Selection of Anti-Sulfadimidine Specific ScFvs from a Hybridoma Cell by Eukaryotic Ribosome DisplayReportar como inadecuado




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Background

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.

Methodology-Principal Findings

In 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-Significance

The 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.



Autor: Yonghua Qi, Congming Wu, Suxia Zhang, Zhanhui Wang, Siyang Huang, Lei Dai, Shaochen Wang, Lining Xia, Kai Wen, Xingyuan Cao, Yong

Fuente: http://plos.srce.hr/



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