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ORIGINAL RESEARCH article

Front. Immunol.
Sec. Systems Immunology
Volume 16 - 2025 | doi: 10.3389/fimmu.2025.1494624
This article is part of the Research Topic Modeling the Immune System: from PK/PD to Systems Immunology View all 5 articles

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

Provisionally accepted
Ágnes Kovács Ágnes Kovács 1Zoltán Hérincs Zoltán Hérincs 2Krisztian Papp Krisztian Papp 2Jakub Zbigniew Kaczmarek Jakub Zbigniew Kaczmarek 3Daniel Nyberg Larsen Daniel Nyberg Larsen 4Pernille Stage Pernille Stage 3László Bereczki László Bereczki 5Eszter Ujhelyi Eszter Ujhelyi 5Tamás Pfeil Tamás Pfeil 6József Prechl József Prechl 2*
  • 1 Department of Biostatistics, University of Veterinary Medicine, Budapest, Hungary
  • 2 Diagnosticum Zrt., Budapest, Hungary
  • 3 Ovodan Biotech A/S, Odense, Denmark
  • 4 University of Southern Denmark, Odense, Denmark
  • 5 G1 Laboratórium, Budapest, Hungary
  • 6 Department of Applied Analysis and Computational Mathematics, Eötvös Loránd University, Budapest, Hungary

The final, formatted version of the article will be published soon.

    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.

    Keywords: antibody, Serology, Microarray, chemical thermodynamics, Curve fitting, SARS-CoV-2, quantitative systems biology

    Received: 11 Sep 2024; Accepted: 03 Feb 2025.

    Copyright: © 2025 Kovács, Hérincs, Papp, Kaczmarek, Larsen, Stage, Bereczki, Ujhelyi, Pfeil and Prechl. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: József Prechl, Diagnosticum Zrt., Budapest, Hungary

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.