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

Xing Liu, Yang Xu, Yong Hua Xiong, Zhui Tu, Yan Ping Li, Zhen Yun He, Yu Lou Qiu, Jin Heng Fu, Shirley J. Gee, Bruce D. Hammock

Research output: Contribution to journalArticle

46 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)7471-7477
Number of pages7
JournalAnalytical Chemistry
Volume86
Issue number15
DOIs
StatePublished - Aug 5 2014

ASJC Scopus subject areas

  • Analytical Chemistry

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    Liu, X., Xu, Y., Xiong, Y. H., Tu, Z., Li, Y. P., He, Z. Y., Qiu, Y. L., Fu, J. H., Gee, S. J., & Hammock, B. D. (2014). VHH phage-based competitive real-time immuno-polymerase chain reaction for ultrasensitive detection of ochratoxin A in cereal. Analytical Chemistry, 86(15), 7471-7477. https://doi.org/10.1021/ac501202d