Analytica chimica acta · 2026
Development of a dual-nanobody sandwich immunochromatographic strip based on a site-directed conjugation strategy for ultrasensitive detection of staphylococcal enterotoxin B (SEB).
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Zheng Hao, Gong Tingting, Chen Kaiyuan, Niu Wenjia, Mao Yulin, Hou Jian, Lu Zhilong, Luo Chen, Sun Tieqiang, Shen Zhiqiang
Аннотация
BACKGROUND: Staphylococcal enterotoxin B (SEB), a highly potent and stable biotoxin, is a leading cause of food poisoning and poses a significant threat to food safety and public health. While lateral flow immunoassay (LFIA) is valued for its low cost and rapid on-site detection capability, it faces challenges in detecting SEB due to the nonspecific binding of staphylococcal protein A to the Fc region of conventional antibodies. To overcome this limitation, a series of paired nanobodies against SEB were successfully selected from an immunized phage-display nanobody library.
RESULTS: The screening was accomplished using an efficient one-step sandwich chemiluminescent immunoassay, which strategically employed Avi-tagged and alkaline phosphatase-fused nanobodies (Nb-Avi and ALP-Nb). Based on the optimal nanobody pair (Nb9A and Nb9C), a sensitive time-resolved fluorescence lateral flow immunoassay was developed for rapid SEB detection. This involved selecting nitrocellulose membrane-compatible nanobody-fusion proteins and establishing an advanced site-directed strategy for labeling nanoparticles with nanobodies. The resulting test strip achieves detection within 15 min, with a remarkably low detection limit of 0.034 ng/mL and a linear range of 0.10-6.3 ng/mL, significantly outperforming most existing methods. Due to the complete absence of an Fc fragment, the nanobodies confer strong resistance to protein A interference in the immunochromatographic system. In practical sample analyses, the method demonstrated consistent recovery rates of 84.40%-114.21%, along with excellent specificity and stability.
SIGNIFICANCE: This study presents an innovative lateral flow immunoassay that overcomes key challenges in achieving highly sensitive and rapid SEB detection by using paired nanobodies. The assay enables rapid, ultrasensitive, and specific detection of SEB, demonstrating high reliability in complex samples,and offers a robust, field-deployable solution for on-site food safety monitoring and public health protection, while also providing a novel strategic framework for detecting other protein toxins.
RESULTS: The screening was accomplished using an efficient one-step sandwich chemiluminescent immunoassay, which strategically employed Avi-tagged and alkaline phosphatase-fused nanobodies (Nb-Avi and ALP-Nb). Based on the optimal nanobody pair (Nb9A and Nb9C), a sensitive time-resolved fluorescence lateral flow immunoassay was developed for rapid SEB detection. This involved selecting nitrocellulose membrane-compatible nanobody-fusion proteins and establishing an advanced site-directed strategy for labeling nanoparticles with nanobodies. The resulting test strip achieves detection within 15 min, with a remarkably low detection limit of 0.034 ng/mL and a linear range of 0.10-6.3 ng/mL, significantly outperforming most existing methods. Due to the complete absence of an Fc fragment, the nanobodies confer strong resistance to protein A interference in the immunochromatographic system. In practical sample analyses, the method demonstrated consistent recovery rates of 84.40%-114.21%, along with excellent specificity and stability.
SIGNIFICANCE: This study presents an innovative lateral flow immunoassay that overcomes key challenges in achieving highly sensitive and rapid SEB detection by using paired nanobodies. The assay enables rapid, ultrasensitive, and specific detection of SEB, demonstrating high reliability in complex samples,and offers a robust, field-deployable solution for on-site food safety monitoring and public health protection, while also providing a novel strategic framework for detecting other protein toxins.