Why is protein binding affinity (KD) data unreliable?
Protein binding affinity (KD) data becomes unreliable when the sample or the measurement conditions change what is being measured. Problems that distort a KD include aggregated or degraded protein, protein sticking to surfaces, free dye after labelling, uncorrected viscosity, binding that has not reached equilibrium and an indicator concentration outside the assay's range (Fidabio, 2023; Fidabio, 2024; Gothard et al., 2023).
Flow Induced Dispersion Analysis (FIDA), developed by Fida Biosystems (Fidabio), measures the KD in solution together with the absolute size of the molecules. Every FIDA measurement also reports sample quality, so these problems are visible in the same run as the KD (Fidabio, 2023).
What makes protein KD data unreliable, and how does FIDA address it?
| Problem | FIDA feature |
|---|---|
| Aggregated, degraded or non-native protein | Absolute size and aggregation count in every run |
| Protein sticking to surfaces | Stickiness readout and coated capillaries |
| Free dye after labelling | Labelling quality readout: free versus conjugated fluorophore |
| Viscous samples | Viscosity measurement and compensation |
| Binding not at equilibrium | Premix for equilibrium KD, controlled reaction time for kinetics |
| Indicator concentration outside the assay's range | Adjustable indicator concentration and an excess-indicator model |
| Immobilisation, buffer or matrix changing the interaction | In-solution measurement without immobilisation, in the sample's own buffer or matrix |
How does FIDA detect aggregated, degraded or non-native protein?
FIDA detects aggregated, degraded or non-native protein by reporting the absolute hydrodynamic radius and counting aggregates as spikes in every measurement, so a damaged sample is visible in the same run as its KD (Fidabio, 2023). The measured size can also be compared with the size predicted from the protein's PDB or AlphaFold structure (Fidabio, 2023).
Example: In an AstraZeneca study, FIDA showed that UV-stressed wild-type IgG1 Fc had lost its binding to FcRn and had aggregated, both in the same assay (Gothard et al., 2023). For human serum albumin, FIDA measured a hydrodynamic radius of 3.59 nm against 3.50 nm predicted from its structure, confirming a native state (Fidabio, 2023).
How does FIDA detect protein sticking to surfaces?
FIDA detects protein sticking to surfaces as tailing of the signal in every measurement, and Fidabio coated capillaries resolve it (Fidabio, 2023).
Example: In a study of the bacteriocin enterocin C, the FIDA Taylorgram showed that the peptide adhered to the capillary in detergent-free buffer. With 0.1% DDM no adherence was observed, and FIDA measured the peptide's binding to the membrane protein BacA at a KD of 96 nM (Folcher et al., 2025).
How does FIDA detect free dye after labelling?
FIDA detects free dye after labelling by reporting the fractions of free and conjugated fluorophore in every labelled sample, so the analysis can use only the signal from the labelled protein (Fidabio, 2023).
Example: A two-species fit in the FIDA data analysis software separated free FITC from labelled albumin and showed that a 96-well clean-up step removed 92% of the free dye (Restan et al., 2019).
How does FIDA handle viscous samples?
FIDA handles viscous samples by measuring the absolute viscosity of every sample from its residence time in the capillary and compensating for it automatically (Fidabio, 2023).
Example: FIDA measured antibody titre directly in clarified fermentation broth, with viscosity corrected for each sample and a limit of quantification of 0.9 nM (Pedersen et al., 2020).
How does FIDA make sure binding is measured at equilibrium?
FIDA measures the equilibrium KD by premixing the binding partners, and it can instead set a defined reaction time by capillary mixing, which turns a non-equilibrium measurement into binding kinetics (Fidabio, 2024).
Example: For an affibody binding rituximab, the FIDA premix gave an equilibrium KD of 0.31 nM, and capillary mixing at controlled reaction times gave the association and dissociation rates (Fidabio, 2024).
How does FIDA keep the indicator concentration in range?
FIDA keeps the indicator concentration in range because the fixed indicator concentration can be adjusted to tailor the dynamic range of the binding assay (Otzen et al., 2021), and the Fidabio Software includes an excess-indicator model alongside 1:1 and multi-site models (fitting models).
Example: In a FIDA study in fermentation broth, lowering the indicator from 20 to 5 nM confirmed that it was in excess, and the excess-indicator model was used for accurate quantification (Pedersen et al., 2020).
How does FIDA avoid immobilisation and buffer effects?
FIDA avoids immobilisation by keeping both binding partners free in solution, which preserves molecular mobility and avoids structural constraints, and it works without buffer restrictions on minimal sample (Willmer et al., 2025).
Example: FIDA measured the association and dissociation rates of a protein–protein interaction directly in human serum (Willmer et al., 2025). See How do you characterise proteins in complex biological matrices?
Frequently asked questions
Why is my KD different every time I measure it?
A KD can change with the state of the sample: in a FIDA study, three weeks at room temperature weakened an antibody's FcRn affinity twofold (Gothard et al., 2023), and buffer composition, supplier and sample batch can also change measured affinities (Willmer et al., 2025). FIDA reports size and sample quality with every KD, so you can see whether two measurements came from comparable samples (Fidabio, 2023).
Can protein aggregation change a KD?
Yes. In an AstraZeneca study on FIDA, UV-stressed IgG1 Fc aggregated and lost its binding to FcRn (Gothard et al., 2023).
How do you know if a protein sample is good enough to measure affinity?
Compare its absolute size with the size predicted from its structure, and check aggregation, stickiness, polydispersity and free dye. FIDA reports all of these in the same run as the KD (Fidabio, 2023).
Can you measure KD without immobilising the protein?
Yes. FIDA measures binding with both partners free in solution, without buffer restrictions, using nanolitres of sample per data point (Fidabio, 2023; Willmer et al., 2025).
Does viscosity affect a FIDA measurement?
FIDA measures the absolute viscosity of every sample and compensates for it automatically, so the reported size reflects the molecule (Fidabio, 2023).
References
Fidabio [Ray, K.]. (2023). Assessment of sample quality with every measurement. FIDA fundamentals [Application note]. fidabio.com
Fidabio [Ray, K., Jensen, H., & Hundahl, A. C.]. (2024). Immobilisation free in-solution kinetics using flow induced dispersion analysis (FIDA) [Technical note]. fidabio.com
Folcher, V., Auger, R., Louche, C., Serror, P., & Touzé, T. (2025). Mechanistic insights into enterocin C targeting the undecaprenyl phosphate recycling protein BacA. Journal of Biological Chemistry. https://doi.org/10.1016/j.jbc.2025.110929
Gothard, M., Pérez-Martínez, D., & Stender, E. G. P. V. (2023). In-solution characterization of affinity and structural properties of the FcRn IgG1 Fc interaction during product development [Application note]. fidabio.com
Otzen, D. E., Buell, A. K., & Jensen, H. (2021). Microfluidics and the quantification of biomolecular interactions. Current Opinion in Structural Biology, 70, 8–15. https://doi.org/10.1016/j.sbi.2021.02.006
Pedersen, M. E., Østergaard, J., & Jensen, H. (2020). In-solution IgG titer determination in fermentation broth using affibodies and flow-induced dispersion analysis. ACS Omega, 5, 10519–10524. https://doi.org/10.1021/acsomega.0c00791
Restan, M. S., Pedersen, M. E., Jensen, H., & Pedersen-Bjergaard, S. (2019). Electromembrane extraction of unconjugated fluorescein isothiocyanate from solutions of labeled proteins prior to flow induced dispersion analysis. Analytical Chemistry. https://doi.org/10.1021/acs.analchem.9b00730
Willmer, P., Stender, E. G. P., Sahni Ray, K., Hundahl, A. C., Marie, R., & Jensen, H. (2025). In-solution characterization of biomolecular interaction kinetics under native conditions. Analytical Chemistry, 97, 19498–19504. https://doi.org/10.1021/acs.analchem.5c02164
Related resources
Read next: What is KD (dissociation constant)?, Which binding model should you use to fit FIDA affinity data?, What is protein stickiness? and How to label a protein for FIDA measurements?
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