OX40/OX40L Inhibitor Binding Assay Kit

Background

OX40 (CD134; TNFRSF4) is a T-cell co-stimulatory receptor that belong to the tumor necrosis factor receptor superfamily (TNFRSF). Binding of OX40 to OX40L (CD252, CD134L, TNFSF4) triggers signal transduction pathways to activate immune response and regulate T-cell activation, proliferation, differentiation, expansion, and survival. OX40 agonists are being developed and tested in clinical trials for cancer treatment. OX40 antagonists, by inhibiting OX40, can dampen T cell activity, potentially reducing inflammation and autoimmune responses. Modulating the OX40/OX40L pathway have implications for treating inflammatory and autoimmune diseases, as well as cancer immunotherapy.

Assay Principle

The OX40-OX40L binding assay kit is a TR-FRET based assay, which is designed to detect the binding status between OX40 and OX40L. Tag6-OX40 and Tag7-OX40L are included in this assay kit. Binding of Tag6-OX40 to Tag7-OX40L 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 OX40-OX40L binding will reduce the HTRF signal. OX40/OX40L Inhibitor Binding Assay Kit

Application

High throughput screening of compounds that inhibit the binding between OX40 and OX40L 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
• Prepare the inhibitor compound solution
• Prepare OX40 solution
• Add inhibitor
• Prepare OX40L solution
• Prepare dye solution
• Incubate the reaction at room temperature for 1 hour.
• Measure fluorescent intensity
• Excitation wavelength at 340 nm and emission at 620 nm.
• Excitation wavelength at 340 nm and emission at 665 nm.
• Calculate sample HTRF signal of each well.
• Calculate percentage activity

Assay Protocol

1. Step 1. Prepare OX40 solution Thaw OX40 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: OX40 protein is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the OX4L protein 80-fold (1 µL OX40L + 79 µL 1X assay buffer). Add 4 µl of diluted protein solution to each well.
2. Step 2. 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).
3. Step 3. Prepare OX40L solution Thaw OX40 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: OX40L protein is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the OX40L protein 600-fold (1 µL OX40L + 599 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.
4. Step 4. Prepare dye solution OX40/OX40L Inhibitor Binding Assay Kit 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 + 1 µl of fluorescence- labeled anti-Tag7 antibody + 198 µl of assay buffer. Add 10 µl of this dye mixture to each well.
5. Step 5. Incubate the reaction at room temperature for 1 hour.
6. Step 6. Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:
7. Step 7. Excitation wavelength at 340 nm and emission at 620 nm.
8. Step 8. Excitation wavelength at 340 nm and emission at 665 nm.

Data Analysis

Calculate sample HTRF signal of each well. Calculate percentage activity In the absence of the compound (positive control), the sample signal (P) is defined as 100% activity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% activity. The percent activity in the presence of each compound is calculated according to the 4 µl 2 µl 6 µl 4 µl 10 µl 20 µl following equation: % activity = (S-N)/(P-N) X100, where S= the sample signal in the presence of the compound.

Assay Validation

Biological Pathway / Process

Immune Co-stimulatory Pathway (T cell activation)

Therapeutic / Disease Area

Oncology / Immunotherapy