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Detection of flagellin by interaction with human recombinant TLR5 immobilized in liposomes

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Abstract

Digestive diseases caused by flagellated bacteria are a huge public health problem worldwide and rapid detection methods are needed for contaminated environments. In this study, we propose a method to detect patterns associated with pathogens based on the properties of the innate immune system. Specifically, we use Toll-like receptor 5 (TLR5), a transmembrane protein that specifically recognizes flagellin (the structural protein of bacterial flagella). TLR5, which was obtained by recombinant production in insect cells, was immobilized into liposomes to form TLR5-proteoliposomes. Through surface plasmon resonance (SPR) and competition flow cytometry assays, the sensitivity of proteoliposomes to recognize Escherichia coli and Salmonella typhimurium flagellin was evaluated. In addition, we compared the results obtained by immobilizing anti-flagellin antibodies into liposomes. The results of the flagellin-affinity tests, expressed as an SPR kinetic rate constant ratio in the equilibrium equation K D = k d/k a, showed values of 13.8 × 10−9 and 7.73 × 10−9 M for the TLR5-proteoliposomes and anti-flagellin antibodies, respectively, against S. typhimurium. The anti-flagellin affinity results for E. coli showed K D of 84.1 × 10−8 M for SPR assays and K D of 3.5 × 10−8 M for competitive flow cytometry, which was used as a detection system without the immobilization of proteoliposomes. This research demonstrates the practical possibility of using proteoliposomes as recognition elements in the generation of systems for the rapid detection of flagellated bacteria, which could help avoid consumption of contaminated food by humans and thereby prevent intestinal infections.

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Acknowledgments

We thank Silob Chile Ltda., the Universidad Tecnica Federico Santa María (USM), and CONICYT through Fondef Project D07i1057 for funding. We also thank Juan Carlos Espinosa (University of Valparaíso), Valeska Simons (University for Development, Santiago, Chile), and Paz Reyes (Fundación Ciencia para la Vida).

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Olguín, Y., Villalobos, P., Carrascosa, L.G. et al. Detection of flagellin by interaction with human recombinant TLR5 immobilized in liposomes. Anal Bioanal Chem 405, 1267–1281 (2013). https://doi.org/10.1007/s00216-012-6523-4

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  • DOI: https://doi.org/10.1007/s00216-012-6523-4

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