and A.R. in surface plasmon resonance if the concentration of biomolecules is high [18]. Therefore, the measurement and assessment of both ellipsometric parameters and enable a more detailed evaluation of interacting biomolecules affinity and association/dissociation rate constants. In this study, we performed a highly sensitive, label-free, and real-time TIRE monitoring of the interaction kinetics between the specific polyclonal human antibody (pAb-SCoV2-S) produced after vaccination and the covalently immobilized spike proteins of the three SARS-CoV-2 variants of concernwild-type (SCoV2-S), B.1.1.7 or so-called alpha (SCoV2-S), and B.1.351 or so-called beta (SCoV2-S). Here, we reported the comparison of the thermodynamic parameters, such as the association rate constant (for 15 min. The titer of the antibody against the S protein of SARS-CoV-2 (pAb-SCoV2-S) was obtained using a chemiluminescent microparticle immunoassay. The titer was then recalculated to a molar concentration, and the primary blood serum pAb-SCoV2-S concentration was obtained at 33.96 nM [26]. Blood serum dilutions with a phosphate-buffered saline (PBS) solution at pH 7.4 (Carl Roth, Karlsruhe, Germany) and at 1:4, 1:10, 1:20, 1:30, and 1:40 ratios were used for the kinetics measurements of the pAb-SCoV2-S interaction with the three types of S protein. The serum sample was stored at ??20 C until analysis. The sample was collected in accordance with the Clorobiocin Lithuania ethics law. This study did not need the approval of the ethics committee (confirmed by the Vilnius Regional Biomedical Research Ethics Committee). 2.2. Modification of the Gold-Coated SPR Sensor Disc with SCoV2-S, SCoV2-S, or SCoV2-S The covalent immobilization of SCoV2-S, SCoV2-S, or SCoV2-S on the gold-coated Clorobiocin Clorobiocin SPR sensor discs surface was performed under standard protocol using an 11-MUA self-assembled monolayer (SAM) Rabbit Polyclonal to ROR2 [21,22,27,28,29]. Briefly, the gold-coated SPR sensor disc was rinsed with hexane and methanol and then immersed in methanol for 3 min for ultrasound treatment. Once dried, the SPR sensor disc was immersed in a 1 mM solution of 11-MUA in methanol for 18 h to form an 11-MUA SAM. Then, the activation of the 11-MUA SAM carboxyl groups required for the Clorobiocin covalent immobilization of SCoV2-S, SCoV2-S, or SCoV2-S was accomplished by injecting the solution consisting of 0.1 M NHS and 0.4 M of EDC, mixed in equal parts, into a TIRE measurement chamber for 15 min. After the activation of the carboxyl groups, the TIRE chamber was rinsed with a PBS solution at a pH level of 7.4. Then, 333 nM SCoV2-S diluted in the PBS was injected into the TIRE chamber and incubated for 60 min. After rinsing with the PBS solution, the surface was exposed to 1 M ethanolamine at a pH level of 8.5 for 10 min to deactivate any active carboxyl groups present in the 11-MUA SAM. The schematic representation of all the steps in the surface modification is presented in Figure 1A. The same immobilization protocol was applied for SCoV2-S and SCoV2-S using the same 333 nM concentration in PBS (Figure 1C). Open in a separate window Figure 1 (A) Schematic representation of SARS-CoV-2 S protein (SCoV2-S, SCoV2-S, or SCoV2-S) covalent immobilization on the gold-coated SPR sensor disc pre-modified with 11-MUA SAM. (B) The principle scheme representing total internal reflection ellipsometry measurements. (C) Schematic representation of SCoV2-S, SCoV2-S, and SCoV2-S covalent immobilization and interaction with specific polyclonal antibodies (pAb-SCoV2-S). 2.3. TIRE Measurements The ellipsometric measurements were conducted using a rotating compensator ellipsometer J. A. Woollam M2000X (Lincoln, NE, USA). All TIRE experiments were carried out at a 70 angle of incident in the spectral range of 200C1000 nm. A.