Three RBD-Fc fusion proteins including wild-type RBD-Prototype, RBD-Delta, RBD-Omicron were used to detect the binding to ACE2 receptor in the absence or presence of sera, respectively. In brief, dissolving the RBD-Fc protein at 0.4 μg/ml in PBS supplemented with 1% BSA (BPBS). Mice sera with serially dilution were added to the RBD-Fc protein solution and incubated at room temperature for 30 min. Then, added the mixture to the 293T/ACE2 cells and incubated at room temperature. After 30 min, cells were washed three times with BPBS to remove unbound proteins. Then, added the PE-conjugated anti-human IgG Fc antibodies (BioLegend, USA) at 1:100 to stain at 4 °C for 30 min. The cells were detected by the NovoCyte Flow Cytometer (ACEA Biosciences) and the binding assay was analyzed with NovoExpress 1.4.1 software.
Pe conjugated anti human igg fc antibody
Manufactured by BioLegend
Sourced in United States
The PE-conjugated anti-human IgG Fc antibody is a reagent used for the detection and measurement of human IgG Fc in various immunoassays. It binds specifically to the Fc region of human IgG, and the PE (phycoerythrin) fluorescent label allows for the visualization and quantification of bound IgG.
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7 protocols using pe conjugated anti human igg fc antibody
1
SARS-CoV-2 RBD-Fc Binding Assay
2
RBD-ACE2 Binding Inhibition Assay
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In the experiment blocking the binding of RBD to ACE2, we used three RBD-Fc proteins, RBD-WT, RBD-Delta, and RBD-Omicron. The 35-day sera of each group were added to 0.3 μg/mL RBD-Fc protein in BPBS (0.1% BSA in PBS) at a 1:270 dilution and incubated for 30 min at room temperature. RBD-Fc with or without sera was subsequently added to ACE2-expressing HEK293T cells (293T/ACE2, constructed by our group58 (link), 5 × 105/mL) and incubated for 30 min at 4 °C. After the cells were washed with ice-cold PBS three times, PE-conjugated anti-human IgG Fc antibodies (BioLegend, USA) were added at 1:100 and stained for 30 min at 4 °C. Cells were harvested and washed three times with ice-cold PBS. The binding of RBD to ACE2 was detected with FCM.
3
Prostate Cancer Cell Binding and Internalization Assay
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C4-2, DU-145, PC-3, LNCaP, and PC-3-PSMA+ were harvested, washed, and re-suspended in FACS buffer (BD, CA, USA) at the density of 5×105-1×106 cells/mL. For binding assays, cells were stained with 200μL 50nM PSMAb (positive control) or PSP for 30 min at 4°C in darkness, followed by washing and staining with 100 μL PE-conjugated anti-human IgG Fc antibody (HP6017, Biolegend, CA, USA) for 30 min at 4°C in the dark. For PSP internalization assay, PE-PSMAb and PE-PSP conjugate was produced by incubating PSMAb and PSP with PE-labeled-anti-IgG-Fc 2nd antibody at the ratio of 1:1 for 1h at 4 °C. Then, the bound antibody was removed by adding trypsin to C4-2/PC-3 cells incubated PE-PSMAb or PE-PSP at 4°C (negative control) or 37°C for 1 h. After washing with FACS buffer, cells were analyzed by FC (Beckman Coulter, Brea, CA, USA). For the PSPS internalization assay, C4-2/PC-3 were incubated with 100nM PSPS or IgG-sulfo-SMCC-protamine (IgG-P)-FAM-siRNA (negative control) for 3 h at 37°C. C4-2/PC-3 cells transfected with FAM-siRNA using Lipofectamine 2000 was applied as a positive control. After washing with FACS buffer, cells were analyzed by Beckman Coulter.
4
SARS-CoV-2 Spike and Nucleocapsid Protein Production
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The recombinant SARS-CoV-2 Spike protein S1 (RBD, His Tag) containing 319–541 amino acids of the Spike protein was recombinantly produced in Human Embryonic Kidney cells (Innovative Research, Inc, MI). The SARS-CoV-2 nucleocapsid (N) protein (PMC 827, NP2) was produced using suspension cells (ProMab Biotechnologies Inc, CA). Anti-SARS-CoV-2 Spike S1 Antibody (human chimeric, IgG isotype) was purchased from GenScript Biotech Corporation (Piscataway, NJ). PE-conjugated anti-human IgG Fc antibody was purchased from BioLegend (San Diego, CA). MagPlex Microsphere (magnetic beads) were purchased from Luminex (Austin, TX).
5
Cell-cell association assay for bispecific antibodies
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Cells were incubated in the presence of serially diluted antibodies for 1 hour at room temperature. Subsequently, the cells were washed twice in PBS buffer (PBS+2%FBS+ 2 mM EDTA) and stained for 25 minutes with PE-conjugated anti-human IgG Fc antibody (HP6017, Biolegend, San Diego, USA) diluted in 1:100 into PBS buffer. The bound antibodies were measured using flow cytometry.
To determine the cell-to-cell association mediated by Y111, CFSE-stained H1975 cells were co-cultured with PKH26-labeled Jurkat cells at a ratio of 1:1 with specified concentrations of the Y111 or CD3 Isotype for 1-hour in a 96-well-plate. The samples were measured with a FACSelesta instrument (BD, San Jose, USA) and analyzed with FlowJo software (BD, San Jose, USA). Co-binding% of two cells mediated by bispecific antibodies was indicated as the percentages of both CFSE and PKH26 double-positive cells among the total cells.
To determine the cell-to-cell association mediated by Y111, CFSE-stained H1975 cells were co-cultured with PKH26-labeled Jurkat cells at a ratio of 1:1 with specified concentrations of the Y111 or CD3 Isotype for 1-hour in a 96-well-plate. The samples were measured with a FACSelesta instrument (BD, San Jose, USA) and analyzed with FlowJo software (BD, San Jose, USA). Co-binding% of two cells mediated by bispecific antibodies was indicated as the percentages of both CFSE and PKH26 double-positive cells among the total cells.
6
HER3 Expression Analysis in Tumor Cells
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Tumor cells were digested with trypsin and counted to ensure that the viability was > 90.0% by trypan blue staining. The harvested cells were washed twice with FACS staining buffer (Thermo Fisher Scientific, Carlsbad, CA 00-4222) and stained with 100 nM anti-HER3 antibody or human IgG1 isotype for 60 min at 4 °C. The cells were washed twice with FACS staining buffer and stained with a PE-conjugated anti-human IgG Fc antibody (1:500, BioLegend, San Diego, CA. 410,708) for 30 min at 4 °C. Fluorescence data were collected using a Fortessa X20 flow cytometer (Becton and Dickinson Company) and analyzed using FlowJo (BD Biosciences, San Jose, CA, USA).
7
SARS-CoV-2 Antibody Profiling in B Cells
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EasySepTM Human B Cell Isolation Kit (Stemcell, 17954) was used to negatively select B cells. Then 1 × 106 B cells were stained with 5 μL PE conjugated anti-human IgG Fc antibody (BioLegend, 410708), 5 μL PerCP/Cyanine5.5 conjugated anti-human IgM antibody (BioLeged, 314512), 5 μL PerCP/Cyanine5.5 conjugated anti-human IgD antibody (BioLeged, 307810) and AF488 conjugated antigens, which consist of a mixture of RBD monomer, RBD dimer and spike proteins of SARS-CoV-2 (prototype, HB-01 strain) that were labeled by Alexa Fluor® 488. Target cells (IgM–IgD–IgG+ antigen+) were sorted by cell sorter (SONY, sh800s) to V-bottom plates containing lysis buffer.
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