Microfluidic Antibody Affinity Profiling for In-Solution Characterisation of Alloantibody - HLA Interactions in Human Serum

Abstract

The detection and characterisation of antibodies in human blood is a key for clinical diagnostics and risk assessmentn for autoimmunity, infectious diseases and transplantation. Antibody titre derived from immunoassays is a commonly used measure for antibody response, but this metric does not resolve readily the two fundamental properties of antibodies in solution, namely their affinity and concentration. This difficulty originates from the fact that the fundamental parameters describing the binding interaction, affinity and ligand concentration, are convoluted into the titre measurement; moreover, the difficulty of controlling the surface concentration and activity of the immobilised ligand can make it challenging to distinguish between avidity and affinity. To address these challenges, we developed microfluidic antibody affinity profiling, an assay which allows the simultaneous determination of both affinity and antibody concentration, directly in solution, without surface immobilisation or antibody purification. We demonstrate these measurements in the context of alloantibody characterisation in organ transplantation, using complex patient sera, and quantify the concentration and affinity of alloantibodies against donor Human Leukocyte Antigens (HLA), an extensively used clinical biomarker to access the risk of allograft rejection. These results outline a path towards detection and in depth profiling of antibody response in patient sera.

Microfluidic Affinity Profiling reveals a Broad Range of Target Affinities for Anti-SARS-CoV-2 Antibodies in Plasma of COVID-19 Survivors

“…Moreover, our binding equilibria are thus achieved under physiologically relevant conditions and our in-solution measurements allow to determine both the antibody affinity and concentration. As discussed previously 20 Points correspond to the maximum probability values in the two-dimensional probability distribution, and shaded regions to the probability density. In line with physical principles of binding, binding is not observed for samples with [Ab] < 2Kd (dark grey region).…”

“…Here we determined affinity and concentration directly in plasma samples of seropositive individuals using Microfluidic Antibody Affinity Profiling (MAAP) 20 . We quantified both parameters in 39 seropositive individuals who presented either mild symptoms or were asymptomatic, demonstrating a comparable immune response which is independent of the symptoms displayed.…”

In vitro measurements of protein–protein interactions show that antibody affinity governs the inhibition of SARS-CoV-2 spike/ACE2 binding in convalescent serum

“…To determine the K D of anti-S1 antibody-S1 binding, we mixed fluorescently labeled S1 at a constant concentration with patient serum at various dilutions and determined R h as an indicator of complex formation (Figures 3A and C). Since the anti-S1 antibody concentration in the two patient sera was unknown, we extracted this information from the equilibrium binding isotherms by using the concentration of antibody-binding sites as a fitting parameter 21,22 . To obtain well-constrained binding-site concentrations, we measured antibody-binding isotherms at three different concentrations of fluorescently labeled S1.…”

“…A MAAP study on a larger number of patient samples showed a similar range of affinities among anti-spike serum antibodies 21 . To investigate the robustness of the K D values derived by the global fits, we also analyzed the data using Bayesian inference 21,22 . While the agreement of Bayesian inference with the global fits was excellent, in both patient samples the posterior probability for the K D distribution provided strong constraints for the upper bound but weak constraints for the lower bound, thus providing an upper limit on the K D value and indicating a tight binding interaction.…”

“…binding. MAAP is an in-solution assay that determines both the effective dissociation constants (K D ) of polyclonal serum antibodies and their concentrations 21,22 . Based on these findings, we suggest that the ratio of the dissociation constants of ACE2-S1 binding and NAb-S1 binding (r = K D,ACE2 / K D,NAb ) together with the experimentally determined concentration of NAbs in serum are excellent measures to predict the inhibitory potential of the polyclonal antibody response.…”