To measure the binding affinity of MerTK agonistic antibody, a surface plasmon resonance BIAcore™-T200 instrument was used. Series S sensor chip Protein A (GE Healthcare) was applied to capture each antibody on different flow cells (FC) to achieve approximately 200 response units (RU), followed by the injection of five-fold serial dilutions of human or mouse MerTK (0.8 nM to 500 nM) in HBS-EP buffer (100 mM HEPES pH7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) Surfactant P20) with a flow rate of 100 ul/min at 25°C. Association rates (kon) and dissociation rates (koff) were calculated using a simple one-to-one Langmuir binding model (BIAcore T200 evaluation software version 2.0). The equilibrium dissociation constant (KD) was calculated as the ratio koff/kon. The result is shown in Figure S4A.
Series s sensor chip protein a
Manufactured by GE Healthcare
Sourced in United States
The Series S sensor chip protein A is a laboratory equipment product designed for use in various analytical applications. It serves as a sensor chip that incorporates protein A, a bacterial protein known for its ability to bind to the Fc region of immunoglobulins. This sensor chip can be utilized in techniques such as surface plasmon resonance (SPR) to study protein-protein interactions.
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Lab products found in correlation
Biacore t200, by GE Healthcare (2 mentions)
Biacore t200 instrument, by GE Healthcare (2 mentions)
Biacore 8k, by GE Healthcare (2 mentions)
Biacore insight evaluation software v 3, by GE Healthcare (1 mentions)
Superdex 200 10 300 column, by GE Healthcare (1 mentions)
Biacore evaluation software, by GE Healthcare (1 mentions)
Protein a sensor chip, by GE Healthcare (1 mentions)
Cm5 sensor chip, by GE Healthcare (1 mentions)
Hbs ep buffer, by Cytiva (1 mentions)
Biacore 8k evaluation software, by GE Healthcare (1 mentions)
8 protocols using series s sensor chip protein a
1
Quantifying MerTK Antibody Binding Affinity
2
Binding Kinetics of Arenavirus Glycoprotein
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Binding of sAD to GP1JUNV–Fc, GP1MACV–Fc, GP1GTOV–Fc, GP1SBAV–Fc, and GP1WWAV–Fc fusion proteins was measured using a Biacore T200 instrument (GE Healthcare). Fusion proteins were first immobilized at a coupling density of ~500 resonance units (RU) on a series S sensor chip protein A (GE Healthcare) in TBS and 0.02% sodium azide buffer. One of the four flow cells on the sensor chip was coupled with GP1LASV–Fc to serve as a blank. sAD was then injected at 5, 50, 250, 500, and 1000 nM concentrations, at a flow rate of 80 μl/min. Single-cycle kinetics was performed for the binding assay. The sensor chip was regenerated using 10 mM glycine-HCl pH 1.5 buffer. The binding of hTfR1 and wwTfR1 to GP1JUNV–Fc and GP1MACV–Fc was similarly measured, at TfR1 concentrations of 500, 250, 125, 12.5, and 1.25 nM.
3
SARS-CoV Receptor Binding Kinetics
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Binding of SARS-CoV-2 RBD or SARS-CoV-1 RBD to ACE2 –Fc and ACE2mod–Fc fusion proteins were measured using a Biacore T200 instrument (GE Healthcare). Fusion proteins were first immobilized at a coupling density of ∼1000 response units (RU) on a series S sensor chip protein A (GE Healthcare) in PBS and 0.02% (w/v) sodium azide buffer. RBD was then injected at 0.16, 0.8, 4, 20, and 100 nM concentrations, at a flow rate of 60 μL/min. Single-cycle kinetics was performed for the binding assay. The sensor chip was regenerated using 10 mM glycine-HCl pH 1.5 buffer.
4
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.
5
Identifying SARS-CoV-2 Neutralizing Antibodies
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Since antibodies recognize antigens mainly depend on the CDR3 region, we synthesize 57 CDR3 peptides of these heavy chains and light chains with neutralizing potential and verify their binding ability to S1 protein analyzed by microscale thermophoresis (MST). Briefly, single-cycle kinetics experiments with a Biacore T200 instrument (GE Healthcare) was used to analyze the binding of the S1 protein to the various CDR3 sequence. Purified S1 protein was first immobilized on a series S sensor chip protein A (GE Healthcare) at 800–1200 response units (RU) in PBS containing 0.02% sodium azide. One cell on the sensor chip was empty to serve as a blank. Then, a series of concentrations (i.e., 0.8, 4, 20, 100, and 500 nM) of soluble CDR3 peptide was injected in PBS at a flow rate of 60 μL/min. The sensor chip was regenerated using 10 mM Glycine-HCl (pH = 1.5) buffer. A 1:1 binding model was used to describe the experimental data. Due to conformational change in these cases, we fitted a two-state binding model that assumes two binding constants.
6
SPR-based Kinetic Analysis of RBD-mAb Binding
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For SPR measurement, the RBD was additionally purified by size exclusion chromatography (SEC) purification with a Superdex200 10/300 column (GE Healthcare). Binding of the RBD to the various mAbs was measured using single-cycle kinetics experiments with a Biacore T200 instrument (GE Healthcare). Purified mAbs were first immobilized at coupling densities of 800-1200 response units (RU) on a series S sensor chip protein A (GE Healthcare) in PBS and 0.02% sodium azide buffer. One of the four flow cells on the sensor chip was empty to serve as a blank. Soluble RBD was then injected at a series of concentrations (i.e., 0.8, 4, 20, 100, and 500 nM) in PBS at a flow rate of 60 μL/min. The sensor chip was regenerated using 10 mM Glycine-HCl pH 1.5 buffer. A 1:1 binding model was used to describe the experimental data and to derive kinetic parameters. For some mAbs, a 1:1 binding model did not provide an adequate description for binding. In these cases, we fitted a two-state binding model that assumes two binding constants due to conformational change. In these cases, we report the first binding constants (KD1).
7
SARS-CoV-2 Spike Protein Binding Kinetics
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SPR experiments were performed in a running buffer composed of 0.01 M Hepes (pH 7.4), 0.15 M NaCl, 3 mM EDTA, and 0.005% v/v surfactant P20 at 25°C using the Biacore 8K (GE Healthcare), with a series S protein A sensor chip (GE Healthcare). The ACE2 receptor or SARS-CoV-2 spike-specific antibodies (CR3022 or S309) were immobilized on the protein A sensor chip (GE Healthcare) at a ligand capture level of ~100 RU. Serial dilutions of purified spike designs were injected, at concentrations ranging from 10 to 1.25 nM. The resulting data were fit to a 1:1 binding model using the Biacore Evaluation Software (GE Healthcare).
8
Quantifying FcγR-Antibody Interactions
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SPR experiments were performed on a BIAcore 8K (BIAcore, GE Healthcare, USA) at 25 °C in HBS‐EP+ buffer (Cytiva, USA). Mouse FcγRs were purchased from Sino Biological (Beijing, CN) with a 6×His tag fused to the C‐terminus of the FcγRs. Briefly, antibodies with hIgG‐Fc were immobilized on a Series S Protein A sensor chip (GE Healthcare, USA) at a 300‐response unit (RU) density. Antibodies with mIgG‐Fc were captured on a CM5 sensor chip (GE Healthcare, USA) on which anti‐mouse antibodies had been immobilized. Serial dilutions of recombinant FcγRs were injected into the flow cells at 30 µL min−1, at concentrations ranging from 400 to 3.125 nM (1:2 successive dilutions). The association time was 90 s, followed by a 120‐s dissociation step. At the end of each cycle, the sensor surface was regenerated by a glycine HCl buffer (10 mM, pH 1.7; 50 µL min−1, 30 s). Background binding to blank immobilized flow cells was subtracted, and KD values were calculated using BIAcore8K evaluation software (GE Healthcare, USA) with the 1:1 Langmuir binding model.
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