Assay methods and systems

• US 20030175815A1
• Filed: 03/26/2003
• Published: 09/18/2003
• Est. Priority Date: 05/21/1999
• Status: Active Grant

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Nikiforov, Theo T.
• Granted Patent: US 7,150,966 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• 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 fluorescently labeled compound is phosphorylated, the method comprising:

providing the compound in a mixture with a polymer having multivalent metal cations associated therewith which polymer is sufficiently large to induce a shift in an amount of polarized fluorescence emitted from the mixture when the multivalent metal cations bind the polymer to the compound; and

monitoring the amount of polarized fluorescence emitted from the mixture to determine whether the multivalent metal cations bind the polymer to the compound, such binding being indicative that the compound is phosphorylated.

2. The method of claim 1, wherein the multivalent metal cations are selected from the group consisting of Fe³⁺, Ca²⁺, Ni²⁺ and Zn²⁺.

3. The method of claim 1, wherein the multivalent metal cations are chelated to the polymer.

4. The method of claim 1, wherein the polymer is between 5 kD and 1000 kD.

5. 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.

6. The method of claim 5, wherein prior to the providing step, the compound is contacted with a kinase enzyme in the presence of a phosphate donor group.

7. The method of claim 5, wherein prior to the providing step, the compound is contacted with a phosphatase enzyme that removes a phosphate group from the compound.

8. The method of claim 5, 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.

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

10. The method of claim 1, wherein the compound comprises a serine, tyrosine, or threonine substrate.

11. The method of claim 1, wherein the multivalent metal cations bind to the phosphorylated compound based at least partially on a difference in charge between the phosphorylated compound and the multivalent metal cations.

12. The method of claim 11, wherein the multivalent metal cations bind to the phosphorylated compound based at least partially on an affinity between the metal cations and a phosphate group associated with the phosphorylated compound.

13. The method of claim 1, wherein the multivalent metal cations bind to the phosphorylated compound based at least partially on an affinity between the metal cations and a phosphate group associated with the phosphorylated compound.

14. The method of claim 1, wherein the multivalent metal cations comprise trivalent metal cations.

15. The method of claim 14, wherein the trivalent metal cations comprise Fe³⁺.