Methods for rapid and efficient protein cross-linking

• US 6,613,582 B1
• Filed: 05/25/2000
• Issued: 09/02/2003
• Est. Priority Date: 05/25/1999
• Status: Expired due to Fees

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Kodadek, Thomas J., Fancy, David A., Johnston, Stephen A.
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): Padmanabhan, Kartic
• Application Number:
  US09/578,950
• Time in Patent Office: 1,195 Days
• Field of Search: 435/4, 435/7.1, 435/7.92, 435/174-176, 435/6, 435/7.9, 435/177, 435/961, 435/964, 435/971, 436/518, 436/164, 436/172, 436/73, 436/805, 436/824, 436/84, 436/519, 436/523, 436/524, 436/525, 436/526, 436/528, 436/536, 436/806, 436/823, 436/905
• US Class Current: 436/524
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: Y10S 435/961 including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 435/964 including enzyme-ligand conjugate production, e.g. reducing rate of nonproductive linkage
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/823 Immunogenic carrier or carrier per se
• CPC Class Codes: Y10S 436/824 Immunological separation techniques
• CPC Class Codes: Y10S 436/905 Photochemical activation of reactions

Abstract

The present application describes novel uses of ruthenium bipyridyls or palladium porphyrins as photo-activatable crosslinking agents. Crosslinking can be between any two molecules including peptides, proteins, or compounds. Crosslinking occurs in the presence of an electron donor such as ammonium persulfate, and requires only moderate intensity visible light. Crosslinking can be between peptides, polypeptides or lead candidate compounds to unknown target molecules. Reagents utilyzing ruthenium bipyridyls and palladium porphyrins crosslinkers for use in diagnostic and detection scenarios are also disclosed.

Claims

1. A method of covalently bonding a first molecule and a second molecule, wherein said first molecule is a protein, comprising:

(i) providing said first protein molecule and said second molecule in proximity to each other;

(ii) contacting said molecules with a metal-ligand complex; and

(iii) subjecting said complex to light,

whereby said light photoactivates said complex, and said complex causes direct bonding of said first protein molecule to said second molecule without incorporation of the metal-ligand complex.

2. The method of claim 1, wherein said protein is an antibody.

3. The method of claim 1, wherein said protein is a peptide.

4. The method of claim 3, wherein said peptide is conjugated to a metal.

5. The method of claim 1, wherein said second molecule is a protein.

6. The method of claim 1, wherein said complex comprises a detectable agent.

7. The method of claim 1, wherein said complex comprises a palladium(II) porphyrin and an electron acceptor.

8. The method of claim 1, wherein said complex comprises Ruthenium (II)(bypyridyl) and an electron acceptor.

9. The method of claims 7 or 8, wherein said electron acceptor is ammonium persulfate or a cobalt(III) complex.

10. The method of claim 1, wherein said light has a wavelength of >

400 nm.

11. The method of claim 1, wherein said first and said second molecules are located in a cell.

12. The method of claim 1, further comprising isolating the complex-linked first and second molecules.

13. The method of claim 12, further comprising identifying said first molecule.

14. The method of claim 12, further comprising identifying said second molecule.