Antibody characterization from COVID-19 patient plasma binding to SARS-COV-2 antigens

In this technical note, we show how the Creoptix WAVE can be used for the characterization of binding affinity and kinetics of antibodies raised against SARS-CoV-2 in clinical human blood plasma samples. With innovative disposable, no-clog microfluidics and high sensitivity, the Creoptix WAVE opens the door for antibody characterization directly from clinical blood plasma samples. The methods described here are also compatible with high concentrations of serum and plasma of SARS-CoV-2 patients’ samples. 

Summary

When the novel Coronavirus disease (COVID-19) emerged worldwide in January 2020, scientists not only scrambled to develop tests but also to understand the biology of this disease. Scientists began tackling the problem with the tools and knowledge already on hand from studying other infectious diseases like Dengue, Zika, and Ebola. However, the Coronavirus proved challenging in ways current technologies were unable to address.

One example is the analysis of molecular interactions between antibodies present in complex matrices like patient blood serum and plasma and SARS-CoV-2 spike protein. Matrix components like serum albumin are often incompatible with label-free surface-based biosensors due to potentially deleterious effects on the microfluidics and high non-specific binding.

Here, we show how the Creoptix WAVE has the potential to overcome these limitations with innovative disposable, no-clog microfluidics. Using the proprietary Grating-Coupled Interferometry (GCI) technology to deliver high sensitivity, the Creoptix WAVE provides very high tolerance against crude matrixes and enables the characterization of antibody kinetics directly from clinical blood plasma samples.

Materials and methods

Three SARS-CoV-2 antigens (Spike ECD, Spike RBD and Nucleocapsid Protein) were immobilized on a 4PCP WAVEchip by standard amine coupling chemistry. An empty surface was activated/deactivated and used as a reference channel. Additionally, BSA was immobilized to full saturation on all channels - including the reference one - by means of amine coupling.

High immobilization densities for the abovementioned viral antigens were chosen to enable subsequent serological detection of anti-SARS-CoV-2 antibodies in patient samples. As a positive control, to ensure the integrity of SARS-CoV-2 antigens, the kinetics of specific monoclonal antibodies were determined. For Spike ECD and Spike RBD, mAb CR3022 was diluted into 5% pooled human plasma (in PBS + 0.3% BSA) and injected in duplicates for 200 seconds at a flow rate of 55 ul/min in a dilution series from 0.4 to 50 nM, followed by injection of running buffer for 600 seconds. For the Nucleocapsid Protein (NP), the anti-NP mAb was diluted in PBS + 0.3% BSA and injected in duplicates for 100 seconds at a flow rate of 55 ul/min in a dilution series from 3 to 50 nM, followed by injection of running buffer for 600 seconds. The surface was regenerated with 25 mM NaOH + 1M NaCl after the injection of each antibody concentration, followed by injection of running buffer.

Inactivated plasma samples from patients with COVID-19 (from the Institute of Clinical Chemistry at the University Hospital of Zurich) were diluted in PBS + 0.3% BSA to a final concentration of 5% (v/v) and injected for 150 seconds at a flow rate of 100 ul/min, followed by injection of running buffer (PBS + 0.3% BSA) for 600 seconds. Blank injections were performed with pooled human COVID-19 negative plasma diluted to 5% (v/v) in PBS + 0.3% BSA. All data were double-referenced with the signal on a reference channel and blank injections and fitted with a 1:1 binding model using the Creoptix WAVEcontrol software.

Results

Figure 1 and Figure 2 show binding curves recorded for 7 concentrations of the monoclonal antibody CR3022 ranging from 0.4 to 50 nM in 5% pooled human plasma diluted in PBS + 0.3% BSA (w/v), binding to SARS-CoV-2 Spike ECD and Spike RBD, respectively. In Figure 3, binding responses of an anti-NP monoclonal antibody binding to SARS-CoV-2 NP for a concentration range of 3 to 50 nM are shown.

[Figure 1 TN201124-Creoptix-antibody-characterization-plasma-binding.jpg] Figure 1 TN201124-Creoptix-antibody-characterization-plasma-binding.jpg

Figure 1: Kinetics of MAB CR3022 on spike ECD, spiked into 5% pooled human plasma

[Figure 2 TN201124-Creoptix-antibody-characterization-plasma-binding.jpg] Figure 2 TN201124-Creoptix-antibody-characterization-plasma-binding.jpg

Figure 2: Kinetics of MAB CR3022 on spike RBD, spiked into 5% pooled human plasma

[Figure 3 TN201124-Creoptix-antibody-characterization-plasma-binding.jpg] Figure 3 TN201124-Creoptix-antibody-characterization-plasma-binding.jpg

Figure 3: Kinetics of anti-NP MAB on SARS-CoV-2 nucleocapsid (NP) (PBS+ 0.3% BSA)

Binding responses of polyclonal anti-SARS-CoV-2 antibodies from patient plasma samples binding to the three viral antigens are shown in Figure 4. Duplicate injections are shown for a range of patient plasma samples shown in different colors, with polyclonal antibodies binding to Spike ECD (panel A), Spike RBD (panel B) and NP (panel C). In panel D, an off rate fit for an exemplary patient sample injected onto Spike RBD in duplicate is shown, using a 1:1 model focused on the slow phase of the dissociation curve. Differences in curvature and response levels between the patient samples are presumably due to different kinetics and antibody titers in these patients, which both are currently being investigated in subsequent studies in collaboration with the laboratory of Prof. Adriano Aguzzi at the University Hospital of Zurich. The data shown here was generated during the development of assays for a large serological study on SARS-CoV-2 immunity in the greater Zurich area.2

[Figure 4 TN201124-Creoptix-antibody-characterization-plasma-binding.jpg] Figure 4 TN201124-Creoptix-antibody-characterization-plasma-binding.jpg

Figure 4: Binding responses of polyclonal antiSARS-CoV-2 antibodies in patient plasma samples

Conclusion

In short, here we show that the Creoptix WAVE enables the kinetic characterization of SARS-CoV-2 antibodies to three specific antigens in diluted human plasma. The robust microfluidics of the system allow similar measurements at higher plasma (and serum) concentrations, thereby opening the door for antibody quantification without the need for dilution, which is often responsible for the perceived limited limit of detection of traditional label-free technologies. 

Key takeaways

Understand the COVID-19 disease and generate novel data to strengthen results and publications with the Creoptix WAVE:

  • Robust microfluidics for novel data: characterize the interactions directly from clinical blood plasma samples using a self-contained disposable cartridge
  • Validate your ELISA results: correlate and map levels of immunity in communities with confidence

Great for:

  • Investigating antibody-dependent enhancement (ADE) upon SARS-CoV-2 infection
  • Gaining insights in early immune response in current infection (IgM)
  • Measuring long-term immunity (IgG)
  • Orthogonal validation of ELISA data

References

1. Huo, J. et al. 2020. Neutralization of SARS-CoV-2 by destruction of the prefusion spike. Cell Host & Microbe doi:10.1016/j.chom.2020.06.010

2. Emmenegger, M. et al. 2020. Early plateau of SARS-CoV-2 seroprevalence identified by tripartite immunoassay in a large population. medRxivdoi:10.1101/2020.05.31.20118554

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