Fluorescent polarization assays involving multivalent metal ions and systems

• US 6,699,655 B2
• Filed: 05/24/2001
• Issued: 03/02/2004
• Est. Priority Date: 05/21/1999
• Status: Expired due to Term

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Nikiforov, Theo T.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/865,044
• Publication Number:
  US 20020146703A1
• Time in Patent Office: 1,013 Days
• Field of Search: 435/4, 435/6, 435/7.1, 435/91.1, 435/91.2, 435/183, 336/22.1, 336/23.7, 336/24-- 2, 422/68.1, 436/536, 530/350
• US Class Current: 435/4
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• 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 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

Methods, systems, kits for carrying out a wide variety of different assays that comprise providing a first reagent mixture which comprises a first reagent having a fluorescent label. A second reagent is introduced into the first reagent mixture to produce a second reagent mixture, where the second reagent reacts with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent. A polyion is introduced into at least one of the first and second reagent mixtures, and the fluorescent polarization in the second reagent mixture relative to the first reagent mixture is determined, this fluorescent polarization being indicative of the rate or extent of the reaction.

Claims

1. A method of determining whether a compound is phosphorylated, the compound comprising a fluorescent label, the method comprising:

providing the compound in a mixture with a binding component, wherein the binding component comprises multivalent metal ions and is sufficiently large to induce a shift in an amount of polarized fluorescence emitted from the compound, when the binding component binds the compound; and

detecting whether the binding component binds to the compound by monitoring the amount of polarized fluorescence emitted from the mixture, binding of the binding component to the compound being indicative that the compound is phosphorylated.

2. The method of claim 1, wherein the binding component comprises a polymeric material having multivalent metal ions associated therewith.

3. The method of claim 2, wherein the multivalent metal ions are selected from the group consisting of Fe³⁺, Ca²⁺, Ni²⁺ and Zn²⁺.

4. The method of claim 2, wherein the multivalent metal ions are chelated to the polymeric material.

5. The method of claim 1, wherein the binding component is between 5 kD and 1000 kD.

6. The method of claim 1, wherein prior to the providing step, the compound is contacted with an enzyme that either adds or removes a phosphate group to or from the compound under conditions suitable for addition or removal of a phosphate group, respectively.

7. The method of claim 6, wherein prior to the providing step, the compound is contacted with a kinase enzyme in the presence of a phosphate donor group.

8. The method of claim 6, wherein prior to the providing step, the compound is contacted with an enzyme that removes a phosphate group from the compound.

9. The method of claim 6, wherein prior to the providing step, the compound is contacted with an enzyme that either adds or removes a phosphate group to or from the compound, in the presence of at least a first test compound.

10. The method of claim 1, wherein the compound comprises a polypeptide.

11. An assay system, comprising:

a first reaction vessel having disposed therein a first reaction mixture comprising a phosphorylated first fluorescent compound, and a binding component, wherein the binding component comprises multivalent metal ions and is sufficiently large to induce a shift in an amount of polarized fluorescence emitted by the compound when bound by the binding component; and

a fluorescence polarization detector positioned in sensory communication with the first mixture in the reaction vessel.

12. The assay system of claim 11, wherein the reaction vessel comprises a well in a multiwell plate.

13. The assay system of claim 11, wherein the binding component comprises a polymeric material.

14. The assay system of claim 11, wherein the binding component is between 5 kD and 1000 kD.

15. The assay system of claim 11, wherein the multivalent metal ions are selected from the group consisting of Fe³⁻

, Ca²⁻

, Ni²⁻

and Zn²⁻

.

16. The assay system of claim 15, wherein the binding component comprises chelating groups for chelating multivalent metal ions to the binding component.

17. The assay system of claim 11, further comprising at least a second reaction vessel having a second mixture disposed therein, the second mixture comprising an unphosphorylated first fluorescent compound and the binding component.

18. The assay system of claim 11, wherein the first reaction mixture comprises an enzyme for adding a phosphate group to the first fluorescent compound.

19. The assay system of claim 11, wherein the first reaction mixture comprised an enzyme for removing a phosphate group from a phosphorylated first compound.