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Development of a Nanobody-AviTag Fusion Protein and Its Application in a Streptavidin–Biotin-Amplified Enzyme-Linked Immunosorbent Assay for Ochratoxin A in Cereal

Sun, Zhichang, Lv, Jingwen, Liu, Xing, Tang, Zongwen, Wang, Xuerou, Xu, Yang, Hammock, Bruce D.
Analytical chemistry 2018 v.90 no.17 pp. 10628-10634
antibodies, barley, biotinylation, detection limit, enzyme-linked immunosorbent assay, food contamination, genetic vectors, inhibitory concentration 50, liquid chromatography, molecular weight, oats, ochratoxin A, rice, tandem mass spectrometry
Ochratoxin A (OTA) is a common food contaminant that threatens 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 (IC₅₀) of 0.14 ng mL–¹ and the limit of detection (LOD = IC₁₀) 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.