SPR-based measurements were performed by BIAcore8000 system (GE Healthcare) with protein A chip (GE Healthcare) at 25°C in single-cycle mode. All proteins used for the kinetic analyses were exchanged to PBST. The concentrated supernatant containing MAbs was captured on the protein A chip at more than 500 response units. Gradient concentrations of the RBD proteins were run across flow cell 2 of the chip, with flow cell 1 set as the control. After each cycle, the sensor chip was regenerated using glycine (pH 1.5). For spike trimer-Fabs binding assay, GX/P2V/2017 S trimer protein was biotinylated and immobilized on an SA chip to approximately 2,000 response units. Gradient concentrations of 11 Fab proteins were run across flow cell 2 of the chip, with flow cell 1 set as the control. After each cycle, the sensor chip was regenerated using glycine (pH 2.5). Binding kinetics were analyzed with the BiacoreTM Insight software (GE Healthcare) using a 1:1 Langmuir binding model. These results were visualized using Origin 2021.
Biacore insight software
Manufactured by GE Healthcare
The BIAcore Insight software is a powerful analytical tool designed for the analysis of biomolecular interactions. It provides a comprehensive platform for data acquisition, processing, and visualization, enabling researchers to gain insights into the kinetics and thermodynamics of various biomolecular systems.
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Lab products found in correlation
Biacore 8k, by GE Healthcare (5 mentions)
Biacore insight, by Cytiva (4 mentions)
Biacore 8k evaluation software, by GE Healthcare (3 mentions)
Protein a chip, by GE Healthcare (1 mentions)
Biacore8000 system, by GE Healthcare (1 mentions)
Originpro 9, by OriginLab (1 mentions)
Biacore 8k system, by GE Healthcare (1 mentions)
Biacore 8k, by Cytiva (1 mentions)
Sensor cm5, by GE Healthcare (1 mentions)
Biacore 8 k control system, by GE Healthcare (1 mentions)
Cm5 chip, by GE Healthcare (1 mentions)
Protein a sensor chip, by GE Healthcare (1 mentions)
9 protocols using biacore insight software
1
SPR-based Kinetic Analysis of Antibody-Antigen Interactions
2
Characterizing SARS-CoV-2 RBD Binding Affinities
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The refolded hACE2, eqACE2 and mutated eqACE2 were transferred into PBST buffer (1.8 mM KH2PO4, 10 mM Na2HPO4 (pH 7.4), 137 mM NaCl, 2.7 mM KCl and 0.05% (v/v) Tween 20) and biotinylated with an NHS-LC-LC-Biotin kit. Then, the biotinylated proteins are immobilized on flow cell 2 of a SA chip. Flow cell 1 was used as the negative control. Then, serially diluted wt or mutated RaTG13-RBD and SARS-COV-2 PT-RBD were flowed over the chip in PBST buffer. Binding affinities were measured using a BIAcore 8 K (GE Healthcare) at 25 °C in the single-cycle mode. Binding kinetics data were collected using BIAcoreTM 8 K control software 3.0.12.15655 and analyzed with BiacoreTM Insight software (GE healthcare) using a 1:1 Langmuir binding model. The SARS-COV-2 PT-RBD, RBDs from VOCs and mutated SARS-COV-2 PT-RBD were immobilized on CM5 chips and the following protocol of SPR assay is the same.
3
SARS-CoV-2 RBD Binding Affinity Assay
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For all measurements, phosphate-buffered saline containing Tween 20 (PBST; 1.8 mM KH2PO4, 10 mM Na2HPO4 pH 7.4, 137 mM NaCl, 2.7 mM KCl and 0.05% (v/v) Tween 20) was used as the running buffer. hACE2, bACE2-Ra, mutant hACE2 and mutant bACE2-Ra were transferred into PBST buffer and immobilized on the CM5 chip. SARS-CoV-2-RBD or variant RBDs were serially diluted and flowed through the CM5 chip in PBST buffer. Binding affinities were measured using a BIAcore 8K system (GE Healthcare) at 25℃ in single-cycle mode. After each cycle, the sensor chips were regenerated with glycine (pH 1.7). Kinetics or steady states were analyzed using BIAcore Insight software (GE Healthcare) with a 1:1 Langmuir binding model. Graphics were generated using OriginPro 9.1 software (OriginLab Corporation, Northampton, USA).
4
Evaluating ACE2-RBD Binding Interactions
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To determine the affinity between ACE2 orthologs and RBDs, ACE2-mFc proteins were immobilized on CM5 sensors (GE Healthcare). As the flow phases, the prototype, Delta and Omicron BA.1 RBDs were doubly diluted into five stages of concentration and then interacted with the CM5 sensor using a single cycle mode generated by the BIAcore 8 K control system (GE Healthcare).
Human, mouse, palm-civet, and least horseshoe bat were chosen as the typical species. To compare the binding differences between prototype RBD mutations and these species, the RBDs were immobilized on CM5 sensors (GE Healthcare), and the peptidase domains of the four ACE2s were serially diluted as the flow phases, using the same mode as mentioned above.
For all measurements, PBST (PBS, pH 7.4, 0.5‰ (v/v) Tween-20) was used as the running buffer. Kinetics or steady states were analyzed using the BIAcore™ Insight software (GE Healthcare) using a 1:1 binding model. The appropriate immobilization levels and concentrations of the solutions were set (Supplementary TablesS4 and S5 ). Graphics were generated using OriginPro 9.1.
Human, mouse, palm-civet, and least horseshoe bat were chosen as the typical species. To compare the binding differences between prototype RBD mutations and these species, the RBDs were immobilized on CM5 sensors (GE Healthcare), and the peptidase domains of the four ACE2s were serially diluted as the flow phases, using the same mode as mentioned above.
For all measurements, PBST (PBS, pH 7.4, 0.5‰ (v/v) Tween-20) was used as the running buffer. Kinetics or steady states were analyzed using the BIAcore™ Insight software (GE Healthcare) using a 1:1 binding model. The appropriate immobilization levels and concentrations of the solutions were set (Supplementary Tables
5
SARS-CoV-2 Protein-ACE2 Binding Kinetics
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The SARS-CoV-2 PT and variants S and RBD proteins were transferred into PBST buffer (1.8 mM KH2PO4, 10 mM Na2HPO4 (pH 7.4), 137 mM NaCl, 2.7 mM KCl, and 0.05% (v/v) Tween 20) and immobilized on CM5 chip. Serially diluted hACE2s were then flowed over the chip in PBST buffer. Binding affinities were measured using a BIAcore 8K (GE Healthcare) at 25 °C in the single-cycle mode. To test the binding affinities of MAbs with different RBDs, MAbs immobilized on CM5 chip and RBDs flowed over the chip in PBST buffer. Binding kinetics were analyzed with BIAcore Insight software (GE Healthcare) using a 1:1 binding model. Gradient concentrations of hACE2 from 200 nM to 12.5 nM with two-fold dilution flowed over the chip in PBST buffer. The CM5 chip (GE Healthcare) was regenerated using 10 mM Glycine-HCl (pH 1.5). KD values of SPR experiments were obtained with BIAcore 8K Evaluation Software (GE Healthcare), using a 1:1 binding model. The values indicate the mean ± SD of three independent experiments.
6
Characterizing ACE2-RBD Binding Kinetics
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The 25 ACE2-mFC fusion proteins were transferred into HBST buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% (v/v) Tween 20) and immobilized on the CM 5 chip (Table S2 ). Then, serially diluted wild-type or mutated RaTG13 RBD proteins flowed over the chip in HBST buffer. PD-L1 protein was used as negative control. Binding affinities were measured using a BIAcore 8K (GE Healthcare) at 25°C in the single-cycle mode. Binding kinetics were analyzed with Biacore™ Insight software (GE healthcare) using a 1:1 Langmuir binding model.
The seven recombinant antibodies (2 μg/mL) were first captured on flow cell 2 of the protein A sensor chip (GE Healthcare) at more than 500 response units (Table S4 ). Flow cell 1 was used as the negative control. Then, serially diluted SARS-CoV-2 RBD or RaTG13 RBD proteins flowed over the chip in PBST buffer. Response Units (RU) were measured with a BIAcore 8K (GE Healthcare) at 25°C in single-cycle mode. The antibodies were regenerated with 10 mM glycine-HCl (pH 1.5). The equilibrium dissociation constants (KD) of each pair of interactions were calculated using BIAcore® 8K Evaluation Software (GE Healthcare) by fitting to a 1:1 Langmuir binding model.
The seven recombinant antibodies (2 μg/mL) were first captured on flow cell 2 of the protein A sensor chip (GE Healthcare) at more than 500 response units (
7
SARS-CoV-2 RBD Binding Kinetics
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The biotinylated SARS-CoV-2 and VOCs RBD proteins were transferred into PBST buffer (1.8 mM KH2PO4, 10 mM Na2HPO4 (pH 7.4), 137 mM NaCl, 2.7 mM KCl, and 0.05% (v/v) Tween 20) and immobilized on SA chip. Serially diluted hACE2s were then flowed over the chip in PBST buffer. Binding affinities were measured using a BIAcore 8K (GE Healthcare) at 25 °C in the single-cycle mode. Binding kinetics were analyzed with Biacore Insight software (GE Healthcare) using a 1:1 Langmuir binding model.
KD values of SPR experiments were obtained with BIAcore 8K Evaluation Software (GE Healthcare), using a 1:1 binding model. The values indicate the mean ± SD of three independent experiments.
KD values of SPR experiments were obtained with BIAcore 8K Evaluation Software (GE Healthcare), using a 1:1 binding model. The values indicate the mean ± SD of three independent experiments.
8
SARS-CoV-2 Spike RBD Binding Kinetics
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The ACE2-mFC fusion proteins were transferred into PBST buffer (1.8 mM KH2PO4, 10 mM Na2HPO4 (pH 7.4), 137 mM NaCl, 2.7 mM KCl, and 0.05% (v/v) Tween 20) and immobilized on flow cell 2 of CM5 chip. Flow cell 1 was used as the negative control. Serially diluted RBDs from original SARS-CoV-2 and VOCs were then flowed over the chip in PBST buffer. Binding affinities were measured using a BIAcore 8K (GE Healthcare) at 25°C in the single-cycle mode. Binding kinetics were analyzed with Biacore™ Insight software (GE healthcare) using a 1:1 Langmuir binding model.
9
Binding Affinity Determination by SPR
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KD values for the binding study-SPR experiment were obtained using BIAcore Insight software (GE Healthcare), using a 1:1 binding model. The values shown are the mean ± SD of three replicates. All experiments were performed in biologically independent replicates.
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