Newsletter | Fidabio

G protein-coupled receptors (GPCRs) are the largest family of membrane proteins. They are involved in many vital physiological cell mechanisms, amongst others hormones signalling, cell cycle regulation, neurotransmitters signalling and smell receptors. GPCRs-targeting drugs represent 27% of the global commercial drugs. Therefore, new technologies in this area will advance new discoveries and ultimately new treatments for GPCR related disorders. In this work, it is shown that Flow-Induced-Dispersion-Analysis (FIDA) can be used to fully characterize the binding between Nanodisc-embedded β2-adrenergic receptor and Nanobody (Nb80). In addition to the ability to analyse the functions of the complex, the built-in quality control validates sample integrity and the platform also provides information on the oligomeric state of the receptor. This application is another example of the accuracy of our capillary-based technology for measuring complex bindings through absolute and accurate size determinations of analytes in a pressure-driven flow system.

Materials & Methods
Fida 1 platform with 480 nm LED fluorescence detection for binding

experiments (Fida Biosystems ApS). FIDA standard capillary (i.d.: 75 µm, LT: 100 cm, Leff: 84 cm). HEPES buffer 10mM pH 7.4, 0.5 mg/ml BSA was used as the working buffer. Nanobody Nb80 was used as the indicator. Nb80 was labeled with an Alexa Fluor® 488 Protein Labeling Kit from ThermoFisher Scientific. NanoDisc-embedded β2-adrenergic receptor (β2AR) was used as the analyte (0-1 µM). Sample analysis was performed by filling the capillary with the analyte, followed by an injection of 39 nL of preincubated indicator+analyte, which was mobilized towards the detector at 400 mbar.

Conclusion
These results show how the Fidabio platform may be used to provide functional characterisation of complex membrane proteins and, in the specific case, determine the activity of GPCR embedded in Nano-Discs, based on in-solution and non-invasive analyses. 

For more information, please refer to our application note on the topic.

Over the past couple of years, assessments of immune responses in numerous patient samples have been conducted using our Fidabio technology. The clear advantages are testing and characterization under physiological relevant conditions as well as absolute and quantitative measurements.

As part of Morten Enghave Pedersen’s industrial PhD project, he has established a new application involving characterization of immunogenicity in patients receiving treatment with the anti-TNF-α drug, adalimumab. In only 2 weeks, he has analyzed more than 1,000 data points, and thereby identified patients that had generated neutralizing antibodies and differentiated these from patients with therapeutically active adalimumab.

Morten is in the process of further expanding this unique application.

The figure below shows the development data of the immunogenicity model.

The interactions between TNF-α and adalimumab were fully characterized using the FIDA 1 instrument. The apparent size of TNF-α-alexa488 (100 nM) was measured in a titration series with adalimumab (0-1000 nM).  Here, he observed the formation of both lower and higher-order TNF-α-adalimumab complexes, due to multiple stoichiometries (seen as a hook effect). The binding isotherm was applied to the data set (solid line), revealing an apparent Kd of 1.52 nM and complex size of 8.9 nm. Finally, size-based characterization of the TNF-α-adalimumab complexes allows for immunogenicity assessments in patients receiving adalimumab treatment, by following the apparent size of TNF-α. Hence, the presence of neutralizing antibodies will be reported as a low apparent size of TNF-α, whereas occurrences of binding antibodies will result in high TNF-α size.

We are looking forward to publishing the entire data set. Please get with us, if in the meantime, you are interested in following the project.

New software features enabling fast and agile amendments of sequences

In our continuous effort to improve our software suite, we have just launched a significant new feature that enables users to set up, edit, and adjust sequences on a single parameter level.

It is easy with the Fidabio Assay Design software to set up your experiments and automatically generate the protocols, the methods and sequences needed to run your analyses, incl. the titration series required to establish your binding curves. From time-to-time however, you may want to adjust specific method and sequence parameters. With the new features, we have made such edits as easy as changing the content of a cell in an excel sheet. In the screenshot below editing is done in the table to the left.