Evaluation of kinetics and thermodynamics of interaction between immobilized SARS-CoV-2 nucleoprotein and specific antibodies by total internal reflection ellipsometry.

During the pandemic, different methods for SARS-CoV-2 detection and COVID-19 diagnostics were developed, including antibody and antigen tests. For a better understanding of the interaction mechanism between SARS-CoV-2 virus proteins and specific antibodies, total internal reflection ellipsometry based evaluation of the interaction between SARS-CoV-2 nucleoprotein (SCoV2-rN) and anti-SCoV2-rN antibodies was performed. Results show that the appropriate mathematical model, which takes into account the formation of an intermediate complex, can be applied for the evaluation of SCoV2-rN/anti-SCoV2-rN complex formation kinetics. The calculated steric factor indicated that SCoV2-rN/anti-SCoV2-rN complex formation has very strict steric requirements. Estimated Gibbs free energy (ΔGAssoc) for SCoV-rN and anti-SCoV-rN binding was determined as -34 kJ/mol. The reported findings are useful for the design of new analytical systems for the determination of anti-SCoV2-rN antibodies and for the development of new anti-SARS-CoV-2 medications.

Evaluation of affinity sensor response kinetics towards dimeric ligands linked with spacers of different rigidity: Immobilized recombinant granulocyte colony-stimulating factor based synthetic receptor binding with genetically engineered dimeric analyte derivatives.

The modelling of protein-protein binding kinetics is important for the development of affinity-sensors and the prediction of signaling protein based drug efficiency. Therefore, in this research we have evaluated the binding kinetics of several genetically designed protein models: (i) three different ligands based on granulocyte colony-stimulating factor GCSF homo-dimeric derivatives linked by differed by linkers of different length and flexibility; (ii) an antibody-like receptor (GCSF-R) based on two GCSF-receptor sites immobilized to Fc domains, which are common parts of protein structures forming antibodies. Genetically engineered GCSF-R is similar to an antibody because it, like the antibody, has two binding sites, which both selectively bind with GCSF ligands. To design the affinity sensor model studied here, GCSF-R was immobilized on a thin gold layer via self-assembled monolayer conjugated with Protein-G. Binding kinetics between immobilized GCSF-R and all three different recombinant GCSF-based homo-dimeric derivatives were evaluated by total internal reflection ellipsometry. Association constants were determined by fitting mathematical models to the experimental data. It was clearly observed that both (i) affinity and (ii) binding kinetics depend on the length and flexibility of the linker that connects both domains of a GCSF-based ligand. The fastest association between immobilized GCSF-R and GCSF-based ligands was observed for ligands whose GCSF domains were interconnected by the longest and the most flexible linker. Here we present ellipsometry-based measurements and models of the interaction kinetics that advance the understanding of bidentate-receptor-based immunosensor action and enables us to predict the optimal linker structure for the design of GCSF-based medications.