TITLE: Antibody characterizations by BioLayer Interferometry (BLI)
AUTHORS: Rabiatul Adawiyah, Patricia Ng, Bei Wang, Cheng-I Wang

[STEPS]
?. [ACE2 competition assay by BioLayer Interferometry (BLI)]
ACE2 competition assay by BioLayer Interferometry (BLI)
?. [Epitope binning by BioLayer Interferometry (BLI)]
Epitope binning by BioLayer Interferometry (BLI)
?. [Fab affinity measurement by BioLayer Interferometry (BLI)]
Fab affinity measurement by BioLayer Interferometry (BLI)
?. [Avidity binding by BioLayer Interferometry (BLI)]
Avidity Binding by BioLayer Interefometry
?. [Avidity binding by BioLayer Interferometry (BLI)]
Hydrate each biosensor with 200 µl of kinetics buffer in the sensor tray for at least 15-20 min prior to every run. For each avidity measurement, run a reference sensor in parallel.
?. [Avidity binding by BioLayer Interferometry (BLI)]
Set up the sample plate with each well containing 200 µl of reagent as described below:o Column 1: Buffer – Kinetics buffero Column 2: Loading – 1 µg/ml of Fc-RBD or Fc-RBD mutant diluted in kinetics buffero Column 3: Loading – Kinetics buffer (for ref sensor loading)o Column 4: Wash – Kinetics buffero Column 5: Quench – 0.25 mg/ml of Human Fco Column 6: Wash/Dissociation – Kinetics buffero Column 7: Association Sample – Purified IgG clone in two-fold dilutions starting from 25 nM, diluted in kinetics buffer. Ensure that the last well in row H contains 0 nM (ie. Kinetics buffer only) to serve as the reference well.o Column 11: Regeneration – 10 mM Glycine pH 2.3o Column 12: Neutralization – PBST 0.1%
?. [Avidity binding by BioLayer Interferometry (BLI)]
The assay protocol is defined in the data acquisition software as below. Replicate steps as “new assay” for the reference sensor, taking note to change the loading sample plate column. Plate temperature and shaking speed is set to 25°C and 1000 rpm respectively.
?. [Avidity binding by BioLayer Interferometry (BLI)]
Run the assay according to the protocol set. Each avidity measurement (including reference) should take about 1 hr 40 min.
?. [Avidity binding by BioLayer Interferometry (BLI)]
Once completed, process the data according to the following parameters in the analysis software (v. 9.0.0.4):o Step 1: Data Selection – Sensor selection. Select Sample plate row H as reference well and Sensor tray column 2 as reference sensor.o Step 2: Subtraction – Select “Double reference” to subtract both reference well and reference sensor.o Step 3: Align Y-axis to “Baseline” and set time range as the last 10 secs of the Baseline step before association.o Step 4: Inter-step correction to remove any bulk shifts or artefacts for a better line fit.o Step 5: Process data with Savitzky-Golay Filter.oAfter processing, calculate KD based on the 1:2 bivalent model fitting.
?. [Avidity binding by BioLayer Interferometry (BLI)]
Repeat measurement for each IgG clone and RBD mutant. Regenerated sensors can be used for another 1 or 2 more times, depending on whether the signal reached baseline levels after regeneration and whether loading signal with 1 µg/ml Fc-RBD remains consistent with previous runs. Otherwise, use fresh sensors.
?. [ACE2 competition assay by BioLayer Interferometry (BLI)]
To immobilize ACE2-Fc onto AR2G sensors, first hydrate each biosensor with 200 µl of water in the sensor tray for at least 15-20 min prior to the run.
?. [ACE2 competition assay by BioLayer Interferometry (BLI)]
Prepare EDC:NHS activation buffer (20 mM EDC + 10 mM NHS in water). Note that, after mixing, the activation buffer will only be viable for about 1 hour. It is best to prepare them fresh right before starting the run.
?. [ACE2 competition assay by BioLayer Interferometry (BLI)]
Set up the sample plate with each well containing 200 µl of reagent as described below:o Column 1: Buffer – Watero Column 2: Activation – EDC:NHS buffero Column 3: Loading – 10 µg/ml ACE2-Fc in 10mM sodium acetate pH 4.0o Column 4: Quench – 1M Ethanolamine pH 8.5o Column 5: Buffer – Kinetics buffero Column 6: Association 1 – 5 µg/ml Tag-less RBD, prepared in kinetics buffero Column 7: Association 2 – Panel of IgG clones, 10 µg/ml each, prepared in kinetics buffero Column 9: Association 1 – Kinetics buffer (This serves as a control to ensure there is no cross-reactivity between ACE2 and the antibodies in the absence of antigen).o Column 10: Loading – Kinetics buffero Column 11: Regeneration – 10 mM Glycine pH 2.3o Column 12: Neutralization – PBST 0.1%
?. [ACE2 competition assay by BioLayer Interferometry (BLI)]
Set up the assay definition as below. Plate temperature and shaking speed is set to 25°C and 1000 rpm respectively. Replicate steps of Cycle 1 and “append to current assay” to use the same sensor in Cycle 2 as reference to check for any non-specific binding.
?. [ACE2 competition assay by BioLayer Interferometry (BLI)]
Run the assay according to the protocol set.
?. [ACE2 competition assay by BioLayer Interferometry (BLI)]
After the run is complete, extract and process the raw data:o Step 1: Find the timepoints of interest to select data for analysis.o Step 2:  Normalize Cycle 1 data (set Y-value to 0).o Step 3: Normalize Cycle 2 data (set Y-value to 0).o Step 4: Subtract normalized Cycle 2 data from normalized Cycle 1 data.o Step 5: Plot subtracted data on Prism.
?. [Epitope binning by BioLayer Interferometry (BLI)]
Epitope binning consists of first immobilizing antibody 5A6 onto AR2G biosensors, followed by a classical sandwich assay with consecutive associations to target RBD and a panel of second antibodies. To start, hydrate each biosensor with 200 µl of water in the sensor tray for at least 15-20 min prior to the immobilization run.
?. [Epitope binning by BioLayer Interferometry (BLI)]
Prepare EDC:NHS activation buffer (20 mM EDC + 10 mM NHS in water). Note that, after mixing, the activation buffer will only be viable for about 1 hour. Thus, it is best to mix them right before starting the run.
?. [Epitope binning by BioLayer Interferometry (BLI)]
Set up the sample plate with each well containing 200 µl of reagent as described below:o Column 1: Buffer – Watero Column 2: Activation – EDC:NHS buffero Column 3: Loading – 7.5 µg/ml 5A6 antibody in 10mM sodium acetate pH 6.0o Column 4: Quench – 1M Ethanolamine pH 8.5o Column 5: Buffer – Kinetics buffero Column 6: Association 1 – 5 µg/ml Tag-less RBD, prepared in kinetics buffero Column 7: Association 2 – Panel of IgG clones, 10 µg/ml each, prepared in kinetics buffer. Include 5A6 and at least one irrelevant IgG as controls to ensure self-blocking (i.e. 5A6-5A6 sandwich should not have any binding signal) and no non-specific binding to irrelevant proteins.o Column 9: Association 1 – Kinetics buffer (This serves as another control to ensure there is no cross-reactivity between antibodies in the absence of antigen).o Column 10: Loading – Kinetics buffero Column 11: Regeneration – 10 mM Glycine pH 2.3o Column 12: Neutralization – PBST 0.1%
?. [Epitope binning by BioLayer Interferometry (BLI)]
Set up the assay protocol for the immobilization run as below. Plate temperature and shaking speed is set to 25°C and 1000 rpm respectively.
?. [Epitope binning by BioLayer Interferometry (BLI)]
Run the assay according to the protocol set. Immobilization of 5A6 IgG should take about 20 min.
?. [Epitope binning by BioLayer Interferometry (BLI)]
Once immobilization is successful, prepare to run the sandwich assay according to the assay definition below. Replicate steps of Cycle 1 and “append to current assay” to use the same 5A6-immobilized sensor in Cycle 2 as reference to check for any non-specific binding. Repeat cycles in the sandwich assay as necessary to screen against the full panel of IgGs.
?. [Epitope binning by BioLayer Interferometry (BLI)]
After the run is complete, extract and process the raw data:o Step 1: Find the time points of interest to select data for analysis.o Step 2: Normalize Cycle 1 data (set Y-value to 0).o Step 3: Normalize Cycle 2 data (set Y-value to 0).o Step 4: Subtract normalized Cycle 2 data from normalized Cycle 1 data.o Step 5: Plot subtracted data on Prism.
?. [Fab affinity measurement by BioLayer Interferometry (BLI)]
Hydrate each biosensor with 200 µl of kinetics buffer in the sensor tray for at least 15-20 min prior to every run. For each Fab affinity measurement, run a reference sensor in parallel.
?. [Fab affinity measurement by BioLayer Interferometry (BLI)]
Set up the sample plate with each well containing 200 µl of reagent as described below:o Column 1: Buffer – Kinetics buffero Column 2: Loading – 1 µg/ml of Fc-RBD diluted in kinetics buffero Column 3: Loading – Kinetics buffer (for ref sensor loading)o Column 4: Wash/Dissociation – Kinetics buffero Column 5: Association Sample – Purified Fab clone in two-fold dilutions starting from 100 nM, diluted in kinetics buffer. Ensure that the last well in row H contains 0 nM (i.e. Kinetics buffer only) to serve as the reference well.o Column 11: Regeneration – 10 mM Glycine pH 2.3o Column 12: Neutralization – PBST 0.1%
?. [Fab affinity measurement by BioLayer Interferometry (BLI)]
The assay protocol is defined in the data acquisition software as below. Replicate steps as “new assay” for the reference sensor, taking note to change the loading sample plate column. Plate temperature and shaking speed is set to 25°C and 1000 rpm respectively.
?. [Fab affinity measurement by BioLayer Interferometry (BLI)]
Run the assay according to the protocol set. Each Fab affinity measurement (including reference) should take about an hour.
?. [Fab affinity measurement by BioLayer Interferometry (BLI)]
Once completed, process the data according to the following parameters in the analysis software (v. 9.0.0.4):o Step 1: Data Selection – Sensor selection. Select Sample plate row H as reference well and Sensor tray column 2 as reference sensor.o Step 2: Subtraction – Select “Double reference” to subtract both reference well and reference sensor.o Step 3: Align Y-axis to “Baseline” and set time range as the last 10 secs of the Baseline step before association.o Step 4: Inter-step correction to remove any bulk shifts or artefacts for a better line fit.o Step 5: Process data with Savitzky-Golay Filter.o After processing, calculate KD based on the 1:1 grouped fitting.
?. [Fab affinity measurement by BioLayer Interferometry (BLI)]
Repeat measurement for each subsequent Fab clones. Regenerated sensors can be used for another 1 or 2 more times, depending on whether the signal reached baseline levels after regeneration and whether loading signal with 1 µg/ml Fc-RBD remains consistent with previous runs. Otherwise, use fresh sensors.