Nanobody-Nanoluciferase Fusion Protein-Enabled Immunoassay for Ochratoxin A in Coffee with Enhanced Specificity and Sensitivity

Kunlu Bao; Xing Liu; Yujing Liao; Zilong Liu; Hongmei Cao; Long Wu; Qi Chen

doi:10.3390/toxins14100713

Nanobody-Nanoluciferase Fusion Protein-Enabled Immunoassay for Ochratoxin A in Coffee with Enhanced Specificity and Sensitivity

Toxins (Basel). 2022 Oct 19;14(10):713. doi: 10.3390/toxins14100713.

Authors

Kunlu Bao¹, Xing Liu¹, Yujing Liao¹, Zilong Liu¹, Hongmei Cao¹, Long Wu¹, Qi Chen^{2

1}

Affiliations

¹ Key Laboratory of Tropical and Vegetables Quality and Safety for State Market Regulation, School of Food Science and Engineering, Hainan University, Haikou 570228, China.
² National Engineering Research Center for Bioengineering Drugs and the Technologies, Institute of Translational Medicine, Nanchang University, Nanchang 330031, China.

Abstract

Ochratoxin A (OTA), one of the best-known mycotoxins, causes problems concerning food safety with potential toxic effects in humans and animals. So, it is crucial to develop simple and sensitive methods for the detection of OTA. Herein, a nanoluciferase-nanobody fusion protein (Nb28-Nluc)-retaining antibody recognition and enzymatic activity was first prepared, which was then applied as a bifunctional tracer to construct a one-step bioluminescent enzyme-linked immunosorbent assay (BLEIA) for OTA in coffee samples. On the basis of Nb28-Nluc, the BLEIA can be completed with a one-step incubation and detection, with only a substrate replacement from 3,3',5,5'-tetramethylbenzidine (TMB) to a Nluc assay reagent (Furimazine). Under the optimal experimental conditions, the proposed one-step BLEIA achieved a detection limit of 3.7 ng/mL (IC₁₀) within 3 h. Moreover, the BLEIA method showed good repeatability and accuracy in the spike recovery experiments with recoveries of 83.88% to 120.23% and relative standard deviations (RSDs) of 5.2% to 24.7%, respectively. Particularly, the BLEIA displayed superior performances, such as fewer operations and more rapid and sensitive detection as compared with Nb28-based enzyme-linked immunosorbent assay. Therefore, the proposed one-step BLEIA has great potential for the sensitive and accurate screening of OTA in food samples.

Keywords: BLEIA; Nb28-Nluc; bioluminescent; food safety; ochratoxin A.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Coffee
Food Contamination / analysis
Humans
Immunoassay
Mycotoxins* / analysis
Ochratoxins* / analysis
Sensitivity and Specificity

Substances

ochratoxin A
Coffee
Ochratoxins
Mycotoxins

Grants and funding

This research was funded by the Natural Science Foundation of Hainan Province (No. 219QN149 and No. 322MS016) and the National Natural Science Foundation of China (No. 31901800 and No. 32260623), and the specific research fund of the Innovation Platform for Academicians of Hainan Province (No. YSPTZX202152).And the APC wans funded by the National Natural Science Foundation of China (No. 32260623).