Dual bead assays using cleavable spacers and/or ligation to improve specificity and sensitivity including related methods and apparatus

• US 20030054376A1
• Filed: 03/14/2002
• Published: 03/20/2003
• Est. Priority Date: 07/07/1997
• Status: Abandoned Application

• Current Assignee: Nagaoka & Co., Ltd.
• Original Assignee: Nagaoka & Co., Ltd.
• Inventors: Phan, Brigitte Chau, Virtanen, Jorma Antero, Mullis, Kary Banks
• Application Number:
  US10/099,256
• Publication Number:
  US 20030054376A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex

Abstract

Methods for deceasing non-specific bindings of beads in dual bead assays and related optical bio-discs and disc drive systems. The methods include determining the suitability of a test solid phase for purposes of use in a dual bead assay. The method also includes identifying whether a target agent is present in a biological sample and involves mixing capture beads, reporter beads, and a biological sample. The mixing is performed under binding conditions to permit formation of a dual bead complex if the target agent is present in the sample. The reporter bead and capture bead are each bound to the target agent. Cleavable spacers or displacement linkers may be used in forming the dual bead complexes. The methods also include placing the capture beads and the reporter beads spatially proximally, performing a ligation reaction employing a ligase, and isolating the dual bead complex from the mixture to obtain the isolate. The isolate is exposed to the capture field on a disc and the capture field is having a capture agent that binds to the dual bead complex. The ligation reaction enables covalent binding between capture probe and reporter probe. The ligation also reaction enhances the sensitivity of the dual bead assay.

Claims

1. A method using a detachable linker to identify whether a target is present in a biological sample, said method comprising the steps of:

preparing a dual bead complex including at least one reporter bead and at least one capture bead, the beads being linked together by a cleavable spacer;

mixing said dual bead complex with a biological sample to be tested for a target;

allowing any target present in the sample to form an association with said dual bead complex;

cleaving the cleavable spacers of the dual bead complexes so that only complexes associated with the target remain in the dual bead formation;

isolating the remaining dual bead complexes from solution to obtain an isolate;

exposing the isolate to a capture field on an optical bio-disc, the capture field having a capture agent that binds to the dual bead complex; and

detecting the presence of the dual bead complex in the disc to indicate that the target is present in the sample.

2. The method according to claim 1 wherein said cleavable spacer includes at least one transfer probe and at least one reporter probe.

3. The method according to claim 1 wherein said capture bead has at least one transport probe.

4. The method according to claim 1 wherein said reporter bead has at least one signal probe.

5. The method according to claim 1 wherein said mixing step is performed in the disc.

6. The method according to claim 1 wherein said capture bead has at least one transport probe and said reporter bead has at least one signal probe.

7. The method according to claim 6 including the further step of performing a ligation reaction to introduce a covalent bond between the transport probe and the signal probe to thereby strengthen the bond between the capture bead and the reporter bead.

20. An optical bio-disc adapted to implement the method recited in any one of claims 1, 816, or 17, said optical bio-disc comprising:

a substrate having encoded information associated therewith, said encoded information being readable by a disc drive assembly to control rotation of the disc;

a target zone associated with said substrate, said target zone disposed at a predetermined location relative to said substrate;

an active layer associated with said target zone; and

a plurality of capture agents attached to said active layer so that when said substrate is rotated, said capture agents remain attached to said active layer to thereby maintain a number of said capture agents within said target zone so that when a dual bead complex is introduced into said target zone, said capture agent sequesters said dual bead complex therein to thereby allow detection of captured dual bead complex.

8. A method using a displaceable member to identify whether a target is present in a biological sample, said method comprising the steps of:

preparing a dual bead complex including at least one reporter bead and at least one capture bead, the beads being linked together by a displaceable spacer;

mixing said dual bead complex with a biological sample to be tested for a target;

allowing any target present in the sample to form an association with said dual bead complex;

displacing the displaceable spacers of the dual bead complexes so that only complexes associated with the target remain in the dual bead formation;

isolating the remaining dual bead complexes from solution to obtain an isolate;

exposing the isolate to a capture field on an optical bio-disc, the capture field having a capture agent that binds to the dual bead complex; and

detecting the presence of the dual bead complex in the disc to indicate that the target is present in the sample.

9. The method according to claim 8 wherein said displaceable spacer includes at least one transfer probe and at least one reporter probe.

10. The method according to claim 8 wherein said capture bead has at least one transport probe.

11. The method according to claim 8 wherein said reporter bead has at least one signal probe.

12. The method according to claim 8 wherein said mixing step is performed in the disc.

13. The method according to claim 8 wherein said capture bead has at least one transport probe and said reporter bead has at least one signal probe.

14. The method according to claim 13 including the further step of performing a ligation reaction to introduce a covalent bond between the transport probe and the signal probe to thereby strengthen the bond between the capture bead and the reporter bead.

15. The method according to claim 8 wherein said displacing step is preformed by use of a displacement probe.

16. A method using ligation to identify whether a target is present in a biological sample, said method comprising the steps of:

preparing a plurality of capture beads each of having at least one transport probe affixed thereto;

preparing a plurality of reporter beads each having at least one signal probe affixed thereto;

mixing said capture beads, said reporter beads, and a sample to be tested for the presence of a target;

allowing any target present in the sample to bind to the transport and reporter probes thereby forming a dual bead complex including at least one reporter bead and one capture bead; and

performing a ligation reaction to introduce a covalent bond between the transport probes and the reporter probes to thereby strengthen the bond between the capture bead and the reporter bead so that when the dual bead complexes are processed in a fluidic circuit of a rotating optical bio-disc, said strengthened bond withstands any rotational forces acting thereon.

17. The method according to claim 16 including the further steps of isolating the dual bead complex from solution to obtain the isolate;

exposing the isolate to a capture field on an optical bio-disc, the capture field having a capture agent that binds to the dual bead complex; and

detecting the presence of the dual bead complex in the disc to indicate that the target agent is present in the sample.

18. The method according to claim 16 wherein said mixing, allowing, and performing steps are carried out in said optical bio-disc.

19. The method according to claim 17 wherein said isolating, exposing, and detecting steps are performed in association with said optical bio-disc.