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Characterization of Anti-Citrinin Specific ScFvs Selected from Non-Immunized Mouse Splenocytes by Eukaryotic Ribosome Display.

Cheng H, Chen Y, Yang Y, Chen X, Guo X, Du A - PLoS ONE (2015)

Bottom Line: The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA.These specific scFvs offer a potential novel immunoassay method for CIT residues.This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display.

View Article: PubMed Central - PubMed

Affiliation: Institute of Preventive Veterinary Medicine & Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, China.

ABSTRACT
Single chain variable fragments (scFvs) against citrinin (CIT) were selected from a scFv library constructed from the splenocytes of non-immunized mice by an improved eukaryotic ribosome display technology in this study. Bovine serum albumin (BSA)/ CIT-BSA and ovalbumin (OVA)/ CIT-OVA were used as the antigens to select specific anti-CIT scFvs. Eukaryotic in situ RT-PCR method was used to recover the selected mRNA after every affinity selection. After six rounds of ribosome display, expression vector pTIG-TRX carrying specific scFv DNAs were constructed and transformed into Escherichia coli BL21 (DE3) for protein expression. Thirteen positive clones were selected out of which three (designated 23, 68 and 109) showed high binding activity and specificity to CIT by indirect ELISA, while no clone showed binding activity with carrier proteins. The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA. These specific scFvs offer a potential novel immunoassay method for CIT residues. This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display.

No MeSH data available.


Related in: MedlinePlus

Agarose gel electrophoresis of the selected anti-CIT scFv genes over six rounds of ribosome display.After selection, the mRNA was amplified by in situ RT-PCR and the products were analyzed by agarose gel electrophoresis. A: The selected anti-CIT scFv genes over ribosome display. Lane 1: The selected anti-CIT scFv genes over the first round of ribosome display; Lane 2: The selected anti-CIT scFv genes over the second round of ribosome display; Lane 3: The selected anti-CIT scFv genes over the third round of ribosome display; Lane M: 100bp DNA ladder marker. B: The selected anti-CIT scFv genes over ribosome display. Lane 4: The selected anti-CIT scFv genes over the fourth round of ribosome display; Lane 5: The selected anti-CIT scFv genes over the fifth round of ribosome display; Lane 6: The selected anti-CIT scFv genes over the sixth round of ribosome display; Lane M: 100bp DNA ladder marker.
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pone.0131482.g007: Agarose gel electrophoresis of the selected anti-CIT scFv genes over six rounds of ribosome display.After selection, the mRNA was amplified by in situ RT-PCR and the products were analyzed by agarose gel electrophoresis. A: The selected anti-CIT scFv genes over ribosome display. Lane 1: The selected anti-CIT scFv genes over the first round of ribosome display; Lane 2: The selected anti-CIT scFv genes over the second round of ribosome display; Lane 3: The selected anti-CIT scFv genes over the third round of ribosome display; Lane M: 100bp DNA ladder marker. B: The selected anti-CIT scFv genes over ribosome display. Lane 4: The selected anti-CIT scFv genes over the fourth round of ribosome display; Lane 5: The selected anti-CIT scFv genes over the fifth round of ribosome display; Lane 6: The selected anti-CIT scFv genes over the sixth round of ribosome display; Lane M: 100bp DNA ladder marker.

Mentions: The progress of selection was monitored by estimating the enrichment of PCR products on agarose gel-electrophoresis. After the first round of selection, in situ RT-PCR recovery was performed in the wells carrying selected ARM complexes using the primer D2. A weak DNA band was observed (Fig 7). Six rounds of affinity selection were performed and primer D2 was used in the first and fourth rounds, primer D3 was used in the second and fifth rounds while primer D4 was used in the third and sixth rounds. Although the use of primer D2, D3, D4 in the in situ reverse transcription reaction would result in getting progressively shorter DNA fragments, the shortening only affects the spacer domain of the Cκ chain. A full-length library was regenerated by SOE-PCR after the third cycle [37]. The enrichment of PCR products continued during the next rounds of panning (Fig 7).


Characterization of Anti-Citrinin Specific ScFvs Selected from Non-Immunized Mouse Splenocytes by Eukaryotic Ribosome Display.

Cheng H, Chen Y, Yang Y, Chen X, Guo X, Du A - PLoS ONE (2015)

Agarose gel electrophoresis of the selected anti-CIT scFv genes over six rounds of ribosome display.After selection, the mRNA was amplified by in situ RT-PCR and the products were analyzed by agarose gel electrophoresis. A: The selected anti-CIT scFv genes over ribosome display. Lane 1: The selected anti-CIT scFv genes over the first round of ribosome display; Lane 2: The selected anti-CIT scFv genes over the second round of ribosome display; Lane 3: The selected anti-CIT scFv genes over the third round of ribosome display; Lane M: 100bp DNA ladder marker. B: The selected anti-CIT scFv genes over ribosome display. Lane 4: The selected anti-CIT scFv genes over the fourth round of ribosome display; Lane 5: The selected anti-CIT scFv genes over the fifth round of ribosome display; Lane 6: The selected anti-CIT scFv genes over the sixth round of ribosome display; Lane M: 100bp DNA ladder marker.
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Related In: Results  -  Collection

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pone.0131482.g007: Agarose gel electrophoresis of the selected anti-CIT scFv genes over six rounds of ribosome display.After selection, the mRNA was amplified by in situ RT-PCR and the products were analyzed by agarose gel electrophoresis. A: The selected anti-CIT scFv genes over ribosome display. Lane 1: The selected anti-CIT scFv genes over the first round of ribosome display; Lane 2: The selected anti-CIT scFv genes over the second round of ribosome display; Lane 3: The selected anti-CIT scFv genes over the third round of ribosome display; Lane M: 100bp DNA ladder marker. B: The selected anti-CIT scFv genes over ribosome display. Lane 4: The selected anti-CIT scFv genes over the fourth round of ribosome display; Lane 5: The selected anti-CIT scFv genes over the fifth round of ribosome display; Lane 6: The selected anti-CIT scFv genes over the sixth round of ribosome display; Lane M: 100bp DNA ladder marker.
Mentions: The progress of selection was monitored by estimating the enrichment of PCR products on agarose gel-electrophoresis. After the first round of selection, in situ RT-PCR recovery was performed in the wells carrying selected ARM complexes using the primer D2. A weak DNA band was observed (Fig 7). Six rounds of affinity selection were performed and primer D2 was used in the first and fourth rounds, primer D3 was used in the second and fifth rounds while primer D4 was used in the third and sixth rounds. Although the use of primer D2, D3, D4 in the in situ reverse transcription reaction would result in getting progressively shorter DNA fragments, the shortening only affects the spacer domain of the Cκ chain. A full-length library was regenerated by SOE-PCR after the third cycle [37]. The enrichment of PCR products continued during the next rounds of panning (Fig 7).

Bottom Line: The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA.These specific scFvs offer a potential novel immunoassay method for CIT residues.This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display.

View Article: PubMed Central - PubMed

Affiliation: Institute of Preventive Veterinary Medicine & Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, China.

ABSTRACT
Single chain variable fragments (scFvs) against citrinin (CIT) were selected from a scFv library constructed from the splenocytes of non-immunized mice by an improved eukaryotic ribosome display technology in this study. Bovine serum albumin (BSA)/ CIT-BSA and ovalbumin (OVA)/ CIT-OVA were used as the antigens to select specific anti-CIT scFvs. Eukaryotic in situ RT-PCR method was used to recover the selected mRNA after every affinity selection. After six rounds of ribosome display, expression vector pTIG-TRX carrying specific scFv DNAs were constructed and transformed into Escherichia coli BL21 (DE3) for protein expression. Thirteen positive clones were selected out of which three (designated 23, 68 and 109) showed high binding activity and specificity to CIT by indirect ELISA, while no clone showed binding activity with carrier proteins. The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA. These specific scFvs offer a potential novel immunoassay method for CIT residues. This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display.

No MeSH data available.


Related in: MedlinePlus