Inhibition assay of CD279 using 65684-1-TR by ELISA. Mouse PD-1 (Eg0986) was coated at 5 ug/mL. Serial diluted 65684-1-TR and 5 ug/ml biotin-labelled mouse PD-L1 was applied, and the binding signal of PD-L1 to PD-1 was detected by HRP-SA. Signal was developed by TMB substrate. The IC50 is 0.1993 ug/ml. This data was developed using the same antibody clone with 65684-1-PBS in a different storage buffer formulation.

1x10^6 untreated or anti-CD3/CD28 treated mouse splenocytes were surface stained with 0.25 ug Anti-Mouse PD-1/CD279 (RMP1-14) Rat IgG2a RecAb (65684-1-TR, Clone:RMP1-14) and PE anti-rat IgG2a Antibody. Cells were then stained with CoraLite® Plus 647 Anti-Mouse CD3. Cells were not fixed. This data was developed using the same antibody clone with 65684-1-PBS in a different storage buffer formulation.

1x10^6 anti-CD3/CD28 treated mouse splenocytes were surface stained with 0.25 ug Anti-Mouse PD-1/CD279 (RMP1-14) Rat IgG2a RecAb (65684-1-TR, Clone: RMP1-14) (red) or Rat IgG2a Isotype Control (2A3) (65209-1-Ig, Clone: 2A3) (blue), and PE anti-rat IgG2a Antibody. 1x10^6 untreated mouse splenocytes were surface stained with 0.25 ug Anti-Mouse PD-1/CD279 (RMP1-14) Rat IgG2a RecAb (65684-1-TR, Clone:RMP1-14) (black, dashed) and PE anti-rat IgG2a Antibody. Cells were not fixed. This data was developed using the same antibody clone with 65684-1-PBS in a different storage buffer formulation.

Inhibition assay of PD-1(CD279) using 65684-1-TR by Biolayer interferometry (BLI). Biolayer interferometry (BLI) inhibition results demonstrate that 65684-1-TR effectively blocks the PD-1/PD-L1 immune checkpoint interaction. In the assay, mouse PD-1 (Eg0918) was immobilized, saturated with 65684-1-TR, and then challenged with mouse PD-L1 (Eg0985). The compound achieved an inhibition rate of 0.237, corresponding to 76.3% suppression of PD-L1 binding, supporting its potential as a functional immune checkpoint inhibitor. This data was developed using the same antibody clone with 65684-1-PBS in a different storage buffer formulation.

Biolayer interferometry (BLl) epitope binding assay of 65684-1-TR and 98080-1-RR. Biolayer interferometry (BLl) epitope binning assay revealed distinct binding epitopes for 65684-1-TR and 98080-1-RR. 5 ug/ml mouse PD-1 (Eg0918) was immoblized onto anti-His probe, followed by binding to 65684-1-TR until saturation, and then the binding signal of 98080-1-RR with binded PD-1 complex was detected. The inhibition rate (the rate of inhibited response to uninhibited response) is 0.969. This data was developed using the same antibody clone with 65684-1-PBS in a different storage buffer formulation.

Biolayer interferometry (BLl) kinetic assays of 65684-1-TR against Mouse PD-1/CD279 were performed. The affinity constant is 64.5 pM.