Microfluidic Diffusional Sizing (MDS) Measurements of Secretory Neutralizing Antibody Affinity Against SARS-CoV-2

Abstract

SARS-CoV-2 has rampantly spread around the globe and continues to cause unprecedented loss through ongoing waves of (re)infection. Increasing our understanding of the protection against infection with SARS-CoV-2 is critical to ending the pandemic. Serological assays have been widely used to assess immune responses, but secretory antibodies, the essential first line of defense, have been studied to only a limited extent. Of particular interest and importance are neutralizing antibodies, which block the binding of the spike protein of SARS-CoV-2 to the human receptor angiotensin-converting enzyme-2 (ACE2) and thus are essential for immune defense. Here, we employed Microfluidic Diffusional Sizing (MDS), an immobilization-free technology, to characterize neutralizing antibody affinity to SARS-CoV-2 spike receptor-binding domain (RBD) and spike trimer in saliva. Affinity measurement was obtained through a contrived sample and buffer using recombinant SARS-CoV-2 RBD and monoclonal antibody. Limited saliva samples demonstrated that MDS applies to saliva neutralizing antibody measurement. The ability to disrupt a complex of ACE2-Fc and spike trimer is shown. Using a quantitative assay on the patient sample, we determined the affinity and binding site concentration of the neutralizing antibodies.

Keywords: Microfluidic diffusional sizing; Neutralizing antibody; SARS-CoV-2; Saliva.

Fig. 1

A Schematic diagram of the microfluidic chamber. Arrows indicate the direction of the liquid flow. The sample (analyte) and the auxiliary fluid are introduced into the microfluidic chamber through the inlets. The two streams of the liquid exploit laminar flow without convective mixing. The exchange of the molecules between the streams is through diffusion. Larger molecular complexes diffuse relatively slower than the smaller molecules. Therefore, the size (in hydrodynamic radius) of the molecule complexes can be measured through the ratio of the fluorescent intensity at the end of the chamber where the two stream splits. B Hydrodynamic radius for recombinant anti-spike monoclonal antibody and Alexa Fluor^TM 647 labeled spike RBD (10 nM) measured in PBS-T or 90% pooled saliva (supplemented with 10% PBS-T). Incubation of 10-nM RBD with 13 different titration points (one with no antibody and 12 geometrically spaced antibody concentrations) for 30 min prior to reading in duplicate.

Fig. 2

Schematic depict of the principles of the neutralizing antibody detection by microfluidic diffusional sizing. A When the labeled ACE2 is mixed with the spike trimer, the two binding partners form a complex that diffuses relative slower than the ACE2 alone. That results in an increase of the R_h. B When the labeled ACE2 is mixed with spike trimer pre-incubated with the neutralizing antibody, the spike trimer can no longer bind to the labeled ACE2 as it is blocked by the antibody. Therefore, the measured R_h will be equivalent to the R_h of the labeled ACE2 alone.

Fig. 3

Affinity-based neutralization measurements (rANT) in pooled saliva. ACE2-Fc (5 nM) alone (blue symbol) and a mixture of 5-nM ACE2-Fc and 30-nM spike trimer (red symbol) were prepared in pooled saliva. Inhibition of ACE2-Fc/spike trimer incubated with 180-nM monoclonal antibody spiked into pooled saliva is shown in green. *** indicates a statistical difference (p < 0.001).

Fig. 4

Qualitative measurements for neutralization in patient saliva (rANT). Samples were incubated for 1 A or 16 h B at 4 °C prior to measurement. Control measurements for ACE2 alone (blue symbols) and the ACE2 and spike trimer (red symbols) that were prepared in pooled saliva are shown. Measurements for patient saliva samples are indicated as E1P1, E14P4, and E18P5. The open symbols indicate the second measurement, which has approximately 1-h longer incubation time due to the time needed for taking each measurement sequentially.

Fig. 5

Bayesian K_D and binding site concentration assay analysis of ACE2 and spike protein. Indicated are the binding curve for the interaction (left panel), the probability density plot indicating the convergence of the K_D and spike binding site concentration based on the analysis (middle panel) and the modal distribution of the K_D derived from the fit of the data (right panel).

Fig. 6

qANT analysis of E1P1 saliva. Three neutralization titration curves were measured for the disruption of a pre-formed complex of ACE2 (5 nM) and spike at 30 nM (green symbols), 15 nM (blue symbols), or 7.5 nM (red symbols).