PD-1/PD-L1 Inhibitor Binding Assay Kit

Background

Immune checkpoint blockade is a groundbreaking approach in cancer immunotherapy that enhances the immune system’s ability to recognize and destroy cancer cells. The immune checkpoint PD- 1(CD279)/PD-L1 (CD274 or B7-H1) is an attractive target for cancer immunotherapy. The interaction of PD-1 with PD-L1 induces T cell apoptosis and allows cancer to evade the immune response by suppressing the adaptive immune system. Immunotherapy drugs work by blocking the interaction between PD-1 and PD-L1, and enhancing the anti-tumor immune response.

Assay Principle

The PD-1/PD-L1 binding assay kit is a TR-FRET based assay, which is designed to detect the binding status between PD-1 and PD-L1. Tag6-PD-1 and Tag7-PD-L1 are included in this assay kit. Binding of Tag6-PD-1 to Tag7-PD-L1 brings the Terbium (Tb, HTRF donor) and the fluorophore d2 (HTRF acceptor) in a proximity distance, and activation of Tb results in fluorescence resonance energy transfer (FRET). Thus, the binding status can be quantitively measured by calculating the ratio of the emission fluorescence intensity of the acceptor (665 nm) and donor (620 nm). Interference of the PD-1/PD-L1 binding will reduce the HTRF signal.

Application

PD-1/PD-L1 Inhibitor Binding Assay Kit High throughput screening of compounds that inhibit the binding between PD-1 and PD-L1 for drug discovery.

Instrument Required

A HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.

Kit Components

Materials Not Supplied

• Microplate reader, HTRF® certified microplate reader
• 0.5 M DTT
• Adjustable micro-pipettor
• Sterile Tips

Assay Protocol

1. Step 1. Prepare the inhibitor compound solution If the inhibitor compound is dissolved in water, make a solution of the compound 10-fold higher than the final concentration in 1X assay buffer (since you will add 2 µl to the 20 µl reaction). If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 10-fold dilution in 1X assay buffer (at this step, the compound concentration is 10-fold higher than the final concentration and the DMSO concentration is 10%). To determine an IC50 or to test lower concentrations of the compound, prepare as series of further dilutions in 1X assay buffer containing 10% DMSO (the final concentration of the DMSO will be 1% in all samples).
2. Step 2. Prepare PD-1 solution Thaw PD-1 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: PD-1 protein is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the PD-1 protein 32-fold (1 µL PD-1 + 31 µL 1X assay buffer). Add 4 µl of diluted protein solution to each well.
3. Step 3. Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well. Incubate at room temperature for 30 minutes (optional).
4. Step 4. Prepare PD-L1 solution Thaw PD-L1 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: PD-L1 protein is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the PD-L1 protein 400-fold (1 µL PD-L1 + 399 µL 1X assay buffer). Add 4 µl of diluted protein solution to each positive control well and inhibitor test well. Add 4 µl of assay buffer to each of negative control well.
5. Step 5. Prepare dye solution Dilute Terbium-labeled anti-Tag6 antibody and fluorescence-labeled anti-Tag7 antibody 1:200 in assay buffer. For example: 1 µl of Terbium-labeled anti-Tag6 antibody + 2 µl of fluorescence- labeled anti-Tag7 antibody + 197 µl of assay buffer. Add 10 µl of this dye mixture to each well.
6. Step 6. Incubate the reaction at room temperature for 1 hour.
7. Step 7. Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:
8. Step 8. Excitation wavelength at 340 nm and emission at 620 nm.
9. Step 9. Excitation wavelength at 340 nm and emission at 665 nm.

Data Analysis

Calculate HTRF signal of each well. Calculate percentage activity In the absence of the compound (positive control), the sample HTRF signal (P) is defined as 100% activity. In the absence of enzyme (negative control), the sample HTRF signal (N) is defined as 0% activity. The percent activity in the presence of each compound is calculated according to the following equation: % activity = (S-N)/(P-N) X100, where S= the sample HTRF signal in the presence of the compound.

Assay Validation

Biological Pathway / Process

Immune Checkpoint (T cell exhaustion pathway)

Therapeutic / Disease Area

Oncology / Immunotherapy