In-depth immunochemical characterization of the serum antibody response using a dual-titration microspot assay
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 s...
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| Format: | Article |
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Frontiers Media S.A.
2025-02-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1494624/full |
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| author | Ágnes Kovács Ágnes Kovács Zoltán Hérincs Krisztián Papp Jakub Zbigniew Kaczmarek Daniel Nyberg Larsen Daniel Nyberg Larsen Pernille Stage László Bereczki Eszter Ujhelyi Tamás Pfeil Tamás Pfeil József Prechl |
| author_facet | Ágnes Kovács Ágnes Kovács Zoltán Hérincs Krisztián Papp Jakub Zbigniew Kaczmarek Daniel Nyberg Larsen Daniel Nyberg Larsen Pernille Stage László Bereczki Eszter Ujhelyi Tamás Pfeil Tamás Pfeil József Prechl |
| author_sort | Ágnes Kovács |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-dcf1aa2c1f084903924c2c44dc4cfa24 |
| institution | DOAJ |
| issn | 1664-3224 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj-art-dcf1aa2c1f084903924c2c44dc4cfa242025-08-20T03:11:03ZengFrontiers Media S.A.Frontiers in Immunology1664-32242025-02-011610.3389/fimmu.2025.14946241494624In-depth immunochemical characterization of the serum antibody response using a dual-titration microspot assayÁgnes Kovács0Ágnes Kovács1Zoltán Hérincs2Krisztián Papp3Jakub Zbigniew Kaczmarek4Daniel Nyberg Larsen5Daniel Nyberg Larsen6Pernille Stage7László Bereczki8Eszter Ujhelyi9Tamás Pfeil10Tamás Pfeil11József Prechl12Department of Biostatistics, University of Veterinary Medicine Budapest, Budapest, HungaryDepartment of Applied Analysis and Computational Mathematics, Eötvös Loránd University, Budapest, HungaryR&D Laboratory, Diagnosticum Zrt, Budapest, HungaryR&D Laboratory, Diagnosticum Zrt, Budapest, HungaryResearch and Development Department, Ovodan Biotech A/S, Odense, DenmarkResearch and Development Department, Ovodan Biotech A/S, Odense, DenmarkDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, DenmarkResearch and Development Department, Ovodan Biotech A/S, Odense, DenmarkG1 Labordiagnosztikai Kft, Budapest, HungaryG1 Labordiagnosztikai Kft, Budapest, HungaryDepartment of Applied Analysis and Computational Mathematics, Eötvös Loránd University, Budapest, HungaryHUN-REN–ELTE Numerical Analysis and Large Networks Research Group, Budapest, HungaryR&D Laboratory, Diagnosticum Zrt, Budapest, HungaryAntigen 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.https://www.frontiersin.org/articles/10.3389/fimmu.2025.1494624/fullantibodyserologymicroarraychemical thermodynamicscurve fittingSARS-CoV-2 |
| spellingShingle | Ágnes Kovács Ágnes Kovács Zoltán Hérincs Krisztián Papp Jakub Zbigniew Kaczmarek Daniel Nyberg Larsen Daniel Nyberg Larsen Pernille Stage László Bereczki Eszter Ujhelyi Tamás Pfeil Tamás Pfeil József Prechl In-depth immunochemical characterization of the serum antibody response using a dual-titration microspot assay Frontiers in Immunology antibody serology microarray chemical thermodynamics curve fitting SARS-CoV-2 |
| title | In-depth immunochemical characterization of the serum antibody response using a dual-titration microspot assay |
| title_full | In-depth immunochemical characterization of the serum antibody response using a dual-titration microspot assay |
| title_fullStr | In-depth immunochemical characterization of the serum antibody response using a dual-titration microspot assay |
| title_full_unstemmed | In-depth immunochemical characterization of the serum antibody response using a dual-titration microspot assay |
| title_short | In-depth immunochemical characterization of the serum antibody response using a dual-titration microspot assay |
| title_sort | in depth immunochemical characterization of the serum antibody response using a dual titration microspot assay |
| topic | antibody serology microarray chemical thermodynamics curve fitting SARS-CoV-2 |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1494624/full |
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