Application Note Accurate Affinity Profiling of a SARS-CoV-2 Antibody in Serum Published on May 19th, 2020 Authors: Viola Denninger, Sebastian Fiedler, Alison Ilsley, Heike Fiegler, and Sean Devenish 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. Download the application note Watch our short webinar on accurate solution phase affinity profiling of a SARS-CoV-2 antibody in serum: Introduction In the quest to accurately identify individuals who are seropositive as well as finding the most effective vaccines against the recently emerged coronavirus SARS-CoV-2, it is fundamental to thoroughly characterize the immune response in the course of the infection or after vaccination. In particular, 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. Like other members of the coronavirus family, SARS-CoV-2 is a positive-sense single-stranded RNA virus that is predominantly made up of four main structural proteins: the envelope (E), membrane (M), nucleoprotein (N) and spike (S) proteins. The spike protein is crucial for virus entry into the host cell. It is composed of two subunits: S1, which binds to the host cell receptor ACE2 (Figure 1); and S2, which mediates the subsequent fusion of the virus with the cell membrane. 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. Quantitative analyses of antibodies to determine seropositivity in patient samples are routinely performed using enzyme-linked immunosorbent assays (ELISA). In ELISA tests 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 in turn allows a better understanding of antibody maturation and persistence of immunity, and in the future 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 [Protocol] SARS-CoV-2 Antibody Profiling User Guide Read protocol Methods SARS-CoV-2 RBD (40592-V08H, Sino Biological) was reconstituted in 400 µL sterile water to a concentration of 0.25 mg/mL. For labeling, the protein was diluted into labeling buffer (0.2 M NaHCO3 pH 8.3) and mixed with Alexa Fluor™ 647 NHS ester (Thermo Fisher Scientific) at a dye-to-protein ratio of 10:1. Following incubation overnight at 4 °C, labeled RBD was purified via size exclusion chromatography using a Superdex 75 Increase 10/300 GL column with PBS (pH 7.4) as elution buffer. For affinity measurements in serum, SARS-CoV-2 (2019-nCoV) spike antibody (40150-R007, Sino Biological) was diluted in human serum (H5667, Sigma), to achieve a two-fold concentration series ranging from 490 pM to 1 uM. Antibody dilutions were subsequently mixed in a 1:1 ratio with a 40 nM solution of Alexa Fluor 647 labeled SARS-CoV-2 RBD, to obtain a final RBD concentration of 20 nM. All samples were incubated for 30 min at 4 °C prior to measurement and kept at 4 °C throughout the experiment. For affinity measurements in buffer, antibody and Alexa Fluor 647 labeled protein were diluted in PBS with 0.05% Tween 20 instead of serum. Concentrations and incubation times were identical to experiments in serum. Samples were measured on the Fluidity One-W Serum using the 1.5 – 8 nm size-range setting. Measurements were performed in triplicate at room temperature. To correct for background fluorescence caused by serum, independent measurements of human serum were performed, and a background subtraction was applied to individual data points obtained in serum. The binding affinity, KD, was automatically generated by non-linear least squares fitting to Equation 1 (see Appendix). Since absolute sizes indicated that two molecules of SARS-CoV-2 RBD bound to one antibody, the stoichiometric parameter, n, was set to 0.5. Results 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 fitting using Equation 1. 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. Conclusion 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. References Jang, S., Hillyer, C. & Du, L. Neutralizing Antibodies against SARS-CoV-2 and Other Human Coronaviruses. Trends in Immunology 2020, 41(5), 335-359. (publication) 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. (publication) 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. (publication) 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. (publication) The Fluidity One-W Serum is for research use only. Alexa Fluor, NanoDrop One and Pierce are all trademarks of ThermoFisher Scientific. Download application note Fill out the form below to download the application note "Accurate solution phase affinity profiling of SARS-CoV-2 antibody in serum". 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