BLI was carried out on an OctetRED384 (Pall FortéBio) using streptavidin-coated biosensors (Pall FortéBio) to immobilise PvDBPII enzymatically monobiotinylated on a C-terminal AviTag™. Assays were carried out in 96-well format in black plates (Greiner). For epitope binning studies (Figure 1E-G ), a six-step sequential assay was performed: Baseline (PBS, 30 s); Protein immobilisation (neat supernatant, 120 s); Wash (PBS, 60 s); first mAb (mAb1) binding (300 nM mAb1, 120 s); Wash (PBS, 60 s); second mAb (mAb2) binding (150 nM mAb2, 120 s). “Relative binding” in Figure 1E shows the ratio (SignalmAb2 with mAb1 bound)/(SignalmAb2 with no mAb1) where “SignalmAb2” was normalised for the amount of PvDBPII bound to the biosensor, such that “SignalmAb2” = the raw signal in “mAb2 binding” divided by the raw signal in the “Protein immobilisation” phase. The resulting “binding profile” for any given mAb corresponds to the column of “relative binding values” under that mAb in the “relative binding” table. To establish the epitope bins, binding profiles between each mAb pair were correlated using a Pearson product-moment correlation coefficient, the values of which are shown in the “binding profile correlation” matrix in Figure 1F . mAb pairs whose binding profile correlation was > 0.7 were grouped into the same epitope bin (Figure 1G ).
Black plates
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Black plates are a type of laboratory equipment used for various scientific applications. They are typically made of a durable material, such as plastic or metal, and are designed to provide a dark background for experiments or analyses. The black color of the plates helps to enhance contrast and visibility, making them useful for various types of optical and imaging experiments.
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4 protocols using black plates
1
Epitope Binning of Anti-PvDBPII Monoclonal Antibodies
2
Fragment Library Screening and Enzymatic Assay
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A fragment library from the Drug Discovery Unit (DDU, University of Dundee) was screened using an OctetRED384 (ForteBio‐Molecular Devices, Fremont, CA, USA). The protein was immobilised on hexahistidine tag high affinity sensors (ForteBio), and the experiment carried out in black plates (Greiner). We used TEV protease as a control and to identify promiscuous binders, and ATP as the control ligand. During the assay performance, we noticed a low binding signal (lower than 0.1 nm) in the presence of ATP. Selected hits were tested for enzyme inhibition using the coupled enzymatic assay at a fixed concentration of 500 μm , in the presence of 2 mm d ‐Ala. One compound with an inhibition effect greater than 20% was followed up for Ki determination.
3
Epitope Binning of Anti-PvDBPII Monoclonal Antibodies
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BLI was carried out on an OctetRED384 (Pall FortéBio) using streptavidin-coated biosensors (Pall FortéBio) to immobilise PvDBPII enzymatically monobiotinylated on a C-terminal AviTag™. Assays were carried out in 96-well format in black plates (Greiner). For epitope binning studies (Figure 1E-G ), a six-step sequential assay was performed: Baseline (PBS, 30 s); Protein immobilisation (neat supernatant, 120 s); Wash (PBS, 60 s); first mAb (mAb1) binding (300 nM mAb1, 120 s); Wash (PBS, 60 s); second mAb (mAb2) binding (150 nM mAb2, 120 s). “Relative binding” in Figure 1E shows the ratio (SignalmAb2 with mAb1 bound)/(SignalmAb2 with no mAb1) where “SignalmAb2” was normalised for the amount of PvDBPII bound to the biosensor, such that “SignalmAb2” = the raw signal in “mAb2 binding” divided by the raw signal in the “Protein immobilisation” phase. The resulting “binding profile” for any given mAb corresponds to the column of “relative binding values” under that mAb in the “relative binding” table. To establish the epitope bins, binding profiles between each mAb pair were correlated using a Pearson product-moment correlation coefficient, the values of which are shown in the “binding profile correlation” matrix in Figure 1F . mAb pairs whose binding profile correlation was > 0.7 were grouped into the same epitope bin (Figure 1G ).
4
Competitive Binding Kinetics of SARS-CoV-2 RBD
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(BLI) All BLI was carried out on an OctetRED96 (Sartorius) using Anti-Penta-HIS (HIS1K) Biosensors (Sartorius) to immobilize the recombinant native RBD with a 6×his tag. Assays were carried out in 96-well format in black plates (Greiner). For ACE2 or mAb competition studies, the experiment followed a five-step sequential assay: Baseline (PBS, 60 s); immobilization of the recombinant RBD (10 μg/mL, 120 s); Baseline2 (kinetic buffer, 60 s); mAb1 binding (100 nM mAb1 in the kinetic buffer, 250 s); ACE2 or mAb2 binding (100 nM ACE2 or mAb2 in the kinetic buffer, 250 s) in the presence of the mAb1 (100 nM in the kinetic buffer). Kinetic buffer is PBS supplemented with 0.1% BSA and 0.02% tween 20. The level of competition was estimated by the ratios between mAb1 signals in the presence over absence of ACE2 or of mAb2.
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