UniOne® TR-FRET Human PD1/PD-L1 Binding Kit

The assay kit utilizes homogeneous time-resolved fluorescence (TR-FRET) technology to measure the interaction between PD1 and PD-L1. This method enables simple, rapid, and high-throughput screening of inhibitors and antibody blockers.

As shown in the figure, the interaction between PD1 and PD-L1 is detected using an Eu-labeled anti-Tag1 antibody (TR-FRET donor) and an Ac-labeled anti-Tag2 antibody (TR-FRET acceptor). The binding of PD1 and PD-L1 brings the donor and acceptor antibodies into proximity, allowing the excitation of the donor antibody to trigger fluorescence resonance energy transfer (FRET) to the acceptor antibody, resulting in a specific emission signal at 665 nm. Pembrolizumab, as a positive control, blocks the interaction between PD1 and PD-L1, preventing FRET signal generation. The stronger the blocking effect of the screened drug on the PD1/PD-L1 interaction, the lower the signal. The specific signal is proportional to the extent of the PD1/PD-L1 interaction. This homogeneous assay is simple to perform and requires no washing steps.

Note: Aliquot immediately upon first thaw and store at the recommended temperature. Avoid storage after dilution and repeated freeze-thaw cycles.

1. Reagent Preparation

1.1 Thaw all reagents to room temperature before use (equilibrate at room temperature for at least 30 min). The reaction volume for the 384-well shallow plate is 20 μL (reagent volumes for the reaction system are shown in the table). Calculate the required volume for the experiment before preparation and prepare as needed; the following preparation is for reference only, using 500 reactions as an example.

Table 1. Reagent Preparation

1.2 Gradient Dilution of Test Samples

Using Pembrolizumab as an example, the diluent is 1× Detection buffer. To minimize the impact of matrix effect interference, it is recommended to dilute with a solution having the same matrix as the test sample; adjust the test sample according to its actual concentration.

Table 2. Gradient Dilution of Positive Drug Pembrolizumab (Adjust according to actual conditions)

2. Sample Addition and Controls

2.1 Sample addition order for assay wells: Add 2 μL of test sample, 4 μL of Tag2-PD1 protein working solution, 4 μL of Tag1-PD-L1 protein working solution, and 10 μL of mixed Mix sequentially into the 384-well shallow plate;

2.2 Maximum Control: 2 μL of 1× Detection buffer, 4 μL of Tag2-PD1 protein working solution, 4 μL of Tag1-PD-L1 protein working solution, and 10 μL of mixed Mix;

2.3 Quality Control Well (NC): 10 μL of 1× Detection buffer plus 10 μL of Mix.

3. Detection

Detect on a microplate reader compatible with TR-FRET. Excitation wavelength is 320/340 nm, and emission wavelengths are 620 nm and 665 nm.

[Result Calculation]

1) Calculate the signal value (Ratio): Divide the 665 nm fluorescence signal by the 620 nm fluorescence signal, then multiply by 10,000.

Ratio = (665/620) ×10000

2) Calculate Net signal based on the signal value:

Net signal = (Std-NC)/NC×100

3) Calculate CV (%):

CV (%) = Standard Deviation/Mean Ratio × 100%

[Data Example]

The following data cannot replace the data obtained in experiments and is only for illustration; results may vary depending on the plate reader.

Note: Recommended microplate (384-well plate, white, shallow well)