method. The pre-filtered (0.45 μm) supernatants were loaded
in manual mode (for volumes >50 mL) onto a 1 mL
pre-packed protein A cartridge. Following washing, the antibodies were eluted by pH shift and directly loaded onto a
10 mL P6 cartridge for desalting. The protein fraction was
stored at 4
C for further analysis
3.6 Biolayer
Interferometry
Protocols were set up using the Acquisition and Analysis User
Guide for the OctetRed 96 (Fortebio, Pall) and according to the
methods described in detail in [19].
1. Purified pro-KLK7 samples were activated for 16 h at 4
C
using 0.02 U/g bovine Enterokinase (Sigma).
2. The activated KLK7 was biotinylated using 1 mM EZ-link
Sulfo-NHS-LC-LC-Biotin (Thermo Fisher). The protein was
cross-linked in at molar ratio of 3:1 (linker: KLK7) for 30 min
at room temperature.
3. Non-reacted biotinylating reagent was removed by gel filtration in a Zeba spin desalting column (Thermo Fisher) according to the manufacturer’s protocol. The biotinylated KLK7 was
diluted to 12.5 μg/mL. Antibodies were diluted in a range of
concentration from 1 to 500 nM. The sample plate was
prepared as described in Fig 6. Each well was filled with
200 μL of buffer or sample.
4. Prepare a plate with eight streptavidin biosensors in row 1 and
preincubate the tips in 200 μL PBS for 10 min. Start the
acquisition software (8.2) of the OctetRed 96 and set up and
kinetic experiment with regeneration of the tips according to
the Octet Data Acquisition User Guide. Detailed protocols are
described by Kamaraswamy and Tobias [19].
5. The setup for the biolayer interferometric kinetic measurements is described in detail in Table 1. Preincubated streptavidin biosensors were equilibrated for 60 s in PBS before the
biotine-KLK7 was immobilized on the sensor for
300 s. Baseline, association, and dissociation to the antibody
were monitored over time as shown in Table 1. The tips were
subsequently regenerated by a threefold cycle of regeneration
in 10 mM 1Â glycine buffer, pH 1.5 followed by neutralization
in PBS. The tips were reused to measure the remaining
KLK7—anti-KLK7 antibody kinetics.
6. The data were processed using the Octet Data Analysis software package (8.2). The association step was aligned to the
baseline. The relative signal of antibody binding to the immobilized KLK7 is shown in Fig 7. Make sure that the antibody
does not show nonspecific binding to the tips by monitoring
the signal of a tip which is not charged with biotinylated KLK7.
Expression of Recombinant Antibodies in Different Formats
71
in manual mode (for volumes >50 mL) onto a 1 mL
pre-packed protein A cartridge. Following washing, the antibodies were eluted by pH shift and directly loaded onto a
10 mL P6 cartridge for desalting. The protein fraction was
stored at 4
C for further analysis
3.6 Biolayer
Interferometry
Protocols were set up using the Acquisition and Analysis User
Guide for the OctetRed 96 (Fortebio, Pall) and according to the
methods described in detail in [19].
1. Purified pro-KLK7 samples were activated for 16 h at 4
C
using 0.02 U/g bovine Enterokinase (Sigma).
2. The activated KLK7 was biotinylated using 1 mM EZ-link
Sulfo-NHS-LC-LC-Biotin (Thermo Fisher). The protein was
cross-linked in at molar ratio of 3:1 (linker: KLK7) for 30 min
at room temperature.
3. Non-reacted biotinylating reagent was removed by gel filtration in a Zeba spin desalting column (Thermo Fisher) according to the manufacturer’s protocol. The biotinylated KLK7 was
diluted to 12.5 μg/mL. Antibodies were diluted in a range of
concentration from 1 to 500 nM. The sample plate was
prepared as described in Fig 6. Each well was filled with
200 μL of buffer or sample.
4. Prepare a plate with eight streptavidin biosensors in row 1 and
preincubate the tips in 200 μL PBS for 10 min. Start the
acquisition software (8.2) of the OctetRed 96 and set up and
kinetic experiment with regeneration of the tips according to
the Octet Data Acquisition User Guide. Detailed protocols are
described by Kamaraswamy and Tobias [19].
5. The setup for the biolayer interferometric kinetic measurements is described in detail in Table 1. Preincubated streptavidin biosensors were equilibrated for 60 s in PBS before the
biotine-KLK7 was immobilized on the sensor for
300 s. Baseline, association, and dissociation to the antibody
were monitored over time as shown in Table 1. The tips were
subsequently regenerated by a threefold cycle of regeneration
in 10 mM 1Â glycine buffer, pH 1.5 followed by neutralization
in PBS. The tips were reused to measure the remaining
KLK7—anti-KLK7 antibody kinetics.
6. The data were processed using the Octet Data Analysis software package (8.2). The association step was aligned to the
baseline. The relative signal of antibody binding to the immobilized KLK7 is shown in Fig 7. Make sure that the antibody
does not show nonspecific binding to the tips by monitoring
the signal of a tip which is not charged with biotinylated KLK7.
Expression of Recombinant Antibodies in Different Formats
71