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Appl Microbiol Biotechnol. 2018 May;102(9):4193-4201. doi: 10.1007/s00253-018-8919-3. Epub 2018 Mar 17.

Detecting ligand interactions in real time on living bacterial cells.

Author information

1
Ridgeview Instruments AB, Vänge, Sweden. joao@ridgeview.eu.
2
Department of Immunology, Pathology and Genetics, Uppsala University, Uppsala, Sweden. joao@ridgeview.eu.
3
Science for Life Laboratory, Department of Medicine Solna, Karolinska Institute, and Department of Infectious Diseases, Karolinska University Hospital, Solna, Sweden.
4
Ridgeview Instruments AB, Vänge, Sweden.
5
Department of Immunology, Pathology and Genetics, Uppsala University, Uppsala, Sweden.

Abstract

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 LigandTracer®, 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.

KEYWORDS:

Antibodies; Drug kinetics; Living bacteria; Real-time interactions

PMID:
29550990
PMCID:
PMC5895670
DOI:
10.1007/s00253-018-8919-3
[Indexed for MEDLINE]
Free PMC Article

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