The binding affinities of antibodies to SARS-CoV-2 RBD and S trimers (WA1/2020/B.1.1.7/B.1.351/P.1/B.1.617.1/B.1.617.2) were tested using a BIAcore 8K system (Cytiva) together with CM5 biosensor chips (Cytiva). Antigens were diluted in pH 5.0 Acetate (Cytiva) and covalently coupled on chips using an Amine Coupling Kit (Cytiva). After reaching a 70 RU coupling level, the excess antigens were washed away and the unbound sites were blocked with ethanolamine. Antibodies were 2-fold serially diluted from 1.250 to 0.039 μg/mL in HBS-EP buffer (Cytiva) and then injected for 120 s at 30 μL/min. After that, the binding was dissociated with HBS-EP buffer for 120 s, followed by chip regeneration with pH 1.5 Glycine (Cytiva). Parameters including Ka, Kd, and KD values were calculated employing a monovalent analyte model with BIAevaluation software.
Hbs ep buffer
Manufactured by Cytiva
Sourced in United States, Sweden
HBS-EP+ buffer is a concentrated solution that is used to prepare running buffers for surface plasmon resonance (SPR) analysis. It is designed to provide a stable and consistent environment for protein-protein, protein-small molecule, and other molecular interactions to be measured in an SPR system.
Automatically generated - may contain errors
Lab products found in correlation
Biacore t200, by Cytiva (10 mentions)
Amine coupling kit, by Cytiva (6 mentions)
Biacore 8k, by Cytiva (6 mentions)
Cm5 sensor chip, by Cytiva (5 mentions)
Octet data analysis ht 12, by Sartorius (5 mentions)
Biacore 3000, by Cytiva (4 mentions)
Cm5 sensor chip, by GE Healthcare (3 mentions)
His capture kit, by Cytiva (3 mentions)
Biacore x100, by Cytiva (3 mentions)
Biacore insight, by Cytiva (3 mentions)
Streptavidin sa sensor chips, by Cytiva (3 mentions)
Zeba micro spin desalting column, by Thermo Fisher Scientific (3 mentions)
Biacore 8k system, by Cytiva (2 mentions)
Series s cm5 chip, by Cytiva (2 mentions)
Biacore 8k, by GE Healthcare (2 mentions)
Biacore 8k instrument, by Cytiva (2 mentions)
Protein a chip, by Cytiva (2 mentions)
Insight evaluation software, by Cytiva (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Biacore t100 biosensor, by Cytiva (2 mentions)
55 protocols using hbs ep buffer
1
Antibody Binding Affinities to SARS-CoV-2 Variants
2
Binding Kinetics of Anti-CD20 Antibodies
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The binding kinetics of chimeric anti-CD20 mutant antibodies for each shFcγR (shFcγRI, shFcγRIIa, shFcγRIIIa-158V, shFcγRIIIa-158F, and shFcγRIIIb) were analyzed by surface plasmon resonance (SPR) measurement using a T100 biosensor instrument and CM5 sensor chips (BIAcore; GE Healthcare, Pittsburgh, PA), as described previously [24 (link)]. Briefly, assays were performed with anti-tetra-His antibody-immobilized CM5 sensor chips using an Amine Coupling Kit (BIAcore). The individual hexa-His-tagged shFcγRs were captured by the immobilized anti-tetra-His antibodies at a flow rate of 5 μL/min. Antibodies were diluted in HBS-EP+ Buffer (BIAcore) at various concentrations (for shFcγRI and shFcγRIIIa-158V: from 4 to 267 nM; for shFcγRIIa, shFcγRIIIa-158F, and shFcγRIIIb: from 8 to 534 nM), and each diluted antibody was injected into the shFcγRs-coated sensor chip at a flow rate of 30 μL/min. The experiments were performed with HBS-EP+ as the running buffer at 25°C. The shFcγRs and antibodies bound to the sensor surface were removed by injecting 10 mM HCl. The data obtained by the injection of antibodies were corrected for the blank control prior to data analysis. The dissociation constant (KD) for each shFcγR was calculated by steady-state analysis using BIAcore T100 kinetic evaluation software (BIAcore).
3
Real-Time Protein Binding Kinetics
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Real-time binding interaction studies were performed using a Biacore X100 (GE Healthcare, USA)36 (link). Recombinant human EGFR protein (Fc chimera) (ab155726, Abcam) was immobilized on a CM5 sensor chip (GE Healthcare) using an amine-coupling method. Injection of EGFR was terminated when surface plasmon resonance reached ~ 3000 RU. For EGFR kinetics assays, EGF (Cat. No. 53003-018, Invitrogen) at 0, 0.813, 1.63, 3.25, and 6.5 μg/ml, or chondroitin sulfate C (CS-C) at 0, 15.6, 31.3, 62.5, and 125 μg/ml were sequentially injected at a flow rate of 30 μL/min for 120 s at 25 °C; dissociation time was set for 130 s (for EGF) or 180 s (for CS-C). Recombinant EGF protein (Fc chimera) (Cat. No. 10605-H01H, Sino Biological) was immobilized on a CM5 sensor chip. Injection of EGF was terminated when the surface plasmon resonance reached ~ 1600 RU. For EGF kinetics assays, EGFR protein (Fc chimera) at 0, 6.24, 12.5, 25, and 50 μg/ml, or CS-C at 0, 15.6, 31.3, 62.5, and 125 μg/ml were sequentially injected at a flow rate of 30 μL/min for 120 s at 25 °C; dissociation time was set to 3 min. Binding reactions proceeded in HBS-EP buffer (BIAcore) for EGF and EGFR, or Tris-HCl (pH 7.5) for CS-C. To evaluate the binding affinity, the equilibrium dissociation constant (KD) was calculated for individual analytes using Biacore X100 Evaluation software (GE Healthcare), assuming a 1:1 binding ratio.
4
Real-time Binding Interaction Analysis
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Biacore T200 (GE Healthcare, USA) was used for real-time binding interaction studies. Binding reactions were done in HBS-EP buffer from BIAcore, containing 10 mM hepes, 150 mM NaCl, 3 mM EDTA and 0.05% surfactant p20, pH 7.4. At least 3 independent experiments were performed.
5
Kinetic analysis of toxin-scFv interactions
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The association and dissociation kinetic constants of the 10FG2 were determined as in [20 (link)] with some modifications. Serial dilutions of 10FG2 in HBS-EP buffer (Biacore) were injected into chips containing the immobilized Cll1, Cn2, Css2, Cll2, CeII9, or Ct1a toxins. Samples of 100 µL were injected over each chip at a flow rate of 50 µL min−1. The protein concentrations ranged from one nM to 50 nM for each assay. The delay phase lasted 600 seconds or 900 seconds. The kinetic constants were determined using the corresponding sensorgrams, which were corrected by subtracting the values from both the reference flow cell and the blank buffer injection. The Langmuir (1:1) model from BIA-evaluation software version 3.1 was used to determine the kinetic constants and the affinity constant (KD values are the ratios of association over dissociation constants). The time of residence (TR) of the scFvs on the different toxins was determined as the 1/koff value expressed in min.
6
Measuring gp140 Binding Kinetics
7
Affinity-based Antibody Purification
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All reagents were purchased at the highest purity that was commercially available. 1-[3-(Dimethylamino)-propyl]-3-ethylcarbodiimide (EDC), bovine serum albumin (BSA; fraction V), ovalbumin (OVA; grade V) and the PURE1A Protein A Antibody Purification Kit were purchased from Sigma. Goat polyclonal anti-rabbit IgG-HRP conjugate (secondary antibody) was from Abcam. Affinity resin EAH Sepharose 4B was purchased from Amersham Biosciences. Nitrocellulose transfer membrane Protran from Schleicher & Schuell and ECL detection reagents from Amersham Biosciences were used in the dot blot and western blot experiments. Microplates (96-well), LockWell MaxiSorp from Nunc, 3,5-tetramethylbenzidine (TMB) enzyme substrate from Sigma, and a microplate reader, Multiskan EX from Thermo, were used for the ELISA experiments. UV measurements were carried out on a Varian Cary 50 Bio spectrophotometer. Antibodies against benzyl-penicillin (anti-PenG) were produced and purchased from COVALAB S.A.S. (Villeurbanne, France). HBS-EP buffer was purchased from Biacore (GE Healthcare). The milk was produced from Granarolo (Granarolo S.p.a. Italy).
8
Immobilizing E. coli in Agarose
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The competent E. coli strain BL21(DE3)pLysS (Promega, Madison, WI) was used in this study. E. coli cells carrying either the EGFP or NTF2-EGFP vector were cultured overnight at ~30 C° in Lysogeny broth (LB) medium with 1 mM ampicillin. The medium was diluted to 0.2–0.3 OD600nm with fresh LB medium the next morning. After growing to 0.6–0.8 OD600nm the medium was centrifuged at 6000 g for ~10 s. After removing the old medium the cells were resuspended with fresh LB medium and mixed with low-melting point agarose dissolved in PBS medium at ~32 C°. A volume of 0.5 μl of the 1% agarose/medium mixture was transferred to a microscope slide and covered by a coverslip, which was gently pressed to achieve a layer thickness of ~1 μm between the microscope slide and the coverslip. This process resulted in an orientation of E. coli cells parallel to the glass interface. The slide borders were sealed with nail polish. The preparation of yeast and U2OS samples has been described elsewhere [8 (link)]. In addition, EGFP was purified as reported [10 (link)] and dissolved in Biacore's HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) for solution measurements.
9
Biosensor Assay for Toxin Binding
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For these assays, we used chips CM5, the Amine Coupling Kit (Biacore), and a Biacore biosensor system (Biacore X, Uppsala, Sweden). For each toxin, 250 ng was dissolved in 100 μL of 10 mM 2-(N-morpholino) ethanesulfonic acid (pH 6). A total of 10 μL of toxin solution was bound to cell 2 of the CM5 sensor chip previously activated at a flow rate of 5 μL min−1. Approximately 100 resonance units (RU) were coupled. After coupling, during the assays, the cell 1 (nothing bound) was used as a control. The protein solutions of scFvs were serially diluted in HBS-EP buffer (Biacore); 100 μL of samples of scFvs were injected over each chip (CsEM1a or CsEd coupled) at a flow rate 50 μL min−1. Biosensor measurements were performed at 25 °C. The scFv protein concentrations ranging from 0.5 nM to 180 nM were assayed. The delay phase lasted 1000 s. The chip surfaces were regenerated with 10 mM Glycine-HCl pH = 2. The kinetic constants were determined using the corresponding sensorgrams, which were corrected by subtracting the values from both the reference flow cell and the blank buffer injection. The Langmuir (1:1) model from BIA-evaluation software version 3.1 was used for kinetic constants determination.
10
Binding Kinetics of Gremlin to VEGFR2
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SPR measurements were performed on BIAcore X instrument (GE Healthcare). The extracellular domain D1-7 of human VEGFR2 (ECD-VEGFR2) (ReliaTech GmbH) was immobilized at approximately 0.036-0.064 pmol/mm2 onto CM5 sensorchips (BIAcore). The chips were pre-activated with a mixture of 0.2 M N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 0.05 M N-hydroxysuccinimide (35 μL; flow rate: 10 μL/minute). After ECD-VEGFR2 immobilization (70 μL of a solution of 0.345 μM sVEGFR2 in 10 mM sodium acetate pH 4.5 at flow rate 10 μL/minute), the remaining dextran active moieties were deactivated with 1.0 M ethanolamine at pH 8.5 (35 μL, flow rate 10 μL/minute). The activated/deactivated dextran was used as reference. Increasing concentrations (from 100 ng/mL to 4 μg/mL) of gremlinWT or gremlinC141A were injected in HBS-EP buffer (BIAcore) for 10 minutes (sample volume: 50 μL; flow rate: 5 μL/minute; dissociation time: 2 minutes). The response (in response units) was monitored as a function of time. For each concentration of the ligand, the SPR response at equilibrium was used to build the normalized dose-response binding isotherms of gremlin with ECD-VEGFR2. Binding isotherm points were fitted to the Langmuir equation for monovalent binding to determine equilibrium affinity constants, by using SOLAR2.0 software (http://www.chem4tech.it/ ).
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