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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

Detection of soluble scFvs in periplasmatic extracts by Western Blot.M: low molecular weight protein marker, lanes1–3: Periplasmatic extracts of scFvs SAS14, SAS68, and SAS71.
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pone-0006427-g004: Detection of soluble scFvs in periplasmatic extracts by Western Blot.M: low molecular weight protein marker, lanes1–3: Periplasmatic extracts of scFvs SAS14, SAS68, and SAS71.

Mentions: The selected specific scFv fragment after the fourth round was ligated with the expression vector pCANTAB5E for soluble scFvs expression. After electrotransformation, around 100 colonies from the selected library were isolated and soluble proteins of these clones were expressed using the nonsuppressor strain E.coli HB2151. The periplasmic extracts from individual clones were tested by indirect ELISA. The result showed that few clones showed positive to SM2 in ribosome-ELISA before panning (Fig. S1), however, several clones from the fourth selected library had a good conjugation activity to specific SM2-OVA. Among these clones, the three clones (SAS14, SAS68, SAS71) exhibiting the highest ELISA signals to SM2-OVA were not significantly different to the anti-SM2 MAb (Fig. 3). The result suggested that the conjugation activity of scFvs was similar to that of parent MAb. The three specific soluble scFvs were confirmed by SDS-PAGE and Western-blotting analysis using anti-E tag antibodies. Approximately 30 kDa scFv protein was expressed from each of the three selected clones in the periplasmic extract sample compared to negative control (Fig. 4).


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)

Detection of soluble scFvs in periplasmatic extracts by Western Blot.M: low molecular weight protein marker, lanes1–3: Periplasmatic extracts of scFvs SAS14, SAS68, and SAS71.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006427-g004: Detection of soluble scFvs in periplasmatic extracts by Western Blot.M: low molecular weight protein marker, lanes1–3: Periplasmatic extracts of scFvs SAS14, SAS68, and SAS71.
Mentions: The selected specific scFv fragment after the fourth round was ligated with the expression vector pCANTAB5E for soluble scFvs expression. After electrotransformation, around 100 colonies from the selected library were isolated and soluble proteins of these clones were expressed using the nonsuppressor strain E.coli HB2151. The periplasmic extracts from individual clones were tested by indirect ELISA. The result showed that few clones showed positive to SM2 in ribosome-ELISA before panning (Fig. S1), however, several clones from the fourth selected library had a good conjugation activity to specific SM2-OVA. Among these clones, the three clones (SAS14, SAS68, SAS71) exhibiting the highest ELISA signals to SM2-OVA were not significantly different to the anti-SM2 MAb (Fig. 3). The result suggested that the conjugation activity of scFvs was similar to that of parent MAb. The three specific soluble scFvs were confirmed by SDS-PAGE and Western-blotting analysis using anti-E tag antibodies. Approximately 30 kDa scFv protein was expressed from each of the three selected clones in the periplasmic extract sample compared to negative control (Fig. 4).

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