VHH Phage-Based Competitive Real-Time Immuno-Polymerase Chain Reaction for Ultrasensitive Detection of Ochratoxin A in Cereal
Open Access
- 3 July 2014
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 86 (15), 7471-7477
- https://doi.org/10.1021/ac501202d
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.Keywords
This publication has 31 references indexed in Scilit:
- Deleterious Effects of Mycotoxin Combinations Involving Ochratoxin AToxins, 2013
- Fate of the teratogenic and carcinogenic ochratoxin A in human perfused placentaToxicology Letters, 2012
- Isolation of Alpaca Anti-Hapten Heavy Chain Single Domain Antibodies for Development of Sensitive ImmunoassayAnalytical Chemistry, 2011
- An Update on Direct Genotoxicity as a Molecular Mechanism of Ochratoxin A CarcinogenicityChemical Research in Toxicology, 2011
- Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune ResponseAnalytical Chemistry, 2011
- Noncompetitive Phage Anti-Immunocomplex Real-Time Polymerase Chain Reaction for Sensitive Detection of Small MoleculesAnalytical Chemistry, 2010
- A real-time immuno-PCR method for detecting 3,3′,4,4′-tetrachlorobiphenylMicrochimica Acta, 2010
- Characterization of phage peptide interaction with antibody using phage mediated immuno-PCRJournal of Immunological Methods, 2007
- An immunoassay for ochratoxin A without the mycotoxinFood Control, 2006
- Phage display mediated immuno-PCRNucleic Acids Research, 2006