Preparation of divalent camelid single-domain antibody and its application in immunoassays for Salmonella detection in food.

IF 3.8 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Analytical and Bioanalytical Chemistry Pub Date : 2024-12-01 Epub Date: 2024-10-26 DOI:10.1007/s00216-024-05610-4

Yanhong Chen, Yingjie Huang, Ruo Yang, Yongliang Cui, Yanru Wang, Xiaohong Wang, Jia Wang

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引用次数: 0

Abstract

Salmonella-related foodborne infections are commonly caused by the serovars of S. Typhimurium, which can be detected using antibody-based immunoassays. The monovalent variable domain of the camelid heavy chain antibody (VHH) performs excellently in constructing multivalent VHH variants, which generally exhibit higher affinities with antigens and consequently enhance the assay sensitivity. In this study, the divalent variants of VHHs (diVHHs) targeting S. Typhimurium were generated by encoding the monovalent VHH genes assembled in tandem with a flexible linker peptide (G₄S)₂. Soluble diVHHs were produced in a prokaryotic expression system and purified with a yield of 4.22 mg/L. Benefiting from their stability and antigen-binding abilities towards tested Salmonella serovars, diVHH-based immunoassays were developed. The diVHH-based sandwich immunoassay, using diVHH as capture antibody, exhibited a detection limit of 1.04×10² CFU/mL and enabled as low as 10 CFU/mL S. Typhimurium to be detected after 6 h of enrichment in lettuce. Furthermore, this assay can be applied to spiked lettuce, chicken, and pork samples, showing acceptable recoveries ranging from 83 to 106%. This study presented feasible strategies for VHH multivalence and established a superior sensitivity VHH-based immunoassay for monitoring and analyzing Salmonella contamination in food.

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二价骆驼单域抗体的制备及其在食品中沙门氏菌检测免疫测定中的应用

与沙门氏菌有关的食源性感染通常是由鼠伤寒沙门氏菌的血清型引起的，可使用抗体免疫测定法检测。驼科动物重链抗体（VHH）的单价可变结构域在构建多价 VHH 变体方面表现出色，这些变体通常与抗原具有更高的亲和力，从而提高了检测灵敏度。在本研究中，通过编码单价 VHH 基因与柔性连接肽（G4S）2 的串联，生成了针对鼠伤寒杆菌的 VHH 二价变体（diVHH）。可溶性 diVHHs 在原核表达系统中产生并纯化，产量为 4.22 mg/L。利用 diVHHs 的稳定性和与被测沙门氏菌血清的抗原结合能力，开发出了基于 diVHH 的免疫测定方法。基于 diVHH 的夹心免疫测定使用 diVHH 作为捕获抗体，检测限为 1.04×102 CFU/mL，在莴苣中富集 6 小时后可检测到低至 10 CFU/mL 的鼠伤寒沙门氏菌。此外，该检测方法还可用于加标莴苣、鸡肉和猪肉样品，回收率在 83% 至 106% 之间。这项研究提出了可行的 VHH 多价策略，并建立了一种灵敏度极高的基于 VHH 的免疫测定方法，用于监测和分析食品中的沙门氏菌污染。

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来源期刊

Analytical and Bioanalytical Chemistry 化学-分析化学

CiteScore

8.00

自引率

4.70%

发文量

638

审稿时长

2.1 months

期刊介绍： Analytical and Bioanalytical Chemistry’s mission is the rapid publication of excellent and high-impact research articles on fundamental and applied topics of analytical and bioanalytical measurement science. Its scope is broad, and ranges from novel measurement platforms and their characterization to multidisciplinary approaches that effectively address important scientific problems. The Editors encourage submissions presenting innovative analytical research in concept, instrumentation, methods, and/or applications, including: mass spectrometry, spectroscopy, and electroanalysis; advanced separations; analytical strategies in “-omics” and imaging, bioanalysis, and sampling; miniaturized devices, medical diagnostics, sensors; analytical characterization of nano- and biomaterials; chemometrics and advanced data analysis.