Microfluidic characterisation reveals broad range of SARS-CoV-2 antibody affinity in human plasma.

Funder: Herchel Smith FundFunder: St John’s College CambridgeFunder: Centre for Misfolding Diseases, CambridgeFunder: Swiss FCS and the Forschungskredit of the University of ZurichFunder: Frances and Augustus Newman FoundationFunder: BBRSCFunder: NOMIS FoundationThe clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, K d, of anti-receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect-based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations

Microfluidic characterisation reveals broad range of SARS-CoV-2 antibody affinity in human plasma

The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti–SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, Kd, of anti–receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect–based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations

Single-Molecule Sizing through Nanocavity Confinement.

An approach relying on nanocavity confinement is developed in this paper for the sizing of nanoscale particles and single biomolecules in solution. The approach, termed nanocavity diffusional sizing (NDS), measures particle residence times within nanofluidic cavities to determine their hydrodynamic radii. Using theoretical modeling and simulations, we show that the residence time of particles within nanocavities above a critical time scale depends on the diffusion coefficient of the particle, which allows the estimation of the particle's size. We demonstrate this approach experimentally through the measurement of particle residence times within nanofluidic cavities using single-molecule confocal microscopy. Our data show that the residence times scale linearly with the sizes of nanoscale colloids, protein aggregates, and single DNA oligonucleotides. NDS thus constitutes a new single molecule optofluidic approach that allows rapid and quantitative sizing of nanoscale particles for potential applications in nanobiotechnology, biophysics, and clinical diagnostics

α-Synuclein oligomers form by secondary nucleation.

Oligomeric species arising during the aggregation of α-synuclein are implicated as a major source of toxicity in Parkinson's disease, and thus a major potential drug target. However, both their mechanism of formation and role in aggregation are largely unresolved. Here we show that, at physiological pH and in the absence of lipid membranes, α-synuclein aggregates form by secondary nucleation, rather than simple primary nucleation, and that this process is enhanced by agitation. Moreover, using a combination of single molecule and bulk level techniques, we identify secondary nucleation on the surfaces of existing fibrils, rather than formation directly from monomers, as the dominant source of oligomers. Our results highlight secondary nucleation as not only the key source of oligomers, but also the main mechanism of aggregate formation, and show that these processes take place under conditions which recapitulate the neutral pH and ionic strength of the cytosol

Direct digital sensing of protein biomarkers in solution.

Funder: Please see main manuscript file.The detection of proteins is of central importance to biomolecular analysis and diagnostics. Typical immunosensing assays rely on surface-capture of target molecules, but this constraint can limit specificity, sensitivity, and the ability to obtain information beyond simple concentration measurements. Here we present a surface-free, single-molecule microfluidic sensing platform for direct digital protein biomarker detection in solution, termed digital immunosensor assay (DigitISA). DigitISA is based on microchip electrophoretic separation combined with single-molecule detection and enables absolute number/concentration quantification of proteins in a single, solution-phase step. Applying DigitISA to a range of targets including amyloid aggregates, exosomes, and biomolecular condensates, we demonstrate that the assay provides information beyond stoichiometric interactions, and enables characterization of immunochemistry, binding affinity, and protein biomarker abundance. Taken together, our results suggest a experimental paradigm for the sensing of protein biomarkers, which enables analyses of targets that are challenging to address using conventional immunosensing approaches

Direct digital sensing of protein biomarkers in solution.

Funder: Please see main manuscript file.The detection of proteins is of central importance to biomolecular analysis and diagnostics. Typical immunosensing assays rely on surface-capture of target molecules, but this constraint can limit specificity, sensitivity, and the ability to obtain information beyond simple concentration measurements. Here we present a surface-free, single-molecule microfluidic sensing platform for direct digital protein biomarker detection in solution, termed digital immunosensor assay (DigitISA). DigitISA is based on microchip electrophoretic separation combined with single-molecule detection and enables absolute number/concentration quantification of proteins in a single, solution-phase step. Applying DigitISA to a range of targets including amyloid aggregates, exosomes, and biomolecular condensates, we demonstrate that the assay provides information beyond stoichiometric interactions, and enables characterization of immunochemistry, binding affinity, and protein biomarker abundance. Taken together, our results suggest a experimental paradigm for the sensing of protein biomarkers, which enables analyses of targets that are challenging to address using conventional immunosensing approaches

Continuous population-level monitoring of SARS-CoV-2 seroprevalence in a large European metropolitan region.

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae