Assay methods and systems

• US 20040033531A1
• Filed: 06/27/2003
• Published: 02/19/2004
• Est. Priority Date: 05/21/1999
• Status: Active Grant

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Technologies Corp (Revvity, Inc.)
• Inventors: Nikiforov, Theo T.
• Granted Patent: US 7,122,659 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• 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 kit, comprising:

a fluorescently labeled phosphorylatable compound; and

a polymer having multivalent metal cations associated therewith.

2. The kit of claim 1, wherein the multivalent metal cations comprise trivalent metal cations.

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

4. The kit of claim 1, wherein the phosphorylatable compound is selected from a group consisting of a serine, threonine or tyrosine substrate.

5. The kit of claim 1, wherein the multivalent metal cations are chelated to the polymer.

6. The kit of claim 1, wherein the polymer is between 5 kD and 1000 kD.

7. The kit of claim 1, further comprising a kinase enzyme.

8. The kit of claim 1, further comprising ATP.

9. The kit of claim 1, further comprising a buffer solution.

10. The kit of claim 1, wherein the multivalent metal cations comprise Fe³⁺.

11. The kit of claim 1, further comprising a multiwell plate.

12. A kit, comprising:

a fluorescently labeled phosphorylatable compound; and

a binding component having multivalent metal cations associated therewith.

13. The kit of claim 12, wherein the multivalent metal cations comprise trivalent metal cations.

14. The kit of claim 12, wherein the multivalent metal cations are selected from a group consisting of Fe 3+, Ca²⁺, Ni²⁺ and Zn²⁺.

15. The kit of claim 12, wherein the phosphorylatable compound is selected from a group consisting of a serine, threonine or tyrosine substrate.

16. The kit of claim 12, wherein the binding component comprises a polymer.

17. The kit of claim 16, wherein the multivalent metal cations are chelated to the polymer.

18. The kit of claim 12, wherein the polymer is between 5 kD and 1000 kD.

19. The kit of claim 12, further comprising a kinase enzyme.

20. The kit of claim 12, further comprising ATP.

21. The kit of claim 12, further comprising a buffer solution.

22. The kit of claim 12, further comprising a multiwell plate.