UniOne® TR-FRET Human IL33/ST2 Binding Kit

The reagent kit employs homogeneous time-resolved fluorescence technology (TR-FRET) for measuring the interaction between Human IL33 (Interleukin-33) and Human ST2 (also known as IL1RL1, Interleukin 1 Receptor Like 1). This method enables simple and rapid high-throughput screening of inhibitors and antibody blockers.

As illustrated, the interaction between IL33 and ST2 is detected using Eu-labeled anti-Tag1 antibody (TR-FRET donor) and Accepter-labeled Tag2 antibody (TR-FRET acceptor). The binding of IL33 and ST2 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. The positive control drug Itepekimab blocks the binding of IL33 and ST2, preventing FRET signal generation. The stronger the blocking effect of the screened drug on the IL33-ST2 interaction, the lower the signal. The signal is proportional to the degree of IL33-ST2 interaction. No washing steps are required.

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1. Reagent Preparation

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

Table 1. Reagent Preparation

1.2 Sample Serial Dilution

Using Itepekimab as an example, dilute with 1× Detection buffer. To minimize matrix interference, it is recommended to use a solution with the same matrix as the sample. Adjust dilution based on actual sample concentration.

Table 2. Serial Dilution of Positive Control (Itepekimab) (Adjust as needed)

2. Sample Loading and Controls

2.1 Sample wells: Add sequentially to 384-well shallow plates—2 μL diluted sample, 4 μL Tag2-IL33 protein working solution, 4 μL Tag1-ST2 protein working solution, and 10 μL Detection Mix.

2.2 Maximum signal control: Add sequentially—2 μL 1× Detection buffer, 4 μL Tag2-IL33 protein working solution, 4 μL Tag1-ST2 protein working solution, and 10 μL Detection Mix.

2.3 Negative control (NC): Add sequentially—10 μL 1× Detection buffer and 10 μL Detection Mix.

After loading, centrifuge and seal with plate film. Incubate at room temperature for 2 hours.

3.Detection

Read on a TR-FRET-compatible microplate reader. Excitation wavelength: 320/340 nm; emission wavelengths: 620 nm and 665 nm.

[Data Analysis]

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

Ratio = (665/620) × 10000

2) Calculate Net signal:

Net signal = (Std - NC)/NC × 100

3) Calculate CV (%):

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

[Example Data]

The following data is for illustration only and may vary depending on the plate reader used.

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