Toll-like receptor assays
Methods of identifying compounds that modulate the interaction between a TLR and a molecule that interacts with the TLR by direct binding or by inclusion in a complex that associates with the TLR are described. Methods of identifying molecules that interact with a TLR are also described.
1. A method of identifying a compound that modulates binding of a cognate ligand to Toll-like receptor 2 (TLR2), the method comprising:
(a) providing a sample comprising a chimeric polypeptide comprising a TLR2 polypeptide or ligand-binding fragment thereof, and a fluorescent protein;
(b) contacting the sample with a cognate TLR2 ligand and a test compound, thereby forming a test sample;
(c) incubating the test sample for a time and under conditions sufficient for the TLR2 ligand to bind to the TLR2 polypeptide in the absence of the test compound; and
(d) determining binding between the TLR2 polypeptide and the TLR2 ligand in the test sample,
wherein the TLR2 ligand is bound to a bead and wherein a difference in binding in the test sample compared to a control indicates that the test compound is a candidate compound for modulating binding of a cognate ligand to TLR2.
2. The method of claim 1 , wherein the TLR2 ligand or the TLR2 polypeptide is biotinylated.
3. The method of claim 1 , wherein the fluorescent protein is Green fluorescent protein (GFP) or a fluorescent variant thereof.
4. The method of claim 1 , wherein the TLR2 ligand is biotinylated and the bead comprises avidin, streptavidin, or a deglycosylated form of avidin.
5. The method of claim 1 , wherein the TLR2 polypeptide is bound to a solid surface.
6. The method of claim 5 , wherein the TLR2 polypeptide is biotinylated and the solid surface comprises avidin, streptavidin, or a deglycosylated form of avidin.
7. The method of claim 1 , wherein one or more of the TLR2 polypeptide and the TLR2 ligand is labeled.
8. The method of claim 7 , wherein the label comprises europium or allophycocyanin.
9. The method of claim 1 , wherein the binding between the TLR2 polypeptide and TLR2 ligand is detected by time resolved fluorimetry.
10. The method of claim 1 , wherein the binding between the TLR2 polypeptide and the TLR2 ligand is detected with an antibody that specifically binds to the TLR2 polypeptide.
11. The method of claim 10 , wherein the antibody is labeled with a lanthanide chelate fluorophore and time-resolved fluorimetry is used to detect the binding.
12. The method of claim 1 , wherein a test compound that decreases the binding between the TLR2 polypeptide and the TLR2 ligand is a candidate compound for inhibiting binding of cognate ligand to TLR2.
13. The method of claim 1 , wherein a test compound that increases the binding between the TLR2 polypeptide and the TLR2 ligand is a candidate compound for increasing binding of cognate ligand to TLR2.
14. The method of claim 1 , wherein the sample further comprises a Toll-like receptor 1 (TLR1) polypeptide, a Toll-like receptor 6 (TLR6) polypeptide, or both.