Journal: Scientific Reports

Article Title: A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

doi: 10.1038/s41598-024-82288-9

Figure Lengend Snippet: Representative SPR sensorgrams obtained by Biacore of antibody mAb I3RB18 Fab (I3RB119), and BsAbs binding to recombinant human antigen CD123 SP1 ( A ) and its truncated variant CD123 SP2 ( B ).

Article Snippet: To detect free (unbound) antibody in solution using the (ECLIA) assay, MSD SA-STD plates were blocked with 50 μL per well of assay buffer (PBS containing 0.2% BSA, 0.05% Tween 20) for at least 5 min. Plates were turned over to remove assay buffer and tapped on paper towels and 50 μL per well of 0.7 μg/mL biotinylated human CD123 SP1 (huCD123 SP1, R&D Systems cat 301-R3/CF, lot# LV091112A or LV081110A) or human CD123 SP2 BlueSky Lot #120,503-CP05057h or 6519A) in assay buffer were added and the plates were allowed to incubate overnight in the refrigerator.

Techniques: Binding Assay, Recombinant, Variant Assay

Journal: Scientific Reports

Article Title: A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

doi: 10.1038/s41598-024-82288-9

Figure Lengend Snippet: Biacore and MSD-CAT affinity data for the binding of anti-CD123 to human antigen-full length (CD123 SP1) and its truncated variant (CD123 SP2). For the SPR experiment recombinant CD123 SP1 or CD123 SP2 were used for complex formation. For the MSD-CAT experiments reported in the last column, complex formation was obtained by incubating antibodies or Fabs with cells expressing human CD123 SP1 or CD123 SP2, or cynomolgus full length CD123. Detection of antibody in the cell-antibody equilibrated mixture was performed by capturing free antibody in the supernatant with biotinylated recombinant CD123 SP1 or CD123 SP2 which has been previously captured on a streptavidin MSD-plate.

Article Snippet: To detect free (unbound) antibody in solution using the (ECLIA) assay, MSD SA-STD plates were blocked with 50 μL per well of assay buffer (PBS containing 0.2% BSA, 0.05% Tween 20) for at least 5 min. Plates were turned over to remove assay buffer and tapped on paper towels and 50 μL per well of 0.7 μg/mL biotinylated human CD123 SP1 (huCD123 SP1, R&D Systems cat 301-R3/CF, lot# LV091112A or LV081110A) or human CD123 SP2 BlueSky Lot #120,503-CP05057h or 6519A) in assay buffer were added and the plates were allowed to incubate overnight in the refrigerator.

Techniques: Binding Assay, Variant Assay, Recombinant, Expressing

Journal: Scientific Reports

Article Title: A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

doi: 10.1038/s41598-024-82288-9

Figure Lengend Snippet: Representative binding curves for MSD-CAT affinity analysis for cell surface expressed CD123, SP1 ( A ) and CD123 SP2 ( B ). The Y-axis shows % free anti-receptor molecules (BsAb, Fab or mAb) or CD123 negative control mAb (I3CB15) while the X-axis shows the concentration of cell surface antigen. Each curve represents a different but fixed concentration of mAb, bispecific or Fab as indicated.

Article Snippet: To detect free (unbound) antibody in solution using the (ECLIA) assay, MSD SA-STD plates were blocked with 50 μL per well of assay buffer (PBS containing 0.2% BSA, 0.05% Tween 20) for at least 5 min. Plates were turned over to remove assay buffer and tapped on paper towels and 50 μL per well of 0.7 μg/mL biotinylated human CD123 SP1 (huCD123 SP1, R&D Systems cat 301-R3/CF, lot# LV091112A or LV081110A) or human CD123 SP2 BlueSky Lot #120,503-CP05057h or 6519A) in assay buffer were added and the plates were allowed to incubate overnight in the refrigerator.

Techniques: Binding Assay, Negative Control, Concentration Assay

Journal: Scientific Reports

Article Title: A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

doi: 10.1038/s41598-024-82288-9

Figure Lengend Snippet: Binding curves for MSD-CAT affinity analysis for cyno CD123. The Y-axis shows % free anti-receptor molecules (mAb or its Fab) while the X-axis shows the concentration of cell surface antigen. Each curve represents a different but fixed concentration of mAb, or Fab as indicated. The figure also shows a plot of the control commercial antibody, 7G3, showing cross reactivity to human CD123 (SP1).

Article Snippet: To detect free (unbound) antibody in solution using the (ECLIA) assay, MSD SA-STD plates were blocked with 50 μL per well of assay buffer (PBS containing 0.2% BSA, 0.05% Tween 20) for at least 5 min. Plates were turned over to remove assay buffer and tapped on paper towels and 50 μL per well of 0.7 μg/mL biotinylated human CD123 SP1 (huCD123 SP1, R&D Systems cat 301-R3/CF, lot# LV091112A or LV081110A) or human CD123 SP2 BlueSky Lot #120,503-CP05057h or 6519A) in assay buffer were added and the plates were allowed to incubate overnight in the refrigerator.

Techniques: Binding Assay, Concentration Assay, Control

Journal: Scientific Reports

Article Title: A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

doi: 10.1038/s41598-024-82288-9

Figure Lengend Snippet: Representative SPR sensorgrams obtained by Biacore of antibody mAb I3RB18 Fab (I3RB119), and BsAbs binding to recombinant human antigen CD123 SP1 ( A ) and its truncated variant CD123 SP2 ( B ).

Article Snippet: For generation of monoclonal anti-CD123 antibodies, recombinant human CD123 SP1 ECD-His tag protein (R&D Systems, 301-R3/CF, lot# LV081110A) was used for phage panning and hit screening.

Techniques: Binding Assay, Recombinant, Variant Assay

Journal: Scientific Reports

Article Title: A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

doi: 10.1038/s41598-024-82288-9

Figure Lengend Snippet: Biacore and MSD-CAT affinity data for the binding of anti-CD123 to human antigen-full length (CD123 SP1) and its truncated variant (CD123 SP2). For the SPR experiment recombinant CD123 SP1 or CD123 SP2 were used for complex formation. For the MSD-CAT experiments reported in the last column, complex formation was obtained by incubating antibodies or Fabs with cells expressing human CD123 SP1 or CD123 SP2, or cynomolgus full length CD123. Detection of antibody in the cell-antibody equilibrated mixture was performed by capturing free antibody in the supernatant with biotinylated recombinant CD123 SP1 or CD123 SP2 which has been previously captured on a streptavidin MSD-plate.

Article Snippet: For generation of monoclonal anti-CD123 antibodies, recombinant human CD123 SP1 ECD-His tag protein (R&D Systems, 301-R3/CF, lot# LV081110A) was used for phage panning and hit screening.

Techniques: Binding Assay, Variant Assay, Recombinant, Expressing

Journal: Scientific Reports

Article Title: A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

doi: 10.1038/s41598-024-82288-9

Figure Lengend Snippet: Representative binding curves for MSD-CAT affinity analysis for cell surface expressed CD123, SP1 ( A ) and CD123 SP2 ( B ). The Y-axis shows % free anti-receptor molecules (BsAb, Fab or mAb) or CD123 negative control mAb (I3CB15) while the X-axis shows the concentration of cell surface antigen. Each curve represents a different but fixed concentration of mAb, bispecific or Fab as indicated.

Article Snippet: For generation of monoclonal anti-CD123 antibodies, recombinant human CD123 SP1 ECD-His tag protein (R&D Systems, 301-R3/CF, lot# LV081110A) was used for phage panning and hit screening.

Techniques: Binding Assay, Negative Control, Concentration Assay

Journal: Scientific Reports

Article Title: A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

doi: 10.1038/s41598-024-82288-9

Figure Lengend Snippet: Binding curves for MSD-CAT affinity analysis for cyno CD123. The Y-axis shows % free anti-receptor molecules (mAb or its Fab) while the X-axis shows the concentration of cell surface antigen. Each curve represents a different but fixed concentration of mAb, or Fab as indicated. The figure also shows a plot of the control commercial antibody, 7G3, showing cross reactivity to human CD123 (SP1).

Article Snippet: For generation of monoclonal anti-CD123 antibodies, recombinant human CD123 SP1 ECD-His tag protein (R&D Systems, 301-R3/CF, lot# LV081110A) was used for phage panning and hit screening.

Techniques: Binding Assay, Concentration Assay, Control