All surface plasma resonance assays were performed on a Biacore 3000 (GE Healthcare) with a running buffer of 10 mM HEPES pH 7.5 and 150 mM NaCl supplemented with 0.05% Tween 20 at 25°C. Initial peptide scanning was performed by the binding of a series of SARS-CoV-2 S2 synthetic peptides (GenScript) to immobilized CV3–25 IgG (~5800 RU) on a Protein A sensor chip (Cytiva). For the kinetic binding measurements of S2 peptides #289 (15-mer), #289 (11-mer) and the 26mer (1140–1165) to CV3–25, ~5800 RU of CV3–25 IgG was first immobilized on a protein A chip (Cytiva) and 2-fold serial dilutions of the S2 peptides were then injected with concentrations ranging from 6.25 to 200 nM. After each cycle the Protein A sensor chip was regenerated with 0.1 M Glycine pH 2.0. CV3–1 IgG was used as a negative control. All sensorgrams were corrected by subtraction of the corresponding blank channel in addition to the buffer background and the kinetic constant determined using a 1:1 Langmuir model with the BIAevaluation software (GE Healthcare). Goodness of fit of the curve was evaluated by the Chi2 value with a value below 3 considered acceptable.
Protein a chip
Manufactured by Cytiva
Sourced in United States
The Protein A chip is a lab equipment product designed for protein purification and analysis. It provides a solid-phase support for the immobilization of Protein A, a bacterial protein that has a strong affinity for the Fc region of antibodies. This allows for the selective capture and purification of antibodies from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Biacore 8k instrument, by Cytiva (3 mentions)
Biaevaluation software, by GE Healthcare (2 mentions)
Biacore 3000, by GE Healthcare (2 mentions)
Sars cov 2 s2 synthetic peptides, by GenScript (2 mentions)
Protein a sensor chip, by Cytiva (2 mentions)
Biacore s200 system, by Cytiva (2 mentions)
Biacore evaluation software, by Cytiva (2 mentions)
Hbs ep buffer, by Cytiva (2 mentions)
Insight evaluation software, by Cytiva (2 mentions)
Ovalbumin (ova), by InvivoGen (1 mentions)
Biacore t200, by Cytiva (1 mentions)
Cm5 sensor chip, by Cytiva (1 mentions)
Biaevaluation software, by Cytiva (1 mentions)
Biacore 3000, by Cytiva (1 mentions)
9 protocols using protein a chip
1
Binding Kinetics of SARS-CoV-2 S2 Peptides
2
Quantifying RBD-ACE2 Binding Kinetics
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Binding interactions between RBDs and ACE2 molecules were measured by SPR using a Biacore S200 system (Cytiva) (17 (link)). Briefly, ACE2-Fc was immobilized to a protein A chip (Cytiva). Serial dilutions of purified recombinant RBD-His flowed through (protein concentrations ranging from 20 to 3,200 nM) in a running buffer composed of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid, and 0.05% tween 20. Biacore Evaluation Software (Cytiva) was used for calculating the binding kinetics.
3
Antibody Kinetics Analysis by SPR
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Antibody association and dissociation rates were determined by Surface Plasmon Resonance (SPR) measurement using a Biacore 8K instrument (Cytiva, Marlborough, MA, USA). Each antibody at 0.5 μg/mL in the HBS-P (0.01 M HEPES, 0.15 M NaCl and 0.05% Surfactant P20) running buffer was captured by a Protein A chip (Cytiva, Marlborough, MA, USA). To measure the binding kinetics, ovalbumin (InvivoGen, San Diego, CA, USA) from 11 nM to 100 nM in 3-fold serial dilutions, and a blank buffer for baseline subtraction, were injected at 30 μL/min for 120 s, followed by a 10 min dissociation period. Regeneration of the Protein A surface was achieved via 30 s of 10 mM glycine (pH 1.5) at 50 μL/min between each running cycle. All kinetic experiments were performed at 25 °C.
4
Binding Affinity of MAb 22.9-1 to SARS-CoV-2 Variants
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The binding affinity of the MAb 22.9-1 for the RBDs of the WT, Delta, and Omicron variants were determined by SPR binding assays with the Biacore 8K instrument and protein A chip (Cytiva). HBS-EP+ buffer (Cytiva) was used as the running buffer and sample diluent. Monoclonal antibody was captured over the protein A surface for 60 s at a flow rate of 10 μL/min. The RBD antigens were injected into each cycle at a flow rate of 30 μL/min; this was followed by contact for 120 s and dissociation for 400 s. The chip surface was regenerated each cycle with glycine solution (pH 1.5) at a flow rate of 30 μL/min for 30 s. The data were analyzed, and affinity KD values were calculated with the Biacore Insight Evaluation software.
5
Kinetic analysis of ACE2-RBD binding
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Binding kinetics were determined using a Biacore T200 instrument (Cytivia). All experiments were performed in HBS-EP buffer, pH 7.4. 10 nM ACE2-Fc protein was captured onto a Protein A Chip (Cytiva) at a flow rate of 10 µl/min for ∼1 min. A series of solutions ranging from 0 to 300 nM glycosylated and de-glycosylated RBD proteins were subsequently injected at a flow rate of 30 µl/min onto the ACE2-Fc captured surface. Data were corrected by double-referencing against a control flow cell containing no ACE2-Fc capture and against the flow cell with buffer injection. Sensogram curves were analyzed using the BiaEval 3.0 manufacturer's software. The dissociation constant (KD), association rate constant (kon), and dissociation rate constant (koff) values were calculated by fitting the kinetic association and dissociation curves to a 1:1 binding model. Extracted data were plotted via in-house Python scripts.
6
Surface Plasmon Resonance Binding Kinetics
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Surface plasmon resonance assay using a Biacore S200 system (Cytiva) were carried out as described previously (17 (link)). Briefly, ACE2-Fc was immobilized to a protein A chip (Cytiva). The running buffer composed of 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20. Serial dilutions of purified recombinant RBD-His were injected ranging in concentration from 20 to 320 nM. In a different approach, RBD-His was immobilized to a CM5 sensor chip through chemical cross-linking (Cytiva). Serial dilutions of purified recombinant ACE2-His were injected ranging in concentration from 100 to 1,600 nM. Binding kinetics were calculated using Biacore evaluation software (Cytiva).
7
Kinetic Analysis of SARS-CoV-2 RBD Binding
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All surface plasma resonance assays were performed on a Biacore 3000 (Cytiva) with a running buffer of 10 mM HEPES pH 7.5 and 150 mM NaCl supplemented with 0.05% Tween 20 at 25°C. For the kinetic measurements of SARS-CoV-2 VOCs’ RBDs to EH3 and EH8, either of the IgGs (∼250-350 response unit, RU) was first immobilized on a protein A chip (Cytiva) and 2-fold serial dilutions of the RBDs were then injected with concentrations ranging from 6.25 to 200 nM. After each cycle the sensor chip was regenerated with 0.1 M Glycine pH 2.5. All sensorgrams were corrected by subtraction of the corresponding blank channel in addition to the buffer background and the kinetic constant determined using a 1:1 Langmuir model with the BIAevaluation software (Cytiva). Goodness of fit of the curve was evaluated by the Chi2 value with a value below 3 considered acceptable. The sensorgrams are shown in Figures S2 A and S2B and the kinetic constants are summarized in Table 1 .
8
SARS-CoV-2 S2 Peptide Binding Kinetics
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All surface plasma resonance assays were performed on a Biacore 3000 (GE Healthcare) with a running buffer of 10 mM HEPES pH 7.5 and 150 mM NaCl supplemented with 0.05% Tween 20 at 25°C. Initial peptide scanning was performed by the binding of a series of SARS-CoV-2 S2 synthetic peptides (GenScript) to immobilized CV3-25 IgG (∼5800 RU) on a Protein A sensor chip (Cytiva). For the kinetic binding measurements of S2 peptides #289 (15-mer), #289 (11-mer) and the 26mer (1140–1165) to CV3-25, ∼5800 RU of CV3-25 IgG was first immobilized on a protein A chip (Cytiva) and 2-fold serial dilutions of the S2 peptides were then injected with concentrations ranging from 6.25 to 200 nM. After each cycle the Protein A sensor chip was regenerated with 0.1 M Glycine pH 2.0. CV3-1 IgG was used as a negative control. All sensorgrams were corrected by subtraction of the corresponding blank channel in addition to the buffer background and the kinetic constant determined using a 1:1 Langmuir model with the BIAevaluation software (GE Healthcare). Goodness of fit of the curve was evaluated by the Chi2 value with a value below 3 considered acceptable.
9
Binding Affinity of MAb 22.9-1 to SARS-CoV-2 Variants
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The binding affinity of the MAb 22.9-1 for the RBDs of the WT, Delta, and Omicron variants were determined by SPR binding assays with the Biacore 8K instrument and protein A chip (Cytiva). HBS-EP+ buffer (Cytiva) was used as the running buffer and sample diluent. Monoclonal antibody was captured over the protein A surface for 60 s at a flow rate of 10 μL/min. The RBD antigens were injected into each cycle at a flow rate of 30 μL/min; this was followed by contact for 120 s and dissociation for 400 s. The chip surface was regenerated each cycle with glycine solution (pH 1.5) at a flow rate of 30 μL/min for 30 s. The data were analyzed, and affinity KD values were calculated with the Biacore Insight Evaluation software.
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