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

Qi Y, Wu C, Zhang S, Wang Z, Huang S, Dai L, Wang S, Xia L, Wen K, Cao X, Wu Y, Shen J - PLoS ONE (2009)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: National Center for Veterinary Drug Safety Evaluation, College of Veterinary Medicine, China Agricultural University, Beijing, China.

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.

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.

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.

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Related in: MedlinePlus

Agarose gel electrophoresis of amplified VH chain, VL chain and assembled scFv fragments.Lane M: 100 bp plus DNA marker, lane 1: VH fragments, lane 2: VL fragments, lane3: about 800 bp assembled scFv fragments.
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pone-0006427-g001: Agarose gel electrophoresis of amplified VH chain, VL chain and assembled scFv fragments.Lane M: 100 bp plus DNA marker, lane 1: VH fragments, lane 2: VL fragments, lane3: about 800 bp assembled scFv fragments.

Mentions: VH and VL fragments were amplified by RT-PCR from hybridoma cell lines secreting anti-SM2 MAb and assembled into full-length scFvs library with the (Gly4Ser)3-linker sequence. The amplified VH and VL fragments were the expected size (about 340 bp and 325 bp). (Fig. 1). The assembled approximate 0.8 kb full-length scFv fragments were used for the construction of templates of ribosome display.


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

Qi Y, Wu C, Zhang S, Wang Z, Huang S, Dai L, Wang S, Xia L, Wen K, Cao X, Wu Y, Shen J - PLoS ONE (2009)

Agarose gel electrophoresis of amplified VH chain, VL chain and assembled scFv fragments.Lane M: 100 bp plus DNA marker, lane 1: VH fragments, lane 2: VL fragments, lane3: about 800 bp assembled scFv fragments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2712767&req=5

pone-0006427-g001: Agarose gel electrophoresis of amplified VH chain, VL chain and assembled scFv fragments.Lane M: 100 bp plus DNA marker, lane 1: VH fragments, lane 2: VL fragments, lane3: about 800 bp assembled scFv fragments.
Mentions: VH and VL fragments were amplified by RT-PCR from hybridoma cell lines secreting anti-SM2 MAb and assembled into full-length scFvs library with the (Gly4Ser)3-linker sequence. The amplified VH and VL fragments were the expected size (about 340 bp and 325 bp). (Fig. 1). The assembled approximate 0.8 kb full-length scFv fragments were used for the construction of templates of ribosome display.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: National Center for Veterinary Drug Safety Evaluation, College of Veterinary Medicine, China Agricultural University, Beijing, China.

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.

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.

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.

Show MeSH
Related in: MedlinePlus