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Selection of anti-sulfadimidine specific ScFvs from a hybridoma cell by eukaryotic ribosome display.
- Source :
-
PloS one [PLoS One] 2009 Jul 29; Vol. 4 (7), pp. e6427. Date of Electronic Publication: 2009 Jul 29. - Publication Year :
- 2009
-
Abstract
- 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.<br />Methodology/principal Findings: 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.<br />Conclusions/significance: 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.
- Subjects :
- Amino Acid Sequence
Animals
Base Sequence
Blotting, Western
DNA Primers
Escherichia coli genetics
Immunoglobulin Variable Region genetics
Molecular Sequence Data
Polymerase Chain Reaction
Protein Biosynthesis
Rabbits
Reverse Transcriptase Polymerase Chain Reaction
Sequence Homology, Amino Acid
Transcription, Genetic
Hybridomas immunology
Immunoglobulin Variable Region immunology
Ribosomes immunology
Sulfamethazine pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 4
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 19641611
- Full Text :
- https://doi.org/10.1371/journal.pone.0006427