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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

Alignment of the amino acid sequence of selected specific anti-SM2 scFvs.CDRs and the (Gly4Ser)3 linker are boxed. The regions of CDR1-CDR3 were deduced according to Kabat database.
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pone-0006427-g005: Alignment of the amino acid sequence of selected specific anti-SM2 scFvs.CDRs and the (Gly4Ser)3 linker are boxed. The regions of CDR1-CDR3 were deduced according to Kabat database.

Mentions: The deduced amino acid sequences of the above-mentioned three scFvs and complementary determining regions (CDR) were shown in Fig. 5. By using the DNA sequences, the VH and VL gene families of the scFvs were designated based on Werner Müller's database (DNAPLOT software). The heavy chains of scFvs SAS68 and SAS71 belong to the VH1 gene family and that of SAS14 belongs to the VH2 gene family. The light chains of clones SAS68 and SAS71 belong to the Vk IGKV12/13 subgroup and that of SAS14 belongs to the Vk IGKV4/5 subgroup. Sequencing alignment by the Vector NTI software program showed that the VH of the clones SAS68 and SAS71 have very similar sequences (90.44% homology) and the VL of SAS14, SAS68 and SAS71 shared 82.81% homology. Although the linking sequence of scFv SAS71 showed some mutations, these did not influence protein structure obviously. This indicated that a random mutation was induced by the second ribosome display, but mutation affected scFv structures were selected. Sequencing data indicated that we succeeded in achieving our desired goals of library generation with the affinity maturation method.


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)

Alignment of the amino acid sequence of selected specific anti-SM2 scFvs.CDRs and the (Gly4Ser)3 linker are boxed. The regions of CDR1-CDR3 were deduced according to Kabat database.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006427-g005: Alignment of the amino acid sequence of selected specific anti-SM2 scFvs.CDRs and the (Gly4Ser)3 linker are boxed. The regions of CDR1-CDR3 were deduced according to Kabat database.
Mentions: The deduced amino acid sequences of the above-mentioned three scFvs and complementary determining regions (CDR) were shown in Fig. 5. By using the DNA sequences, the VH and VL gene families of the scFvs were designated based on Werner Müller's database (DNAPLOT software). The heavy chains of scFvs SAS68 and SAS71 belong to the VH1 gene family and that of SAS14 belongs to the VH2 gene family. The light chains of clones SAS68 and SAS71 belong to the Vk IGKV12/13 subgroup and that of SAS14 belongs to the Vk IGKV4/5 subgroup. Sequencing alignment by the Vector NTI software program showed that the VH of the clones SAS68 and SAS71 have very similar sequences (90.44% homology) and the VL of SAS14, SAS68 and SAS71 shared 82.81% homology. Although the linking sequence of scFv SAS71 showed some mutations, these did not influence protein structure obviously. This indicated that a random mutation was induced by the second ribosome display, but mutation affected scFv structures were selected. Sequencing data indicated that we succeeded in achieving our desired goals of library generation with the affinity maturation method.

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