Accurate solution phase affinity profiling of a SARS-CoV-2 antibody in serum

ATA Scientific Pty Ltd
By Viola Denninger, Sebastian Fiedler, Alison Ilsley, Heike Fiegler and Sean Devenish
Tuesday, 13 October, 2020

The ability to accurately characterize the immune response against SARS-CoV-2 is of vital importance in managing the current COVID-19 pandemic.

Measuring antibody affinity under physiologically relevant conditions in complex mixtures like serum remains challenging but is critically important to furthering our understanding of the immune response and protection window in patients and vaccinated individuals. Using Microfluidic Diffusional Sizing (MDS), we have characterized an anti-spike S1 antibody by measuring its binding affinity to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein directly in serum.


In the quest to accurately identify individuals who are seropositive and effective vaccines against SARS-CoV-2, it is fundamental to thoroughly characterize the immune response during infection or after vaccination. Specifically, the virus-neutralizing capacity of the immune system is of vital interest, and accurate tests to evaluate the affinity and quantity of neutralizing antibodies (NAbs) in serum samples of COVID-19 patients or vaccinated individuals are key.

For SARS-CoV-2 virions, the spike protein is crucial for virus entry into the host cell. It is composed of two subunits: S1, which binds to ACE2 (Figure 1); and S2, which mediates the entry of the virion into the cell. Due to its key role mediating the first step of viral invasion of host cells, the RBD (receptor binding domain) of S1 has proven to be the target of NAbs raised against other viruses of the corona family, and is likely to also be an important target in the case of SARS-CoV-2.1

Figure 1: Antibodies that bind the RBD are expected to neutralize the SARS-CoV-2 virus. The RBD region of the spike S1 subunit binds to the ACE2 receptor on the cell membrane before the spike S2 subunit mediates membrane fusion. Binding of antibodies to the RBD can prevent receptor binding and subsequent invasion of the host cell.

In ELISA tests, which are used to determine seropositivity in patient samples, the reported titer for each sample is dependent on both concentration and affinity of the antibody. However, the contribution of each of these parameters to the detected signal cannot be accurately decoupled. The ability to determine these two important parameters independently is crucial for a deeper understanding of the immune response. This knowledge would therefore allow a better understanding of antibody maturation and persistence of immunity and could aid in convalescent plasma therapy research.

Measuring antibody affinity in human samples ideally makes use of undiluted serum to maximize the range of antibody concentrations that can be used to generate the equilibrium binding curve. Most established technologies for measuring protein binding, however, rely on surface immobilization of one of the binding partners. This can cause significant difficulties when working with complex samples such as serum due to non-specific binding of other proteins within the serum to the analytical surface, leading to false positives or at least low signal-to-noise ratios.2,3

Here, we apply MDS to measure the affinity of an anti-spike S1 antibody to fluorescently labeled SARS-CoV-2 RBD directly in serum. This in-solution technology enables the detection of antigen–antibody interactions by measuring the changes in hydrodynamic radius (Rh) of the labeled antigen upon binding to the antibody. As a result, MDS allows the accurate detection and characterization of antibodies directly in serum, thus eliminating the constraints of surface-bound technologies.4


To assess the binding affinity of the anti-spike S1 antibody to the RBD of the SARS-CoV-2 spike protein, the antibody was titrated against a constant concentration of 20 nM Alexa Fluor 647 labeled recombinantly expressed RBD. As a control, the titration experiment was first performed in buffer. Figure 2A shows the affinity binding curve measured in PBS with 0.05% Tween 20, yielding a KD of 9.6 ± 1.7 nM.

Figure 2: Equilibrium binding curves of anti-spike S1 antibody to 20 nM Alexa Fluor 647 labeled SARS-CoV-2 RBD in (A) buffer (PBS with 0.05% Tween 20) and (B) human serum. Measurements were performed in triplicate. For serum measurements, serum background fluorescence was subtracted from raw data before the KD was determined by non-linear least squares.

For the measurements in human serum, the same antibody concentrations were titrated against 20 nM Alexa Fluor 647 labeled RBD, with the antibody diluted in serum. Figure 2B depicts the corresponding binding curve after background subtraction. Dependent on the dilution factor of the unlabeled anti-spike S1 antibody, the respective serum concentrations ranged from 91–97%, and, despite the high concentrations of serum in these samples, the KD value determined for this interaction matches that in PBS.


Here we show that by using MDS on the Fluidity One-W Serum we can accurately detect and characterize the binding affinity of antibodies to virus proteins directly in human serum. Thus, this technology could be used for in-depth analysis of the humoral immune response against SARS-CoV-2 to support the development of reliable antibody tests and vaccines in the fight against the COVID-19 pandemic.

  1. Jang, S., Hillyer, C. & Du, L. Neutralizing Antibodies against SARS-CoV-2 and Other Human Coronaviruses. Trends in Immunology 2020, 41(5), 335-359.
  2. Güven, E., Duus, K., Lydolph, M. C., Jørgensen, C. S., Laursen, I., & Houen, G. Non-specific binding in solid phase immunoassays for autoantibodies correlates with inflammation markers. Journal of Immunological Methods 2014, 403(1-2), 26-36.
  3. Waritani, T., Chang, J., McKinney, B., & Terato, K. An ELISA protocol to improve the accuracy and reliability of serological antibody assays. MethodsX 2017, 4, 153-165.
  4. Arosio, P., Müller, T., Rajah, L., Yates, E.V., Aprile, F.A., Zhang, Y., Cohen, S.I., White, D.A., Herling, T.W., De Genst, E.J. & Linse, S. Microfluidic diffusion analysis of the sizes and interactions of proteins under native solution conditions. ACS nano 2016, 10(1), 333-341.

The Fluidity One-W Serum is for research use only.

Alexa Fluor, NanoDrop One and Pierce are all trademarks of Thermo Fisher Scientific.

Top image credit: ©

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