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基于纳米抗体的间接竞争 ELISA 法用于灵敏检测动物尿液中的 19-去甲睾酮。

Nanobody-Based Indirect Competitive ELISA for Sensitive Detection of 19-Nortestosterone in Animal Urine.

机构信息

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.

Guangzhou Institute of Food Inspection, Guangzhou 510080, China.

出版信息

Biomolecules. 2021 Jan 27;11(2):167. doi: 10.3390/biom11020167.

DOI:10.3390/biom11020167
PMID:33513883
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7912623/
Abstract

Nanobody (Nb), a new type of biorecognition element generally from , has the characteristics of small molecular weight, high stability, great solubility and high expression level in . 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 (IC) 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.

摘要

纳米抗体（Nb）是一种新型的生物识别元件，通常来源于骆驼，具有分子量小、稳定性高、溶解性好和表达水平高等特点。本研究以 19-去甲睾酮（19-NT）为靶标药物，采用噬菌体展示技术从骆驼免疫文库中筛选出 3 种针对 19-NT 的特异性 Nb。获得的 Nb 表现出优异的热稳定性和有机溶剂耐受性。性能最佳的纳米抗体 Nb2F7 被用于开发用于检测 19-NT 的灵敏间接竞争酶联免疫吸附测定法（ic-ELISA）。在优化条件下，ic-ELISA 的标准曲线拟合得到半抑制浓度（IC）为 1.03 ng/mL，检测限（LOD）为 0.10 ng/mL。同时，该检测方法与类似物的交叉反应性较低，在添加的样品中 19-NT 的回收率范围为 82.61%至 99.24%。ic-ELISA 与超高效液相色谱/质谱（UPLC-MS/MS）方法的相关系数为 0.9975，表明基于纳米抗体的 ic-ELISA 可作为一种快速分析动物尿液样品中 19-NT 的有用工具。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/e9106b1cff45/biomolecules-11-00167-g011.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/2880281942fa/biomolecules-11-00167-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/a211cd9659f1/biomolecules-11-00167-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/340894996e5a/biomolecules-11-00167-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/682eb2c64aa2/biomolecules-11-00167-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/a6d17924d40b/biomolecules-11-00167-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/c0acb561ffb4/biomolecules-11-00167-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/4a5ccdc27b75/biomolecules-11-00167-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/4a12a1e03ad0/biomolecules-11-00167-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/7f7a6af17fe1/biomolecules-11-00167-g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/91abb020cf30/biomolecules-11-00167-g010.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/e9106b1cff45/biomolecules-11-00167-g011.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/2880281942fa/biomolecules-11-00167-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/a211cd9659f1/biomolecules-11-00167-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/340894996e5a/biomolecules-11-00167-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/682eb2c64aa2/biomolecules-11-00167-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/a6d17924d40b/biomolecules-11-00167-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/c0acb561ffb4/biomolecules-11-00167-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/4a5ccdc27b75/biomolecules-11-00167-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/4a12a1e03ad0/biomolecules-11-00167-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/7f7a6af17fe1/biomolecules-11-00167-g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/91abb020cf30/biomolecules-11-00167-g010.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/730f/7912623/e9106b1cff45/biomolecules-11-00167-g011.jpg

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