HEK-293F cells (Invitrogen) were transfected using BG505 gp160 pCI plasmid expression vector and lipofectamine (thermofisher) in OptiMEM as previously described (38 (link)). After 48 hours, HEK-293F were harvested and opsonized for 1 hour at 37°C with serial dilutions of the nanobody-IgG1 antibodies. After incubation, the cells were washed twice using PBS 2%FCS and subsequently stained for 30 minutes at 4°C with Goat F(ab’)2 anti-Human IgG-PE (Southernbiotech) and analyzed using flow cytometry.
Goat f ab 2 anti human igg pe
Manufactured by Southern Biotech
Goat F(ab')2 Anti-Human IgG-PE is a secondary antibody conjugate. It consists of the F(ab')2 fragment of goat antibodies specific to human IgG, labeled with the fluorescent dye Phycoerythrin (PE).
Automatically generated - may contain errors
Lab products found in correlation
Anti ha apc antibody, by BioLegend (2 mentions)
Hek293f cell, by Thermo Fisher Scientific (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Prism 9, by GraphPad (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Anti complement c3b ic3b apc antibody clone 3e7 c3b, by BioLegend (1 mentions)
96 well round bottom plate, by Corning (1 mentions)
Cd8 antibody, by BioLegend (1 mentions)
Facscelesta, by BD (1 mentions)
5 protocols using goat f ab 2 anti human igg pe
1
HEK-293F Transfection and Nanobody Binding
2
Epitope Mapping of Antibodies
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Antibodies used in epitope mapping studies were titrated against yeast displaying GP38 to adequately calculate EC50s and EC80s for each antibody. Yeast were induced to express non-mutagenized GP38 as noted above. Antibodies were titrated from 100 nM in two-fold, 12-point serial dilutions. Once an OD600 of 0.1 was achieved, the non-mutagenized GP38-expressing yeast were mixed with each antibody dilution and incubated on ice for one hour. Yeast cells were washed two times with PBSF and subsequently stained for 30 minutes on ice with a cocktail of anti-HA APC antibody (Biolegend, Clone: 16B12, dilution 1:100), goat F(ab′)2 anti-human IgG PE (SouthernBiotech, dilution 1:100), and PI (Invitrogen, 1:100 dilution). After staining, samples were run on a FACSCanto II flow cytometer (BD Biosciences). PE MFIs were plotted against antibody concentrations; EC50 and EC80 concentrations were calculated using GraphPad Prism 9.
3
Complement-mediated Platelet Activation
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Platelets (5x106) were incubated with equal volumes of anti-platelet antibody (0.001-20 µg/mL) and pooled complement-rich human serum, for 30 min. at 37°C while shaking. For the antibody combinations, the dilution series started with 10 µg/mL of both antibodies, resulting in a total antibody concentration of 20 µg/mL. The platelets were washed four times with PBS + 0.5% BSA + 5 mM EDTA and stained for 20 min. for flow cytometry with Goat F(ab')2 Anti-Human IgG-PE (1/250, Southern Biotech), anti-complement C3b/iC3b-APC Antibody Clone: 3E7/C3b (1/250, Biolegend) and C1q Polyclonal Antibody-FITC (1/25, Thermo Fisher Scientific). The samples were split and anti-C1q-FITC was measured separately due to the high fluorescence intensity of the anti-IgG-PE. The flow cytometry data were analyzed using FlowJo vX for Windows (BD Biosciences). The platelets were gated based on the FSC-A/SSC-A and single cells were selected (FSC-H/FSC-A). The geometric-mean fluorescence intensities of all parameters were calculated.
4
Screening CCHFV Gc Mutant Library
5
Flow Cytometry Antibody Screening Protocol
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All washes and dilutions of cells, antibodies, and reagents were performed using flow buffer (1X PBS, 1% BSA, 0.1% NaN3, pH 7.4). Staining was performed in a round-bottom 96-well plate (Corning) seeded at 100,000 cells/well and all incubations were performed at 4 °C or on ice. For primary and secondary screens, the cells were incubated for 30 min with pre-diluted test antibodies (secondary screen and dose-curves) or 1:5 diluted HEK 293 supernatants containing antibodies (for primary screens and diversity screens) in a total volume of 50 μL. The cells were washed twice with 200 µL flow buffer. The cells were then incubated for 30 min with detection antibody (Goat F(ab')2 Anti-Human IgG-PE, Southern Biotech) at 0.625 µg/mL in flow buffer. Following 2 more washes, the cells were resuspended in a final volume of 150 µL of flow buffer. The cells were analyzed on a BD FACSCelesta or a Guava easyCyte 8-HT flow cytometer. At least 3000 events were collected, and PE geometric mean fluorescence intensity was plotted as a fold over background (cells incubated with secondary detection antibody only). In some secondary screens involving human or cynomolgus PBMCs, an additional CD4 antibody (BioLegend) and/or CD8 antibody (BioLegend) was included to further characterize cell binding.
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