PD-1 [Biotinylated] : PD-L1 Inhibitor Screening ELISA Assay Pair

Details for Product No. ABIN2762507
Target
Reactivity
Human
Detection Method
Colorimetric
Method Type
Sandwich ELISA
Detection Range
0.01875-0.3 μg/mL
Minimum Detection Limit
0.01875 μg/mL
Application
Screening Assay (ScA), ELISA
Options
Purpose This inhibitor screening ELISA pair is designed to facilitate the identification and characterization of new PD-1 pathway inhibitors.
Brand MABSol®
Detection Method Colorimetric
Characteristics
  • This pair is useful for screening for inhibitors of human PD-1 binding to human PD-L1.
  • By employing the pre-labeled PD-1 protein, we eliminate the often time-consuming labeling process, and greatly simplify the experimental procedures.
  • Both biotinylated PD-1 and PD-L1 proteins are expressed in the HEK293 cells. The use of human cells as expression host confers authentic post-translational modifications essential for their binding activities.
  • We include anti-PD1 neutralizing mAb as a standard positive control. This would allow the users to validate their experiments.
  • Our biotin labeling platform produces biotinylated proteins with high detection sensitivity and minimal lot-to-lot variation. These features ensure that the assay kit has desired detection sensitivity and the results are reproducible.
Components - Human PD-L1
- Human PD-1-Biotin
- Streptavidin-HRP
- Anti-PD-1 Neutralizing Antibody
Material not included - Coating Buffer: PBS (Phosphate Buffered Saline), pH 7.4, 12 mL is sufficient for 96 tests.
- Wash Buffer: PBS with 0.05 % (v/v) Tween-20, 500 mL is sufficient for 96 tests.
- Blocking Buffer: Wash Buffer with 2 % (w/v) bovine serum albumin, 35 mL is sufficient for 96 tests.
- Dilution Buffer: Wash Buffer with 0.5 % (w/v) bovine serum albumin, 50 mL is sufficient for 96 tests.
- Substrate Stock: Solution 10 mg/mL TMB in Dimethyl sulfoxide, 1 mL is sufficient for 96 tests. Protect from light.
- Substrate Dilution Buffer: 50 mM disodium hydrogen phosphate (Na2HPO4) and 25 mM citric acid, adjust pH to 5.5 with 1 M Sodium hydroxide (NaOH), 25 mL is sufficient for 96 tests.
- TMB Substrate Working Solution:
For each plate dilute 250 μL substrate stock solution in 25 mL substrate dilution buffer and add 12 μL 5 % H2O2 (pipette 10 μL 30 % H2O2 into 50 μL distilled water), mix well.
Notes:
1) The TMB Substrate Working Solution should be freshly prepared and used within 15 minutes.
2) If you choose to use other commercially available ready-to-use TMB substrate solutions, you should follow the manufacturer's instruction
- Stop Solution: 1 M sulfuric acid (aqueous), 6 mL is sufficient for 96 tests.
- High binding surface 96-well microplate, clear flat bottom
- Microplate sealing film
- Pipettes and pipette tips
- UV/Vis microplate spectrophotometer (absorbance 450nm, correction wavelength set to 600 nm).
Target
Background Immune checkpoint pathway is a focal point of today's cancer research. PD-1 is one of the best characterized checkpoint proteins. The binding between PD-1 and its ligand PD-L1 suppresses T-cell activation and allows cancer cells to escape from body's immune surveillance. Therefore, the pharmaceutical inhibition of PD-1 or its ligand has been considered a promising strategy by many oncologists.
Application Notes This pair is useful for screening for inhibitors of human PD-1 binding to human PD-L1.
Comment

METHOD VERIFICATION
PD-1 [BIOTINYLATED]:PD-L1 BINDING IN THE ABSENCE OF INHIBITORS:
Immobilized human PD-L1 protein at 2 μg/mL (100 μL/well) can bind human PD-1-Biotin with a linear range of 0.01875-0.3 μg/mL when detected by Streptavidin-HRP. Background was subtracted from data points before curve fitting.

INHIBITION OF PD-1 [BIOTINYLATED] : PD-L1 BINDING BY ANTI-PD-1 NEUTRALIZING ANTIBODY
Serial dilutions of anti-PD-1 neutralizing antibody (1:2 serial dilutions, from 10 μg/mL to 0.078 μg/mL) was added into PD-L1 : PD-1-Biotin binding reactions. The assay was performed according to the above described protocol. Background was subtracted from data points prior to log transformation and curve fitting.
Preparation of Standard Serial Dilutions
1) Pipette 480 μL Dilution Buffer into tube Std-1 and 150 μL Dilution Buffer into the remaining tubes (tube Std.-2 to tube Std.-8).
2) Add 20 μL anti-PD-1 neutralizing antibody stock solution (250 μg/mL) to tube Std.-1 to make the final concentration 10 μg/mL. Mix well.
3) To make a 1:2 serial dilutions, pipette 150 μL solution to the next tube as illustrated in Fig.3.
IMPORTANT: Mix thoroughly at each step during the dilution process.

Sample Volume 50 μL
Protocol This assay employs a simple colorimetric ELISA platform, which measures the binding between immobilized human PD-L1 and in-house developed biotinylated PD-1 protein. This product is uniquely suitable for rapid high-throughput screening of putative PD-1 and PD-L1 inhibitors. Briefly, we provide you with a human PD-1-Biotin protein, a human PD-L1 protein, an anti-PD-1 neutralizing antibody (as method verified Std.), and Streptavidin-HRP reagent. Your experiment will include 4 simple steps:
  • Coat the plate with human PD-L1.
  • Add your molecule of interest to the tests.
  • Add human PD-1-Biotin to bind the coated human PD-L1.
  • Add Streptavidin-HRP followed by TMB or other colorimetric HRP substrate.
Finally, the ability of your compound to inhibit PD-1 : PD-L1 binding will be determined by comparing OD readings among different experimental groups
Assay Procedure

Your experiment will include 4 simple steps: a) Coat the plate with human PD-L1, b) add your molecule of interest to the reactions, c) add biotinylated human PD-1 to bind with PD-L1, and d) add Streptavidin-HRP followed by TMB or other colorimetric HRP substrate to measure the binding activities. The ability of your compound to inhibit PD-1 : PD-L1 binding will be determined by comparing OD readings among different experimental groups.

Restrictions For Research Use only
Format Lyophilized
Reconstitution Reconstitute the provided lyophilized materials to stock solutions with PBS as recommended. Solubilize for 15 to 30 minutes at room temperature with occasional gentle mixing. Avoid vigorous shaking or vortexing. The reconstituted stock solutions should be stored at -80°C Avoid freeze-thaw cycles.
Storage -20 °C
Storage Comment Room temperature (RT) for 1 month in lyophilized state. -20°C for 1 year in lyophilized state. -80°C for 2 months under sterile conditions after reconstitution.
Images
Image no. 1 for PD-1 [Biotinylated] : PD-L1 Inhibitor Screening ELISA Assay Pair (ABIN2762507) INHIBITION OF PD-1 [BIOTINYLATED] : PD-L1 BINDING BY ANTI-PD-1 NEUTRALIZING ANTIBODY
Image no. 2 for PD-1 [Biotinylated] : PD-L1 Inhibitor Screening ELISA Assay Pair (ABIN2762507) BINDING OF BIOTINYLATED HUMAN PD-1 TO IMMOBILIZED HUMAN PD-L1 IN A FUNCTIONAL ELISA A...