Selection of anti-sulfadimidine specific ScFvs from a hybridoma cell by eukaryotic ribosome display.
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.


RESULTS
In our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM(2)) 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 (SM(2)-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 SM(2) 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
The selection of anti-SM(2) specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs.
Qi
Yonghua
Y
National Center for Veterinary Drug Safety Evaluation, College of Veterinary Medicine, China Agricultural University, Beijing, China.
Wu
Congming
C
Zhang
Suxia
S
Wang
Zhanhui
Z
Huang
Siyang
S
Dai
Lei
L
Wang
Shaochen
S
Xia
Lining
L
Wen
Kai
K
Cao
Xingyuan
X
Wu
Yongning
Y
Shen
Jianzhong
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006825:Hybridomas / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000276:immunology*; D017386:Sequence Homology, Amino Acid; D013418:Sulfamethazine / Q000494:pharmacology*; D014158:Transcription, Genetic
D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D013418:Sulfamethazine