Monoclonal antibodies binding data for SARS-CoV-2 proteins
Description
Figure 1: Illustrate the schematic workflow of antibodies generational and characterizations. Figure 2: Describe the IgG binding of the synthetic antibodies. Figure 3: Binding kinetics of the antibodies. To estimate further the binding kinetics and specificity of IgG formatted IgG. Figure 4 Western blot (WB) analysis of Nsp1, Nsp8 and Nsp12 antibodies with respective purified proteins. Data set for Figure 2 and Figure 3 in Graphpad prism 9.2.0
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EC50_IgGs 384-well microplates were coated overnight at 4 oC with 2 μg/mL protein or GST/MHT control proteins in PBS pH 7.4. Wells were blocked with 0.2% BSA in PBS for one hour and washed 4 times with 0.05% Tween in PBS (PT buffer). Serial diluted IgG into PBT were transferred to antigen-coated plates and incubated at room temperature for 30 min, washed with PBST four times and incubated for 30 min with anti-β-HRP antibody conjugate (1:7500 dilution in PBT). The plate was washed, developed and read as describe above. The data were fit to standard four-parameter logistic equations using GraphPad Prism (GraphPad Software, La Jolla, CA) and EC50 values were estimated. Binding Kinetics (BLI) BLI experiments were performed on an Octet HTX instrument (Sartorius) at 1000 rpm and 25 °C, were 2-20 microgram per milliliter of SARS-CoV2 antigens were covalently captured on AR2G biosensors (Sartorius, 18-5092) by amine-reactive coupling chemistry, and unbound reactive N-hydroxysulfosuccinimide (NHS) esters on the biosensors were quenched for 600 s by 1M ethanolamine (pH 8.5)[3, 4]. The biosensor probe were equilibrating with assay buffer (PBS, 1% BSA, 0.05% Tween 20), dipped for 600 s into wells containing serial 3-fold dilutions of IgGs and were transferred back into assay buffer for 600 s of dissociation. The binding response were analyzed by subtracting data from reference, and data were globally fitted with 1:1 binding model using ForteBio’s Data Analysis software 9.0. Western blot analyses To check the specificity antibodies, western blot were carried out. 0.3 μg of purified SARS-CoV-2 and Maltose Binding Protein (MBP) was loaded onto a pre-cast 15% SDS-PAGE gel including a Precision Plus Protein Dual Color Standard (Bio-Rad). After gel electrophoresis (running buffer: 25 mM Tris HCl, 193 mM glycine, 0.1% sodium dodecyl sulfate (SDS), pH 8.3), the blots were transfer and onto nitrocellulose membranes (Bio-Rad) (transfer buffer: 20% methanol, 25 mM Tris HCl, 193 mM glycine) at 4 °C 2 h. The transferred membrane was blocked in 5% skim milk for 60 min and membrane was incubated in synthetic antibody (primary antibody) for 2 h. Secondary Rabbit anti-mouse IgG HRP conjugated (Cruz Biotechnologies) was used for 1 h. The were then washed and incubated with HRP substrate (GE Healthcare Life Sciences, Pittsburgh, PA) for 1 min before recording the chemiluminescent signal using a ChemiDoc MP gel imaging system (GE).