The affinities and kinetics were analyzed using the BIAcore 8 K (GE Healthcare) performed at 25 °C in single-cycle mode. The proteins were transferred into phosphate-buffered saline/Tween 20 (PBST; 10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4, and 0.005% [v/v] Tween 20) buffer. The S304 NAb was captured on a protein A chip (GE Healthcare). Gradient concentrations of prototypic RBD from 50 nM to 3.125 nM and Omicron RBD from 25 nM to 1.5625 nM with twofold dilution flowed over the chip in PBST buffer. The protein A chip (GE Healthcare) was regenerated using 10 mM Glycine-HCl (pH 1.5). The affinity values were calculated using a 1:1 (Langmuir) binding fit model with Biacore Insight Evaluation software v.3.0 (GE Healthcare).
Biacore insight evaluation software v 3
Manufactured by GE Healthcare
Biacore Insight Evaluation software v.3.0 is a data analysis software designed for use with Biacore surface plasmon resonance (SPR) instruments. The software provides tools for analyzing and interpreting SPR data, including kinetic and affinity analysis, concentration determination, and advanced data visualization.
Automatically generated - may contain errors
2 protocols using biacore insight evaluation software v 3
1
Kinetic Analysis of SARS-CoV-2 Antibody Binding
2
Quantifying Nanobody-RBD Interactions
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The affinity of anti-RBD nanobodies and RBD antigen was measured with Biacore 8 K. A biosensor chip, Series S Sensor Chip Protein A (Cat. # 29127556, GE), was used to affinity capture a certain amount of Fc-tagged nanobodies to be tested and then flow through a series of COVID-19 S.P. RBD (Cat. # 40592-V08B, SinoBiological) under a concentration gradient on the surface of the chip (dilution ratio: 2, conc. levels: at least 5 (excluding curves with irregularities or high background)). Biacore 8 K (GE) was used to detect the reaction signal in real-time to obtain the binding and dissociation curves.
To measure the competitive response of anti-RBD nanobodies and hACE2, Fc-tagged nanobodies were immobilized on chip, then flowed with a 50 nM of RBD (Cat. # 40592-V08B, SinoBiological) and a 100 nM of hACE2 (Cat. #1010B-H08H, SinoBiological). the reaction signal in real-time were detected to obtain the binding and dissociation curves (theoretical ACE2 Rmax > 220 RU and kinetically simulated ACE2 binding > 160 RU for all). The buffer used in the experiment is an HBS-EP + solution (pH 7.4, Cat. # BR100669, GE). The data obtained in the experiment was fitted with Biacore Insight Evaluation Software v3.0, GE software with a (1:1) binding model to obtain the affinity value.
To measure the competitive response of anti-RBD nanobodies and hACE2, Fc-tagged nanobodies were immobilized on chip, then flowed with a 50 nM of RBD (Cat. # 40592-V08B, SinoBiological) and a 100 nM of hACE2 (Cat. #1010B-H08H, SinoBiological). the reaction signal in real-time were detected to obtain the binding and dissociation curves (theoretical ACE2 Rmax > 220 RU and kinetically simulated ACE2 binding > 160 RU for all). The buffer used in the experiment is an HBS-EP + solution (pH 7.4, Cat. # BR100669, GE). The data obtained in the experiment was fitted with Biacore Insight Evaluation Software v3.0, GE software with a (1:1) binding model to obtain the affinity value.
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