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Antibody- and aptamer-based competitive fluorescence polarization/anisotropy assays for ochratoxin A with
1State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. qiangzhao@rcees.ac.cn.
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.
Insights
Two new fluorescence assays detect Ochratoxin A (OTA), a food contaminant and potential carcinogen. These methods utilize antibody or aptamer recognition for sensitive and selective OTA analysis in food matrices.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Food Safety
Background:
- Ochratoxin A (OTA) is a prevalent mycotoxin found in foodstuffs, posing a significant human health risk due to its carcinogenic potential.
- Sensitive and selective detection methods are crucial for monitoring OTA contamination in food products.
Purpose of the Study:
- To develop and compare two competitive fluorescence polarization/anisotropy (FP/FA) assays for the detection of Ochratoxin A.
- To evaluate the performance of antibody-based and aptamer-based assays for OTA quantification.
Main Methods:
- Development of two competitive FP/FA assays using tetramethylrhodamine (TMR)-labeled OTA as a tracer.
- Utilizing either an antibody or an aptamer as the affinity ligand for OTA recognition.
- Assessing assay performance based on affinity, signal change, and detection limits.
Main Results:
- Both antibody and aptamer assays demonstrated selective OTA detection.
- The antibody-based assay exhibited stronger affinity and higher signal changes compared to the aptamer-based assay.
- Both assays achieved a detection limit of 2.4 nM OTA, with the aptamer assay benefiting from streptavidin conjugation.
Conclusions:
- Competitive FP/FA assays provide a sensitive and rapid method for OTA detection.
- Antibody-based assays offer advantages in signal response due to larger molecular size.
- These assays are suitable for analyzing OTA in complex matrices like diluted red wine.
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