数字农科院2.0

Alpaca nanobody-based sandwich ELISA for specific detection of Salmonella enteritidis in chicken

文献类型： 外文期刊

作者： Liang Yuan;Shibo Wang;Yani Ren;Zhenghua Ji;Xinru Jiang;Beili Hao;Shuangshuang Hao;Xiaoxi Han;Changwen Li;Yicong Chang;Rui Li;Fangping Liu

作者机构：

关键词： Alpaca;Chicken;ELISA;Nanobody;Salmonella enteritidis;SipD;Virulence factor

期刊名称： Food Control

ISSN： 0956-7135

年卷期： 2025 年 181 卷

页码：

收录情况： SCIE（2025版）

摘要： Salmonella enteritidis (S. enteritidis) is a foodborne pathogen that is widely present in the environment and can be transmitted to humans through poultry products, which affects food safety and human health. The SipD protein is a key virulence factor of the type III secretion system of S. enteritidis, and contains conserved sequences. Alpacas have immense potential for application as novel animal resources, and the nanobodies produced in their bodies have unique properties, including high affinity, thermal stability, and ease of production. The aim of this study was to prepare alpaca nanoantibodies targeting the conserved SipD protein for the development of a sandwich ELISA method for the detection of S. enteritidis. The alpaca was immunized with purified SipD protein to construct a nanobody library. Two specific nanobodies (Nb3 and Nb89) subsequently were screened from the library via phage display technology. The Nb3 with better affinity activity was then coupled with horseradish peroxidase, and a sandwich ELISA was successfully developed with a detection limit of 9.5 × 104 CFU/mL, which specifically detected S. enteritidis without cross-reactivity with other pathogens. The spiked recovery rates in matrices ranged from 85.5 % to 109.4 % with coefficients of variation below 10 %. Molecular docking and molecular dynamics simulations revealed that the SipD protein primarily binds to the complementarity-determining regions of nanobodies, forming a stable complex. In conclusion, we prepared two SipD nanobodies and developed a sandwich ELISA for the detection of S. enteritidis in chickens. These results provide a new strategy for the detection of S. enteritidis in poultry products.

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