Development of a Nanobody-AviTag Fusion Protein and Its Application in a Streptavidin–Biotin-Amplified Enzyme-Linked Immunosorbent Assay for Ochratoxin A in Cereal
| Autor: | Bruce D. Hammock, Xuerou Wang, Yang Xu, Zhichang Sun, Xing Liu, Zongwen Tang, Jingwen Lv |
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| Rok vydání: | 2018 |
| Předmět: |
Ochratoxin A
Streptavidin Recombinant Fusion Proteins Biotin Enzyme-Linked Immunosorbent Assay 02 engineering and technology 01 natural sciences Article Analytical Chemistry chemistry.chemical_compound Limit of Detection In vivo IC50 Detection limit Chromatography Prevention 010401 analytical chemistry Assay sensitivity Single-Domain Antibodies 021001 nanoscience & nanotechnology Ochratoxins 0104 chemical sciences chemistry Biotinylation Other Chemical Sciences Edible Grain 0210 nano-technology |
| Zdroj: | Anal Chem Analytical chemistry, vol 90, iss 17 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.8b03085 |
| Popis: | Ochratoxin A (OTA) is a common food contaminant that threatens the consumers’ safety and health. A sensitive and selective biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) for OTA using a nanobody-AviTag fusion protein (Nb-AviTag) was developed in this study. The prokaryotic expression vector Nb28-AviTag-pAC6 for Nb-AviTag was constructed, followed by transformation to the AVB101 cells for antibody expression and in vivo biotinylation. The purified Nb28-AviTag was used to establish the BA-ELISA and the procedures for this Nb-AviTag-based BA-ELISA were optimized. The Nb-AviTag-based BA-ELISA exhibited the half maximal inhibitory concentration (IC(50)) of 0.14 ng mL(−1) and the limit of detection (LOD = IC(10)) of 0.028 ng mL(−1) for OTA basing on the optimized experiment parameters. The assay sensitivity was improved 4.6 times and 4.3 times compared to Nb-based ELISA, respectively. This method had LODs of 1.4 μg kg(−1) in barley, 0.56 μg kg(−1) in oats, and 0.84 μg kg(−1) in rice for OTA. The average recovery percent was in a range of 84–137%, and the relative standard derivation percent ranged from 0.64% to 7.8%. The content of OTA in contaminated cereal samples was determined by both the developed Nb-AviTag-based method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results demonstrated that the Nb-AviTag was a robust and promising bioreceptor in highly sensitive detection of OTA and other low molecular weight compounds using BA system. |
| Databáze: | OpenAIRE |
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