Selection of Anti-Sulfadimidine Specific ScFvs from a Hybridoma Cell by Eukaryotic Ribosome Display

Yonghua Qi,Congming Wu,Lei Dai,Jianzhong Shen,Suxia Zhang,Kai Wen,Siyang Huang,Xingyuan Cao,Shaochen Wang,Yongning Wu,Zhanhui Wang,Lining Xia

doi:10.1371/journal.pone.0006427

Abstract

BackgroundRibosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes.Methodology/Principal FindingsIn our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM2) by using a ribosome library technology. A DNA library of scFv antibody fragments was constructed for ribosome display, and then mRNA–ribosome–antibody (MRA) complexes were produced by a rabbit reticulocyte lysate system. The synthetic sulfadimidine-ovalbumin (SM2-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning. After four rounds of ribosome display, the expression vector pCANTAB5E containing the selected specific scFv DNA was constructed and transformed into Escherichia coli HB2151. Three positive clones (SAS14, SAS68 and SAS71) were screened from 100 clones and had higher antibody activity and specificity to SM2 by indirect ELISA. The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis.Conclusions/SignificanceThe selection of anti-SM2 specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs.

Highlights

Sulfadimidine, derivatives of r-aminobenzenesulfonamide, is widely used in veterinary and human medicine for prophylactic and therapeutic purposes
Antibody library construction variable heavy (VH) and VL fragments were amplified by room temperature (RT)-PCR from hybridoma cell lines secreting anti-SM2 monoclonal antibodies (MAb) and assembled into full-length scFvs library with the (Gly4Ser)3-linker sequence
The selected specific scFv fragment based on mRNA of retained MRA complexes bound to SM2-OVA at the plate wells was recovered after several washing with increasing stringency during the individual rounds of selection by in situ RTPCR and single-primer PCR (SP-PCR)

Summary

Introduction

Sulfadimidine, derivatives of r-aminobenzenesulfonamide, is widely used in veterinary and human medicine for prophylactic and therapeutic purposes. It is used as additive of animal feed due to their growth promotion properties. The monitoring programs, especially immunochemical screening methods have been widely used to evaluate antibiotics derived residues in food matrixes. Current conventional methods for the analysis of sulfonamides derived residue are microbiological tests and analytical methods, such as thin-layer chromatography or high-performance liquid chromatography. These methods require well equipped laboratory, trained personnels, high capital expenditure and time-consuming sample preparation steps. Ribosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes

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Results

Conclusion