Method for direct measuring antibody affinity in biological sample.

Abstract

We are reporting a new method to measure the affinity of specific antibodies in biological sample without the need of purification, or direct labeling. It was used to measure the affinity of anti-RBD (SARS-CoV2) IgG in plasma from Covid-19 recovered patient and vaccinated populations. Among the vaccinated population, we also measured the affinities of vaccine-induced antibodies against the RBD of the ancestral SARS-Cov-2 virus and the Omicron variants. The method has provided a quantitative way to evaluate antibodies produced by vaccination. The new method involves a classical Solution phase Equilibrium Titration step and an Imaging based Detection step (SET-ID). In the Solution phase Titration step, the antibody was kept at constant concentration (lower than the expected KD value) and mixed with various concentrations of antigen solution; In the Imaging Detection step, the amount of free antibody (not bound to antigen) at each antigen concentration was probed with small amount of microparticles coated with antigen and then the labeled secondary antibody. In this presentation, we will discuss the critical experimental condition, validation, and limitations. For verification, the binding curves of an antibody were compared using a well-established fluorescence spectroscopic-based method and the SET-ID method. The incubation time and the number of microparticles used in the second step were evaluated experimentally and theoretically to ensure minimal perturbation to the solution phase equilibrium, while the fraction of free antibody presented in the solution can still be measured by the signal on the microparticles.