Biodetectors targeted to specific ligands

• US 6,638,752 B2
• Filed: 10/30/1998
• Issued: 10/28/2003
• Est. Priority Date: 04/19/1996
• Status: Expired due to Term

• Current Assignee: Xenogen Corporation (Revvity, Inc.)
• Original Assignee: Xenogen Corporation (Revvity, Inc.)
• Inventors: Contag, Christopher H., Benaron, David A., Contag, Pamela R.
• Primary Examiner(s): Smith, Lynette R. F.
• Assistant Examiner(s): Zeman, Robert A.
• Application Number:
  US09/183,566
• Publication Number:
  US 20020086424A1
• Time in Patent Office: 1,824 Days
• Field of Search: 435/69.7, 435/69.6, 435/252.3, 435/69.1, 435/8
• US Class Current: 435/252.3
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/56988 HIV or HTLV

Abstract

The present invention relates to biodetectors for detecting and quantifying molecules in liquid, gas, or matrices. More specifically, the present invention relates to biodetectors comprising a molecular switching mechanism to express a reporter gene upon interaction with target substances. The invention further relates to methods using such biodetectors for detecting and quantifying selected substances with high specificity and high sensitivity.

Claims

1. A biodetector for the detection of a selected substance, said biodetector comprising:

a signal converting element, comprising (i) an extracellular ligand-specific binding domain which specifically binds said selected substance, wherein said ligand-specific binding domain comprises an epitope-binding fragment of an antibody, and (ii) an intracellular signal transforming domain of PhoQ, wherein binding of said substance to said epitope-binding fragment of said ligand-specific binding domain activates said intracellular signal transforming domain providing an activated intracellular signal transforming domain;

a transducer, wherein (i) said transducer has an inactive form and an active form which are distinct from each other, and (ii) said activated intracellular signal transforming domain converts said inactive form of said transducer into said active form of said transducer;

a transcription control element comprising the phoN promoter, wherein expression mediated by said transcription control element is activated by said active form of said transducer; and

a reporter gene operatively linked to said transcription control element, wherein expression of said reporter gene mediated by said transcription control element causes expression of a reporter gene product that provides a detectable signal, wherein said detectablen signal is detected optically by bioluminescence detection or fluorescence detection.

2. The biodetector of claim 1, wherein said detectable signal is detectable by bioluminescence detection.

3. The biodetector of claim 2, wherein said reporter gene encodes a luciferase.

4. The biodetector of claim 3, wherein said luciferase is encoded by a luc gene.

5. The biodetector of claim 3, wherein said luciferase is encoded by a lux gene.

6. The biodetector of claim 1, wherein said epitope-binding fragment of an antibody is selected from the group consisting of a single chain variable fragment (ScFv), a Fab fragment, a F(ab′

)₂ fragment, an epitope-binding fragment of a polyclonal antibody, an epitope-binding fragment of a monoclonal antibody, an epitope-binding fragment of a humanized antibody, an epitope-binding fragment of a chimeric antibody, and an epitope-binding fragment of an anti-idiotypic antibody.

7. The biodetector of claim 6, wherein said epitope-binding fragment of an antibody comprises a single chain variable fragment (ScFv).

8. The biodetector of claim 1, wherein said biodetector comprises an intact bacterial cell.

9. The biodetector of claim 8, wherein said biodetector comprises a Gram-positive bacterial cell.

10. The biodetector of claim 9, wherein said bacterial cell is selected from the group consisting of Streptococcus, Staphylococcus, Listeria, Clostridium, Bacillus, and Corynebacteria.

11. The biodetector of claim 8, wherein said biodetector comprises a Gram-negative bacterial cell.

12. The biodetector of claim 11, wherein said bacterial cell is selected from the group consisting of Escherichia, Salmonella, Pseudomonas, Helicobacter, Shigella, Proteus, Bordetella, Neisseria, Haemophilus, Bacteriodes, Vibrio, Brucella, Campylobacter, Klebsiella, and Yersinia.

13. A library of biodetectors, comprising

at least about 1000 biodetectors of claim 8, wherein the extracellular ligand-specific binding domain of each of said biodetectors comprises a different antibody fragment,

a reporter gene operatively linked to said transcription control element, wherein expression of said reporter gene mediated by said transcription control element causes expression of a reporter gene product that provides a detectable signal, wherein said detectable signal is detected optically by means selected from the group consisting of bioluminescence detection and fluorescence detection.

14. An ordered array of the library of biodetectors of claim 13.

15. The biodetector of claim 1, wherein said detectable signal is detectable by fluorescence detection.

16. The biodetector of claim 15, wherein said reporter gene encodes a fluorescent protein.

17. The biodetector of claim 8, wherein said bacterial cell comprises a luciferase operon.

18. The biodetector of claim 1, wherein said bioluminescence detection or fluorescence detection is performed using a charge coupled device camera.