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Detecting ligand interactions in real time on living bacterial cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Ridgeview Instruments AB, Vange, Sweden.ORCID iD: 0000-0002-4509-4106
Karolinska Inst, Dept Med Solna, Sci Life Lab, Solna, Sweden;Karolinska Univ Hosp, Dept Infect Dis, Solna, Sweden.
Karolinska Inst, Dept Med Solna, Sci Life Lab, Solna, Sweden;Karolinska Univ Hosp, Dept Infect Dis, Solna, Sweden.
Ridgeview Instruments AB, Vange, Sweden.
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2018 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 102, no 9, p. 4193-4201Article in journal (Refereed) Published
Abstract [en]

Time-resolved analysis assays of receptor-ligand interactions are fundamental in basic research and drug discovery. Adequate methods are well developed for the analysis of recombinant proteins such as antibody-antigen interactions. However, assays for time-resolved ligand-binding processes on living cells are still rare, in particular within microbiology. In this report, the real-time cell-binding assay (RT-CBA) technology LigandTracerA (R), originally designed for mammalian cell culture, was extended to cover Gram-positive and Gram-negative bacteria. This required the development of new immobilization methods for bacteria, since LigandTracer depends on cells being firmly attached to a Petri dish. The evaluated Escherichia coli CJ236 and BL21 as well as Staphylococcus carnosus TM300 strains were immobilized to plastic Petri dishes using antibody capture, allowing us to depict kinetic binding traces of fluorescently labeled antibodies directed against surface-displayed bacterial proteins for as long as 10-15 h. Interaction parameters, such as the affinity and kinetic constants, could be estimated with high precision (coefficient of variation 9-44%) and the bacteria stayed viable for at least 16 h. The other tested attachment protocols were inferior to the antibody capture approach. Our attachment protocol is generic and could potentially also be applied to other assays and purposes.

Place, publisher, year, edition, pages
SPRINGER , 2018. Vol. 102, no 9, p. 4193-4201
Keywords [en]
Real-time interactions, Drug kinetics, Living bacteria, Antibodies
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-352571DOI: 10.1007/s00253-018-8919-3ISI: 000429800600027PubMedID: 29550990OAI: oai:DiVA.org:uu-352571DiVA, id: diva2:1237122
Funder
Swedish Research CouncilEU, European Research Council, 675555Available from: 2018-08-07 Created: 2018-08-07 Last updated: 2018-08-07Bibliographically approved

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