Blocking Assay:Article Title: Humanized anti-PD-1 antibody and the use thereof
Article Snippet: .. This result indicates that the murine antibody PD1-M944 has a good function of blocking the binding of PD-1 to its ligand PD-L1. (2) Blocking the Binding of Recombinant Human PD-1 to PD-L2: Recombinant human PD-1 protein at a concentration of 1 μg/mL was coated on a 96-well plate at 100 μL per well, overnight at 4° C., overnight, washed the next day and blocked at room temperature for 1 h. 100 μL of 0.5 μg/mL of biotinylated protein PD-L2-Fc-biotin (Sino Biological, Inc.) was co-incubated with different concentrations (0.5 μg/mL, 2.5 μg/mL and 12.5 μg/mL) of PD1-M944 or Nivolumab (Sino Biological, Inc.). .. The plates were washed to remove unbound antibodies, incubated with antibiotic streptavidin/HRP (Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd), repeatedly washed, and the substrate chromogenic solution was added for color development, and OD450 was detected after termination.
Article Title: Humanized anti-PD-1 antibody and the use thereof
Article Snippet: .. 1.4 Murine Antibody PD1-M944 Functional Assay (1) Blocking the Binding of Recombinant Human PD-1 to PD-L1: Recombinant human PD-1 protein at a concentration of 1 μg/mL was coated on a 96-well plate at 100 μL per well overnight at 4° C. The plates were washed the next day and blocked at room temperature for 1 h. 100 μL of 10 μg/mL of biotinylated protein PD-L1-Fc-biotin (SinoBiological, Inc.) was co-incubated with different concentrations (0.4 μg/mL, 2 μg/mL and 10 μg/mL) of PD1-M944 or Nivolumab (Sino Biological, Inc). ..
Binding Assay:Article Title: Humanized anti-PD-1 antibody and the use thereof
Article Snippet: .. This result indicates that the murine antibody PD1-M944 has a good function of blocking the binding of PD-1 to its ligand PD-L1. (2) Blocking the Binding of Recombinant Human PD-1 to PD-L2: Recombinant human PD-1 protein at a concentration of 1 μg/mL was coated on a 96-well plate at 100 μL per well, overnight at 4° C., overnight, washed the next day and blocked at room temperature for 1 h. 100 μL of 0.5 μg/mL of biotinylated protein PD-L2-Fc-biotin (Sino Biological, Inc.) was co-incubated with different concentrations (0.5 μg/mL, 2.5 μg/mL and 12.5 μg/mL) of PD1-M944 or Nivolumab (Sino Biological, Inc.). .. The plates were washed to remove unbound antibodies, incubated with antibiotic streptavidin/HRP (Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd), repeatedly washed, and the substrate chromogenic solution was added for color development, and OD450 was detected after termination.
Article Title: Humanized anti-PD-1 antibody and the use thereof
Article Snippet: .. 1.4 Murine Antibody PD1-M944 Functional Assay (1) Blocking the Binding of Recombinant Human PD-1 to PD-L1: Recombinant human PD-1 protein at a concentration of 1 μg/mL was coated on a 96-well plate at 100 μL per well overnight at 4° C. The plates were washed the next day and blocked at room temperature for 1 h. 100 μL of 10 μg/mL of biotinylated protein PD-L1-Fc-biotin (SinoBiological, Inc.) was co-incubated with different concentrations (0.4 μg/mL, 2 μg/mL and 10 μg/mL) of PD1-M944 or Nivolumab (Sino Biological, Inc). ..
Recombinant:Article Title: Humanized anti-PD-1 antibody and the use thereof
Article Snippet: .. This result indicates that the murine antibody PD1-M944 has a good function of blocking the binding of PD-1 to its ligand PD-L1. (2) Blocking the Binding of Recombinant Human PD-1 to PD-L2: Recombinant human PD-1 protein at a concentration of 1 μg/mL was coated on a 96-well plate at 100 μL per well, overnight at 4° C., overnight, washed the next day and blocked at room temperature for 1 h. 100 μL of 0.5 μg/mL of biotinylated protein PD-L2-Fc-biotin (Sino Biological, Inc.) was co-incubated with different concentrations (0.5 μg/mL, 2.5 μg/mL and 12.5 μg/mL) of PD1-M944 or Nivolumab (Sino Biological, Inc.). .. The plates were washed to remove unbound antibodies, incubated with antibiotic streptavidin/HRP (Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd), repeatedly washed, and the substrate chromogenic solution was added for color development, and OD450 was detected after termination.
Article Title: Engineering affibody domains as anti-idiotypic masks for nivolumab-based prodrugs
Article Snippet: Protein concentrations were measured by absorbance at 280 nm using a spectrophotometer (Thermo Fisher, USA). .. Recombinant Human PD-1 Protein (ECD, His Tag) was purchased from Sino Biological (China), and recombinant tobacco etch virus (TEV) protease was produced and purified in-house following standard methods. .. E. coli BL21 (DE3) cells transformed with pPALU ( ) plasmids encoding for an affibody library (~10 11 variants) were grown overnight (O/N) in Luria-Bertani (LB) medium supplemented with carbenicillin (100 μg/ml).
Article Title: Engineering affibody domains as anti-idiotypic masks for nivolumab-based prodrugs.
Article Snippet: Protein concentrations were measured by absorbance at 280 nm using a spectrophotometer (Thermo Fisher, USA). .. Recombinant Human PD-1 Protein (ECD, His Tag) was purchased from Sino Biological (China), and recombinant tobacco etch virus (TEV) protease was produced and purified inhouse following standard methods. .. D ow nloaded from https://academ ic.oup.com /peds/advance-article/doi/10.1093/protein/gzag010/8655119 by guest on 18 April 2026 UN CO RR EC TE D MA NU SC RI PT Bacterial culture and library expression Escherichia coli BL21 (DE3) cells transformed with pPALU9 plasmids encoding for an affibody library (~1011 variants) were grown overnight (O/N) in Luria-Bertani (LB) medium supplemented with carbenicillin (100 μg/mL).
Article Title: Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors
Article Snippet: .. For the detection of AAV-encoded nivolumab, plate coating was performed with 100 ng/well using recombinant human PD-1 protein (Sino Biological) in H 2 O overnight, while the remaining procedure was identical, only varying in secondary antibody concentration and incubation time (αhuFc-HRP 1:10,000 for 3 h and 1:500 for 2 h for murine αPD-1 and AAV-Nivolumab, respectively). .. Standard values were generated using the therapeutic drug Opdivo® (Bristol Myers Squibb).
Article Title: Humanized anti-PD-1 antibody and the use thereof
Article Snippet: .. 1.4 Murine Antibody PD1-M944 Functional Assay (1) Blocking the Binding of Recombinant Human PD-1 to PD-L1: Recombinant human PD-1 protein at a concentration of 1 μg/mL was coated on a 96-well plate at 100 μL per well overnight at 4° C. The plates were washed the next day and blocked at room temperature for 1 h. 100 μL of 10 μg/mL of biotinylated protein PD-L1-Fc-biotin (SinoBiological, Inc.) was co-incubated with different concentrations (0.4 μg/mL, 2 μg/mL and 10 μg/mL) of PD1-M944 or Nivolumab (Sino Biological, Inc). ..
Article Title: PD-L1 targeted peptide demonstrates potent antitumor and immunomodulatory activity in cancer immunotherapy
Article Snippet: .. Human PD-L1 natural ORF mammalian expression plasmid (Cat: HG10084-UT), rabbit anti-human PD-1 antibody (Clone D67, Cat: 10377-RD67), recombinant human PD-L1 protein (Cat: 10084-H08H), human PD-L1/B7-H1/CD274 protein (Cat: 10084-HNAH), biotinylated mouse PD-1/PDCD1 protein (His tag) and recombinant human PD-1 protein (Cat: 10377-H08H) were purchased from Sino Biological Inc (Beijing). .. GM-CSF (Cat: 315-03) and Murine TNF-α (Cat: 315-01A) were purchased from PEPROTECH.
Concentration Assay:Article Title: Humanized anti-PD-1 antibody and the use thereof
Article Snippet: .. This result indicates that the murine antibody PD1-M944 has a good function of blocking the binding of PD-1 to its ligand PD-L1. (2) Blocking the Binding of Recombinant Human PD-1 to PD-L2: Recombinant human PD-1 protein at a concentration of 1 μg/mL was coated on a 96-well plate at 100 μL per well, overnight at 4° C., overnight, washed the next day and blocked at room temperature for 1 h. 100 μL of 0.5 μg/mL of biotinylated protein PD-L2-Fc-biotin (Sino Biological, Inc.) was co-incubated with different concentrations (0.5 μg/mL, 2.5 μg/mL and 12.5 μg/mL) of PD1-M944 or Nivolumab (Sino Biological, Inc.). .. The plates were washed to remove unbound antibodies, incubated with antibiotic streptavidin/HRP (Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd), repeatedly washed, and the substrate chromogenic solution was added for color development, and OD450 was detected after termination.
Article Title: Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors
Article Snippet: .. For the detection of AAV-encoded nivolumab, plate coating was performed with 100 ng/well using recombinant human PD-1 protein (Sino Biological) in H 2 O overnight, while the remaining procedure was identical, only varying in secondary antibody concentration and incubation time (αhuFc-HRP 1:10,000 for 3 h and 1:500 for 2 h for murine αPD-1 and AAV-Nivolumab, respectively). .. Standard values were generated using the therapeutic drug Opdivo® (Bristol Myers Squibb).
Article Title: Humanized anti-PD-1 antibody and the use thereof
Article Snippet: .. 1.4 Murine Antibody PD1-M944 Functional Assay (1) Blocking the Binding of Recombinant Human PD-1 to PD-L1: Recombinant human PD-1 protein at a concentration of 1 μg/mL was coated on a 96-well plate at 100 μL per well overnight at 4° C. The plates were washed the next day and blocked at room temperature for 1 h. 100 μL of 10 μg/mL of biotinylated protein PD-L1-Fc-biotin (SinoBiological, Inc.) was co-incubated with different concentrations (0.4 μg/mL, 2 μg/mL and 10 μg/mL) of PD1-M944 or Nivolumab (Sino Biological, Inc). ..
Virus:Article Title: Engineering affibody domains as anti-idiotypic masks for nivolumab-based prodrugs
Article Snippet: Protein concentrations were measured by absorbance at 280 nm using a spectrophotometer (Thermo Fisher, USA). .. Recombinant Human PD-1 Protein (ECD, His Tag) was purchased from Sino Biological (China), and recombinant tobacco etch virus (TEV) protease was produced and purified in-house following standard methods. .. E. coli BL21 (DE3) cells transformed with pPALU ( ) plasmids encoding for an affibody library (~10 11 variants) were grown overnight (O/N) in Luria-Bertani (LB) medium supplemented with carbenicillin (100 μg/ml).
Article Title: Engineering affibody domains as anti-idiotypic masks for nivolumab-based prodrugs.
Article Snippet: Protein concentrations were measured by absorbance at 280 nm using a spectrophotometer (Thermo Fisher, USA). .. Recombinant Human PD-1 Protein (ECD, His Tag) was purchased from Sino Biological (China), and recombinant tobacco etch virus (TEV) protease was produced and purified inhouse following standard methods. .. D ow nloaded from https://academ ic.oup.com /peds/advance-article/doi/10.1093/protein/gzag010/8655119 by guest on 18 April 2026 UN CO RR EC TE D MA NU SC RI PT Bacterial culture and library expression Escherichia coli BL21 (DE3) cells transformed with pPALU9 plasmids encoding for an affibody library (~1011 variants) were grown overnight (O/N) in Luria-Bertani (LB) medium supplemented with carbenicillin (100 μg/mL).
Produced:Article Title: Engineering affibody domains as anti-idiotypic masks for nivolumab-based prodrugs
Article Snippet: Protein concentrations were measured by absorbance at 280 nm using a spectrophotometer (Thermo Fisher, USA). .. Recombinant Human PD-1 Protein (ECD, His Tag) was purchased from Sino Biological (China), and recombinant tobacco etch virus (TEV) protease was produced and purified in-house following standard methods. .. E. coli BL21 (DE3) cells transformed with pPALU ( ) plasmids encoding for an affibody library (~10 11 variants) were grown overnight (O/N) in Luria-Bertani (LB) medium supplemented with carbenicillin (100 μg/ml).
Article Title: Engineering affibody domains as anti-idiotypic masks for nivolumab-based prodrugs.
Article Snippet: Protein concentrations were measured by absorbance at 280 nm using a spectrophotometer (Thermo Fisher, USA). .. Recombinant Human PD-1 Protein (ECD, His Tag) was purchased from Sino Biological (China), and recombinant tobacco etch virus (TEV) protease was produced and purified inhouse following standard methods. .. D ow nloaded from https://academ ic.oup.com /peds/advance-article/doi/10.1093/protein/gzag010/8655119 by guest on 18 April 2026 UN CO RR EC TE D MA NU SC RI PT Bacterial culture and library expression Escherichia coli BL21 (DE3) cells transformed with pPALU9 plasmids encoding for an affibody library (~1011 variants) were grown overnight (O/N) in Luria-Bertani (LB) medium supplemented with carbenicillin (100 μg/mL).
Purification:Article Title: Engineering affibody domains as anti-idiotypic masks for nivolumab-based prodrugs
Article Snippet: Protein concentrations were measured by absorbance at 280 nm using a spectrophotometer (Thermo Fisher, USA). .. Recombinant Human PD-1 Protein (ECD, His Tag) was purchased from Sino Biological (China), and recombinant tobacco etch virus (TEV) protease was produced and purified in-house following standard methods. .. E. coli BL21 (DE3) cells transformed with pPALU ( ) plasmids encoding for an affibody library (~10 11 variants) were grown overnight (O/N) in Luria-Bertani (LB) medium supplemented with carbenicillin (100 μg/ml).
Article Title: Engineering affibody domains as anti-idiotypic masks for nivolumab-based prodrugs.
Article Snippet: Protein concentrations were measured by absorbance at 280 nm using a spectrophotometer (Thermo Fisher, USA). .. Recombinant Human PD-1 Protein (ECD, His Tag) was purchased from Sino Biological (China), and recombinant tobacco etch virus (TEV) protease was produced and purified inhouse following standard methods. .. D ow nloaded from https://academ ic.oup.com /peds/advance-article/doi/10.1093/protein/gzag010/8655119 by guest on 18 April 2026 UN CO RR EC TE D MA NU SC RI PT Bacterial culture and library expression Escherichia coli BL21 (DE3) cells transformed with pPALU9 plasmids encoding for an affibody library (~1011 variants) were grown overnight (O/N) in Luria-Bertani (LB) medium supplemented with carbenicillin (100 μg/mL).
Bioprocessing:Article Title: Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors
Article Snippet: .. For the detection of AAV-encoded nivolumab, plate coating was performed with 100 ng/well using recombinant human PD-1 protein (Sino Biological) in H 2 O overnight, while the remaining procedure was identical, only varying in secondary antibody concentration and incubation time (αhuFc-HRP 1:10,000 for 3 h and 1:500 for 2 h for murine αPD-1 and AAV-Nivolumab, respectively). .. Standard values were generated using the therapeutic drug Opdivo® (Bristol Myers Squibb).
Incubation:Article Title: Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors
Article Snippet: .. For the detection of AAV-encoded nivolumab, plate coating was performed with 100 ng/well using recombinant human PD-1 protein (Sino Biological) in H 2 O overnight, while the remaining procedure was identical, only varying in secondary antibody concentration and incubation time (αhuFc-HRP 1:10,000 for 3 h and 1:500 for 2 h for murine αPD-1 and AAV-Nivolumab, respectively). .. Standard values were generated using the therapeutic drug Opdivo® (Bristol Myers Squibb).
Functional Assay:Article Title: Humanized anti-PD-1 antibody and the use thereof
Article Snippet: .. 1.4 Murine Antibody PD1-M944 Functional Assay (1) Blocking the Binding of Recombinant Human PD-1 to PD-L1: Recombinant human PD-1 protein at a concentration of 1 μg/mL was coated on a 96-well plate at 100 μL per well overnight at 4° C. The plates were washed the next day and blocked at room temperature for 1 h. 100 μL of 10 μg/mL of biotinylated protein PD-L1-Fc-biotin (SinoBiological, Inc.) was co-incubated with different concentrations (0.4 μg/mL, 2 μg/mL and 10 μg/mL) of PD1-M944 or Nivolumab (Sino Biological, Inc). ..
Expressing:Article Title: PD-L1 targeted peptide demonstrates potent antitumor and immunomodulatory activity in cancer immunotherapy
Article Snippet: .. Human PD-L1 natural ORF mammalian expression plasmid (Cat: HG10084-UT), rabbit anti-human PD-1 antibody (Clone D67, Cat: 10377-RD67), recombinant human PD-L1 protein (Cat: 10084-H08H), human PD-L1/B7-H1/CD274 protein (Cat: 10084-HNAH), biotinylated mouse PD-1/PDCD1 protein (His tag) and recombinant human PD-1 protein (Cat: 10377-H08H) were purchased from Sino Biological Inc (Beijing). .. GM-CSF (Cat: 315-03) and Murine TNF-α (Cat: 315-01A) were purchased from PEPROTECH.
Plasmid Preparation:Article Title: PD-L1 targeted peptide demonstrates potent antitumor and immunomodulatory activity in cancer immunotherapy
Article Snippet: .. Human PD-L1 natural ORF mammalian expression plasmid (Cat: HG10084-UT), rabbit anti-human PD-1 antibody (Clone D67, Cat: 10377-RD67), recombinant human PD-L1 protein (Cat: 10084-H08H), human PD-L1/B7-H1/CD274 protein (Cat: 10084-HNAH), biotinylated mouse PD-1/PDCD1 protein (His tag) and recombinant human PD-1 protein (Cat: 10377-H08H) were purchased from Sino Biological Inc (Beijing). .. GM-CSF (Cat: 315-03) and Murine TNF-α (Cat: 315-01A) were purchased from PEPROTECH.
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