How to measure protein binding in serum or plasma?

To measure protein binding directly in serum or plasma, you can use FIDA (Flow Induced Dispersion Analysis). FIDA measures binding in solution, without immobilising either partner, so you can study an interaction in the biological matrix itself rather than in a purified system.

How does FIDA measure binding in serum and plasma?

FIDA follows the apparent size (hydrodynamic radius) of a fluorescently labelled molecule, the indicator, as it binds its partner in the sample. Both molecules stay free in solution. When the indicator binds, it appears larger, and this change in apparent size is used to determine the interaction and the concentration of the binding partner. See What is FIDA? for how the measurement works.

Does the serum background affect the measurement?

No, the serum background does not interfere with the measurement. FIDA reads the size from the indicator's peak, and the serum only raises the flat baseline underneath it. In a FIDA measurement, the analyte solution is recorded as the baseline signal and the indicator as a Gaussian peak (see the image below).

For example, the binding of a monoclonal anti-dsDNA antibody to a labelled dsDNA indicator gave K_D values of 236, 278 and 362 nM in 0, 20 and 85% healthy donor plasma, which correlated well, as shown in the application note Detection of Auto-Antibodies in Systemic Lupus Erythematosus Patients.

How much serum or plasma can you use?

It depends on the assay and on the fluorescence background of your sample. FIDA measurements have been run in serum and plasma at concentrations from 2% up to 100%. To compare samples with each other, keep the serum or plasma concentration the same in all of them.

For example, the application note FIDA in a clinical setting used 10% plasma or serum as the analyte, and 3% when the fluorescence background was high. The application note Protein Oligomerization: Easy and Accurate determination in plasma and buffer followed the oligomeric state of a therapeutic protein in 90% plasma, and the case study Case studies: 50x faster screening with FIDA & 100% Plasma binding measurements measured binding in 100% plasma.

What can you measure in serum and plasma?

With FIDA, you can measure binding affinity, protein concentration, binding kinetics, drug activity and immunogenicity in serum and plasma.

How to measure antibody affinity and concentration in patient serum?

FIDA determines the affinity (K_D) and the concentration of an antibody in the same serum sample. Because the measurement is in solution, it reports true affinity rather than avidity.

For example, the publication HLA antibody affinity determination: From HLA-specific monoclonal antibodies to donor HLA specific antibodies (DSA) in patient serum (HLA, 2023) determined both values for donor-specific HLA antibodies in 20 pre-transplant patient sera, with K_D from 0.055 to 24.7 nM and concentrations from 11.2 to 1223 nM.

How to measure binding kinetics in serum?

FIDA measures the association and dissociation rate constants (k_on and k_off) in serum without immobilisation, with both partners free in solution. How FIDA measures kinetics is explained in How to measure binding kinetics in solution?

For example, the publication In-solution Characterization of Biomolecular Interaction Kinetics under Native Conditions (Analytical Chemistry, 2025) measured binding kinetics for protein-protein and protein-small molecule pairs, including an interaction in 90% human serum.

How to quantify proteins in plasma?

FIDA quantifies a protein in plasma in one step, from the change in apparent diffusivity of an indicator as it binds the target protein. It needs a single affinity interaction, with no surface chemistry or secondary antibodies.

For example, the publication Flow Induced Dispersion Analysis Rapidly Quantifies Proteins in Human Plasma Samples (Analyst, 2015) quantified human serum albumin in 2% human plasma in as little as 3 minutes, with results comparable to the standard bromocresol purple assay, and detected antibody binding at concentrations as low as 1 nM.

How to measure drug activity and anti-drug antibodies in patient serum?

FIDA measures active drug and anti-drug antibodies in patient serum in a single assay. The apparent size of the drug's fluorescently labelled target shows how much active drug is present and whether anti-drug antibodies have formed.

For example, the publication Assessment of immunogenicity and drug activity in patient sera by flow-induced dispersion analysis (Scientific Reports, 2022) analysed serum from 40 arthritis patients treated with adalimumab by tracking the size of TNF-α, and detected more immunogenicity than a comparison ELISA.

Frequently asked questions

How do you set up a serum or plasma assay in the Fidabio software?

The Fidabio software includes serum, plasma and other additives in the pipetting scheme it calculates for the assay. This is covered in the Software Basics course, section Assay design, on the Fidabio Learning Hub.

Related resources

Working in cell lysate, fermentation broth or another crude sample? How do you characterise proteins in complex biological matrices? explains what makes these samples hard to measure and how to set up a FIDA assay in them.

Working with patient samples? The application note FIDA in a clinical setting follows sphingosine 1-phosphate binding to serum albumin and HDL in plasma from healthy donors and surgical trauma patients, and the publication Flow-Induced Dispersion Analysis for Probing Anti-DsDNA Antibody Binding Heterogeneity in Systemic Lupus Erythematosus Patients (Analytical Chemistry, 2016) reveals how autoantibody binding differs between lupus patients, measured in up to 85% plasma.

Prefer to watch? In the webinar Early detection and monitoring of Parkinson's disease. A story of Neurotidal, Francoise Dekker, Ph.D., shows how amyloid fibril size is measured directly in patient blood and spinal fluid. In the webinar How to screen de novo designed protein binders in unpurified lysate using flow induced dispersion analysis?, Magnus Kjæregaard, Ph.D., shows how binders are screened in crude lysate, with sample dilution to handle its autofluorescence.

More examples in plasma: the application note Correlating Supramolecular Behaviour in Buffer and Plasma shows how a potential therapeutic molecule behaves in buffer and in human plasma, where it binds human serum albumin.

Want to know if FIDA can measure your interaction in serum or plasma? Book a discovery call.