FcγRIIa (131R), FcγRIIb, FcγRIIIa (158V), FcγRIIIa (158F) or FcγRIIIb (R&D Systems) was immobilized through standard amine coupling chemistry to a BiaCore CM5 chip (GE Healthcare Bio-Sciences). The Kd was determined using the steady state affinity model. The binding data generated from the assay run was used to determine the relative binding activity of the test samples in comparison to the reference standard by dividing the Kd of the sample by that of the reference standard.
Fcγriib
Manufactured by R&D Systems
Sourced in United States
FcγRIIb is a cell surface receptor that binds to the Fc region of immunoglobulins. It plays a role in the regulation of the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Fcγriiib, by R&D Systems (2 mentions)
Fcγriia, by R&D Systems (2 mentions)
Biacore cm5 chip, by GE Healthcare (1 mentions)
Ni nta biosensor, by Molecular Devices (1 mentions)
Streptavidin biosensor, by Molecular Devices (1 mentions)
Octet k2 system, by Molecular Devices (1 mentions)
Fcγriiia, by R&D Systems (1 mentions)
Human ige, by Abcam (1 mentions)
Octet red384, by Molecular Devices (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence procedure, by Bio-Rad (1 mentions)
Nickel plates, by Qiagen (1 mentions)
Fcγri, by R&D Systems (1 mentions)
5 protocols using fcγriib
1
FcγR Binding Kinetics Analysis
2
Investigating IgE-FcεRI Disruption by IgE TRAP and Omalizumab
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To assess the ability of IgETRAP and omalizumab to disrupt IgE-FcεRI complexes, His-tagged human FcεRI (SinoBiological, China; 5 μg mL−1) was immobilized on Ni-NTA biosensors (Pall ForteBio, USA) for 600 s before human IgE (Abcam, UK; 100 nM) was allowed to bind sensor-immobilized FcεRI for 120 s. IgETRAP- and omalizumab-mediated dissociation of the IgE-FcεRI complexes was then measured for 600 s. To determine the binding potential of IgETRAP and omalizumab to IgG receptors, recombinant human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB (R&D Systems, USA; 5 μg mL−1) in 10 mM acetate buffer (pH 5) was immobilized on activated AR2G biosensors for 300 s. The binding kinetics of IgETRAP and omalizumab to the sensor-immobilized IgG receptors were measured for 300 s. To assess Interaction of IgETRAP and omaizumab to C1q protein, biotinylated IgETRAP and omalizumab (10 μg mL−1) were immobilized on streptavidin biosensors (Pall ForteBio, USA) and binding kinetics of C1q protein were analyzed45 (link). All experiments were performed at 30 °C with the sample plate shaker speed set at 1000 rpm using an Octet RED384 or Octet K2 system (Pall ForteBio, USA).
3
Pg-induced Osteoclast Protein Expression
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Osteoclasts cultured for 10 days were treated with formalin‐fixed Pg or opsonized (PgLO/PgHI) for 48 hours. Whole cell lysates were prepared in RIPA buffer containing protease inhibitor cocktail following brief sonication (30 sec pulse, ×2). Equal protein concentrations were electrophoresed on 8%‐10% SDS‐polyacrylamide denaturing gels. After transfer to Immobilon‐P transfer membranes (Millipore, MA), blocked for nonspecific binding by incubating in 5% milk for 1 hour and with specific antibodies overnight at 4°C, over gentle agitation. Incubation in secondary antibodies was done for 1 hour in 5% milk before detection with an enhanced chemiluminescence procedure (Bio‐Rad, CA). Western blots were scanned and quantified by densitometry using ImageJ. The primary antibodies were used at: TLR2 (R and D Systems Cat# AF2616, RRID:http://scicrunch.org/resolver/AB_416645 ), TLR4 (R and D Systems Cat# AF1478, RRID:http://scicrunch.org/resolver/AB_354816 ) @ 1/2500 (R and D Systems, IN), FcγR IIB (R and D Systems Cat# AF1330, RRID:http://scicrunch.org/resolver/AB_354737 ), FcγR III (R and D Systems Cat# AF1597, RRID:http://scicrunch.org/resolver/AB_354882 ) @ 1/5000 and β‐Actin @ 1/10,000 (Santa Cruz Biotechnology Cat# sc‐47778 HRP, RRID:http://scicrunch.org/resolver/AB_2714189 ). The secondary antibodies (Santa Cruz, TX) were used @ 1/10 000 dilution.
4
IgG Variant Binding to Complement and Fc Receptors
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The IgG variants were tested for their binding to the human complement C1q and several human Fc receptors by ELISA: C1q complement (Calbiochem), FcγRIIIA-V158 (R&D system), FcRn-p3, FcγRIIA-R131 (R&D system), and FcγRIIB (R&D system). ELISA tests were performed in PBS for all effector molecules except for FcRn, which was realized in P6. Maxisorp immunoplates were coated with 0.5 μg C1q complement/well in PBS, 0.1 μg FcγRIIIA-V158/well in PBS, or 0.1 μg FcRn-p3/well in P6. Immobilizer nickel chelate plates (Nunc) were coated with 0.1 μg FcγRIIA-R131/well or 0.4 μg FcγRIIB/well in 0.01 M KCl. After coating overnight at 4°C, plates were washed two times with PBS/0.05% Tween-20 (or P6/0.05% Tween-20 for FcRn) and saturated with PBS/4% BSA (or P6/4% BSA for FcRn) for 2 h at 37°C. In parallel, supernatants were diluted in PBS to a final IgG concentration of 0.5 μg/ml (or diluted in P6 to 0.3 μg/ml for the FcRn-binding test) and mixed with HRP F(ab′)2 goat anti-human F(ab′)2 at the same concentration for 2 h at room temperature. F(ab′)2-aggregated IgGs were then incubated under gentle agitation for 1 h at 30°C on the saturated ELISA plates without dilution for C1q, FcγRIIA-R131, and FcγRIIB (i.e., IgGs at 0.5 μg/ml), diluted in PBS to 0.25 μg/ml for FcγRIIIA-V158 or diluted in P6 to 0.0375 μg/ml for FcRn-p3. Plates were then revealed with TMB (Pierce) and absorbance read at 450 nm.
5
FcγR Binding Assay by ELISA
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Antibody binding to FcγR was measured by ELISA as described previously (55 (link)). Briefly, FcγRI, FcγRIIa, FcγRIIb and FcγRIIIa His6-tagged receptors (R&D Systems Minneapolis, MN) were coated on nickel plates (Qiagen) at 2 μg/ml or 4 μg/ml. Five-fold serial dilutions starting at 5 μg/ml of bNAbs were added. Binding was detected by a goat Anti-Human IgG (Fab specific) antibody goat at 1 in 10,000 (Sigma). Results were visualized with tetramethylbenzidine (TMB).
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