Binding interactions in dipstick assays

• US 20040072176A1
• Filed: 04/07/2003
• Published: 04/15/2004
• Est. Priority Date: 07/07/2000
• Status: Active Grant

• Current Assignee: Diagnostics for the Real World, Ltd.
• Original Assignee: Diagnostics for the Real World, Ltd.
• Inventors: Dineva, Magda Anastassova, Lee, Helen, Hu, Hsiang Yun
• Granted Patent: US 8,431,336 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates

Abstract

Use of dipsticks to test for the presence of target nucleic acid in a sample solution is described. The dipsticks comprise a contact end for contacting the sample solution and a capture zone, remote from the contact end, to which a capture probe is immobilised. The capture probe is capable of hybridising to the target nucleic acid. The sample solution is contacted with the contact end of the dipstick and travels by capillary action to the capture zone. If target nucleic acid is present in the sample solution it is captured and can be detected at the capture zone. The capture probe is immobilised to the capture zone by a spacer. Use of the spacer increases the stability of the interaction between the capture probe and the target nucleic acid and thus improves the sensitivity of target nucleic acid detection. Detection probes with spacers are also described.

Claims

1. A dipstick for testing for the presence of target nucleic acid in a sample solution which comprises:

a chromatographic strip having a contact end for contacting the sample solution; and

a capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end, the capture probe being capable of hybridising to the target nucleic acid or to a hook capture probe bound to the target nucleic acid, wherein the capture probe is linked to a capture probe spacer and the capture probe spacer is linked to the capture zone, thereby immobilising the capture probe at the capture zone and spacing the capture probe from the capture zone.

2. A dipstick according to claim 1 in which the capture probe spacer comprises a protein.

3. A dipstick according to claim 2 in which the protein is BSA, thyroglobulin, or a derivative thereof.

4. A dipstick according to claim 2 or 3 in which the protein is adsorbed directly to the capture zone.

5. A dipstick according to any of claims 2 to 4 in which the capture probe is linked to the protein by a linker.

6. A dipstick according to claim 1 in which the capture probe spacer comprises a non protein.

7. A dipstick according to any of claims 2 to 4 in which the capture probe spacer further comprises a non protein.

8. A dipstick according to claim 7 in which the capture probe is coupled to the non protein and the non protein is coupled to the protein thereby spacing the capture probe from the protein.

9. A dipstick according to claim 8 in which the non protein is coupled to the protein by a linker.

10. A dipstick according to any of claims 6 to 9 in which the non protein is at least the length of three nucleotides, preferably at least the length of six nucleotides.

11. A dipstick according to any of claims 6 to 10 in which the non protein comprises a nucleobase capable of forming a stacking interaction with a base pair formed when the capture probe has hybridised to the target nucleic acid or hook capture probe.

12. A dipstick according to any of claims 6 to 11 in which the non protein comprises a nucleotide.

13. A dipstick according to any of claims 6 to 12 in which the non protein comprises 1′

,2′

-Dideoxyribose phosphate.

14. A dipstick according to any of claims 6 to 13 in which the non protein comprises 3-Hydroxypropyl phosphate.

15. A dipstick according to any of claims 6 to 14 in which the non protein comprises Hexaethyleneglycol phosphate.

16. A dipstick according to claim 12 in which the non protein consists only of one or more nucleotides.

17. A dipstick according to any preceding claim in which the capture probe spacer is linked to one end of the capture probe.

18. A dipstick according to claim 17 in which the end of the capture probe not linked to the capture probe spacer is coupled to one or more nucleotides, preferably at least three nucleotides, which do not hybridise to the target nucleic acid when the capture probe has hybridised to the target nucleic acid or which do not hybridise to the hook capture probe when the capture probe has hybridised to the hook capture probe.

19. A dipstick according to any of claims 1 to 16 in which the capture probe spacer is linked to a part of the capture probe between the ends of the capture probe.

20. A dipstick according to claim 19 in which one or both ends of the capture probe are coupled to one or more nucleotides, preferably at least three nucleotides, which do not hybridise to the target nucleic acid when the capture probe has hybridised to the target nucleic acid or which do not hybridise to the hook capture probe when the capture probe has hybridised to the hook capture probe.

21. A dipstick according to any preceding claim which further comprises a detection probe, releasably immobilised at a probe zone located between the contact end and the capture zone of the chromatographic strip, the detection probe being capable of hybridising to the target nucleic acid to allow detection of the target nucleic acid.

22. A dipstick according to claim 21 in which the detection probe is coupled to a label which allows direct detection of the target nucleic acid when the detection probe has hybridised to the target nucleic acid.

23. A dipstick according to claim 21 in which the detection probe is coupled to a detection ligand which can be bound by a detection ligand binding moiety to allow indirect detection of the target nucleic acid when the detection probe has hybridised to the target nucleic acid.

24. A dipstick according to claim 22 or 23 in which the label or detection ligand is linked to a detection probe spacer and the detection probe spacer is linked to the detection probe, thereby coupling the label or the detection ligand to the detection probe and spacing the label or the detection ligand from the detection probe.

28. A dipstick according to any of claims 24 to 27 in which the detection probe spacer comprises a protein.

29. A dipstick according to claim 28 in which the protein is BSA, thyroglobulin, or a derivative thereof.

30. A dipstick according to claim 28 or 29 in which the the label or the detection ligand is attached to or part of a bead and the protein is adsorbed directly to the bead.

31. A dipstick according to any of claims 28 to 30 in which the protein is linked to the detection probe by a linker.

32. A dipstick according to any of claims 24 to 27 in which the detection probe spacer comprises a non protein.

33. A dipstick according to any of claims 28 to 30 in which the detection probe spacer further comprises a non protein.

34. A dipstick according to claim 33 in which the detection probe is coupled to the non protein and the non protein is coupled to the protein thereby spacing the detection probe from the protein.

35. A dipstick according to claim 34 in which the non protein is coupled to the protein by a linker.

36. A dipstick according to any of claims 33 to 35 in which the non protein is at least the length of three nucleotides.

37. A dipstick according to any of claims 33 to 36 in which the non protein comprises a base capable of forming a stacking interaction with a base pair formed when the detection probe has hybridised to the target nucleic acid.

38. A dipstick according to any of claims 33 to 37 in which the non protein comprises a nucleotide.

39. A dipstick according to any of claims 33 to 38 in which. the non protein comprises 1′

,2′

-Dideoxyribose phosphate.

40. A dipstick according to any of claims 33 to 39 in which the non protein comprises 3-Hydroxypropyl phosphate.

41. A dipstick according to any of claims 33 to 40 in which the non protein comprises Hexaethyleneglycol phosphate.

42. A dipstick according to claim 38 in which the non protein consists only of one or more nucleotides.

43. A dipstick according to any of claims 24 to 42 in which the detection probe spacer is linked to one end of the detection probe.

44. A dipstick according to claim 43 in which the end of the detection probe not linked to the detection probe spacer is coupled to one or more nucleotides, preferably at least three nucleotides, which do not hybridise to the target nucleic acid when the detection probe has hybridised to the target nucleic acid.

45. A dipstick according to any of claims 24 to 42 in which the detection probe spacer is linked to a part of the detection probe between the ends of the detection probe.

46. A dipstick according to claim 45 in which one or both ends of the detection probe are coupled to one or more nucleotides, preferably at least three nucleotides, which do not hybridise to the target nucleic acid when the detection probe has hybridised to the target nucleic acid.

47. A kit for testing for the presence of target nucleic acid in a sample solution which comprises:

a dipstick according to any of claims 1 to 20; and

a detection probe capable of hybridising to the target nucleic acid thereby allowing detection of target nucleic utilising the detection probe.

48. A kit according to claim 47 in which the detection probe is coupled to a label allowing direct detection of the detection probe hybridised to the target nucleic acid, or a detection ligand allowing indirect detection of the detection probe hybridised to the target nucleic acid by a detection ligand binding moiety, wherein the label or the detection ligand is linked to a detection probe spacer and the detection probe spacer is linked to the detection probe thereby coupling the label or the detection ligand to the detection probe and spacing the label or the detection ligand from the detection probe.

25. A dipstick for testing for the presence of target nucleic acid in a sample solution which comprises:

a chromatographic strip having a contact end for contacting the sample solution;

a capture moiety, immobilised at a capture zone remote from the contact end, the capture moiety being capable of binding directly or indirectly to the target nucleic acid; and

a detection probe, releasably immobilised at a probe zone located between the contact end and the capture zone, the detection probe being capable of hybridising to the target nucleic acid and the detection probe being coupled to a label allowing direct detection of the target nucleic acid utilising the detection probe, or the detection probe being coupled to a detection ligand allowing indirect detection of the target nucleic acid utilising the detection probe,

wherein the label or the detection ligand is linked to a detection probe spacer and the detection probe spacer is linked to the detection probe, thereby coupling the label or the detection ligand to the detection probe and spacing the label or the detection ligand from the detection probe.

26. A dipstick according to claim 25 in which the capture moiety comprises a capture probe capable of hybridising to the target nucleic acid or to a hook capture probe bound to the target nucleic acid.

27. A dipstick according to claim 25 in which the capture moiety is capable of binding to a capture ligand coupled to a capture probe bound to the target nucleic acid.

49. A kit for testing for the presence of target nucleic acid in a sample solution which comprises:

a dipstick according to claim 27;

and

a capture probe capable of hybridising to the target nucleic acid, or to a hook capture probe bound to the target nucleic acid, wherein the capture probe is coupled to a capture ligand which can be bound by the capture moiety.

50. A kit according to claim 49 in which the capture ligand is linked to a capture probe spacer and the capture probe spacer is linked to the capture probe thereby coupling the capture ligand to the capture probe and spacing the capture ligand from the capture probe.

51. A kit for testing for the presence of target nucleic acid in a sample solution which comprises:

i) a dipstick comprising a chromatographic strip having a contact end for contacting the sample solution and a capture probe capable of hybridising to the target nucleic acid, or to a hook capture probe bound to the target nucleic acid, the capture probe being immobilised at a capture zone of the chromatographic strip remote from the contact end; and

ii) a detection probe capable of hybridising to the target nucleic acid, the detection probe being coupled to a label allowing direct detection of the target nucleic acid utilising the detection probe, or the detection probe being coupled to a ligand allowing indirect detection of the target nucleic acid utilising the detection probe, wherein the label or the detection ligand is linked to a detection probe spacer and the detection probe spacer is linked to the detection probe thereby coupling the label or the detection ligand to the detection probe and spacing the label or the detection ligand from the detection probe.

52. A kit for testing for the presence of target nucleic acid in a sample solution which comprises:

i) a dipstick comprising a chromatographic strip having a contact end for contacting the sample solution and a capture moiety immobilised at a capture zone of the chromatographic strip remote from the contact end, the capture moiety being capable of binding directly or indirectly to the target nucleic acid;

ii) a capture probe capable of hybridising to the target nucleic acid, or to a hook capture probe bound to the target nucleic acid, wherein the capture probe is coupled to a capture ligand which can be bound by the capture moiety; and

iii) a detection probe capable of hybridising to the target nucleic acid, the detection probe being coupled to a label allowing direct detection of the target nucleic acid utilising the detection probe, or the detection probe being coupled to a detection ligand allowing indirect detection of the target nucleic acid utilising the detection probe, wherein the label or the detection ligand is linked to a detection probe spacer and the detection probe spacer is linked to the detection probe thereby coupling the label or the detection ligand to the detection probe and spacing the label or the detection ligand from the detection probe.

53. A kit for testing for the presence of target nucleic acid in a sample solution which comprises:

i) a dipstick comprising a chromatographic strip having a contact end for contacting the sample solution and a capture moiety immobilised at a capture zone of the chromatographic strip remote from the contact end;

ii) a capture probe capable of hybridising to the target nucleic acid, or to a hook capture probe bound to the target nucleic acid, wherein the capture probe is linked to a capture probe spacer and the capture probe spacer is linked to a capture ligand thereby coupling the capture ligand to the capture probe and spacing the capture ligand from the capture probe; and

iii) a detection probe capable of hybridising to the target is nucleic acid, the detection probe being coupled to a label allowing direct detection of the target nucleic acid utilising the detection probe, or the detection probe being coupled to a detection ligand allowing indirect detection of the target nucleic acid utilising the detection probe.

54. A method for immobilising a probe to a solid phase which comprises providing a probe coupled to a protein and adsorbing the protein to the solid phase.

55. A method according to claim 54 which further comprises coupling the probe to the protein.

56. A method according to claim 55 which further comprises coupling the probe to a linker and coupling the linker to the protein.

57. A method according to claim 56 in which the probe is coupled to the linker before the linker is coupled to the protein.

58. A method according to claim 56 or 57 in which the probe is coupled to the linker by reaction of a phosphoramidite group attached to the linker with a hydroxyl group of the probe or by reaction of a hydroxyl group of the linker with a phosphoramidite group attached to the probe.

59. A method according to any of claims 56 to 58 in which the linker is coupled to the protein by reaction of a primary amino group attached to the linker with a carboxyl group of the protein.

60. A method according to any of claims 56 to 59 in which the linker is coupled to a non nucleobase part of the probe.

61. A method according to any of claims 54 to 60 in which the solid phase comprises a membrane.

62. A method according to claim 61 in which the membrane is a nitrocellulose membrane.

63. A method according to any of claims 54 to 60 in which the solid phase comprises a particle such as a bead.

64. A method according to any of claims 54 to 63 in which the probe comprises a nucleic acid or nucleic acid analogue.

65. A probe immobilised to a solid phase by adsorption to the solid phase of a protein coupled to the probe.

66. A probe for detecting or capturing a target nucleic acid which comprises a nucleic acid or nucleic acid analogue capable of hybridising to the target nucleic acid, and a label allowing direct detection of the target nucleic acid utilising the probe, or a ligand allowing capture or indirect detection of the target nucleic acid utilising the probe, wherein the label or the ligand is linked to a spacer and the spacer is linked to the nucleic acid or nucleic acid analogue thereby spacing the label or the ligand from the nucleic acid or nucleic acid analogue.