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VHH phage-based competitive real-time immuno-polymerase chain reaction for ultrasensitive detection of ochratoxin A in cereal.

Liu X, Xu Y, Xiong YH, Tu Z, Li YP, He ZY, Qiu YL, Fu JH, Gee SJ, Hammock BD - Anal. Chem. (2014)

Bottom Line: The clone VHH-28 resulted in the lowest 50% inhibitory concentration of 0.31 ng/mL in the phage enzyme-linked immunosorbent assay (ELISA) and was selected to develop the VHH phage-based real-time immuno-PCR (RT-IPCR).This method was compared with conventional ELISA, and validation results indicated the reliability of VHH phage-based RT-IPCR in the detection of OTA in cereal samples.This study provides a new idea for the ultrasensitive detection of mycotoxins and other toxic small molecular weight compounds.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Food Science and Technology and ‡Sino-Germany Joint Research Institute, Nanchang University , 235 Nanjing East Road, Nanchang 330047, People's Republic of China.

ABSTRACT
Phage display-mediated immuno-polymerase chain reaction (PD-IPCR) is an ultrasensitive detection technology that combines the advantages of immuno-PCR and phage display. The phage particle, which displayed antibody fragments including single-chain fragment variable (scFv), variable domain of heavy-chain antibodies (VHH), and antigen-binding fragment (Fab) on the surface can be directly used in IPCR, supplying both the detection antibody and deoxyribonucleic acid (DNA) template. In this work, we used ochratoxin A (OTA) as a model system to study the capacity of PD-IPCR in the detection of toxic small molecular weight compounds, especially mycotoxins. An alpaca-derived VHH library was constructed and subjected to four cycles of panning. In total, 16 clones with four unique sequences were selected by competitive binding with OTA. The clone VHH-28 resulted in the lowest 50% inhibitory concentration of 0.31 ng/mL in the phage enzyme-linked immunosorbent assay (ELISA) and was selected to develop the VHH phage-based real-time immuno-PCR (RT-IPCR). The detection limit of the VHH phage-based RT-IPCR was 3.7 pg/L, with a linear range of 0.01-1000 pg/mL. This method was compared with conventional ELISA, and validation results indicated the reliability of VHH phage-based RT-IPCR in the detection of OTA in cereal samples. This study provides a new idea for the ultrasensitive detection of mycotoxins and other toxic small molecular weight compounds.

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Cross-reactivity of VHH phage-based competitive RT-IPCR. The 96-wellPCR plates were coated with 20 μL/well of OTA–OVA conjugate(4 μg/mL). Serial 10-fold dilutions of (a) ochratoxin A or (b–f)tested compounds (ochratoxin B, fumonisin B1, deoxynivalenol,aflatoxin B1, and zearalenone) in 2.5% methanol–PBSwere mixed with an equal volume of VHH-28 (4 × 109 cfu/mL) in PBS. The mixture (20 μL/well) was then added towells and incubated at 37 °C for 1 h. The bound phage were detectedby RT-PCR.
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fig5: Cross-reactivity of VHH phage-based competitive RT-IPCR. The 96-wellPCR plates were coated with 20 μL/well of OTA–OVA conjugate(4 μg/mL). Serial 10-fold dilutions of (a) ochratoxin A or (b–f)tested compounds (ochratoxin B, fumonisin B1, deoxynivalenol,aflatoxin B1, and zearalenone) in 2.5% methanol–PBSwere mixed with an equal volume of VHH-28 (4 × 109 cfu/mL) in PBS. The mixture (20 μL/well) was then added towells and incubated at 37 °C for 1 h. The bound phage were detectedby RT-PCR.

Mentions: The specificityof the assay was testedfor the phage clone VHH-28 by use of five common mycotoxins: ochratoxinB (OTB), fumonisin B1 (FB1), deoxynivalenol(DON), aflatoxin B1 (AB1), and zearalenone (ZEN)(Figure 5). The cross-reactivity of the assaywas obtained by comparing 50% inhibitory concentrations (IC50).32 Negligible cross-reactivity was observed,except for OTB, which showed 3.5% cross-reactivity, indicating theexcellent selectivity of VHH-28 in VHH phage-based competitive RT-IPCRfor OTA.


VHH phage-based competitive real-time immuno-polymerase chain reaction for ultrasensitive detection of ochratoxin A in cereal.

Liu X, Xu Y, Xiong YH, Tu Z, Li YP, He ZY, Qiu YL, Fu JH, Gee SJ, Hammock BD - Anal. Chem. (2014)

Cross-reactivity of VHH phage-based competitive RT-IPCR. The 96-wellPCR plates were coated with 20 μL/well of OTA–OVA conjugate(4 μg/mL). Serial 10-fold dilutions of (a) ochratoxin A or (b–f)tested compounds (ochratoxin B, fumonisin B1, deoxynivalenol,aflatoxin B1, and zearalenone) in 2.5% methanol–PBSwere mixed with an equal volume of VHH-28 (4 × 109 cfu/mL) in PBS. The mixture (20 μL/well) was then added towells and incubated at 37 °C for 1 h. The bound phage were detectedby RT-PCR.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306448&req=5

fig5: Cross-reactivity of VHH phage-based competitive RT-IPCR. The 96-wellPCR plates were coated with 20 μL/well of OTA–OVA conjugate(4 μg/mL). Serial 10-fold dilutions of (a) ochratoxin A or (b–f)tested compounds (ochratoxin B, fumonisin B1, deoxynivalenol,aflatoxin B1, and zearalenone) in 2.5% methanol–PBSwere mixed with an equal volume of VHH-28 (4 × 109 cfu/mL) in PBS. The mixture (20 μL/well) was then added towells and incubated at 37 °C for 1 h. The bound phage were detectedby RT-PCR.
Mentions: The specificityof the assay was testedfor the phage clone VHH-28 by use of five common mycotoxins: ochratoxinB (OTB), fumonisin B1 (FB1), deoxynivalenol(DON), aflatoxin B1 (AB1), and zearalenone (ZEN)(Figure 5). The cross-reactivity of the assaywas obtained by comparing 50% inhibitory concentrations (IC50).32 Negligible cross-reactivity was observed,except for OTB, which showed 3.5% cross-reactivity, indicating theexcellent selectivity of VHH-28 in VHH phage-based competitive RT-IPCRfor OTA.

Bottom Line: The clone VHH-28 resulted in the lowest 50% inhibitory concentration of 0.31 ng/mL in the phage enzyme-linked immunosorbent assay (ELISA) and was selected to develop the VHH phage-based real-time immuno-PCR (RT-IPCR).This method was compared with conventional ELISA, and validation results indicated the reliability of VHH phage-based RT-IPCR in the detection of OTA in cereal samples.This study provides a new idea for the ultrasensitive detection of mycotoxins and other toxic small molecular weight compounds.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Food Science and Technology and ‡Sino-Germany Joint Research Institute, Nanchang University , 235 Nanjing East Road, Nanchang 330047, People's Republic of China.

ABSTRACT
Phage display-mediated immuno-polymerase chain reaction (PD-IPCR) is an ultrasensitive detection technology that combines the advantages of immuno-PCR and phage display. The phage particle, which displayed antibody fragments including single-chain fragment variable (scFv), variable domain of heavy-chain antibodies (VHH), and antigen-binding fragment (Fab) on the surface can be directly used in IPCR, supplying both the detection antibody and deoxyribonucleic acid (DNA) template. In this work, we used ochratoxin A (OTA) as a model system to study the capacity of PD-IPCR in the detection of toxic small molecular weight compounds, especially mycotoxins. An alpaca-derived VHH library was constructed and subjected to four cycles of panning. In total, 16 clones with four unique sequences were selected by competitive binding with OTA. The clone VHH-28 resulted in the lowest 50% inhibitory concentration of 0.31 ng/mL in the phage enzyme-linked immunosorbent assay (ELISA) and was selected to develop the VHH phage-based real-time immuno-PCR (RT-IPCR). The detection limit of the VHH phage-based RT-IPCR was 3.7 pg/L, with a linear range of 0.01-1000 pg/mL. This method was compared with conventional ELISA, and validation results indicated the reliability of VHH phage-based RT-IPCR in the detection of OTA in cereal samples. This study provides a new idea for the ultrasensitive detection of mycotoxins and other toxic small molecular weight compounds.

Show MeSH