Assay methods and systems

• US 6,689,565 B2
• Filed: 01/24/2002
• Issued: 02/10/2004
• 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:
  US10/057,812
• Publication Number:
  US 20020197619A1
• Time in Patent Office: 747 Days
• Field of Search: 435/4, 435/6, 435/7.1, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 204/155
• US Class Current: 435/6.14
• 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 first reagent which comprises a fluorescent label;

a second reagent which reacts with the first reagent to produce a fluorescent product having a substantially different charge from the first reagent;

a polycationic component that binds to the fluorescent product to change a level of fluorescence polarization of the fluorescent product.

2. The kit of claim 1, wherein the first reagent comprises a phosphorylatable compound, and the product comprises a phosphorylated first reagent.

3. The kit of claim 1, wherein the second reagent comprises a kinase enzyme.

4. The kit of claim 1, wherein the first reagent comprises a phosphorylatable compound, and the product comprises a dephosphorylated first reagent.

5. The kit of claim 1, wherein the phosphorylatable compound comprises a peptide that comprises a phosphorylation site.

6. The kit of claim 1, wherein the second reagent comprises a phosphatase enzyme.

7. The kit of claim 1, wherein the first reagent comprises a fluorescently labeled phosphorylated peptide.

8. The kit of claim 1, wherein the first reagent comprises a first polypeptide sequence, and the product comprises a fragment of the polypeptide sequence, the fragment having a different charge than the first polypeptide sequence.

9. The kit of claim 1, wherein the second reagent comprises a proteolytic reagent.

10. The kit of claim 9, wherein the proteolytic reagent comprises a protease enzyme.

11. The kit of claim 1, wherein the polycationic component associates with the product.

12. The kit of claim 11, wherein the polycationic component associates with the product in a charge-dependent manner.

13. The kit of claim 1, wherein the polycationic component bears a charge that is opposite to a charge of the product.

14. The kit of claim 1, wherein the polycationic component has polyvalent metal ions associated therewith.

15. The kit of claim 14, wherein the polyvalent metal ions are selected from a group consisting of Fe³⁺, Ca²⁺, Ni²⁺, and Zn²⁺.

16. The kit of claim 1, wherein the polycationic component is a polyamino acid.

17. The kit of claim 16, wherein the polyamino acid comprises a protein that associates with the fluorescent product in a charge dependent manner.

18. The kit of claim 16, wherein the polyamino acid is selected from polylysine, polyhistidine, polyarginine, or copolymers thereof.

19. A kit, comprising:

a first reagent comprising a fluorescent label;

a kinase enzyme that transfers a phosphate from ATP to the first reagent to produce a phosphorylated florescent product;

a polymer having multivalent metal cations associated therewith, wherein the metal cations bind the polymer to the fluorescent product; and

instructions for;

mixing the fluorescent product and the polymer in a reaction mixture; and

measuring a level of fluorescence polarization of the reaction mixture, wherein the polymer is sufficiently large to induce a shift in an amount of polarized fluorescence emitted from the reaction mixture when the multivalent metal cations bind the polymer to the fluorescent product.

20. The kit of claim 19, wherein the first reagent comprises a peptide that comprises a phosphorylation site.

21. The kit of claim 19, wherein the first reagent comprises a fluorescently labeled phosphorylated peptide.

22. The kit of claim 19, wherein the multivalent metal cations are selected from a group consisting of Fe³⁺, Ca²⁺, Ni²⁺ and Zn⁺.

23. The kit of claim 19, wherein the instructions include instructions for mixing the first reagent and the kinase enzyme in the presence of ATP to produce the phosphorylated fluorescent product.

24. The kit of claim 19, wherein the instructions include instructions for mixing the first reagent, the kinase enzyme and the polymer in the reaction mixture.

25. The kit of claim 19, wherein the instructions include instructions for mixing the first reagent with the produce the fluorescent product prior to mixing the fluorescent product and the polymer.

26. A kit, comprising:

a phosphorylatable compound that comprises a fluorescent label;

a kinase enzyme that transfers a phosphate from ATP to the phosphorylatable compound to produce a phosphorylated fluorescent product;

a polymer having multivalent metal cations associated therewith, wherein the metal cations bind the polymer to the phosphorylated fluorescent product.

27. The kit of claim 26, wherein the multivalent metal cations comprise trivalent metal cations.

28. The kit of claim 26, wherein the multivalent metal cations are selected from a group consisting of Fe³⁺, Ca²⁺, Ni²⁺ and Zn²⁺.

29. The kit of claim 26, wherein the phosphorylatable compound is selected from a group consisting of a serine, threonine or tyrosine substrate.

30. The kit of claim 29, wherein the multivalent metal cations are chelated to the polymer.

31. The kit of claim 26, wherein the polymer is between 5 kD and 1000 kD.