, 2019;Liu, Zhang, et al, 2016), and others (Dong et al, 2021;Li, Hou, et al, 2021;Xie et al, 2018) (Table 3).…”

“…In recent years, with the support of phage display that provides a powerful tool for the development of scFvs in veterinary drug immunoassays, scFvs against a variety of veterinary drugs have been screened, including β‐agonists (J. X. Dong et al., 2012; Dong, Xie, et al., 2014; Lee et al., 2018; Lu et al., 2020; H. Wang et al., 2010), fluoroquinolones (Tao et al., 2013; Wang, Dong, et al., 2016; Wen et al., 2012), β‐lactams (X. He et al., 2017; He, Wei, et al., 2019; Liu, Zhang, et al., 2016), and others (Dong et al., 2021; C. Li et al., 2016; Li, Hou, et al., 2021; Xie et al., 2018) (Table 3).…”