Using designed polypeptides to develop novel phage-mediated triple antibody sandwich immunoassay for the specific detection of Escherichia coli O157:H7 in milk

文献类型： 外文期刊

第一作者： Xue, Chang

作者： Xue, Chang;Ouyang, Qin;Zhang, Cunzheng;Xue, Chang;Fu, Jinmei;Tao, Tingting;Zhu, Shouhu;Han, Bingsong;Wang, Yulong;Ye, Yuhui;Zhang, Cunzheng;Fu, Jinmei;Zhang, Cunzheng;Zhu, Shouhu;Zhang, Cunzheng;Hammock, Bruce D.;Hammock, Bruce D.;Luo, Ruifeng

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关键词： Polypeptides; Phage-displayed nanobody; Serotype discrimination; Triple antibody sandwich immunoassay; Escherichia coli O157:H7

期刊名称：SENSORS AND ACTUATORS B-CHEMICAL （ 影响因子：7.7； 五年影响因子：7.4 ）

ISSN：

年卷期： 2025 年 441 卷

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收录情况： SCI

摘要： In this study, a phage-mediated triple antibody sandwich immunoassay (TAS-ELISA) was developed and exploited for the sensitive detection of Escherichia coli O157:H7 (E. coli O157:H7), which could be potentially tailored to individual serotypes testing. Bioinformatics analysis was conducted to elucidate the potential epitopes of E. coli secreted protein A (EspA) and Outer membrane protein A (OmpA). Peptide fragments were selected and designed to serve as antigens in the screening of phage-displayed antibody library, two nanobodies (Nbs) that exhibit specific capabilities for distinct epitopes recognition were obtained. Using monoclonal antibody as capture and two Nbs as tracer, a phage-mediated triple antibody sandwich immunoassay was developed. The results revealed that the limit of detection (LOD) for E. coli O157:H7 was 1.89 x 10(3) CFU/mL with no significant cross-reactivity to other bacterial, which was 41.8-fold higher than that of the sandwich immunoassay based on mAb. Furthermore, molecular docking provides insight into the specific binding interactions between these two distinct epitopes and nanobodies. Method validation revealed that the developed TAS-ELISA has great potential for the specific determination of E. coli O157:H7 in food and could be further refined for precise serotype discrimination by identifying multiple unique epitopes. This study provides an alternative strategy to precisely detect E. coli O157:H7 in food.

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