How do you measure antibody or protein titre directly in fermentation broth?
Antibody or protein titre can be measured directly in fermentation broth, without purifying the sample, with Flow Induced Dispersion Analysis (FIDA), developed by Fida Biosystems (Fidabio). FIDA works out how much antibody or protein the broth contains by measuring the size of a fluorescently labelled molecule that binds it, such as labelled protein A: the more product there is in the broth, the more of the labelled molecule is bound and the larger it appears. FIDA does this directly in crude samples such as fermentation media.
As an example, in a published FIDA assay the titre of the antibody rituximab was measured in clarified CHO cell fermentation broth with 4 minutes of analysis time and a limit of quantification of 0.9 nM (135 ng/mL), and the titres were in good agreement with protein A purification followed by UV absorbance (Pedersen et al., 2020).
How does FIDA measure titre in fermentation broth?
FIDA measures titre with a fluorescently labelled molecule that binds the product, called the indicator. For an antibody, the indicator can be labelled protein A or a labelled anti-IgG affibody. On its own the indicator is small. When it binds the antibody, the complex is larger, so its hydrodynamic radius (Rh), the size of the molecule in solution, goes up. The more antibody in the sample, the more indicator is bound and the larger the measured size, which is how FIDA turns a size into a concentration. In the IgG quantification in F12 fermentation media, for instance, labelled protein A grew from a hydrodynamic radius of 5.5 nm on its own to about 11 nm as one or two IgG molecules bound it.
FIDA only detects the fluorescent indicator, so the other proteins in the broth do not affect the result unless they bind the indicator. To read off the titre, FIDA compares the measured size with a calibration curve made with known amounts of product, and no second detection antibody is needed, just as in other complex biological samples. The size itself is an absolute measurement, calculated from physics rather than from a reference standard.
What makes titre hard to measure in broth, and how does FIDA solve it?
| Challenge in fermentation broth | How FIDA solves it |
|---|---|
| Purification before measurement | FIDA measures the antibody or protein directly in the fermentation broth, with no purification step. |
| Other proteins and components in the broth | FIDA only sees the fluorescent molecule that binds your product, so other proteins in the broth do not change the result. |
| The broth's own fluorescence | The broth's fluorescence only lifts the background signal; FIDA still reads the size from the peak of the fluorescent molecule. |
| Product sticking to vials and the capillary | Every FIDA run reports whether the sample sticks or is lost, and coated capillaries stop it sticking. |
| Thick, viscous media | FIDA measures how viscous each sample is and corrects the size measurement for it. |
| Small sample volumes | Each FIDA measurement uses about 40 nL of sample, and Fida Evolve can take samples from wells holding as little as 2 µL. |
| Many samples or clones | FIDA runs samples automatically from vials or 96-well plates, works with pipetting robots, and Fida Evolve connects to lab automation systems. |
| Choosing clones on more than expression level | FIDA measures how much antibody each clone makes (titre) and how tightly it binds (KD) in the same assay. |
Can FIDA measure titre without purification?
Yes. FIDA measures the titre of an antibody or protein directly in unpurified samples such as fermentation media, serum, plasma and cell lysate, and it has no buffer restrictions.
Example: FIDA has quantified exosomes directly in spent fermentation medium, using a fluorescently labelled anti-exosome antibody as the indicator. The antibody's size did not change between 0% and 90% spent medium, which showed that the measurement works in the crude medium (Quantification and Characterization of Exosomes).
How does FIDA measure only the product, and not everything else in the broth?
FIDA measures only the product because it only detects the fluorescent indicator, which is chosen to bind the product. Other proteins in the broth do not change the measured size unless they bind the indicator (how FIDA handles complex samples).
Example: Dr. Jan Nowak, who runs a core facility at Aarhus University, tested this on Fida Evolve by measuring a labelled binder in clean buffer and then in the same buffer spiked with bacterial lysate, including its metabolites and DNA. The baseline stayed flat with no extra signal, and the binding was still measurable in the crude sample (The new Fida Evolve and what early users think).
How do you set up a FIDA titre assay in broth?
To set up a FIDA titre assay, make the calibration samples and the broth samples with the same percentage of medium, so they can be compared directly. If the broth is strongly fluorescent, dilute it until the indicator's signal stands out clearly (measuring in complex samples). Before running any samples, the Fidabio Software can simulate the expected binding curve and suggest the run settings.
Example: To measure human IgG in F12/DMEM medium, known amounts of IgG from 0 to 1000 nM were added to 80% medium together with 100 nM labelled protein A. The binding curve from these samples then served as the calibration curve for reading the titre of unknown samples (IgG quantification in F12 fermentation media).
How does FIDA show if the product is sticking to surfaces?
FIDA shows if the product is sticking to surfaces because every run reports stickiness and sample loss. If the sample sticks, coated capillaries stop it.
Example: When the same sample was measured with and without a mild detergent, FIDA showed a clearly smaller peak area without the detergent, which revealed sample lost to sticking. FIDA reports this peak area in every run, with no extra steps (Sample loss: why measure it and how to interpret it).
How does FIDA deal with viscous fermentation medium?
FIDA measures the viscosity of every sample and corrects the size for it, so thick media and additives such as glycerol do not distort the result. The Fida instrument can also be used on its own to measure viscosity accurately.
Example: When fluorescein was measured in buffer containing 10% glycerol, FIDA gave the correct size with viscosity compensation switched on. With compensation switched off, the same sample came out artificially large (working in viscous media with FIDA).
How much sample does a FIDA titre measurement need?
A FIDA titre measurement needs very little sample: each measurement uses about 40 nL, and Fida Evolve can take samples from wells holding as little as 2 µL.
Example: FIDA ranked 12 designed protein binders directly in 30-fold diluted heat-treated cell lysate, using only microlitre volumes. The 3 binders FIDA picked out were later confirmed with purified protein, with KD values of 44 to 727 nM (Using FIDA for de novo binders).
Can FIDA titre measurements be automated?
Yes. FIDA runs titre measurements automatically from vials or 96-well plates, its autosampler works with pipetting robots, and on Fida Evolve a robotic arm can change the 96-well plates and a built-in SiLA driver connects the instrument to lab automation systems.
Example: FIDA measured the size, polydispersity, aggregation and titre of AAV2 gene therapy particles in one automated run, using 40 nL per data point, with the autosampler holding up to two temperature-controlled 96-well plates. The titre was read from the peak area, which is proportional to particle concentration (360-degree automated characterisation of AAVs).
Can FIDA rank clones on titre and binding at the same time?
Yes. FIDA ranks clones on how much product they make (titre) and how tightly the product binds its target (KD) at the same time, directly from unpurified culture supernatant, and it checks the product's integrity in the same measurement.
Example: In the Fidabio Clone Selection Assay, FIDA ranked 96 nanobody clones from a 96-well plate by how much labelled antigen each had bound, using 39 nL per measurement. A second titration then separated each clone's expression level from its binding strength, without His or other tags. The best clone, once purified, bound its antigen with a KD of 2.2 nM.
Frequently asked questions
Can you measure antibody titre without purifying the fermentation broth?
Yes. FIDA can measure antibody titre without purifying the fermentation broth, because it measures the antibody directly in crude samples such as fermentation media. For example, FIDA measured human IgG directly in 80% F12/DMEM fermentation medium with labelled protein A as the indicator, and the same measurement gave the binding strength of protein A to IgG, a KD of about 60 nM (IgG quantification in F12 fermentation media).
How does FIDA turn a change in size into a titre?
FIDA turns a change in size into a titre by measuring how much of a fluorescent indicator is bound to the product and comparing it with a calibration curve made with known amounts of product. For example, FIDA quantified human serum albumin in 2% human plasma in about 3 minutes, and the result matched the bromocresol purple assay (Poulsen et al., 2015).
Can one FIDA assay measure different IgG antibodies?
Yes. One FIDA assay can measure different IgG antibodies when its indicator binds a part that all IgGs share. For example, an anti-IgG affibody that binds the antibody's constant region makes the method work for any human IgG antibody (Pedersen et al., 2020).
How much sample does a FIDA titre measurement need?
A FIDA titre measurement needs very little sample: each measurement uses about 40 nL, and Fida Evolve can take samples from wells holding as little as 2 µL. For example, designed protein binders have been ranked directly in diluted cell lysate using only microlitre volumes (Using FIDA for de novo binders).
Can you measure titre and binding of clones in the same assay?
Yes. FIDA can measure how much product each clone makes and how tightly it binds in the same assay. For example, 96 nanobody-producing E. coli clones were screened directly in unpurified supernatant and ranked on titre and affinity together (Clone Selection from unpurified samples).
References
Fidabio [Herms, C., & Wasilczyk, M.]. (2023). Sample loss: Why measure it and how to interpret it [Blog post]. fidabio.com
Fidabio [Hundahl, A. C., Hedeby, H., & Jensen, H.]. (2023). Clone selection from unpurified samples [Poster]. fidabio.com
Fidabio [Marabini, R.]. (n.d.). Fidabio Clone Selection Assay: Protein expression level and affinity directly in fermentation media without purification [Application note]. fidabio.com
Fidabio [Mirza, N.]. (n.d.). Quantification and characterization of exosomes [Application note]. fidabio.com
Fidabio [Pedersen, M. E.]. (n.d.). Selective quantification of IgG directly in F12 fermentation media [Application note]. fidabio.com
Fidabio [Pedersen, M. E., Goral, J., & Jensen, H.]. (n.d.). 360-degree automated characterization of AAVs: The safe solution for gene therapy [Poster]. fidabio.com
Fidabio [Wasilczyk, M.]. (2025). Supporting the next wave of protein design: Using FIDA for de novo binders [Blog post]. fidabio.com
Fidabio [Wasilczyk, M., & Nowak, J. S.]. (2026). The new Fida Evolve: What is it, how does it differ, and what do early users think? [Blog post]. fidabio.com
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
Poulsen, N. N., Andersen, N. Z., Østergaard, J., Zhuang, G., Petersen, N. J., & Jensen, H. (2015). Flow induced dispersion analysis rapidly quantifies proteins in human plasma samples. Analyst. pubs.rsc.org
Related resources
Read next: How do you characterise proteins in complex biological matrices?, FIDA readouts and Why is protein binding affinity (KD) data unreliable? Prof. Timothy Jenkins, head of the Center for Translational Protein Design at the Technical University of Denmark (DTU), describes how his group uses FIDA to screen designed binders in crude samples in From snakebite to cancer: designing protein binders faster than we can measure them.
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