Antibody- and aptamer-based competitive fluorescence polarization/anisotropy assays for ochratoxin A with tetramethylrhodamine-labeled ochratoxin A

Abstract

Ochratoxin A (OTA) is one of the mycotoxins that often contaminate a variety of food stuffs, and it is a potential carcinogen for humans. Taking advantage of selective affinity binding and simple, rapid, and sensitive fluorescence polarization (FP)/fluorescence anisotropy (FA) analysis, here, we report two competitive FP/FA assays for OTA using tetramethylrhodamine (TMR)-labeled OTA as a fluorescence tracer and either antibody or aptamer as an affinity ligand to recognize OTA. In the absence of OTA, the TMR-labeled OTA binds with a large-sized affinity ligand, showing a high FA value due to the slow rotation of the affinity complex. When OTA is present, OTA competes with the TMR-labeled OTA tracer in binding limited amount of affinity ligand, causing more free TMR-labeled OTA and a significant FA decrease. We found that the antibody showed a stronger affinity towards TMR-labeled OTA compared to the aptamer. The antibody-based FA assay showed higher signal changes than the aptamer based FA assay due to the larger size of antibody over aptamer. The antibody-based competitive FA assay enabled the detection of 2.4 nM OTA, while the aptamer-based FA assay also achieved a detection limit of 2.4 nM OTA at 10 °C with the help of streptavidin conjugation to increase the molecular size and to improve aptamer affinity. These two competitive FA assays were selective, showing capability for analysis in diluted red wine.