Abstract
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.
Highlights
Published: 27 January 2021Nandrolone phenylpropionate (NPP), one of commonly used steroid hormones in foodborne animal, has strong effect of protein assimilation
Based on Nbs these against advantages, Nbswere have been applied in various immunoasnanobody phage says library. To explore their potential in immunoassay, we evaluated for small molecule analytes [14], including Alternaria mycotoxin tenuazonic the acid [15], physical-chemical aflatoxin properties
The were immunoassay results nanobody phagedetection library. Toand explore their potential immunoassay, we evaluated were applied in actual samples validated by theinultra-performance liquid the physical-chemical properties of Nbs and the best one nanobody Nb2F7 was used to dechromatography/mass spectrometry (UPLC-MS/MS)
Summary
Nandrolone phenylpropionate (NPP), one of commonly used steroid hormones in foodborne animal, has strong effect of protein assimilation. It has been extensively used in livestock breeding for accelerating muscle growth with supernormal pattern and fat storage reduction for improving the economic benefit of feed [1]. The secondary metabolite 19-NT is main residual marker which has strong stability and long half-life period in animals [2]. The metabolic residues of anabolic steroid were frequently detected in animal-derived food. It is urgent to develop an economic, sensitive and efficient detection method for the metabolite of anabolic androgenic steroid
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