How do you measure antibody binding affinity in solution?
Antibody 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 antibody–antigen and antibody–receptor interactions from the increase in hydrodynamic radius (Rh) of a fluorescent binding partner as the complex forms, with neither partner immobilised. Because the readout is an absolute size, the same measurement reports the size of the complex, which distinguishes monovalent binding from avidity-stabilised, higher-order complexes. Measurements are performed in buffer, serum and plasma.
For example, FIDA characterised the binding of adalimumab to fluorescently labelled TNF-α in buffer and in human plasma. Free TNF-α measured 3.20 nm, complexes reached 10 nm at 60–120 nM adalimumab, and a 1:1 binding model gave an apparent KD of 1.52 nM in buffer and 1.01 nM in 10% plasma (Pedersen et al., 2021).
What can FIDA measure for antibodies?
FIDA measures the affinity, avidity, complex size, receptor binding and concentration of antibodies from the same size readout, in solution and with nothing immobilised.
| Question | What FIDA measures | Example |
|---|---|---|
| How strongly the antibody binds its antigen | FIDA determines the KD of the antibody–antigen interaction from the size increase of the fluorescent partner, with both partners free in solution. | FIDA determined an apparent KD of 1.52 nM for adalimumab binding TNF-α in buffer (Pedersen et al., 2021). |
| Whether the complex is monovalent or multivalent | FIDA measures the size of the complex, which indicates how many molecules of each partner are bound and whether higher-order or agglutinated complexes have formed. | FIDA measured β-lactoglobulin growing from 2.09 nm to 9.23 nm with polyclonal anti-β-lactoglobulin IgG, above the size expected for the quaternary complex (Stender & Sahni Ray, 2023). |
| Whether binding reflects affinity or avidity | FIDA controls how long the partners are in contact before detection, which separates the primary binding event from slower, avidity-stabilised complex formation. | FIDA measured adalimumab–TNF-α complexes of approximately 8.0 nm within 1.8 minutes of mixing, against up to 10 nm in pre-incubated samples (Pedersen et al., 2021). |
| How strongly the Fc region binds its receptor | FIDA determines the affinity of IgG Fc variants for the neonatal Fc receptor (FcRn) at pH 6.0. | FIDA determined a KD of 9.5 µM for wild-type IgG1 Fc and a 6.5-fold higher affinity for the YTE variant (Gothard et al., 2023). |
| How fast the antibody binds and dissociates | FIDA determines the association and dissociation rate constants on the same instrument, in solution. | FIDA measures association and dissociation rate constants from the same size readout (binding kinetics in solution). |
| How much antibody is present | FIDA determines antibody concentration together with affinity from the same titration, including in unpurified supernatant and fermentation broth. | FIDA ranked 96 VHH-producing clones on titre and affinity together, in 10% culture supernatant in PBS (Clone Selection from unpurified samples). |
| Whether the sample is fit for measurement | FIDA reports the size and aggregation of the fluorescent partner in every measurement, so changes in sample quality are recorded alongside the KD. | FIDA confirmed that labelled antigens were monodisperse at 1.9–2.6 nm and ruled out an aggregate fraction that would otherwise have explained the binding data (Ferapontov et al., 2023). |
What is the difference between antibody affinity and avidity?
Affinity is the binding strength of a single antigen-binding site for its epitope, reported as KD; avidity is the overall binding strength of a multivalent interaction, in which several binding sites engage simultaneously. An IgG carries two antigen-binding sites, and many antigens present more than one epitope, so antibody–antigen interactions can form complexes containing several molecules of each partner. Such complexes dissociate more slowly than a single binding site would, and the apparent binding strength then exceeds the intrinsic affinity.
FIDA distinguishes the two because it measures the size of the complex as well as the extent of binding: a 1:1 complex and a higher-order, avidity-stabilised complex have different hydrodynamic radii. For example, when adalimumab and the TNF-α trimer were mixed inside the capillary and measured within 1.8 minutes, the complex size reached a plateau of approximately 8.0 nm and higher-order complexes did not form, whereas pre-incubated samples formed complexes of up to 10 nm. The authors concluded that avidity-stabilised complexes form on a longer timescale than the primary complexes (Pedersen et al., 2021).
Surface-based measurements can be susceptible to avidity artefacts, such as the bridging described for polyubiquitin-binding assays, which can lead to overestimated binding affinities (limitations of surface-based kinetic assessments). In a study of HLA antibodies in patient serum, the authors attributed the slightly lower binding strengths measured by FIDA to monovalent affinity in solution, compared with avidity on a surface (antibody affinity in serum).
How does FIDA characterise multivalent and agglutinating antibody complexes?
FIDA characterises multivalent antibody complexes by controlling the complex lifetime, the time the partners are in contact before detection, which separates the primary binding event from slower agglutination. In capillary mixing, the antibody and the fluorescent antigen meet inside the capillary, and the applied pressure sets how long the complex has to form (ways of setting up FIDA assays).
For example, fluorescently labelled bovine β-lactoglobulin (BLG, 26 nM) was titrated with polyclonal anti-BLG IgG up to 6.3 µM. The size of BLG increased from 2.09 nm to 9.23 nm, above the approximately 6.6 nm expected for the 336 kDa quaternary complex, and aggregate spikes indicated higher-order agglutinates. At complex lifetimes of 1.2, 1.8 and 7.7 minutes, FIDA determined KD values of 13, 36 and 51 nM, and aggregation increased with IgG concentration and with time (Stender & Sahni Ray, 2023).
FIDA has also measured how many antibodies bind an engineered multivalent antigen. On DNA nanoscaffold antigens carrying up to three haptens, FIDA showed that antibody binding scaled with hapten valency, with up to three antibodies per scaffold and no large immune complexes, and gave a KD of approximately 20 nM for an anti-NP monoclonal antibody binding the monovalent scaffold (Ferapontov et al., 2023).
How is Fc receptor binding measured with FIDA?
FIDA determines the affinity of IgG Fc for the neonatal Fc receptor (FcRn) in solution at pH 6.0, the acidic pH at which FcRn binds IgG. FcRn binding is commonly characterised in drug development as a surrogate for the influence of Fc engineering and product stability on antibody half-life.
For example, in the Bioassay Development laboratory at AstraZeneca in Cambridge, UK, fluorescently labelled FcRn (150 nM) was titrated with IgG1 Fc variants up to 6.6 µM in phosphate buffer at pH 6.0. FIDA determined a KD of 9.5 µM for wild-type IgG1 Fc; the half-life-extending YTE mutation increased the affinity 6.5-fold, and YTE combined with TM increased it 10-fold. A complete binding curve consumed less than 1 µL of labelled FcRn, and three weeks at room temperature weakened the wild-type affinity twofold (Gothard et al., 2023). The same study recorded loss of binding together with aggregation after UV stress, discussed in why binding affinity data can be unreliable.
How is nanobody binding measured?
FIDA measures nanobody (VHH) and single-domain antibody binding with the antigen carrying the fluorescent label, so the nanobody itself is measured unlabelled and many candidates can be compared against one labelled antigen. The small size of a nanobody is an advantage here, because the relative size increase on binding a labelled antigen is large.
For example, FIDA determined the affinity of two VHHs raised against tau for tau monomer on a Fida 1 instrument, with Alexa647-labelled VHH at 50 nM titrated with 2N4R tau at 25 °C. The monomer KD was above 4 µM for VHH(510M) and approximately 0.1 µM for VHH(50M), and the authors read the weak monomer binding as consistent with selectivity for pathological tau seeds through avidity (Gupta et al., 2025).
Nanobody-expressing clones can be ranked on affinity and expression level together, directly in unpurified culture supernatant: 96 VHH-producing E. coli clones were screened in 10% supernatant in PBS, and a reverse titration with the labelled antigen fixed at 50 nM solved for both the VHH concentration and the KD (Clone Selection from unpurified samples). Antibody titre in fermentation broth is covered in measuring antibody and protein titre in fermentation broth, and antibodies and nanobodies selective for amyloid oligomers and fibrils in measuring amyloid fibril length and binding in solution.
Can antibody affinity be measured in serum and plasma?
Yes. FIDA measures antibody affinity in serum and plasma with a fluorescently labelled antigen, so only the labelled antigen and its complexes contribute to the signal and the surrounding serum proteins do not. For example, the apparent KD of adalimumab for TNF-α was 1.01 nM in 10% plasma and 1.73 nM in 20% plasma, compared with 1.52 nM in buffer (Pedersen et al., 2021). Affinity and concentration of HLA antibodies in pre-transplant patient serum, and anti-drug antibodies measured together with residual drug activity, are covered in measuring serum and plasma samples.
How are bispecific antibodies characterised?
FIDA characterises bispecific antibodies by measuring the size of a labelled antigen as it forms a binary complex with the antibody and a ternary complex with the second antigen, which gives the affinity of each binding arm and the cooperativity between them. The assay set-up and the results obtained are described in how to analyse bispecific antibodies.
How is an antibody binding assay set up with FIDA?
An antibody binding assay with FIDA is set up with the smaller partner, usually the antigen or the receptor, as the fluorescent indicator at a constant concentration, and the antibody titrated in to saturation. Labelling the smaller partner gives the larger relative size increase on binding, as for TNF-α, which grew from 3.20 nm to up to 10 nm with adalimumab, and it allows the antibody itself to be measured unlabelled. Choice of label is covered in labelling strategies and the general titration design in how to perform a binding affinity assay. Association and dissociation rate constants are determined on the same instrument (binding kinetics in solution), and a binding curve consumes microlitre volumes of indicator (sample usage).
Frequently asked questions
How is antibody binding affinity measured in solution?
Antibody binding affinity is measured in solution with FIDA by titrating the antibody into a constant concentration of a fluorescently labelled antigen and fitting the increase in hydrodynamic radius as the complex forms, with neither partner immobilised. For example, FIDA determined an apparent KD of 1.52 nM for adalimumab binding TNF-α in buffer (Pedersen et al., 2021).
What is the difference between antibody affinity and avidity?
Affinity is the binding strength of a single antigen-binding site, whereas avidity is the combined strength of several binding sites engaged at once, which makes a multivalent complex appear to bind more strongly. FIDA separates the two by measuring the size of the complex and by controlling the complex lifetime. For example, adalimumab–TNF-α complexes measured within 1.8 minutes reached approximately 8.0 nm, whereas pre-incubated samples formed avidity-stabilised complexes of up to 10 nm (Pedersen et al., 2021).
Can FIDA measure antibody binding to FcRn?
Yes. FIDA determines the affinity of IgG Fc for the neonatal Fc receptor (FcRn) in solution at pH 6.0. For example, FIDA determined a KD of 9.5 µM for wild-type IgG1 Fc and showed that the YTE mutation increased the affinity 6.5-fold, from less than 1 µL of labelled FcRn per binding curve (Gothard et al., 2023).
Does the antibody need a fluorescent label?
Not usually. The fluorescent label is placed on the antigen or the receptor and the antibody is titrated in unlabelled, so antibody candidates do not need to be modified; either partner can carry the label when that suits the system better. For example, 96 VHH-producing clones were ranked on affinity and titre against one labelled antigen in unpurified culture supernatant (Hundahl et al., 2023), whereas the affinity of anti-tau VHHs for tau monomer was measured with the label on the VHH (Gupta et al., 2025).
Can antibody affinity be measured in serum or plasma?
Yes. Because only the labelled antigen is detected, the affinity is measured directly in serum or plasma without separation. For example, the apparent KD of adalimumab for TNF-α was 1.01 nM in 10% plasma, compared with 1.52 nM in buffer (Pedersen et al., 2021).
Can FIDA tell whether an antibody sample has aggregated?
Yes. Every FIDA measurement reports the absolute size and the aggregation of the fluorescent species, so aggregation is recorded alongside the affinity. For example, in an AstraZeneca study UV-stressed wild-type IgG1 Fc lost its binding to FcRn and showed aggregation in the same assay (Gothard et al., 2023).
How many antibodies bind one antigen?
FIDA reports the size of the complex, which indicates how many molecules are bound and whether agglutination has occurred. For example, FIDA showed up to three antibodies bound per nanoscaffold antigen carrying three haptens (Ferapontov et al., 2023), while polyclonal IgG with β-lactoglobulin produced complexes of 9.23 nm, larger than the expected quaternary complex, indicating higher-order agglutinates (Stender & Sahni Ray, 2023).
References
Ferapontov, A., Omer, M., Baudrexel, I., et al. (2023). Antigen footprint governs activation of the B cell receptor. Nature Communications. https://doi.org/10.1038/s41467-023-36672-0
Fidabio. How to overcome the limitations of surface-based kinetic assessments? [Blog post]. fidabio.com
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
Gupta, et al. (2025). Conformation-based detection of tau seeds with a novel VHH. Research Square [Preprint]. https://www.researchsquare.com/article/rs-8274351/v1
Hundahl, A. C., Hedeby, H., & Jensen, H. (2023). Clone selection from unpurified samples [Poster]. fidabio.com
Pedersen, M. E., Haegebaert, R. M. S., Østergaard, J., & Jensen, H. (2021). Size-based characterization of adalimumab and TNF-α interactions using Flow Induced Dispersion Analysis: assessment of avidity-stabilized multiple bound species. Scientific Reports. https://doi.org/10.1038/s41598-021-84113-z
Stender, E. G. P. V., & Sahni Ray, K. (2023). Characterising multivalent complex formation using controlled complex lifetime in Fida 1 [Poster]. fidabio.com
Related resources
Read next: How to perform a binding affinity assay? for the general assay design, How to analyse bispecific antibodies? for bispecific formats, Can FIDA test serum samples? for antibody affinity and concentration in patient serum, and How do you measure antibody and protein titre in fermentation broth? for titre.
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