protein a sensor chip, by Cytiva - Product details

1

SARS-CoV-2 RBD Binding Kinetics

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SPR experiments were performed using Biacore 8K (Cytiva). SA55 or SA58 (human IgG1) was captured by a Sensor Chip Protein A (Cytiva), and SARS-CoV-2 WT or BA.5 RBD (His Tag, Sino Biological) of various concentration (1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, and 50 nM) were injected. The response was recorded at room temperature, and the raw data curves were fitted to a 1:1 binding model using Biacore Insight Evaluation Software (Cytiva, v4.0.8).
For competition assays, SARS-CoV-2 WT or BA.5 RBD (His Tag, Sino Biological) was captured by a Sensor Chip CM5 (Cytiva) with immobilized anti-His using His Capture Kit (Cytiva), and SA55 or SA58 (200 nM) was injected. After equilibrium, the other mAb was also added (SA58+SA55, 200 nM each). The response was recorded by Biacore Insight Evaluation Software (Cytiva, v4.0.8) at room temperature. For control, we also determined the responses using SA55 or SA58 (200 nM) only.

Cao Y., Jian F., Zhang Z., Yisimayi A., Hao X., Bao L., Yuan F., Yu Y., Du S., Wang J., Xiao T., Song W., Zhang Y., Liu P., An R., Wang P., Wang Y., Yang S., Niu X., Zhang Y., Gu Q., Shao F., Hu Y., Yin W., Zheng A., Wang Y., Qin C., Jin R., Xiao J, & Xie X.S. (2022). Rational identification of potent and broad sarbecovirus-neutralizing antibody cocktails from SARS convalescents. Cell Reports, 41(12), 111845.

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2

SPR Analysis of RBD-ACE2 Binding Affinity

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The binding affinity between recombinant His-tagged RBD and Fc-tagged ACE2 was measured by SPR using the Biacore S200 system (Cytiva) as previously described [16 (link),17 (link)]. Each ACE2 protein (in the amount of 4.85 μg) was immobilized onto the Sensor Chip Protein A (Cytiva). Then, the RBD protein at concentrations of 40, 80, 160, 320 and 640 nM was injected over the sensor surface, bound to immobilized ACE2 protein and washed off the surface. The running buffer used for all proteins in the SPR assays consisted of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20, adjusted to pH 7.4. The apparent equilibrium dissociation constant (Kd) was measured using the Biacore analytical software (Cytiva). For this, a one-to-one Langmuir binding model was used for Fc-tagged proteins that were immobilized, with monomeric proteins flown over them. All measurements were independently repeated for biological triplicates.

Hsueh F.C., Shi K., Mendoza A., Bu F., Zhang W., Aihara H, & Li F. (2024). Structural basis for raccoon dog receptor recognition by SARS-CoV-2. PLOS Pathogens, 20(5), e1012204.

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3

Binding Kinetics of SARS-CoV-2 S2 Peptides

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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.

Li W., Chen Y., Prévost J., Ullah I., Lu M., Gong S.Y., Tauzin A., Gasser R., Vézina D., Anand S.P., Goyette G., Chaterjee D., Ding S., Tolbert W.D., Grunst M.W., Bo Y., Zhang S., Richard J., Zhou F., Huang R.K., Esser L., Zeher A., Côté M., Kumar P., Sodroski J., Xia D., Uchil P.D., Pazgier M., Finzi A, & Mothes W. (2021). Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern. bioRxiv.

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4

Surface Plasmon Resonance Characterization

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The surface plasmon resonance experiments were carried out with using Biacore X100 from Cytiva. The measurements were made on protein A sensor chip (Cytiva) with flow rate of 5 μL·min^–1.

Kowalczyk A., Kasprzak A., Ruzycka-Ayoush M., Podsiadły E., Demkow U., Grudzinski I.P, & Nowicka A.M. (2022). Ultrasensitive voltammetric detection of SARS-CoV-2 in clinical samples. Sensors and Actuators. B, Chemical, 371, 132539.

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5

SARS-CoV-2 RBD Binding Kinetics

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The surface plasmon resonance experiments were performed using a Biacore T200 (GE Healthcare). All assays were performed with a running buffer of HBS-EP (Cytiva) at 25°C.
To determine the binding kinetics between the SARS-CoV-2 RBDs and ACE2 / monoclonal antibody (mAb), a Protein A sensor chip (Cytiva) was used. ACE2-Fc or mAb was immobilised onto the sample flow cell of the sensor chip. The reference flow cell was left blank. RBD was injected over the two flow cells at a range of five concentrations prepared by serial twofold dilutions, at a flow rate of 30μl min⁻¹ using a single-cycle kinetics programme. Running buffer was also injected using the same programme for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.

Dejnirattisai W., Huo J., Zhou D., Zahradník J., Supasa P., Liu C., Duyvesteyn H.M., Ginn H.M., Mentzer A.J., Tuekprakhon A., Nutalai R., Wang B., Dijokaite A., Khan S., Avinoam O., Bahar M., Skelly D., Adele S., Johnson S.A., Amini A., Ritter T.G., Mason C., Dold C., Pan D., Assadi S., Bellass A., Omo-Dare N., Koeckerling D., Flaxman A., Jenkin D., Aley P.K., Voysey M., Costa Clemens S.A., Naveca F.G., Nascimento V., Nascimento F., Fernandes da Costa C., Resende P.C., Pauvolid-Correa A., Siqueira M.M., Baillie V., Serafin N., Kwatra G., Da Silva K., Madhi S.A., Nunes M.C., Malik T., Openshaw P.J., Baillie J.K., Semple M.G., Townsend A.R., Huang K.Y., Tan T.K., Carroll M.W., Klenerman P., Barnes E., Dunachie S.J., Constantinides B., Webster H., Crook D., Pollard A.J., Lambe T., Paterson N.G., Williams M.A., Hall D.R., Fry E.E., Mongkolsapaya J., Ren J., Schreiber G., Stuart D.I, & Screaton G.R. (2022). SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses. Cell, 185(3), 467-484.e15.

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6

Binding Kinetics of gp41-246D Antibody

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Surface plasmon 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. The binding kinetics between the gp41 C-C loop and the 246D antibody were obtained in a format where 246D IgG was immobilized onto a Protein A sensor chip (Cytiva) with ~300 response units (RU) and serial dilutions of gp41 (583–618) synthetic peptide were injected with concentrations ranging from 0.488 to 31.25 nM. The protein A chip was regenerated with a wash step of 0.1 M glycine pH 2.0 and reloaded with IgG after each cycle. Kinetic constants were determined using a 1:1 Langmuir model in bimolecular interaction analysis (BIA) evaluation software.

Prévost J., Anand S.P., Rajashekar J.K., Zhu L., Richard J., Goyette G., Medjahed H., Gendron-Lepage G., Chen H.C., Chen Y., Horwitz J.A., Grunst M.W., Zolla-Pazner S., Haynes B.F., Burton D.R., Flavell R.A., Kirchhoff F., Hahn B.H., Smith AB I.I.I., Pazgier M., Nussenzweig M.C., Kumar P, & Finzi A. (2022). HIV-1 Vpu restricts Fc-mediated effector functions in vivo. Cell reports, 41(6), 111624.

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7

Kinetic Binding Analysis of CV3-13 Fab

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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 epitope mapping was performed by the binding of SARS-CoV RBD (residue 306–577) and other SARS-CoV-2 antigens (S1 and S2 obtained from BEI Resources) to immobilized CV3-13 IgG (~5800 RU) on a Protein A sensor chip (Cytiva). For the kinetic measurement of CV3-13 Fab binding to SARS-CoV-2 spike, ~800 RU of his-tagged SARS-CoV-2 S-6P was immobilized on a Ni-pretreated NTA chip (Cytiva). 2-fold serial dilutions of purified CV3-13 Fab were then injected with concentrations ranging from 3.125 to 200 nM. Sensorgrams were corrected by subtraction of the corresponding blank channel as well as for the buffer background and kinetic constants determined using a 1:1 Langmuir model with the BIAevaluation software (GE Healthcare). Goodness of fit of the curve was evaluated by the Chi² of the fit with a value below 3 considered acceptable.

Beaudoin-Bussières G., Chen Y., Ullah I., Prévost J., Tolbert W.D., Symmes K., Ding S., Benlarbi M., Gong S.Y., Tauzin A., Gasser R., Chatterjee D., Vézina D., Goyette G., Richard J., Zhou F., Stamatatos L., McGuire A.T., Charest H., Roger M., Pozharski E., Kumar P., Mothes W., Uchil P.D., Pazgier M, & Finzi A. (2022). A Fc-enhanced NTD-binding non-neutralizing antibody delays virus spread and synergizes with a nAb to protect mice from lethal SARS-CoV-2 infection. Cell Reports, 38(7), 110368.

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8

Kinetic Analysis of CV3-13 Fab Binding to SARS-CoV-2 Spike

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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 epitope mapping was performed by the binding of SARS-CoV RBD (residue 306-577) and other SARS-CoV-2 antigens (S1 and S2 obtained from BEI Resources) to immobilized CV3-13 IgG (∼5800 RU) on a Protein A sensor chip (Cytiva). For the kinetic measurement of CV3-13 Fab binding to SARS-CoV-2 spike, ∼800 RU of his-tagged SARS-CoV-2 S-6P was immobilized on a Ni-pretreated NTA chip (Cytiva). 2-fold serial dilutions of purified CV3-13 Fab were then injected with concentrations ranging from 3.125 to 200 nM. Sensorgrams were corrected by subtraction of the corresponding blank channel as well as for the buffer background and kinetic constants determined using a 1:1 Langmuir model with the BIAevaluation software (GE Healthcare). Goodness of fit of the curve was evaluated by the Chi² of the fit with a value below 3 considered acceptable.

Beaudoin-Bussières G., Chen Y., Ullah I., Prévost J., Tolbert W.D., Symmes K., Ding S., Benlarbi M., Gong S.Y., Tauzin A., Gasser R., Chatterjee D., Vézina D., Goyette G., Richard J., Zhou F., Stamatatos L., McGuire A.T., Charest H., Roger M., Pozharski E., Kumar P., Mothes W., Uchil P.D., Pazgier M, & Finzi A. (2022). A Fc-enhanced NTD-binding non-neutralizing antibody delays virus spread and synergizes with a nAb to protect mice from lethal SARS-CoV-2 infection. Cell Reports, 38(7), 110368.

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9

Standardized SPR Assay for EV Characterization

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We conducted
the SPR experiments
using a Biacore X100 from Cytiva. We used a protein A sensor chip
(Cytiva) that provided high capture capacity on the Fc region of all
human IgG. The measurements were carried out at a flow rate of 5 μL·min^–1. To ensure reproducibility and standardization, we
fixed the parameter values as follows: (i) surface modification of
the biochip with antibodies Ab CD9, Ab CD63, and Ab CD81 (contact
time: 360 s; stabilization: 300 s; and flow rate: 5 μL·min^–1) and (ii) EV interactions with selected antibodies
(contact time: 90 s; dissociation time: 300 s; and flow rate: 5 μL·min^–1). The same sensor chip could be used for several
experiments, provided that the surface was properly regenerated by
removing EVs and captured antibodies. We adopted a two-step regeneration
protocol using 10 mM glycine-HCl pH 1.5 and 50 mM NaOH regeneration
solutions. These solutions were injected at a flow rate of 30 μL·min^–1 for 30 min, followed by a stabilization period of
1 min.

Kowalczyk A., Gajda-Walczak A., Ruzycka-Ayoush M., Targonska A., Mosieniak G., Glogowski M., Szumera-Cieckiewicz A., Prochorec-Sobieszek M., Bamburowicz-Klimkowska M., Nowicka A.M, & Grudzinski I.P. (2023). Parallel SPR and QCM-D Quantitative Analysis of CD9, CD63, and CD81 Tetraspanins: A Simple and Sensitive Way to Determine the Concentration of Extracellular Vesicles Isolated from Human Lung Cancer Cells. Analytical Chemistry, 95(25), 9520-9530.

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10

Binding Kinetics of gp41-246D Antibody

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Surface plasmon 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. The binding kinetics between the gp41 C-C loop and the 246D antibody were obtained in a format where 246D IgG was immobilized onto a Protein A sensor chip (Cytiva) with ~300 response units (RU) and serial dilutions of gp41 (583–618) synthetic peptide were injected with concentrations ranging from 0.488 to 31.25 nM. The protein A chip was regenerated with a wash step of 0.1 M glycine pH 2.0 and reloaded with IgG after each cycle. Kinetic constants were determined using a 1:1 Langmuir model in bimolecular interaction analysis (BIA) evaluation software.

Prévost J., Anand S.P., Rajashekar J.K., Zhu L., Richard J., Goyette G., Medjahed H., Gendron-Lepage G., Chen H.C., Chen Y., Horwitz J.A., Grunst M.W., Zolla-Pazner S., Haynes B.F., Burton D.R., Flavell R.A., Kirchhoff F., Hahn B.H., Smith AB I.I.I., Pazgier M., Nussenzweig M.C., Kumar P, & Finzi A. (2022). HIV-1 Vpu restricts Fc-mediated effector functions in vivo. Cell reports, 41(6), 111624.

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Protein a sensor chip

Lab products found in correlation

12 protocols using protein a sensor chip

SARS-CoV-2 RBD Binding Kinetics

SPR Analysis of RBD-ACE2 Binding Affinity

Binding Kinetics of SARS-CoV-2 S2 Peptides

Surface Plasmon Resonance Characterization

SARS-CoV-2 RBD Binding Kinetics

Binding Kinetics of gp41-246D Antibody

Kinetic Binding Analysis of CV3-13 Fab

Kinetic Analysis of CV3-13 Fab Binding to SARS-CoV-2 Spike

Standardized SPR Assay for EV Characterization

Binding Kinetics of gp41-246D Antibody

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