Biology subjects

Prechl, J.

Publications and source records attributed to Prechl, J..

5 recordsLinked to original sources

bioRxiv2023

Deep physico-chemical characterization of individual serum antibody responses against SARS-CoV-2 RBD 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 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.

immunology↗

bioRxiv2021

Absolute quantitation of serum antibody reactivity using the Richards growth model for antigen microspot titration

In spite of its pivotal role in the characterization of humoral immunity, there is no accepted method for the absolute quantitation of antigen specific serum antibodies. We devised a novel method to quantify polyclonal antibody reactivity, which exploits protein microspot assays and employs a novel analytical approach. Microarrays with a density series of disease specific antigen were treated with different serum dilutions and developed for IgG and IgA binding. By fitting binding data of both dilution series to a product of two generalized logistic functions, we obtained estimates of antibody reactivity of two immunoglobulin classes simultaneously. These estimates are the antigen concentrations required for reaching the inflection point of thermodynamic activity coefficient of antibodies and the limiting activity coefficient of antigen. By providing universal chemical units, this approach may improve standardization of serological testing, the quality control of antibodies and the quantitative mapping of antibody-antigen interaction space.

immunology↗

bioRxiv2018

Quantitative network organization of interactions emerging from the evolution of sequence and structure space of antibodies: the RADARS model

Adaptive immunity in vertebrates represents a complex self-organizing network of protein interactions that develops throughout the lifetime of an individual. While deep sequencing of the immune-receptor repertoire may reveal clonal relationships, functional interpretation of such data is hampered by the inherent limitations of converting sequence to structure to function. In this paper a novel model of antibody interaction space and network, termed radial adjustment of system resolution, RADARS, is proposed. The model is based on the radial growth of interaction affinity of antibodies towards an infinity of directions in structure space, each direction representing particular shapes of antigen epitopes. Levels of interaction affinity appear as free energy shells of the system, where hierarchical B-cell development and differentiation takes place. Equilibrium in this immunological thermodynamic system can be described by a power-law distribution of antibody free energies with an ideal network degree exponent of phi square, representing a scale-free fractal network of antibody interactions. Plasma cells are network hubs, memory B cells are nodes with intermediate degrees and B1 cells represent nodes with minimal degree. Thus, the RADARS model implies that antibody structure space develops against an infinite antigen structure space via interactions that are individually immunologically controlled, but on a systems level are organized by thermodynamic probability distributions. The network of interactions, which control B-cell development and differentiation, represent pathways of antigen removal on systems level. Understanding such quantitative network properties of the system should help the organization of sequence-derived structural data, offering the possibility to relate sequence to function in a complex, self-organizing biological system. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/438804v7_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@883eb7org.highwire.dtl.DTLVardef@cd7d5forg.highwire.dtl.DTLVardef@e225eeorg.highwire.dtl.DTLVardef@1286b49_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

immunology↗

bioRxiv2017

The Generalized Quantitative Model of antibody homeostasis: maintenance of global antibody equilibrium by effector functions

The homeostasis of antibodies can be characterized as a balanced production, binding and elimination regulated by an interaction network, which controls B-cell development and selection. Recently we proposed a quantitative model to describe how the concentration and affinity of interacting partners generates a network.\n\nHere we argue that this physical, quantitative approach can be extended for the interpretation of effector functions of antibodies. We define global antibody equilibrium as the zone of molar equivalence of free antibody and free antigen and immune complex concentrations and of dissociation constant of apparent affinity: [Ab]=[Ag]=[AbAg]=KO_SCPCAPDC_SCPCAP. This zone corresponds to the biologically relevant KO_SCPCAPDC_SCPCAP range of reversible interactions. We show that thermodynamic and kinetic properties of antibody-antigen interactions correlate with immunological functions. The formation of stable, long-lived immune complexes correspond to a decrease of entropy and is a prerequisite for the generation of higher order complexes. As the energy of formation of complexes increases we observe a gradual shift from silent clearance to inflammatory reactions. These rules can also be applied to complement activation-related processes, linking innate and adaptive humoral responses. Affinity of the receptors mediating effector functions shows a corresponding range of affinities, allowing the continuous sampling of antibody-bound antigen over the complete range of concentrations. The generation of multivalent, multicomponent complexes triggers effector functions by cross-linking these receptors on effector cells with increasing enzymatic degradation potential.\n\nThus, antibody homeostasis is a thermodynamic system with complex network properties, nested into the host organism by proper immunoregulatory and effector pathways. Maintenance of global antibody equilibrium is achieved by innate qualitative signals modulating a quantitative adaptive immune system, which regulates molecular integrity of the host by tuning the degradation and recycling of molecules from silent removal to inflammatory elimination.

immunology↗

bioRxiv2017

Thermodynamic Projection Of The Antibody Interaction Network: The Binding Fountain Energy Landscape

The complexity of the adaptive immune system in humans is comparable to that of the central nervous system in terms of cell numbers, cellular diversity and the network of interactions between components. While the application of molecular biological methods and bioinformatics has brought about an ever deepening and sharpening static description of the molecular and cellular components of the system, a unifying theoretical understanding of the laws governing the dynamics of the system is still lacking.\n\nWe have recently developed a quantitative model for the description of antibody homeostasis as defined by the dimensions of antigen concentration, antigen-antibody interaction affinity and antibody concentration. In this paper we develop the concept of a novel thermodynamic representation of multiple molecular interactions in a system, the fountain energy landscape of binding. We show that the hypersurface of the binding fountain corresponds to the antibody-antigen interaction network projected onto an energy landscape defined by conformational entropy and free energy of binding. We demonstrate that thymus independent and thymus dependent antibody responses show distinct patterns of changes in the energy landscape. Overall, the binding fountain energy landscape concept allows a systems biological, thermodynamic perception of the functioning of the clonal humoral immune system.

biophysics↗