Kinetics of Antibody Binding to Membranes of Living Bacteria Measured by a Photonic Crystal-Based Biosensor

Optical biosensors based on photonic crystal surface waves (PC SWs) offer a possibility to study binding interactions with living cells, overcoming the limitation of rather small evanescent field penetration depth into a sample medium that is characteristic for typical optical biosensors. Besides this, simultaneous excitation of s-and p-polarized surface waves with different penetration depths is realized here, permitting unambiguous separation of surface and volume contributions to the measured signal. PC-based biosensors do not require a bulk signal correction, compared to widely used surface plasmon resonance-based devices. We developed a chitosan-based protocol of PC chip functionalization for bacterial attachment and performed experiments on antibody binding to living bacteria measured in real time by the PCSW-based biosensor. Data analysis reveals specific binding and gives the value of the dissociation constant for monoclonal antibodies (IgG2b) against bacterial lipopolysaccharides equal to K-D = 6.2 +/- 3.4 nM. To our knowledge, this is a first demonstration of antibody-binding kinetics to living bacteria by a label-free optical biosensor.

Real-time binding kinetics of ligand-receptor interactions

Ekaterina Rostova

In principle, a major goal in drug development is to design a high-affinity compound with an appropriate pharmacological response, which requires understanding of physical processes underlying the drug-target interaction, affinity and specificity. This drug key information can be obtained by measuring drug binding kinetics in real time as the quantity of a ligand binding to a receptor immobilized on a biosensor chip surface. Almost all drugs independently of their targets interact with a complex cell membrane that incorporate various transmembrane proteins whose impact cannot be disregarded. Therefore, it is highly desirable that preclinical drug development is performed under conditions that are as close as possible to a native cell environment. The above reasons enhance the need for alternative approaches, such as whole-cell assays that are able to examine in vitro potential drug compounds. However, nowadays fragments of cell membranes have to be routinely isolated from the native cell environment due to limitations of currently available label-free technologies. In recent years, attempts were made to measure binding kinetics on living bacteria by surface plasmon resonance-based biosensors. Nevertheless, the experiments were not fully successful because of an insufficient penetration depth of the sensing evanescent field into living bacteria. Label-free biosensors based on metal-free photonic crystals support electromagnetic surface waves with a deeper penetration depth into the sample medium offering a solution for measuring binding interactions with living bacterial and, possibly, also eukaryotic cells. The deeper penetration of the surface waves creates a significant advantage for this non-disruptive technology over conventional immunoassays such as ELISA. For these reasons, label-free biosensors have been attracting a growing interest from pharmaceutical companies and research institutions by avoiding any experimental artefacts induced by labeling and allowing for real-time kinetic measurements. In this work, I contributed to the development of a label-free optical biosensor based on a photonic crystal that can be exploited for living bacteria. Moreover, I designed an experimental protocol and demonstrated how binding kinetics of various ligands, including antibodies and bacteriophages can be measured by the biosensor in vitro. Furthermore, this study attempts to apply the biosensor technology for bacterial viability tests exploring the ways in which the biosensor can detect metabolic activity of bacteria. Additionally, we performed theoretical studies on application of the Hillâs and Mathieuâs equations to study the effect of the photonic crystal design on the wave equation describing electromagnetic field distribution in the photonic crystal. This Thesis work demonstrates capabilities of a versatile biophysical method based on light interaction with living matter. In particular, the device sensitivity allows for measurements on living intact cells in vitro, which is a key technological advantage. Finally, this Thesis provides experimental evidence for future applications of the photonic crystal-based biosensor for antibody and bacteriophage therapies contributing to implementation of this technology in drug development process.

EPFL2017

Functional immobilisation of the nicotinic acetylcholine receptor in tethered lipid membranes

Rudolf Hovius, Per Rigler, Horst Vogel

The nicotinic acetylcholine receptor from Torpedo was immobilised in tethered membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural integrity of the surface-reconstituted receptor was probed using monoclonal antibodies, demonstrating that approximately 65% of the receptors present their ligand-binding site towards the lumen of the flow cell and that at least 85% of these receptors are structurally intact. The conformation of the receptor in tethered membranes was investigated with Fourier transform infrared spectroscopy and found to be practically identical to that of receptors reconstituted in lipid vesicles. The affinity of small receptor ligands was determined in a competition assay against a monoclonal antibody directed against the ligand-binding site which yielded dissociation constants in agreement with radioligand binding assays. The presented method for the functional immobilisation of the nicotinic acetylcholine receptor in tethered membranes might be generally applicable to other membrane proteins.

Elsevier2000

Phage Selection of Bicyclic Peptide Ligands of the Notch1 Receptor

Christian Heinis, Freddy Radtke, Charlotte Marie Urech-Varenne

Precise regulation of the Notch signaling pathway is crucial, as increases or deficiencies in signaling are associated with diseases, including a wide range of cancers. Recent studies have demonstrated that monoclonal antibodies that bind and stabilize the structure of the negative regulatory region (NRR) in the extracellular domain of the Notch receptor can inhibit Notch signaling. In this work, we posed the question whether bicyclic peptides, being around 100-fold smaller than antibodies, can also stabilize the NRR and inhibit Notch signaling. Bicyclic peptides that bind the NRR of human Notch1 were isolated from combinatorial libraries by phage display. Affinity maturation yielded ligands with dissociation constant (K-d) values as low as 150nM. The bicyclic peptides increased the melting temperature of the NRR by up to 8 degrees C, thus substantially stabilizing the protein structure, but they did not inhibit Notch signaling in cellular assays. Although Notch signaling could not be inhibited, this work demonstrates that phage-selected bicyclic peptides can stabilize proteins; this capacity of bicyclic peptides may be exploited for modulating the conformation of other disease targets.

Wiley-Blackwell2015

Real-time binding kinetics of ligand-receptor interactions

Ekaterina Rostova

EPFL2017