Recommended Reading: Immune Checkpoint Bispecific Antibody Targets
| Immune Co-inhibitory Receptors | PD-1 | PD-L1 | PD-L2 | B7-H3 | B7-H4 |
| B7-H5 | BTLA | CD80(B7-1) | CD86(B7-2) | CD96 | |
| CD47 | CD155 | CTLA-4 | LAG-3 | TIGIT | |
| TIM-3 | Nectin-1 | DNAM-1 | Galectin-9 | HVEM | |
| Nectin-2 | Nectin-3 | PVRIG | SIRP alpha | SIRP alphaV2 | |
| SIRP gamma | CD160 |
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| Immune Co-stimulatory Receptors | 4-1BB Ligand | 4-1BB | OX40 Ligand | OX40 | ICOS Ligand (B7-H2) |
| ICOS | GITR Ligand | GITR | CD48 | HVEM | |
| Nectin-2 | CD40 Ligand | CD40 | CD30 ligand | CD30 | |
| CD28 | CD27 Ligand | CD27 | B7-1 | B7-2 | |
| LIGHT | B7-H7 | B7-H6 | 2B4 | CD28H | |
| Nkp30 |
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| Others | LILRA1 | LILRA2 | LILRA3 | LILRA5 | LILRA6 |
| LILRB1 | LILRB2 | LILRB3 | LILRB4 | LILRB5 | |
| LAIR-1 | LAIR-2 | SLAMF1 | SLAMF2(CD48) | SLAMF3(CD229) | |
| SLAMF4(2B4) | SLAMF5(CD84) | SLAMF6(NTB-A) | SLAMF7(CRACC) | SLAMF8(BLAME) | |
| BTN1A1 | BTN3A1 | BTN3A2 | BTN3A3 | CD161 | |
| MARCO | MSP1D1 | Nectin-1 | Nectin-4 | TREM2 | |
| ILDR2 |
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MALS-Verified Native Molecular Weight & Oligomeric State
The purity of Human OX40 Ligand Protein, His Tag (Cat. No. OXL-H52Q8) is more than 95% and the molecular weight of this protein is around 60-80 kDa verified by SEC-MALS.
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The purity of Human CTLA-4, His Tag (Cat. No. CT4-H52H9) is more than 90% and the molecular weight of this protein is around 45-60 kDa verified by SEC-MALS.
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Binding Assay
Immobilized Human PD-1, His Tag (Cat. No. PD1-H5221) at 5 μg/mL, add increasing concentrations of Cadonilimab (PD-1 x CTLA-4 bispecific antibody) and then add Biotinylated Human CTLA-4, Fc,Avitag (Cat. No. CT4-H82F3) at 0.2 μg/mL. Detection was performed using HRP-conjugated Streptavidin (Acro, Cat. No. STN-NH913) with sensitivity of 2.99 ng/mL (Routinely tested).
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Immobilized Human VEGF165, His Tag (Cat. No. VE5-H5248) at 2 μg/mL, add increasing concentrations of Ivonescimab and then add Biotinylated Human PD-1 Protein, Avitag,His Tag (Cat. No. PD1-H82E4) at 2 μg/mL. Detection was performed using HRP-conjugated Streptavidin (Acro, Cat. No. STN-NH913) with sensitivity of 2.17 ng/mL (Routinely tested).
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Anti-BAFFR Antibody, Human IgG1 immobilized on CM5 Chip can bind Human BAFFR Protein, Fc Tag (Cat. No. BAR-H5253) with an affinity constant of 9.66 nM as determined in a SPR assay (Biacore 8K) (Routinely tested).
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Immobilized cell surface PD-1 (5x104 of cells per well) can bind Biotinylated Human PD-L1, Fc,Avitag, His Tag (Cat. No. PD1-H82F3) with an EC50 of 0.082 μg/mL (Routinely tested).
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Immobilized cell surface PD-1 (5x104 of cells per well) can bind Human PD-L1, Fc Tag (Cat. No. PD1-H5258) with an EC50 of 0.029 μg/mL (Routinely tested).
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Blocking Assay
Serial dilutions of Ipilimumab were added into Human CTLA-4, Fc Tag (Cat. No. CT4-H5255): Biotinylated Human B7-1 Protein, Fc,Avitag, premium grade (Cat. No. B71-H82F3) binding reactions. The half maximal inhibitory concentration (IC50) is 0.34215 μg/mL (Routinely tested).
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Serial dilutions of Nivolumab were added into Human PD-L1 Protein, Mouse IgG1 Fc Tag (Cat. No. PD1-H52A3): Biotinylated Human PD-1, Fc, Avitag, His Tag (Cat. No. PD1-H82F2) binding reactions. The half maximal inhibitory concentration (IC50) is 0.38426 μg/mL (QC tested).
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FACS analysis shows that the binding of Human B7-1, Mouse IgG2a Fc Tag (Cat. No. B71-H52A4) to 293 overexpressing CTLA-4 was inhibited by increasing concentration of neutralizing Anti-human CTLA-4 antibody. The concentration of Human B7-1 is 1 μg /mL. The IC50 is 5.5 μg/mL (Routinely tested).
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FACS analysis shows that the binding of Human PD-L2, Mouse IgG1 Fc Tag (Cat. No. PD2-H52A5) to 293 overexpressing PD-1 was inhibited by increasing concentration of neutralizing Anti-PD-1 antibody. The concentration of PD-L2 used is 0.1 μg/mL. The IC50 is 1.3 μg/mL (Routinely tested).
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Biochemical Neutralizing Antibody Screening
The Human PD-1 / PD-L1 Inhibitor Screening Kit (TR-FRET)(Cat. No. FRT-P019)is suitable for the detection of PD1/PD-L1 inhibitors. It shows that both Atezolizumab, Nofazinlima b and Pembrolizumab exhibit good inhibitory activity in this TR-FRET competition assay.
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Inhibition of PD-1-PD-l1 binding by anti-PD-1 neutralizing antibody (catalog # EP-101-c03) measured using the PD-1 [biotinylated] : pd-l1 inhibitor screening ELISA assay pair (catalog # EP-101). Anti-PD-1 neutralizing antibody was diluted from 10 μg/mL to 0.078 μg/ mL (69.628 nM to 0.544 nM) and loaded onto the plate coated by human PD-L1 in the presence of human PD-1-Biotin. Assay was performed according to the protocol in PD-1[Biotinylated]: PD-L1 Inhibitor Screening ELISA Assay Pair. Background was subtracted from data points prior to log transformation and curve fitting (QC tested).
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Cell-Based Neutralizing Antibody Screening
FACS analysis shows that the binding of PE-Labeled Human SIRP alpha, Fc Tag (Cat. No. SIA-HP252) to Jurkat cells overexpressing CD47 was inhibited by increasing concentration of neutralizing anti-human CD47 antibody. The IC50 is 0.37 μg/mL (QC tested).
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FACS analysis shows that the binding of Human PD-L1, Mouse IgG1 Fc Tag, low endotoxin (HPLC-verified) (Cat. No. PD1-H52A3) to HEK293/Human PD-1, GFP Tag Stable Cell Line (Cat. No. CHEK-ATP001) was inhibited by increasing concentration of neutralizing Anti-PD-1 antibody. The concentration of PD-L1 used is 1 μg/mL. The IC50 is 0.145 μg/mL (Routinely tested).
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Analysis of the synergistic effect for anti-human PD-1 and anti-human LAG-3 antibody (RLU). This reporter cell (Cat. No. SCJUR-STF063)was co-incubated with serial dilutions of anti-human PD-1 plus anti-human LAG-3 antibody in the presence of target cells expressing human PD-L1 and MHCⅡ. The EC50 was approximately 0.58 μg/mL.
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Human 4-1BB Ligand (71-254) Protein, His,Flag Tag (Cat. No. 41L-H52D4) induce IL-8 secretion in HT1080 human CD137 cell line. The EC50 for this effect is 0.20-0.44 μg/mL (Routinely tested).
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Immobilized Human CTLA-4, His Tag, active dimer (Cat. No. CT4-H52H9) at 2 μg/mL, add increasing concentrations of CTLA-4 x OX40 Bispecific Antibody in 50% Human serum and then add Biotinylated Human OX40, Avitag,His Tag (Cat. No. TN4-H82E4) at 1 μg/mL. Detection was performed using HRP-conjugated streptavidin with sensitivity of 4 ng/mL (Intact assay, Routinely tested).
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(Cat. No. EPM-V1)Quantitative Analysis Five Different Therapeutic Anti-PD-1 Antibody in Mouse Serum Samples. Serial dilutions of the Therapeutic Anti-PD-1 Antibody (1:2 serial dilutions, from 20 μg/mL to 0.039 μg/mL) was added into PD-1: Biotinylated Anti-PD-1 Antibody binding reactions.
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