View the posters and watch a presentation of each by the winners
Christian Fercher
ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland
Find out more or contact at www.cbns.org.au.
Fluorescence-based immunodiagnostics are an emerging field in biosensor development and exhibit several advantages over traditional detection methods. While various affinity biosensors have been developed to generate a fluorescence signal upon sensing varying analyte concentrations, reagentless, reversible and continuous monitoring of complex biological samples remains challenging. Here, we aimed to genetically engineer biosensors based on single-chain antibody fragments (scFv) that are site-specifically labelled with environmentally sensitive fluorescent unnatural amino acids (UAA).
A rational design approach resulted in quantifiable analyte-dependent changes in peak fluorescence emission wavelength and enabled antigen detection in vitro. Incorporation of a polarity indicator in proximity of the antigen binding interface generated a titratable wavelength blueshift, resulting in nanomolar detection limits. In order to ensure continuous analyte monitoring, scFv candidates with fast binding and dissociation kinetics were selected from a CDR mutant library employing a directed evolution phage display biopanning approach coupled to subsequent high-throughput affinity screening. Initial rankings were further refined towards rapid dissociation kinetics using bio-layer interferometry (BLI) and surface plasmon resonance (SPR).
The most promising candidates were expressed, purified and tested for their potential to detect varying concentrations of the target antigen in a continuous microfluidics-based assay. Variations of dissociation kinetics within an order of magnitude were achieved without compromising specificity of the antibody fragments. This approach is generally applicable to numerous antibody/antigen combinations and currently awaits integration in a wide range of assay platforms for specific one-step protein quantification in complex samples.
Monica Fernández-Quintero
Department of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Austria
Find out more at liedllab.org or email Monica.Fernandez-Quintero@uibk.ac.at
In contrast to this prevalent static view of the binding interface we demonstrate a dynamic perspective not only of the paratope but of whole Fvs and Fabs. We show that antibodies exist as ensembles of paratope states. These paratope states are defined by a characteristic combination of CDR loop conformations and interdomain orientations. They interconvert into each other in the micro-to-millisecond timescale by correlated loop and interdomain rearrangements. We demonstrate that crystal packing effects can distort the paratope state and thus result in misleading X-ray structures. By advancing the repertoire of cutting-edge simulation techniques, for the first time we achieve a complete description of conformations, thermodynamics and kinetics of the whole binding paratope in solution.
These findings have broad implications in the field of antibody design and in the development of biotherapeutics as they provide a new paradigm in the understanding of CDR binding loop states, antibody-antigen recognition, relative VH and VL interface angles and elbow-angle distributions and their respective dynamics. Preliminary findings are already published in six manuscripts, but a considerable number of further publications is upcoming.
These upcoming publications will also address issues like inter-loop correlation and the relationship of Fv-interface dynamics with loop rearrangements:
