Nanobody-Based Indirect Competitive ELISA for Sensitive Detection of 19-Nortestosterone in Animal Urine
Yuan-yuan Yang,
Yu Wang,
Yi-feng Zhang,
Feng Wang,
Yi-fan Liang,
Jin-yi Yang,
Zhen-lin Xu,
Yu-dong Shen,
Hong Wang
Affiliations
Yuan-yuan Yang
Guangdong Provincial Key Laboratory of Food Quality and Safety, National-Local Joint Engineering Research, Center for Processing and Safety Control of Livestock and Poultry Products, College of Food Science, South China Agricultural University, Guangzhou 510642, China
Yu Wang
Guangzhou Institute of Food Inspection, Guangzhou 510080, China
Yi-feng Zhang
Guangdong Provincial Key Laboratory of Food Quality and Safety, National-Local Joint Engineering Research, Center for Processing and Safety Control of Livestock and Poultry Products, College of Food Science, South China Agricultural University, Guangzhou 510642, China
Feng Wang
Guangdong Provincial Key Laboratory of Food Quality and Safety, National-Local Joint Engineering Research, Center for Processing and Safety Control of Livestock and Poultry Products, College of Food Science, South China Agricultural University, Guangzhou 510642, China
Yi-fan Liang
Guangdong Provincial Key Laboratory of Food Quality and Safety, National-Local Joint Engineering Research, Center for Processing and Safety Control of Livestock and Poultry Products, College of Food Science, South China Agricultural University, Guangzhou 510642, China
Jin-yi Yang
Guangdong Provincial Key Laboratory of Food Quality and Safety, National-Local Joint Engineering Research, Center for Processing and Safety Control of Livestock and Poultry Products, College of Food Science, South China Agricultural University, Guangzhou 510642, China
Zhen-lin Xu
Guangdong Provincial Key Laboratory of Food Quality and Safety, National-Local Joint Engineering Research, Center for Processing and Safety Control of Livestock and Poultry Products, College of Food Science, South China Agricultural University, Guangzhou 510642, China
Yu-dong Shen
Guangdong Provincial Key Laboratory of Food Quality and Safety, National-Local Joint Engineering Research, Center for Processing and Safety Control of Livestock and Poultry Products, College of Food Science, South China Agricultural University, Guangzhou 510642, China
Hong Wang
Guangdong Provincial Key Laboratory of Food Quality and Safety, National-Local Joint Engineering Research, Center for Processing and Safety Control of Livestock and Poultry Products, College of Food Science, South China Agricultural University, Guangzhou 510642, China
Nanobody (Nb), a new type of biorecognition element generally from Camelidae, has the characteristics of small molecular weight, high stability, great solubility and high expression level in E. coli. In this study, with 19-nortestosterone (19-NT), an anabolic androgenic steroid as target drug, three specific Nbs against 19-NT were selected from camel immune library by phage display technology. The obtained Nbs showed excellent thermostability and organic solvent tolerance. The nanobody Nb2F7 with the best performance was used to develop a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for 19-NT detection. Under optimized conditions, the standard curve of ic-ELISA was fitted with a half-maximal inhibitory concentration (IC50) of 1.03 ng/mL and a detection limit (LOD) of 0.10 ng/mL for 19-NT. Meanwhile, the developed assay had low cross- reactivity with analogs and the recoveries of 19-NT ranged from 82.61% to 99.24% in spiked samples. The correlation coefficient between ic-ELISA and the ultra-performance liquid chromatography/mass spectrometry (UPLC-MS/MS) method was 0.9975, which indicated that the nanobody-based ic-ELISA could be a useful tool for a rapid analysis of 19-NT in animal urine samples.
