Binding interactions in dipstick assays

• US 8,431,336 B2
• Filed: 07/06/2001
• Issued: 04/30/2013
• Est. Priority Date: 07/07/2000
• Status: Expired due to Term

• Current Assignee: Diagnostics for the Real World, Ltd.
• Original Assignee: Diagnostics for the Real World, Ltd.
• Inventors: Lee, Helen, Dineva, Magda Anastassova, Hu, Hsiang Yun
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US10/332,142
• Publication Number:
  US 20040072176A1
• Time in Patent Office: 4,316 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 immobilized. 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 immobilized 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 a target nucleic acid in a sample solution, wherein the dipstick comprises:

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

a capture probe that is immobilized at a capture zone of the chromatographic strip remote from the contact end, wherein the capture probe comprises an oligonucleotide capable of hybridizing 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 immobilizing the capture probe at the capture zone and spacing the capture probe from the capture zone, wherein;

i) the capture probe spacer is linked to one end of the capture probe, and the end of the capture probe that is not linked to the capture probe spacer is coupled to one or more nucleotides, which do not hybridize to the target nucleic acid when the capture probe is hybridized to the target nucleic acid or do not hybridize to the hook capture probe when the capture probe is hybridized to the hook capture probe;

ii) the capture probe spacer is linked to a part of the capture probe between the ends of the capture probe, and one or both ends of the capture probe are coupled to one or more nucleotides, which do not hybridize to the target nucleic acid when the capture probe is hybridized to the target nucleic acid or do not hybridize to the hook capture probe when the capture probe is hybridized to the hook capture probe;

iii) the capture probe spacer comprises a protein adsorbed directly to the capture zone, wherein the capture probe is covalently linked to the protein by a linker.

2. The dipstick of claim 1, further comprises a detection probe, that is releasably immobilized at a probe zone located between the contact end and the capture zone of the chromatographic strip such that sample solution passing the probe zone mobilizes the detection probe, wherein the detection probe is capable of hybridizing to the target nucleic acid to allow detection of the target nucleic acid.

3. The dipstick according to claim 2, wherein the detection probe is coupled to a label, which allows direct detection of the target nucleic acid when the detection probe is hybridized to the target nucleic acid.

4. The dipstick according to claim 2, wherein 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 is hybridized to the target nucleic acid.

5. The dipstick according to claim 4, wherein 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.

6. A kit for testing for the presence of a target nucleic acid in a sample solution, wherein the kit comprises:

the dipstick of claim 1; and

a detection probe capable of hybridizing to the target nucleic acid thereby allowing detection of the target nucleic acid utilizing the detection probe.

7. The kit according to claim 6, wherein the detection probe is coupled to a label allowing direct detection of the detection probe that is hybridized to the target nucleic acid, or a detection ligand allowing indirect detection of the detection probe that is hybridized 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.