Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.03.14.532012

Deep physico-chemical characterization of individual serum antibody responses against SARS-CoV-2 RBD using a dual titration microspot assay

Kovacs, A.·Herincs, Z.·Papp, K.·Kaczmarek, J. Z.·Larsen, D. N.·Stage, P.·Bereczki, L.·Ujhelyi, E.·Pfeil, T.·Prechl, J.
View original source ↗Export metadata ↓

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 physico-chemical parameters that are both necessary and sufficient to quantitatively characterize the humoral immune response. We define the theory of the approach that is based on physical chemistry. 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 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 thermodynamic variables of binding. We obtained equilibrium affinity constants and chemical potentials 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.

Explore related subjects

immunologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Kovacs, A., Herincs, Z., Papp, K., Kaczmarek, J. Z., Larsen, D. N., Stage, P., Bereczki, L., Ujhelyi, E., Pfeil, T., Prechl, J.. 2023-03-14. Deep physico-chemical characterization of individual serum antibody responses against SARS-CoV-2 RBD using a dual titration microspot assay. https://doi.org/10.1101/2023.03.14.532012

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

ATF3 Preserves Macrophage Cholesterol Homeostasis during Metabolic Stress

Background: Macrophages must coordinate immune activation with maintenance of cellular cholesterol homeostasis, yet the transcriptional mechanisms integrating these processes remain incompletely understood. Activating transcription factor 3 (ATF3) suppresses inflammatory and interferon responses and has been implicated in macrophage lipid metabolism, suggesting a role in coordinating immune and sterol-responsive transcriptional programs. Methods: Wild-type and ATF3-deficient bone marrow-derived macrophages were analyzed by bulk RNA sequencing following Toll-like receptor stimulation and under conditions of sterol loading or depletion. Cholesterol content, cytokine production, and sterol-responsive gene expression were quantified. Human metabolic dysfunction-associated steatotic liver disease (MASLD) datasets and Kupffer cell-specific ATF3 knockout mice were used to assess disease relevance in vivo. Results: ATF3 deficiency enhanced inflammatory and interferon-responsive transcriptional programs while suppressing a sterol-responsive gene module enriched for SREBP2 target genes. This phenotype was conserved across diverse Toll-like receptor stimuli and accompanied by persistent Ch25h upregulation. Despite exhibiting elevated cellular cholesterol, ATF3-deficient macrophages retained transcriptional responsiveness to sterol loading and depletion while demonstrating reduced induction of SREBP2-dependent genes across conditions. IFNAR blockade reduced interferon-responsive gene expression but did not normalize Ch25h or sterol-responsive gene expression. In contrast, CRISPR-mediated reduction of Ch25h partially restored sterol-responsive gene expression in inflammatory ATF3-deficient macrophages. In human MASLD, hepatic ATF3 expression positively correlated with disease severity. In mice, ATF3 was preferentially induced in Kupffer cells during metabolic stress, and Kupffer cell-specific deletion of ATF3 increased liver injury and Kupffer cell lipid accumulation. Conclusions: ATF3 supports sterol-responsive transcriptional programs while restraining inflammatory activation in macrophages. ATF3 loss results in elevated cellular cholesterol, impaired SREBP2-dependent gene expression, and persistent derepression of Ch25h, which contributes to but does not fully explain the sterol phenotype. These findings identify ATF3 as a coordinator of macrophage adaptation to inflammatory and metabolic stress and support a protective role for Kupffer cell ATF3 during metabolic liver disease.

immunology↗
bioRxiv2026

Therapeutic anti-IgE antibodies generate distinct functional immune complex signatures

Therapeutic IgG anti-IgE antibodies are benchmarked by their ability to interfere with IgE-Fc[epsilon]RI binding, but their activity within IgE and IgG-containing immune complexes remains poorly defined. Here we show that omalizumab and ligelizumab form binary IgE-IgG and ternary allergen-containing immune complexes with distinct functional properties. In primary human leukocytes, these complexes exhibited antibody- and composition-dependent patterns of cellular association and activation. Using multiplexed focal molography, we resolved Fc receptor engagement across free and complexed antibody states and identified distinct receptor-binding signatures for omalizumab- and ligelizumab-containing complexes. Notably, allergen incorporation reversed the relative ability of the two antibodies to neutralize IgE-Fc[epsilon]RI interactions and remodelled Fc[gamma]R binding. Thus, immune-complex composition is a determinant of therapeutic antibody function.

immunology↗
bioRxiv2026

Proliferation and mutation as humoral immune response

The affinity maturation process, through which antibodies acquire relatively high affinity for an invading pathogen, involves complex mechanisms whose relative importance is not yet fully understood. This study proposes an analytical model of this process, to investigate the main factors governing proliferation and mutation and assess their respective roles. The model suggests that the immune system should not be expected to follow a single, rigid evolutionary pattern, as a wide range of proliferation and mutation strategies can yield nearly optimal outcomes. Keywords: Antibody, antigen, affinity, B-cell, mutation, proliferation

immunology↗