Development of a nanobody-horseradish peroxidase fusion-based competitive ELISA to rapidly and sensitively detect Enrofloxacin residues in animal-derived foods

IF 4.3 2区化学 Q1 SPECTROSCOPY

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy Pub Date : 2024-10-21 DOI:10.1016/j.saa.2024.125309

Ming Yang , Qiang Xu , Kui Gu , Renqiao Wen , Changyu Zhou , Yu Zhao , Boyan Guo , Wei Xu , Yuanyuan Zhang , Cui Li , Changwei Lei , Hongning Wang

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Abstract

The ability to reliably detect enrofloxacin in animal-derived food products has important health implications. In the present study, a nanobody-horseradish peroxidase fusion specific for ENR was generated to enable a sensitive and rapid competitive ELISA suitable for detecting enrofloxacin in samples of milk and animal tissue. An enrofloxacin hapten generated via the glutaraldehyde method was initially used to immunize an adult Bactrian camel as a means of constructing a phage library. Enrofloxacin−specific nanobodies were then selected through three rounds of biopanning, and HRP-fused versions of these nanobodies were then expressed. Lastly, these nanobodies were used to develop a sensitive cELISA for enrofloxacin detection in milk and animal tissues, with the resultant assay exhibiting an IC₅₀ of 37.41 ng/mL and a linear detection range (IC₂₀-IC₈₀) of 10.89 to 244.34 ng/mL. The limit of detection for this cELISA was 6.48 ng/mL, with 4.66 % cross-reactivity with ciprofloxacin, and recovery rates that ranged from84.99 % to 107.72 % together with an RSD below 10.70 %.

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开发基于纳米抗体-辣根过氧化物酶融合的竞争性酶联免疫吸附试验，快速灵敏地检测动物源食品中的恩诺沙星残留量

能否可靠地检测动物源食品中的恩诺沙星对健康具有重要影响。在本研究中，生成了一种特异于恩诺沙星的纳米抗体-辣根过氧化物酶融合物，从而实现了一种灵敏、快速的竞争性酶联免疫吸附试验，适用于检测牛奶和动物组织样本中的恩诺沙星。通过戊二醛法生成的恩诺沙星单体最初被用来免疫成年双峰驼，作为构建噬菌体库的一种手段。然后，通过三轮生物筛选筛选出恩诺沙星特异性纳米抗体，并表达这些纳米抗体的 HRP 融合版本。最后，利用这些纳米抗体开发出一种灵敏的 cELISA 检测方法，用于检测牛奶和动物组织中的恩诺沙星，其 IC50 值为 37.41 ng/mL，线性检测范围（IC20-IC80）为 10.89 至 244.34 ng/mL。该 cELISA 的检测限为 6.48 纳克/毫升，与环丙沙星的交叉反应率为 4.66%，回收率为 84.99% 至 107.72%，RSD 低于 10.70%。

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来源期刊

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 物理-光谱学

CiteScore

8.40

自引率

11.40%

发文量

1364

审稿时长

40 days

期刊介绍： Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy (SAA) is an interdisciplinary journal which spans from basic to applied aspects of optical spectroscopy in chemistry, medicine, biology, and materials science. The journal publishes original scientific papers that feature high-quality spectroscopic data and analysis. From the broad range of optical spectroscopies, the emphasis is on electronic, vibrational or rotational spectra of molecules, rather than on spectroscopy based on magnetic moments. Criteria for publication in SAA are novelty, uniqueness, and outstanding quality. Routine applications of spectroscopic techniques and computational methods are not appropriate. Topics of particular interest of Spectrochimica Acta Part A include, but are not limited to: Spectroscopy and dynamics of bioanalytical, biomedical, environmental, and atmospheric sciences, Novel experimental techniques or instrumentation for molecular spectroscopy, Novel theoretical and computational methods, Novel applications in photochemistry and photobiology, Novel interpretational approaches as well as advances in data analysis based on electronic or vibrational spectroscopy.