Antibody binding was determined using a FortéBio Bio-Layer Interferometry instrument (Sartorius Octet Red96e) at 25°C with a shake speed of 1000 rpm. Antibodies were diluted to 20 μg/mL in a flat bottom 96-well plate (Greiner) with 0.22 μm filtered phosphate buffered saline pH 7.4 and 0.05% Tween 20 (PBS-T). The antigens were diluted to a concentration of 50 mg/mL using PBS-T. Hydrated Anti-hIgG Fc Capture (AHC) biosensors (Sartorius #18–5060) were equilibrated for 60 s and then antibodies were loaded to biosensors for 300 s. After a 60-s wash and a 180-s baseline step, biosensors were then dipped into the diluted antigens for a 200-s association. Next, antibody and antigens allowed to dissociate for 300 s. Data was analyzed using Data Analysis HT 12.0 software. The negative control antibody, CH65, was indicated as a reference sensor and subtracted from the remaining ligand sensor measurements. Data was then aligned to the average of the baseline step and plotted using GraphPad Prism 9 software.
Anti higg fc capture ahc biosensors
Manufactured by Sartorius
Sourced in Germany
The Anti-hIgG Fc Capture (AHC) biosensors are a type of lab equipment designed for use in biomolecular interaction analysis. They are used to detect and measure interactions between biomolecules, such as proteins, peptides, or small molecules. The AHC biosensors employ an immobilized anti-human IgG Fc capture molecule to capture and detect the binding of analytes to the sensor surface.
Automatically generated - may contain errors
Lab products found in correlation
Flat bottom 96 well plate, by Greiner (2 mentions)
Bio layer interferometry instrument, by Sartorius (2 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (2 mentions)
Data analysis ht 12, by Sartorius (2 mentions)
Octet red96e, by Sartorius (2 mentions)
Prism v9, by GraphPad (1 mentions)
Octet n1, by Sartorius (1 mentions)
Sars cov 2 s1, by Sino Biological (1 mentions)
Sars cov 2 s2, by Sino Biological (1 mentions)
8 protocols using anti higg fc capture ahc biosensors
1
Antibody-Antigen Binding Analysis
2
SARS-CoV-2 Spike Antigen Binding Assay
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A set of 8 anti-hIgG Fc Capture (AHC) Biosensors (Sartorius, cat # 18-5064) were pre-wet in 1x HBS-EP+ buffer (Cytiva, Cat # BR100669) for 10 minutes at room temperature and then loaded on to Octect RED96e (Forte ́Bio). All pre-wet biosensors first underwent 60 seconds of baseline in HBS-EP+ buffer and then were dipped into 200 uL of 25 nM IgGs diluted in HBS-EP+ to capture to a level of 0.6 nm each. IgG G468 (link) was used as an isotype control. All 8 AHC biosensors then underwent another 60 seconds of baseline in buffer followed by a 300-second association in 200 uL of 100 nM SARS-CoV-2 spike antigens (SARS-CoV-2 S-2P, HexaPro, HexaPro-SS, or HexaPro-SS ∆stalk) and another 300-second dissociation back into buffer wells. Data were then reference subtracted (IgG G4) using the software Data Analysis 11.1. Processed data were fit globally to a 1:1 binding model for both association and dissociation.
3
Kinetic Analysis of NKp46-CRT Interaction
4
Quantifying TB31F Binding Affinity
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The binding affinity of purified immunogens to TB31F was measured using a kinetic BLI assay using an Octet-Red96e (Sartorius)16 (link). TB31F was buffer exchanged into HBS-EP buffer (10 mM Na-HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% v/v P20 surfactant) using Zeba spin desalting columns (ThermoFisher Scientific). TB31F at 10 nM was loaded onto Anti-hIgG Fc Capture (AHC) biosensors (Sartorius) over the course of 300 s, until reaching a signal of ~0.6 nm. BLI pins were then immersed in immunogens 2-fold serially diluted in HBS-EP buffer (30 nM to 0.469 nM). After 300 s, pins were immersed in HBS-EP buffer to measure dissociation. Association rate (ka), dissociation rate (kdis), and dissociation constant (KD) were globally fit using a 1:1 binding model in Data Analysis HT 12.0 (Sartorius). Three independent protein preps (biological replicates) were each measured in technical triplicate. Values reported are the average and standard deviation between biological replicates.
5
Antibody Binding Kinetics Analysis
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Antibody binding was determined using a FortéBio Bio-Layer Interferometry instrument (Sartorius Octet Red96e) at 25°C with a shake speed of 1000 rpm. Antibodies were diluted to 20 μg/mL in a flat bottom 96-well plate (Greiner) with .22μm filtered phosphate buffered saline pH 7.4 and 0.05% Tween 20 (PBS-T). The antigens were diluted to a concentration of 50μg/mL using PBS-T. Hydrated Anti-hIgG Fc Capture (AHC) biosensors (Sartorius #18–5060) were equilibrated for 60 second and then antibodies were loaded to biosensors for 300 seconds. After a 60-second wash and a 180-second baseline step, biosensors were then dipped into the diluted antigens for a 200-second association. Next, antibody and antigens allowed to dissociate for 300 seconds. Data was analyzed using Data Analysis HT 12.0 software. The negative control antibody, CH65, was indicated as a reference sensor and subtracted from the remaining ligand sensor measurements. Data was then aligned to the average of the baseline step and plotted using GraphPad Prism 9 software.
6
Binding Kinetics of CTLA-4 and FcγRIIIa
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BLI buffer used in all experiments was 10 mM HEPES, 150 mM NaCl, pH 7.4, with 0.02% Tween 20, and 0.1% BSA. Commercially purchased recombinant human and mouse CTLA-4 proteins (SinoBiological, China) and human FcγRIIIa (V158) protein (R&D systems, United States) were used in the experiments. For determining the binding affinity for huCTLA-4 and muCTLA-4, plant-produced anti-CTLA-4 2C8 mAb was immobilized on anti-hIgG Fc Capture (AHC) biosensors (Sartorius Corporation, Germany). A concentration series of 100, 50, 25, 12.5 nM of huCTLA-4 and a concentration series of 100, 25, 6.25, 3.13 of muCTLA-4 were used to determine the kinetic constants (kon, koff, KD) for huCTLA-4 and muCTLA-4, respectively, using 1:1 binding curve fit. For determining binding affinity for huFcγRIIIa (V 158), the receptor protein was loaded on the HIS1K sensor, and a concentration series of 4000, 2000, 1000, 500, 250, 125, 62.5 nM of anti-CTLA-4 2C8 was used to determine KD using steady state analysis.
7
Kinetic Binding Affinity Assay
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The binding affinity of purified immunogens to REGN10933 and Ace2-Fc was measured using a kinetic BLI assay using an Octet-Red96e (Sartorius). REGN10933 IgG or Ace2-Fc was buffer-exchanged into HBS-EP buffer [10 mM Na-Hepes (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% (v/v) P20 surfactant] using Zeba spin desalting columns (Thermo Fisher Scientific). REGN10933 or Ace2-Fc was loaded onto Anti-hIgG Fc Capture (AHC) biosensors (Sartorius) over the course of 300 s until reaching a signal of ~0.6 nm. BLI pins were then immersed in immunogens twofold serially diluted in HBS-EP buffer (150 to 2.34 nM). After 300 s, pins were immersed in HBS-EP buffer to measure dissociation. Association rate (ka), dissociation rate (kdis), and dissociation constant (KD) were globally fit using a 1:1 binding model in Data Analysis HT 12.0 (Sartorius). Three independent protein preps (biological replicates) were each measured in technical triplicate. Values reported are the average and SD between biological replicates.
8
SARS-CoV-2 Spike Protein Binding Kinetics
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BLI buffer used in all experiments was 10 mM Hepes, 150 mM NaCl, pH 7.4, with 0.02% Tween 20, and 0.1% BSA. Analytes used in kinetic analysis were uncleavable S trimer, RBD-SD1, RBD wild-type, RBD mutants, commercially purchased recombinant SARS-CoV-2 S2 and SARS-CoV-2 S1 (SinoBiological; Cat #s 40,590- and 40,591-V08H, respectively). For determining binding affinities, IgGs were immobilized on Anti-hIgG Fc Capture (AHC) biosensors (Sartorius; Cat # 18–5063) and a concentration series of 200, 100, 50, 25, 12.5, 6.25, 3.125 nM was used to determine the equilibrium dissociation constants (KD values) for RBD-SD1, RBD wild-type, RBD mutants, and S1 using 1:1 binding curve fit. For some RBD mutants that weakened binding and showed biphasic dissociation, only 30 s dissociation curves were used to fit 1:1 binding model. A concentration series of 20, 10, 5, 2.5, 1.25, 3.13, 1.56 nM was used to determine apparent KD for uncleavable S trimer using bivalent model on Octet HT software. For determining 1:1 binding affinity for S2, S2-His-tag was immobilized on Anti-Penta-His (HIS1K) biosensors (Sartorius; Cat #18–5120), and a concentration series of S2 binding mAb Fab at 200, 100, 50, 25, 12.5, 6.25, 3.125 nM was used.
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