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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

Selection and amplification of anti-SM2 scFv gene over four rounds of ribosome display.After selection, the selected product was amplified by SP-PCR and the SP-PCR products were analyzed by agarose gel electrophoresis. lane M: DL2000 DNA marker, lane 1: preliminary translation mixture selected on a PBS-coated well, lane 2: translation mixture selected on a OVA-coated well, lane 3: recovered band from the first ribosome display, lane 4: recovered band from the second round, lane 5: recovered band from the third cycle, lane 6: recovered band from the fourth selected library.
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pone-0006427-g002: Selection and amplification of anti-SM2 scFv gene over four rounds of ribosome display.After selection, the selected product was amplified by SP-PCR and the SP-PCR products were analyzed by agarose gel electrophoresis. lane M: DL2000 DNA marker, lane 1: preliminary translation mixture selected on a PBS-coated well, lane 2: translation mixture selected on a OVA-coated well, lane 3: recovered band from the first ribosome display, lane 4: recovered band from the second round, lane 5: recovered band from the third cycle, lane 6: recovered band from the fourth selected library.

Mentions: The gel-purified scFv fragments were digested and ligated into the vector pRDV. The ligation product was directly used as a template for the amplification of initial ribosome display library. The original scFv library was subjected to in vitro transcription and translation using TNT T7 Quick for PCR DNA kit to generate ternary MRA complexes. The target antigens were immobilized on microtiter plates. The selected specific scFv fragment based on mRNA of retained MRA complexes bound to SM2-OVA at the plate wells was recovered after several washing with increasing stringency during the individual rounds of selection by in situ RT-PCR and SP-PCR. The obtained products were applied to the affinity maturation or next round of panning. Each cycle of ribosome display was performed under the same conditions including the concentration of target antigens and the spanning washing time. As a whole, four cycles of selection on SM2-OVA, as well as one round of affinity maturation were performed. The panning progress was monitored by examining the intensity of SP-PCR products (approximately 1.1 kb) on agarose gel–electrophoresis. The quantity of SP-PCR products continually increased during the next rounds of panning. Based on the result of SP-PCR, enrichment of specific scFvs was clearly confirmed. Meanwhile, no band was observed PBS-coated wells or when the untranslated mixture was used. (Fig. 2).


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)

Selection and amplification of anti-SM2 scFv gene over four rounds of ribosome display.After selection, the selected product was amplified by SP-PCR and the SP-PCR products were analyzed by agarose gel electrophoresis. lane M: DL2000 DNA marker, lane 1: preliminary translation mixture selected on a PBS-coated well, lane 2: translation mixture selected on a OVA-coated well, lane 3: recovered band from the first ribosome display, lane 4: recovered band from the second round, lane 5: recovered band from the third cycle, lane 6: recovered band from the fourth selected library.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2712767&req=5

pone-0006427-g002: Selection and amplification of anti-SM2 scFv gene over four rounds of ribosome display.After selection, the selected product was amplified by SP-PCR and the SP-PCR products were analyzed by agarose gel electrophoresis. lane M: DL2000 DNA marker, lane 1: preliminary translation mixture selected on a PBS-coated well, lane 2: translation mixture selected on a OVA-coated well, lane 3: recovered band from the first ribosome display, lane 4: recovered band from the second round, lane 5: recovered band from the third cycle, lane 6: recovered band from the fourth selected library.
Mentions: The gel-purified scFv fragments were digested and ligated into the vector pRDV. The ligation product was directly used as a template for the amplification of initial ribosome display library. The original scFv library was subjected to in vitro transcription and translation using TNT T7 Quick for PCR DNA kit to generate ternary MRA complexes. The target antigens were immobilized on microtiter plates. The selected specific scFv fragment based on mRNA of retained MRA complexes bound to SM2-OVA at the plate wells was recovered after several washing with increasing stringency during the individual rounds of selection by in situ RT-PCR and SP-PCR. The obtained products were applied to the affinity maturation or next round of panning. Each cycle of ribosome display was performed under the same conditions including the concentration of target antigens and the spanning washing time. As a whole, four cycles of selection on SM2-OVA, as well as one round of affinity maturation were performed. The panning progress was monitored by examining the intensity of SP-PCR products (approximately 1.1 kb) on agarose gel–electrophoresis. The quantity of SP-PCR products continually increased during the next rounds of panning. Based on the result of SP-PCR, enrichment of specific scFvs was clearly confirmed. Meanwhile, no band was observed PBS-coated wells or when the untranslated mixture was used. (Fig. 2).

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