Molecular modification assays

• US 7,632,651 B2
• Filed: 09/30/2005
• Issued: 12/15/2009
• Est. Priority Date: 09/15/1997
• Status: Expired due to Term

• Current Assignee: Molecular Devices
• Original Assignee: MDS Analytical Technologies US Incorporated (Danaher Corporation)
• Inventors: Boge, Annegret, Sportsman, J. Richard, Gaudet, Elizabeth, Yi, George G.
• Primary Examiner(s): Haq; Shafiqul
• Application Number:
  US11/241,872
• Publication Number:
  US 20060121544A1
• Time in Patent Office: 1,537 Days
• Field of Search: 436/544, 436/546, 436/164, 436/172, 436/800, 435/7.1
• US Class Current: 435/7.4
• CPC Class Codes: B01L 3/50853 with covers or lids
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12Q 1/25 involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/0405 manipulating closing or opening means, e.g. stoppers, screw caps, lids or covers
• CPC Class Codes: G01N 2035/0425 Stacks, magazines or elevators for plates
• CPC Class Codes: G01N 2035/0462 Buffers [FIFO] or stacks [LIFO] for holding carriers between operations
• CPC Class Codes: G01N 21/6445 Measuring fluorescence polarisation
• CPC Class Codes: G01N 2333/9121 with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• 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
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 35/028 having reaction cells in the form of microtitration plates
• CPC Class Codes: G01N 35/1011 : Control of the position or alignment of the transfer device
• CPC Class Codes: G01N 35/1074 : arranged in a two-dimensional array

Abstract

Systems for detecting molecular modifications and the presence and/or activity of enzymes and/or other agents involved in facilitating or otherwise regulating such modifications.

Claims

1. A method of detecting the activity of an enzyme that catalyzes formation of a product from a substrate, the method comprising:

contacting the substrate with the enzyme and with a binding partner to form a sample mixture, the binding partner binding selectively either to the substrate relative to the product or to the product relative to the substrate, the binding partner including at least one metal that is required for selective binding of the binding partner to the substrate or to the product via at least one phosphate moiety of the substrate or product, where the substrate and/or product includes an energy transfer donor and the binding partner includes an energy transfer acceptor, or the substrate and/or product includes an energy transfer acceptor and the binding partner includes an energy transfer donor, such that luminescence energy transfer can occur between the donor and the acceptor when the binding partner binds to the substrate or the product, where the enzyme is selected from the group consisting of kinases, phosphatases, nucleotide cyclases, and nucleotide phosphodiesterases, where the binding partner includes a lanthanide chelate and the metal, where the lanthanide chelate includes an organic chelator and a lanthanide, and where the organic chelator includes at least one metal-binding functional group that associates the lanthanide chelate with the metal;

exposing the sample mixture to light capable of inducing luminescence from the energy transfer donor;

measuring a detectable luminescence energy transfer response from the sample mixture, without separating the bound substrate or product from the unbound substrate or product, where the detectable luminescence energy transfer response is indicative of the extent of binding between the substrate or product and the binding partner; and

correlating the response with the activity of the enzyme.

2. The method of claim 1, where the metal is selected from the group consisting of aluminum, iron, gallium, europium, and terbium.

3. The method of claim 2, where the metal is Ga(III).

4. The method of claim 2, where the metal is Fe(III).

5. The method of claim 1, where measuring the detectable luminescence energy transfer response includes measuring luminescence intensity.

6. The method of claim 1, where the energy transfer acceptor is luminescent.

7. The method of claim 1, where the energy transfer acceptor is a quencher.

8. The method of claim 1, where the binding partner includes the energy transfer donor.

9. The method of claim 1, the energy transfer donor having a luminescence lifetime, where the luminescence lifetime is at least about 400 nanoseconds.

10. The method of claim 1, where the energy transfer donor comprises the lanthanide chelate.

11. The method of claim 1, where the metal-binding functional group is a phosphate, sulfonic acid, or carboxylic acid functional group.

12. The method of claim 11, where the metal-binding functional group is a phosphate functional group.

13. The method of claim 1, where the organic chelator includes a sensitizer moiety.

14. The method of claim 13, where the organic chelator has the formula

15. The method of claim 14, where Z is a sensitizer moiety having the formula

16. The method of claim 10, where the energy transfer donor includes a complexed lanthanide ion that is selected from terbium, europium, dysprosium, or samarium.

17. The method of claim 1, where the substrate is a polypeptide, and where the substrate and product are related by phosphorylation or dephosphorylation of the polypeptide.

18. The method of claim 1, further comprising:

contacting the substrate and enzyme with a candidate compound; and

determining the ability of the candidate compound to enhance or inhibit enzyme activity by its effects on the response.

19. The method of claim 1, further comprising:

providing a sample holder having a plurality of sample sites supporting a corresponding plurality of sample mixtures; and

repeating the steps of contacting, exposing, measuring, and correlating for the plurality of sample mixtures.