In-solution binding kinetics
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Perform binding kinetics studies in-solution, overcoming surface-related issues
& saving days of work.
In-solution binding kinetics measurements are a novel way to observe interactions in real time and under native conditions – bringing the laboratory research closer to a real biological environment and making workflows faster & less cumbersome.
FIDA is the only technology that measures in-solution binding kinetics, without the limitations of surface-based methods. Thanks to in-solution kinetics you can avoid surface optimisation issues, steric hindrance, nonspecific interactions, and conformational changes associated with surface-based methods.
Operating under physiologically relevant conditions, FIDA delivers association rate (kon) and dissociation rate (koff) measurements, with unparalleled flexibility and reliability for analyzing complex biomolecular interactions, including those with long off-rates.
What are the practical benefits?
No regeneration:
No surface required means no need for surface regeneration. Thanks to the in-solution capillary-based technology, work with strong binders or slow off rates does not require surface regeneration. FIDA performs binding kinetics in a solution phase, meaning that the molecules are free to move in their native environment. This reduces the constraints imposed by surface immobilization, where binding reactions may be altered due to the steric hindrance at surfaces as well alteration of protein structure upon immobilization.
Speed:
FIDA saves time in a multitude of ways. On top of removing the time-consuming step of surface regeneration, our technology measures the equilibrium of binding events, which means that data collection can be faster as it captures the entire binding curve more efficiently than e.g. SPR. Furthermore, since any FIDA in-solution assay can be developed and validated in a few days max, our users save time normally devoted to assay development and optimisation.
Surface Heterogeneity and Orientation:
Unlike surface immobilization technologies, in-solution kinetics are free from complications related to variable ligand density and activity across an SPR sensor chip, as well as the fixed orientation of immobilized molecules.
Elimination of Surface-Related Issues:
By operating in solution, FIDA technology avoids the common challenges associated with surface-based techniques, such as non-specific binding and surface-induced artifacts. Without the need for surface immobilization, experiments are free from concerns about surface heterogeneity, ligand orientation, and unwanted interactions, leading to more accurate and reliable results.
Absolute Kinetic Data:
In-solution kinetics provide absolute kinetic parameters without relying on assumptions or calibrations This enhances data reliability and ensures that all of the results are reflective of true molecular behaviour, giving our users the confidence to move forward in your research or product development.
Versatility Across Applications:
Whether used for studying protein-protein interactions, enzyme kinetics, or drug-target binding, in-solution measurements provide robust and versatile information. This adaptability makes it suitable for a wide range of applications, from fundamental research to industrial drug development.
Small Sample Size, Big Impact:
In-solution binding kinetics require only small sample volumes, making working with limited or precious materials easier. The low sample consumption also reduces costs and allows for faster optimization of experimental conditions.
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FIDA Measures kon and koff without surface issues & faster than SPR
FIDA can function as a standalone kinetics instrument, providing the same key measurements as SPR, such as kon and koff, but with significant advantages. FIDA operates in native, in-solution conditions, eliminating the need for surface immobilization, which often introduces complications like non-specific binding and artificial orientation constraints. By performing measurements in solution, FIDA not only speeds up the process but also ensures that the data reflects more natural, physiologically relevant conditions. This makes it an efficient and reliable alternative to SPR for kinetic analyses.
How does FIDA data compare to an SPR?
The table below shows FIDA and SPR data, showcasing the agreement of the two systems. Mind that the FIDA data is obtained in solution (more similarity to native protein environment), with less sample amount and with less handing requirements from the operating scientist.
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Comparison of FIDA and SPR kinetics data
Results for model systems agrees well with SPR data.
Causes for Deviations:
(1) Structural integrity may be compromised by surface immobilization;
(2) Different biophysical technologies can generate different data
Comparison data between FIDA and SPR on kinetics for protein – protein interactions (upper 2 rows) and protein small molecule interaction (bottom 2 rows).
KD (FIDA) was measured using a FIDA instrument equipped with a 480 nm fluorescence detector using a standard premix assay.
kon (FIDA) and koff (FIDA) was measured in a cap-mix assay.
SPR data was obtained using a biacore X100 platform.
In both cases a phosphate buffer (pH 7.40) was used.
FIDA as an SPR Capacity Enhancer
In addition to using FIDA as a “stand alone” kinetics analysis instrument, it can also be used to accelerate your current SPR workflows. With the option for initial FIDA binding/no-binding screening combined with early, rough dissociation ranking, FIDA enables the exclusion of up to 75% of non-viable entities, ensuring that the SPR capacity is focused on high-likelihood candidates
FIDA is frequently employed to extend the capabilities of SPR by improving and streamlining the screening process. Users often leverage FIDA for primary and secondary pre-screening, allowing them to quickly eliminate non-viable candidates before committing to more complex and time-intensive SPR experiments. Additionally, FIDA is frequently used for rapid screening of various conditions, facilitating the optimization of new SPR assays with greater speed and efficiency
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FAQs
Would you like to know more? See frequently asked questions below. If you do not find an answer to your question, you can ask a question to one of our scientists.
‘In-solution binding kinetics’ refer to the process which enables you to get key information on k-on and k-off without having to developing an immobilisation assay. Measurement are made directly within a solution, in conditions that closely mimic natural biological environments.
In in-solution technologies (FIDA), molecules, such as proteins, ligands, or antibodies, are allowed to interact freely in their native state in the solution. The measurement occurs without altering their structure by attaching them to a solid surface, which can sometimes distort the behaviour of these molecules.
Association Rate constant(kon): Measures how fast two molecules come together and form a bound complex. By monitoring the binding event in real time, we can observe how quickly the binding happens. In FIDA, we measure absolute kon (direct measurement)
Dissociation Rate constant (koff): This parameter describes how fast the bound complex breaks the binding. This is critical for understanding the stability of the interaction.
FIDA kinetics take advantage of the time-dependent aspect of diffusivity in a variable pressure-driven flow.
Please see the application note under the link for an in-depth technical explanation. Alternatively, see our brochure or watch our on demand webinar, in which Henrik Jensen, Ph.D. personally describes the process on the topic here or book a discovery call for a personalised explanation.
Learn more during an exploratory call with Fidabio team.
We are happy to answer all of your questions. Book an exploratory call to learn more about FIDA and the match between your personal needs and what we can deliver. The call is non-binding and free of any charges, so feel free to fill the form!