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. 2023 Aug 25;24(17):13220.
doi: 10.3390/ijms241713220.

Revealing the SARS-CoV-2 Spike Protein and Specific Antibody Immune Complex Formation Mechanism for Precise Evaluation of Antibody Affinity

Ieva Plikusiene  1   2 , Vincentas Maciulis  1   2 , Vilius Vertelis  2 , Silvija Juciute  1 , Saulius Balevicius  2 , Arunas Ramanavicius  1   2 , Julian Talbot  3 , Almira Ramanaviciene  1
Affiliations

Revealing the SARS-CoV-2 Spike Protein and Specific Antibody Immune Complex Formation Mechanism for Precise Evaluation of Antibody Affinity

Ieva Plikusiene et al. Int J Mol Sci. .

Abstract

The profound understanding and detailed evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (SCoV2-S) protein and specific antibody interaction mechanism is of high importance in the development of immunosensors for COVID-19. In the present work, we studied a model system of immobilized SCoV2-S protein and specific monoclonal antibodies by molecular dynamics of immune complex formation in real time. We simultaneously applied spectroscopic ellipsometry and quartz crystal microbalance with dissipation to reveal the features and steps of the immune complex formation. We showed direct experimental evidence based on acoustic and optical measurements that the immune complex between covalently immobilized SCoV2-S and specific monoclonal antibodies is formed in two stages. Based on these findings it was demonstrated that applying a two-step binding mathematical model for kinetics analysis leads to a more precise determination of interaction rate constants than that determined by the 1:1 Langmuir binding model. Our investigation showed that the equilibrium dissociation constants (KD) determined by a two-step binding model and the 1:1 Langmuir model could differ significantly. The reported findings can facilitate a deeper understanding of antigen-antibody immune complex formation steps and can open a new way for the evaluation of antibody affinity towards corresponding antigens.

Keywords: QCM–D; SARS-CoV-2; affinity interaction; antigen–antibody binding kinetics; immune complex formation; spike protein.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Real-time monitoring of ΔF1 and ΔD1 evolution in time for covalent immobilization of SCoV2-S on 11-MUA SAM (A,B) and ΔF2 and ΔD2 for affinity interaction between SCoV2-S and mAb-SCoV2-S (C,D), and SCoV2-S interaction with anti-BSA antibodies (E,F). Each curve corresponds to a particular harmonic, n.
Figure 2
Figure 2
ΔD vs. ΔF plots for covalent immobilization of SCoV2-S (A) and SCoV2-S/mAb-SCoV2-S immune complex formation (B). The arrows K1 = 0.0548; K2 = 0.0624; K3 = 0.0065 indicate the slopes to red and blue curves.
Figure 3
Figure 3
Time evolution of the normalized refractive index during immobilization of SCoV2-S on 11-MUA SAM (A) and SCoV2-S/mAb-SCoV2-S immune complex formation (B); dots and squares correspond to experimental points and curves to the fit of the mathematical model.
Figure 4
Figure 4
The surface mass density and PBS solution fraction (hydration) evolution in time for SCoV2-S covalent immobilization (A) and for the immune complex of SCoV2-S/mAb-SCoV2-S formation obtained using QCM–D and SE (B).
Figure 5
Figure 5
Viscosity coefficient (η) (black) and shear elasticity modulus (μ) (red) vs. time and effective layer density (ρ = ρ1) for SCoV2-S covalent immobilization (A,B) and for mAb-SCoV2-S affinity interaction (C,D) (ρ = ρ2).
Figure 6
Figure 6
Schematic representations of: the two-step model of SCoV2-S covalent immobilization (A) and affinity-based interaction between covalently immobilized SCoV2-S and mAb-SCoV2-S (B). The proteins enter the initial state 1 at a rate constant ka, then may desorb at a rate constant—kd or bind strongly at rate constant—kr.
See this image and copyright information in PMC

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