SEMI-SYNTHETIC ANTIBODIES AS RECOGNITION ELEMENTS

• US 20110229415A1
• Filed: 08/06/2008
• Published: 09/22/2011
• Est. Priority Date: 08/06/2007
• Status: Abandoned Application

• Current Assignee: University of Kentucky
• Original Assignee: University of Kentucky
• Inventors: Bachas, Leonidas G., Haley, Boyd, Joel, Smita, Daunert, Sylvia
• Application Number:
  US12/672,397
• Publication Number:
  US 20110229415A1
• Time in Patent Office: Days
• US Class Current: 424/9.6
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

The presently-disclosed subject matter is directed to biosensors for detecting molecules of interest, and systems and methods for using same. The biosensors include an antibody and a probe covalently-linked to the antibody. The antibody has an antigen-binding site that selectively binds the molecule of interest and a purine-binding site, which is at a location distinct from that of the antigen-binding site. The probe includes a purine molecule, which is covalently bound at the purine-binding site to the antibody, and a label linked to the purine molecule. Upon binding of the molecule of interest to the biosensor antigen-binding site, the biosensor undergoes a conformational change, which detectably alters a signal of the label such that the molecule of interest can be detected.

Claims

1. A biosensor for detecting a molecule of interest, comprising:

(a) a probe, including a purine molecule and a fluorophore bound to the purine molecule; and

(b) an antibody, including an antigen-binding site that selectively binds the molecule of interest and a purine-binding site, wherein the probe is covalently bound at the purine molecule to the purine-binding site,

wherein binding of the molecule of interest to the antibody causes a conformational change in the antibody, which detectably alters a fluorescence intensity of the fluorophore such that the molecule of interest can be detected.

2. The biosensor of claim 1, wherein the purine molecule is a nucleotide or a nucleoside.

3. The biosensor of claim 1, wherein the purine molecule is an adenine or a guanine.

4. The biosensor of claim 3, wherein the purine molecule is an ATP analog or a GTP analog.

5. The biosensor of claim 4, wherein the purine molecule is 2-azido ATP or 8-azido ATP.

6. The biosensor of claim 1, wherein the fluorophore is an Alexa Fluor dye.

7. The biosensor of claim 6, wherein the probe is a compound of Formula II:

8. The biosensor of claim 1, wherein the antibody is a monoclonal antibody.

9. The biosensor of claim 1, wherein the purine-binding site is located within the variable domain of the antibody and at a location distinct from that of the antigen-binding site.

10. The biosensor of claim 9, wherein the purine-binding site comprises invariant amino acid residues within the variable domain.

11. The biosensor of claim 1, wherein the molecule of interest is Interleukin 6 (IL-6) or osteonectin.

12. The biosensor of claim 1, wherein the fluorescence intensity can be correlated to the concentration of the molecule of interest in a sample.

13. A system for continuous in vivo detection of a molecule of interest in a body of a subject, comprising:

(a) a detection and data collection device; and

(b) a biosensor capable of recognizing the molecule of interest, the biosensor being operably connected with the data and collection device and including;

i. a probe having a purine molecule and a fluorophore bound to the purine molecule; and

ii. an antibody having an antigen-binding site that selectively binds the molecule of interest and a purine-binding site, wherein the probe is covalently bound at the purine molecule to the purine-binding site,

wherein binding of the molecule of interest to the antibody causes a conformational change in the antibody, which detectably alters a fluorescence intensity of the fluorophore such that the molecule of interest can be detected.

14. The system of claim 13, wherein the purine molecule is a nucleotide or a nucleoside.

15. The system of claim 14, wherein the purine molecule is an adenine or a guanine.

16. The system of claim 15, wherein the purine molecule is an ATP analog or a GTP analog.

17. The system of claim 16, wherein the purine molecule is 2-azido ATP or 8-azido ATP.

18. The system of claim 13, wherein the fluorophore is an Alexa Fluor dye.

19. The system of claim 18, wherein the probe is a compound of Formula II:

20. The system of claim 13, wherein the antibody is a monoclonal antibody.

21. The system of claim 13, wherein the purine-binding site is located within the variable domain of the antibody and at a location distinct from that of the antigen-binding site.

22. The system of claim 21, wherein the purine-binding site comprises invariant amino acid residues within the variable domain.

23. The system of claim 13, wherein the molecule of interest is Interleukin 6 (IL-6) or osteonectin.

24. The system of claim 13, wherein the fluorescence intensity can be correlated to the concentration of the molecule of interest in a sample.

25. A method for detecting a molecule of interest, comprising:

(a) contacting a biosensor with a sample comprising the molecule of interest, the biosensor including;

i. a probe, including a purine molecule and a fluorophore bound to the purine molecule; and

ii. an antibody, including an antigen-binding site that selectively binds the molecule of interest and a purine-binding site, wherein the probe is covalently bound at the purine molecule to the purine-binding site;

(b) binding the molecule of interest to the antibody, thereby resulting in a conformational change in the antibody, which detectably alters a fluorescence intensity of the fluorophore;

(c) detecting as a signal the altered fluorescence intensity of the fluorophore; and

(d) collecting and displaying the signal with a detection and data collection device, to thereby detect the molecule of interest.

26. The method of claim 25, wherein the molecule of interest is continuously detected.

27. The method of claim 25, wherein the molecule of interest is within an in vitro sample.

28. The method of claim 25, wherein the molecule of interest is within a blood stream in a body of a subject.

29. The method of claim 25, wherein the biosensor is implanted in a body of a subject.

30. The method of claim 25, wherein the purine molecule is a nucleotide or a nucleoside.

31. The method of claim 30, wherein the purine molecule is an adenine or a guanine.

32. The method of claim 31, wherein the purine molecule is an ATP analog or a GTP analog.

33. The method of claim 32, wherein the purine molecule is 2-azido ATP or 8-azido ATP.

34. The method of claim 25, wherein the fluorophore is an Alexa Fluor dye.

35. The method of claim 34, wherein the probe is a compound of Formula II:

36. The method of claim 25, wherein the antibody is a monoclonal antibody.

37. The method of claim 25, wherein the purine-binding site is located within the variable domain of the antibody and at a location distinct from that of the antigen-binding site.

38. The method of claim 37, wherein the purine-binding site comprises invariant amino acid residues within the variable domain.

39. The method of claim 25, wherein the molecule of interest is Interleukin 6 (IL-6) or osteonectin.

40. The method of claim 25, wherein the fluorescence intensity correlates to the concentration of the molecule of interest in a sample.