Production and characterization of a single-chain Fv antibody–alkaline phosphatase fusion protein specific for ampicillin

Abstract

Anti-AMP scFv were prepared by cloning variable region genes (VH and VL) from hybridoma cell line which secretes antibodies against AMP, and phoA gene was cloned from Escherichia coli strain K12 chromosomal DNA. The resulting scFv and phoA gene were inserted into the expression vector pBV220 to express the scFv-AP fusion protein in E. coli strain BL21. The purified scFv-AP fusion protein was used to develop an ic-CLEIA method for detection of AMP. The limit of detection (IC 15) was 0.11 ± 0.004 ng/mL and the IC 50 was 0.75 ± 0.04 ng/mL, respectively. The assay was 3 times as sensitive as the corresponding ic-ELISA based on the same fusion protein. IC-CLEIA was used to analyse AMP spiked milk samples, and the validation was confirmed by HPLC. Good correlation between ic-CLEIA and HPLC data was obtained (R 2 = 0.9934). It indicated that the developed method can apply to effectively monitor food safety.

Expression, purification, and study on the efficiency of a new potent recombinant scFv antibody against the SARS-CoV-2 spike RBD in E. coli BL21

“…after the promoter induction. Finally, pellets were harvested by centrifugation of each sample at 7000 rpm, 10min at 22 °C, and were processed for further studies [ 18 , 19 ].…”

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).…”

Temperature-Controlled Expression of a Recombinant Human-like Collagen I Peptide in Escherichia coli

“…To achieve better production of recombinant human collagen for various applications, it would be an optimized alternative strategy to express functional fragments of human collagen, according to the results described above, we synthesized a DNA fragment encoding a functional section of type I human collagen and then cloned it into a temperature-controlled expression vector, pBV220 which has been used over the past two decades for protein expression in E. coli [ 20 , 21 , 22 , 23 ].…”