Toll-like receptor assays

• US 8,124,349 B2
• Filed: 01/06/2006
• Issued: 02/28/2012
• Est. Priority Date: 12/16/2003
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Latz, Eicke, Visintin, Alberto, Golenbock, Douglas T.
• Primary Examiner(s): Shafer, Shulamith H
• Application Number:
  US11/326,963
• Publication Number:
  US 20060099650A1
• Time in Patent Office: 2,244 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: A61K 49/0008 Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 33/5035 on sub-cellular localization
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

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.

Claims

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.