UniOne® TR-FRET Human IL23/IL23R Binding Kit

The kit utilizes homogeneous time-resolved fluorescence technology (TR-FRET) to measure the interaction between IL23 (IL-23 alpha & IL-12 beta Heterodimer) and IL23R. This method enables simple, rapid, and high-throughput screening of inhibitors and antibody blockers.

As shown in the figure, the interaction between IL23 and IL23R is detected using Eu-labeled anti-Tag1 antibody (TR-FRET donor) and Ac-labeled anti-Tag2 antibody (TR-FRET acceptor). The binding of IL23 to IL23R brings the donor and acceptor antibodies into proximity, allowing the donor's excitation to trigger fluorescence resonance energy transfer (FRET) to the acceptor, resulting in a specific emission signal at 665 nm. The Icotrokinra inhibitor blocks the binding of IL23 to IL23R, preventing FRET signal generation. The stronger the blocking effect of the screened drug, the lower the signal. This specific signal is proportional to the extent of the IL23-IL23R interaction. The homogeneous assay is simple to perform and requires no washing steps.

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Note: Aliquot all components immediately after first thaw and store at recommended temperatures. Avoid storing diluted solutions 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 a 384-well low-profile plate is 20 μL (reagent volumes per reaction are shown in the table). Calculate the required volume before preparation and prepare as needed; the following preparation is for reference only, using 500 tests as an example.

Table 1. Reagent Preparation

1.2 Serial Dilution of Test Samples

Taking Icotrokinra as an example, the diluent is 1× Detection buffer. To minimize matrix effect interference, it is recommended to use a solution with the same matrix as the test sample for dilution; and the test sample should be adjusted according to its actual concentration.

Table 2. Serial Dilution of Positive Control Icotrokinra (adjust according to actual conditions)

2. Sample Addition and Controls

2.1 Order of addition for experimental wells: Add 2 μL of test sample, 4 μL of Tag2-IL23R protein working solution, 4 μL of Tag1-23 protein working solution, and 10 μL of mixed Mix sequentially into the 384-well low-profile plate;

2.2 Maximum control: 2 μL of 1× Detection buffer diluent, 4 μL of Tag2-IL23R protein working solution, 4 μL of Tag1-23 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.

Note: It is recommended to incubate the inhibitor with the inhibited component first. Since Icotrokinra is an inhibitor of IL23R, Icotrokinra and IL23R are incubated first in this experiment.

3.Detection

Detect using a TR-FRET-compatible microplate reader. Excitation wavelength is 320/340 nm, and emission wavelengths are detected at 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 and 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 data obtained from experiments and is provided only as an example. Results may vary depending on the plate reader.

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