ABSTRACT
Antigen specific humoral immunity can be characterized by the analysis of serum antibodies. While serological assays for the measurement of antibody levels and of neutralization potential against SARS-CoV-2 are available, these are not quantitative in the biochemical sense. Yet, understanding the biology of COVID-19 would need an unambiguous, complete, quantitative, comparable measurement of specific serum antibodies. Here we describe a fluorescent, dual-titration immunoassay, which provides the physico-chemical parameters that are both necessary and sufficient to quantitatively characterize the humoral immune response. We used recombinant Receptor Binding Domain of SARS-CoV-2 as antigen on microspot arrays and varied the concentration of both the antigen and serum antibodies from vaccinated persons to obtain a measurement matrix of binding data. Binding curves were fitted using a novel algorithm to obtain thermodynamic variables of binding. We defined the standard state for a system of serum antibodies and antigen and showed how a normalized generalized logistic function is related to thermodynamic activity, standard concentration and activity coefficient. The utility of the method is demonstrated by defining the composition of tested sera with respect to immunoglobulin classes, affinity, concentration, and thermodynamic activity. The proposed fluorescent dual-titration microspot immunoassay can generate truly quantitative serological data that is suitable for immunological, medical and systems biological analysis.