ELISA was used to evaluate the PD-1 binding activity as previously described32 (link). For ELISA screening, culture supernatant was used as a sample and detected with goat anti-mouse IgG Fcγ-HRP antibody (Jackson ImmunoResearch, 115-035-164) diluted 1:8,000 in 0.05% Tween-20 PBS buffer (PBST). For the mouse PD-1 binding profile, serial dilutions of purified mouse anti-PD-1 mAbs were used. For the chimeric PD-1 binding profile, serial dilutions of purified chimeric anti-PD-1 mAbs were used and detected with goat anti-human IgG Fcγ-HRP antibody (Jackson ImmunoResearch, 109-005-098) diluted 1:10,000 in PBST. The SIGMAFAST OPD (Sigma-Aldrich, P9187) substrate solution was used, and the reaction was stopped by adding 1 M H2SO4. The absorbance was measured at 492 nm by a Cytation 5 cell imaging multi-mode reader (BioTek). Mini-pools providing an A492 ELISA signal of more than 1.0 were considered high hPD-1-specific IgG level pools. These mini-pools were further screened for the anti-PD-1 neutralizing antibody (NAb) by flow cytometry.
Goat anti mouse igg fcγ hrp antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Goat anti-mouse IgG Fcγ-HRP antibody is a secondary antibody that binds to the Fc region of mouse immunoglobulin G (IgG) antibodies. This antibody is conjugated with horseradish peroxidase (HRP), which can be used for detection and visualization applications.
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2 protocols using goat anti mouse igg fcγ hrp antibody
1
PD-1 Binding Assay Protocol
2
Monoclonal Antibody PD-L1 Binding Assay
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Culture supernatant or purified monoclonal antibody (100 µl/well) was added to a cPD-L1 hu Fc-coated ELISA plate (10 ng/well) and incubated at 37 °C for 1 h and then washed three times with 0.05% Tween-20 in PBS (PBST) buffer. For the human PD-L1 cross-reactivity profile, human PD-L1 hu Fc (10 ng/well) was used as an antigen for the ELISA coating. Goat anti-mouse IgG Fcγ-HRP antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) dilution at 1:8000 in PBST (100 µl/well) was added to the assay plate and incubated at 37 °C for 1 h. The SIGMAFAST OPD (o-phenylenediamine dihydrochloride, Sigma Aldrich, Saint Louis, MO, USA) substrate solution (100 µl/well) was added to the plate and incubated at room temperature in the dark for 20 min. The reaction was stopped by adding 2 N H2SO4 (50 µl/well). The absorbance was measured at 492 nm using a Cytation 5-cell imaging multi-mode reader (BioTek, Winooski, VT, USA).
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