How to perform a binding affinity assay?
Protein binding affinity can be measured in solution with Flow Induced Dispersion Analysis (FIDA), developed by Fida Biosystems (Fidabio). FIDA determines the equilibrium dissociation constant (KD) of protein–protein, antibody–antigen, protein–small molecule, peptide and membrane protein interactions from the change in hydrodynamic radius of a fluorescent binding partner, with both partners free in solution. The accessible affinity range extends from pM to mM, association and dissociation rate constants are determined on the same instrument (binding kinetics in solution), and measurements are performed in any buffer as well as in serum, plasma and cell lysate.
For example, FIDA determined the KD of frataxin for the iron–sulfur cluster assembly complex (NFS1–ISD11–ACP–ISCU2) as 102 nM, in agreement with 146 nM obtained by isothermal titration calorimetry (ITC) (Want et al., 2026).
What is a binding affinity assay with FIDA?
A binding affinity assay measures how strongly two molecules bind, reported as the dissociation constant KD. FIDA is a fluorescence-based technique, so a binding affinity assay requires one partner with intrinsic fluorescence or a fluorescent label. This partner is the indicator, and its signal reports the interaction with the binding partner, the analyte.
During the assay the indicator is kept at a constant concentration and the analyte is titrated in at increasing concentrations. The highest analyte concentration should saturate binding, so that the binding curve reaches a plateau at which the complex is fully formed. A single titration yields the hydrodynamic radius of the free indicator, the hydrodynamic radius of the indicator–analyte complex and the dissociation constant, KD.
How does FIDA measure binding affinity?
When two biomolecules interact and form a complex in solution, the complex diffuses more slowly than the free partners, which extends its dispersion profile. FIDA quantifies this from the hydrodynamic radius: using the Stokes-Einstein equation, the FIDA software converts the change in dispersion into an increase in size as the complex forms. The increase in apparent size across the titration reflects the fraction of indicator in complex, and fitting this increase yields KD from the hydrodynamic radius alone. Because the measurement is made directly in solution with nothing immobilised, it applies to protein-protein interactions, protein-small molecule binding and other biomolecular studies.
Where complex formation causes only a small change in hydrodynamic radius, as for a small molecule binding a large protein, FIDA additionally quantifies binding from the binding-related intensity change (BRIC) and, on Fida Neo and Fida Evolve, from the shift in emission wavelength (Lambda Dynamics), within the same measurement (small molecule binding in solution).
Which parameters does a FIDA binding assay determine?
| Parameter | How FIDA determines it |
|---|---|
| Equilibrium dissociation constant (KD) | FIDA determines KD by fitting the change in hydrodynamic radius across a titration, over a range from pM to mM. |
| Association and dissociation rate constants (kon, koff) | FIDA determines the rate constants in solution by comparing an equilibrium binding curve with a pre-equilibrium curve, for interactions with time scales from seconds to hours. |
| Complex size and stoichiometry | FIDA reports the absolute hydrodynamic radius of the free indicator and of the complex, from which the number of bound partners can be assessed. |
| Sample quality | Each FIDA measurement also reports aggregation, adsorption to the capillary (stickiness), sample viscosity and the fraction of unconjugated dye. |
| Binding under native or physiological conditions | FIDA measures binding in any buffer, including detergent- or DMSO-containing buffers, and in serum, plasma or cell lysate without prior purification. |
| Sample consumption | Each FIDA data point consumes approximately 40 nL of indicator, so a complete binding isotherm requires microlitre volumes. |
How is a FIDA binding affinity assay performed?
A FIDA binding affinity assay is performed by keeping a fluorescent indicator at a constant concentration, ideally below the expected KD, and titrating the analyte up to a saturating concentration. The assay is designed in four steps:
- Selection of the indicator. The indicator is detected either through its intrinsic tryptophan fluorescence at 280 nm or through a fluorescent label (labelling strategies for FIDA).
- Design of the titration. The Fidabio software simulates the expected binding isotherm before the experiment, which guides the choice of indicator and analyte concentrations (FIDA software).
- Choice of assay format. Premix is used for equilibrium KD determination, Capmix when the indicator is limited, CapDis when the analyte is limited, and continuous titration (cSPRING) for KD determination in approximately one minute (FIDA assay formats).
- Model fitting. A 1:1 binding model is applied to simple interactions, and the excess-indicator and stoichiometry models to interactions involving more than one binding partner (FIDA binding models).
The sources of error that make affinity data unreliable, and the FIDA readouts that identify each of them, are described in why protein binding affinity data is unreliable.
Which interactions has FIDA characterised?
FIDA has been used to characterise interactions of proteins, antibodies, small molecules, peptides, membrane proteins and nucleic acids, both in buffer and in complex biological samples.
Protein–protein interactions
FIDA determines protein–protein affinities by direct titration and by competition. Example: FIDA determined a KD of 102 nM for frataxin binding to the iron–sulfur cluster assembly complex, compared with 146 nM by ITC, and competition against labelled frataxin was used to rank ferredoxin-2 mutants (Want et al., 2026).
Antibody–antigen interactions
FIDA determines antibody affinity and antibody concentration in the same assay, including in patient serum. Example: FIDA quantified donor-specific HLA antibodies directly in 20 pre-transplant patient sera, with KD values between 0.055 and 24.7 nM and concentrations between 11.2 and 1,223 nM (Hug et al., 2023).
Protein–small molecule interactions
FIDA determines small molecule affinities label-free, from the hydrodynamic radius and intrinsic fluorescence of the target protein. Example: FIDA determined an apparent KD of 1.5 nM for binding of the Alzheimer's disease drug candidate REM127 to SEPT6 from the change in hydrodynamic radius, and 1.6 nM from the change in fluorescence intensity in the same measurement (Princen et al., 2024). By continuous titration, FIDA determined a KD in 45 seconds in fast mode (Willmer et al., 2025).
Membrane protein interactions
FIDA determines affinities for membrane proteins solubilised in detergent or reconstituted in nanodiscs. Example: FIDA determined a KD of 96 nM for binding of the bacteriocin enterocin C to the membrane protein BacA in 0.1% DDM (Folcher et al., 2025).
Low-affinity interactions
FIDA quantifies low-affinity interactions in the millimolar range as well as high-affinity interactions. Example: FIDA determined an apparent association constant of approximately 470 M−1 (KD approximately 2 mM) for β-cyclodextrin binding a fluorescein-labelled tryptophan probe (Hamacek & Perche, 2026).
Binders in unpurified lysate
FIDA identifies binders in unpurified expression lysate before purification. Example: FIDA identified de novo designed binders directly in heat-treated bacterial lysate, and subsequent titrations gave KD values of 2.76 to 10.0 µM for the ALFA-tag binders (Pinheiro et al., 2025).
Can FIDA determine binding kinetics as well as affinity?
Yes. FIDA determines association and dissociation rate constants in solution by comparing an equilibrium binding curve with a curve recorded while the partners react inside the capillary, and the resulting rate constants agree closely with those obtained by SPR (binding kinetics in solution). For example, FIDA determined protein–protein binding kinetics in human serum (Willmer et al., 2025).
Frequently asked questions
How is protein binding affinity measured in solution?
Protein binding affinity is measured in solution with FIDA by titrating the analyte into a constant concentration of a fluorescent indicator and fitting the increase in hydrodynamic radius as the complex forms, with neither partner immobilised. For example, FIDA determined a KD of 102 nM for frataxin binding to the iron–sulfur cluster assembly complex, in agreement with 146 nM by ITC (Want et al., 2026).
Which KD range can FIDA measure?
FIDA measures KD values from pM to mM. For example, FIDA determined KD values down to 0.055 nM for HLA antibodies in patient serum (Hug et al., 2023) and approximately 2 mM for β-cyclodextrin binding (Hamacek & Perche, 2026).
Can binding affinity and binding kinetics be measured on the same instrument?
Yes. FIDA determines the equilibrium dissociation constant (KD) and the association and dissociation rate constants (kon, koff) on the same instrument, in solution. For example, FIDA determined protein–protein binding kinetics in human serum (Willmer et al., 2025).
Does a protein need a fluorescent label for a FIDA binding assay?
Not necessarily. FIDA measures binding label-free from the intrinsic tryptophan fluorescence of the protein, and a fluorescent label is used when intrinsic fluorescence is insufficient or when the measurement is made in a protein-rich matrix such as serum. For example, FIDA determined an apparent KD of 1.5 nM for REM127 binding to SEPT6 label-free (Princen et al., 2024).
How much sample does a FIDA binding assay require?
A FIDA binding assay consumes approximately 40 nL of indicator per data point, so a complete binding isotherm requires microlitre volumes (sample usage). For example, in fast-mode continuous titration FIDA measured three inhibitors in triplicate in under 7 minutes, using 4.2 µL of protein per inhibitor (Willmer et al., 2025).
Related resources
The definition and interpretation of the dissociation constant are covered in what KD, the dissociation constant, is, and the binding affinity readout describes how FIDA reports it.
See our literature base for more.
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