Cloning, expression, and identification of anti‐carbofuran single chain Fv gene

Abstract

Phage display method was used to clone anti-carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF-specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly(4)Ser)(3). The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti-CBF antibody libraries were then constructed. After one round of panning with CBF-ovalbumin (CBF-OVA) as a conjugate, antigen-binding positive recombinant phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS-PAGE and showed HRP-anti-E-tag antibody-recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC(50) value of 1.07 ng/mL. The cross reactivity studies showed that the anti-CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF.

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

“…Most RAb's that have been synthesized from hybridoma cell lines were reported to show characteristics similar to those of their parent mAb's, , while some reported RAb's had altered analytical properties. , The possible reasons for differences between the binding characteristics of the mAb secreted by a hybridoma cell line and those of the corresponding derived RAb were discussed by Kramer et al Using the fusion protein can reduce the time required for immunochemical detection, since the bifunctional fusion protein used in the cdELISA FP omits the use of an enzyme-labeled secondary antibody. The entire incubation time for the cdELISA FP was 80 min, while the incubation time would be 130 min or longer , for a competitive indirect ELISA based on using a traditional PAb or mAb.…”

“…The digested products were purified with a PCR product purification kit. The purified PCR products were then further digested with 1 μL of Not I, 2.5 μL of the provided buffer, 2 μL of 0.1% BSA, and 2 μL of 0.1% Triton X-100, aseptic purified water was added to a final volume of 25 μL, and the mixture was incubated at 37 °C for 3 h. The digested products were purified with a PCR product purification kit and then ligated using the T4 DNA ligation enzyme . The digested scFv (about 60 ng) was combined with the digested pLIP6/GN (about 100 ng), and the mix contained 2 μL of the provided buffer and 1 μL of T4 DNA ligation enzyme.…”

“…The purified PCR products were then further digested with 1 μL of NotI, 2.5 μL of the provided buffer, 2 μL of 0.1% BSA, and 2 μL of 0.1% Triton X-100, aseptic purified water was added to a final volume of 25 μL, and the mixture was incubated at 37 °C for 3 h. The digested products were purified with a PCR product purification kit and then ligated using the T4 DNA ligation enzyme. 15 The digested scFv (about 60 ng) was combined with the digested pLIP6/GN (about 100 ng), and the mix contained 2 μL of the provided buffer and 1 μL of T4 DNA ligation enzyme. Aseptic purified water was added to a final volume of 10 μL, and the mixture was incubated at 22 °C for 1 h. The ligation products were transformed into E. coli strain XL1-Blue competent cells.…”

Generation of functional single-chain fragment variable from hybridoma and development of chemiluminescence enzyme immunoassay for determination of total malachite green in tilapia fish

“…However, due to the complexity of hybridomas (Bradbury, Trinklein, Thie, Wilkinson, Tandon, Anderson, et al, 2018), failure in developing scFv has been most recently encountered in our laboratory. Attempts, such as phage display biopanning (Pan, Wang, Zhang, Liu, Lei, Huang, et al, 2006;Wang, Yang, Liu, Liang, Lei, Shen, et al, 2009) and mass spectrometry-based DNA sequence analysis (Du, Zhou, Li, Sheng, Ducancel, & Wang, 2016;X. Y. Zhang, He, Zhao, Wang, & Jin, 2016) were proposed to avoid such pitfalls.…”

Application of Immunoassays for Rapid Monitor of Carbofuran Residue in Vegetables

“…Enzyme-linked immunosorbent assay (ELISA) is regarded as a low-cost, rapid, and sensitive method, which has a very extensive usage in the detection of environmental and food contaminants (And & James, 1996). In 1997, Abad, Moreno, and Montoya (1997) prepared the first carbofuran monoclonal antibody (mAb) and developed an indirect ELISA, and subsequently many researchers (Abad et al, 1999;Jourdan, Scutellaro, Fleeker, Herzog, & Rubio, 1995;Mickova et al, 2003;Wang et al, 2009;Yao, Liu, Song, Hua, & Xu, 2017;Zhu et al, 2008) developed immunoassays for carbofuran residue detection. The IC 50 value ranged from 0.3 to 36.1 ng/mL.…”