AUTHOR=Kovács Ágnes , Hérincs Zoltán , Papp Krisztián , Kaczmarek Jakub Zbigniew , Larsen Daniel Nyberg , Stage Pernille , Bereczki László , Ujhelyi Eszter , Pfeil Tamás , Prechl József 

TITLE=In-depth immunochemical characterization of the serum antibody response using a dual-titration microspot assay

JOURNAL=Frontiers in Immunology

VOLUME=Volume 16 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2025.1494624

DOI=10.3389/fimmu.2025.1494624

ISSN=1664-3224

ABSTRACT=Antigen specific humoral immunity can be characterized by the analysis of serum antibodies. While serological assays for the measurement of specific antibody levels are available, these are not quantitative in the biochemical sense. Yet, understanding humoral immune responses quantitatively on the systemic level would need a universal, complete, quantitative, comparable measurement method of antigen specific serum antibodies of selected immunoglobulin classes. Here we describe a fluorescent, dual-titration immunoassay, which provides the biochemical parameters that are both necessary and sufficient to quantitatively characterize the humoral immune response. For validation of theory, we used recombinant receptor binding domain of SARS-CoV-2 as antigen on microspot arrays and varied the concentration of both the antigen and the serum antibodies from infected persons to obtain a measurement matrix of binding data. Both titration curves were simultaneously fitted using an algorithm based on the generalized logistic function and adapted for analyzing biochemical variables of binding. We obtained equilibrium affinity constants and concentrations for distinct antibody classes. These variables reflect the quality and the effective quantity of serum antibodies, respectively. The proposed fluorescent dual-titration microspot immunoassay can generate truly quantitative serological data that is suitable for immunological, medical and systems biological analysis.