Assay method utilizing photoactivated chemiluminescent label

• US 5,340,716 A
• Filed: 06/20/1991
• Issued: 08/23/1994
• Est. Priority Date: 06/20/1991
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Products GmbH (Siemens AG)
• Original Assignee: Snytex Incorporated
• Inventors: Kirakossian, Hrair, Ullman, Edwin F., Pease, John S., Wagner, Daniel B., Daniloff, Yuri
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Schmickel, David
• Application Number:
  US07/718,490
• Time in Patent Office: 1,160 Days
• Field of Search: 435/5, 435/6, 435/7.92, 435/125, 435/7.7, 436/501
• US Class Current: 435/6.12
• CPC Class Codes: G01N 33/56977 HLA or MHC typing
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex

Abstract

Methods are disclosed for determining an analyte in a medium suspected of containing the analyte. One method comprises providing (1) a medium suspected of containing the analyte, (2) a label reagent comprising a first specific binding pair (sbp) member associated with a photochemically activatable chemiluminescent compound wherein the first sbp member is capable of binding to the analyte or to a second sbp member to form a complex related to the presence of the analyte. The method further comprises photochemically activating the chemiluminescent compound. The amount of luminescence generated by the chemiluminescent compound is detected. The amount thereof is related to the amount of analyte in the medium. Compositions and kits are also disclosed.

Claims

1. A method for determining an analyte, said method comprising:

providing in combination (1) a medium suspected of containing said analyte, (2) a label reagent comprising a first specific binding pair (sbp) member associated with a photochemically activatable chemiluminescent compound (PACC) wherein said first sbp member is capable of binding to said analyte or to a second sbp member to form a complex related to the presence of said analyte,

photochemically activating said PACC, and

detecting the amount of luminescence generated by said PACC, the amount thereof being related to the amount of analyte in said medium.

2. The method of claim 1 which is a homogenous method and wherein said photochemical activation comprises irradiation of said medium with light.

3. The method of claim 1 which is a heterogenous method and wherein said photochemical activation comprises excitation of said PACC with light.

4. The method of claim 1 wherein said photochemical activation comprises reaction of said PACC with singlet oxygen.

5. The method of claim 4 wherein said singlet oxygen is generated by activation of a photosensitizer.

6. The method of claim 5 wherein said photosensitizer is a dye.

7. The method of claim 1 wherein said detecting comprises detecting said luminescence directly.

8. The method of claim 1 wherein said detecting comprises employing a fluorescent energy acceptor.

9. The method of claim 8 wherein said fluorescent energy acceptor is bound to, or is capable of becoming bound to, an sbp member other than that of said label reagent.

10. The method of claim 1 wherein a surface is employed and an energy acceptor is employed that is bound to, or is capable of becoming bound to, said surface.

11. The method of claim 1 wherein said analyte and said first sbp member are each independently selected from the group consisting of ligands, receptors, and polynucleotides.

12. The method of claim 1 wherein said PACC contains an olefin group and one or more electron donating substituents in conjugation with said olefin group.

13. The method of claim 1 wherein said PACC is selected from the group consisting of 9-alkylidene acridans, enol ethers, 9-alkylidene xanthanes and enamines.

14. The method of claim 1 wherein said analyte is a polynucleotide.

15. The method of claim 1 wherein said first sbp member is an antibody.

16. The method of claim 1 wherein said second sbp member is analogous to said analyte.

17. The method of claim 1 wherein said second sbp member is analogous to or complementary to said analyte or to a third sbp member.

18. The method of claim 17 wherein said third sbp member is bound to a support.

19. The method of claim 1 wherein said complex becomes bound to a support.

20. The method of claim 1 wherein said second sbp member is bound to a support.

21. A method for determining an analyte, said method comprising:

combining in an assay medium (1) a medium suspected of containing an analyte, and (2) a label reagent comprising a member of a specific binding pair (sbp member) bound to a photochemically activatable chemiluminescent compound (PACC) under conditions wherein an sbp member complex involving said label reagent is formed in relation to the presence of analyte in said medium,

irradiating said assay medium with light to activate said PACC, and

examining said assay medium for a signal, the presence or intensity thereof being related to the amount of analyte in said medium.

22. The method of claim 21 wherein an energy acceptor is included in said medium and energy from said PACC is capable of activating said energy acceptor.

23. The method of claim 21 wherein said medium is irradiated with light having a wavelength between 500 nm and 950 nm.

24. The method of claim 21 wherein said assay medium comprises a second sbp member other than said analyte.

25. The method of claim 21 wherein said energy acceptor is fluorescent.

26. The method of claim 25 wherein said fluorescent energy acceptor is bound to, or is capable of becoming bound to, a second sbp member or to a surface included in said medium.

27. The method of claim 21 wherein said analyte and said sbp member are each independently selected from the group consisting of ligands, receptors, and polynucleotides.

28. The method of claim 21 wherein said PACC contains an olefin group and one or more electron donating substituents in conjugation with said olefin group.

29. The method of claim 21 wherein said PACC is selected from the group consisting of 9-alkylidene acridans, enol ethers, 9-alkylidene xanthanes and enamines.

30. The method of claim 21 wherein said sbp member is complementary to said analyte.

31. The method of claim 21 wherein said assay medium comprises a second sbp member complementary to said analyte.

32. The method of claim 31 wherein said second sbp member is bound to, or is capable of becoming bound to, a support and said support is combined with said assay medium prior to, simultaneously with, or subsequent to the addition of said label reagent.

33. The method of claim 21 wherein said sbp member is analogous to said analyte and said assay medium comprises a second sbp member complementary to said analyte and said sbp member.

34. The method of claim 33 wherein said second sbp member is bound to a support.

35. The method of claim 21 wherein said sbp member is complementary to a second sbp member complementary to said analyte and said assay medium comprises said second sbp member.

36. The method of claim 35 wherein said second sbp member is bound to a support.

37. The method of claim 21 wherein said second sbp member is analogous to or complementary to said analyte or to a third sbp member.

38. The method of claim 37 wherein said third sbp member is bound to a support and said support is combined with said assay medium prior to, simultaneously with, or subsequent to the addition of said label reagent.

39. A method for determining an analyte, said method comprising:

combining in an assay medium either simultaneously or wholly or partially sequentially (1) a medium suspected of containing an analyte, (2) a label reagent comprising a first member of a specific binding pair (sbp member) bound to a compound capable of chemiluminescence upon reaction with singlet oxygen ("said compound"), and (3) an insolubilized reagent comprising a second sbp member under conditions wherein an sbp member complex involving said label reagent and said insolubilized reagent is formed in relation to the presence of analyte in said medium,

separating said assay medium and said insolubilized reagent,

activating said compound in said medium or on said insolubilized reagent with light, and

examining said assay medium or said reagent for a signal, the presence or intensity thereof being related to the amount of analyte in said medium.

40. The method of claim 39 wherein said assay medium is irradiated with light of wavelength between 500 and 950 nm to activate said compound.

41. The method of claim 39 wherein said singlet oxygen is generated by irradiation of a dye.

42. The method of claim 41 wherein said dye is selected from the group consisting of methylene blue, rose bengal, porphyrins, and phthalocyanines.

43. The method of claim 39 wherein said analyte and said sbp members are each independently selected from the group consisting of ligands, receptors, and polynucleotides.

44. The method of claim 39 wherein said compound contains an olefin group and one or more electron donating substituents in conjugation with said olefin group.

45. The method of claim 39 wherein said compound is selected from the group consisting of 9-alkylidene acridans, enol ethers, 9-alkylidene xanthanes and enamines.

46. The method of claim 39 wherein said first sbp member is complementary to said analyte.

47. The method of claim 39 wherein said a second sbp member is complementary to said analyte.

48. The method of claim 39 wherein said first sbp member is analogous to said analyte and said second sbp member is complementary to said analyte and said first sbp member.

49. The method of claim 39 wherein said singlet oxygen is generated by irradiating said assay medium with light of wavelength of from 450-950 nm.

50. A method for determining an analyte, said method comprising:

combining in an assay medium either simultaneously or wholly or partially sequentially (1) a medium suspected of containing an analyte, (2) a label reagent comprising a first member of a specific binding pair (sbp member) bound to a compound capable of chemiluminescence upon reaction with singlet oxygen ("said compound"), (3) a singlet oxygen generator, and (4) a reagent comprising a fluorescent energy acceptor bound to, or capable of becoming bound to, a second sbp member, under conditions wherein an sbp member complex involving said label reagent and said energy acceptor is formed in relation to the presence of analyte in said medium and energy from said compound is capable of activating said fluorescent energy acceptor,

activating said singlet oxygen generator, and

examining said assay medium for a signal, the presence or intensity thereof being related to the amount of analyte in said medium.

51. The method of claim 50 wherein said singlet oxygen generator is a dye.

52. The-method of claim 51 wherein said dye is selected from the group consisting of methylene blue, rose bengal, porphyrins, and phthalocyanines.

53. The method of claim 50 wherein said analyte and said sbp members are each independently selected from the group consisting of ligands, receptors, and polynucleotides.

54. The method of claim 50 wherein said chemiluminescent compound contains an olefin group and one or more electron donating substituents in conjugation with said olefin group.

55. The method of claim 50 wherein said compound is selected from the group consisting of 9-alkylidene acridans, enol ethers, 9-alkylidene xanthanes and enamines.

56. The method of claim 50 wherein said first sbp member is complementary to said analyte.

57. The method of claim 50 wherein said a second sbp member is complementary to said analyte.

58. The method of claim 50 wherein said first sbp is analogous to said analyte and said second sbp member is complementary to said analyte and said first sbp member.

59. The method of claim 50 wherein said singlet oxygen generator is activated by irradiating said assay medium with light of wavelength of from 450-950 nm.

60. A method for determining a polynucleotide analyte, said method comprising:

combining in an assay medium, (1) a sample suspected of containing a polynucleotide analyte and (2) a label reagent comprising a photochemically activatable chemiluminescent compound (PACC) bound to a polynucleotide, at least a portion of which is capable of hybridizing with said polynucleotide analyte, under conditions wherein said label reagent hybridizes with said polynucleotide analyte if present,

irradiating said assay medium to activate said PACC, and

measuring the fluorescent energy emitted from said medium, the amount thereof being related to the amount of analyte in said sample.

61. The method of claim 60 wherein said assay medium also includes a fluorescent compound capable of activation by said PACC.

62. The method of claim 61 wherein said fluorescent compound is capable of binding to a polynucleotide.

63. The method of claim 61 wherein said fluorescent compound is bound to a polynucleotide, at least a portion of which is capable of hybridizing with said polynucleotide analyte at a region other than that with which said label reagent hybridizes.

64. The method of claim 61 wherein said fluorescent compound is selected from the group consisting of coumarins and xanthene dyes.

65. The method of claim 60 wherein said PACC contains an olefin group and one or more electron donating substituents in conjugation with said olefin group.

66. The method of claim 60 wherein said PACC is selected from the group consisting of 9-alkylidene acridans, enol ethers, 9-alkylidene xanthanes and enamines.

67. The method of claim 60 wherein said medium is irradiated with light having wavelengths between 450 and 950 nm to activate said PACC.

68. The method of claim 60 wherein said medium comprises a dye capable in the excited state of activating molecular oxygen to singlet oxygen.

69. The method of claim 68 wherein said medium is irradiated with light of wavelength 450-950 nm to excite said dye.

70. The method of claim 60 wherein said polynucleotide analyte is DNA or RNA.

71. The method of claim 61 wherein said polynucleotide analyte is DNA or RNA.

72. A composition comprising a photochemically activated chemiluminescent compound (PACC) associated with a member of a specific binding pair.

73. The composition of claim 72 wherein said PACC contains an olefin group.

74. The composition of claim 72 wherein said PACC contains an olefin group and one or more electron donating substitutents in conjugation with said olefin group.

75. The composition of claim 72 wherein said PACC is selected from the group consisting of 9-alkyline-N-alkyl acridans, enolethers, enamines, and 9-alkylidene xanthenes.

76. The composition of claim 72 wherein said sbp member is selected from the group consisting of receptors, ligands, and polynucleotides.

77. A kit comprising in packaged combination:

(1) a composition comprising a photochemically activatable chemiluminescent compound (PACC), having bound thereto a specific binding pair (sbp) member, and (2) a photosensitizer which is not in said composition.

78. The kit of claim 77 wherein said photosensitizer is a dye.

79. The kit of claim 78 wherein said dye is selected from the group consisting of methylene blue, rose bengal, porphyrins, and phthalocyanines.

80. The kit of claim 77 wherein said sbp member is selected from the group consisting of receptors, ligands, and polynucleotides.

81. The kit of claim 77 wherein said PACC contains an olefin group.

82. The kit of claim 77 wherein said PACC contains an olefin group and one or more electro donating substitutents in conjugation with said olefin group.

83. The kit of claim 77 wherein said PACC is selected from the group consisting of 9-alkylidene-N-alkyl acridans, enolethers, 9-alkylidene xanthenes and enamines.

84. The kit of claim 83 which comprises an energy acceptor.

85. The kit of claim 84 wherein said energy acceptor is bound to an sbp member.

86. The kit of claim 85 where said sbp member is selected from the group consisting of receptors, ligands, and polynucleotides.