Method for detecting a target substance

• US 20080268545A1
• Filed: 06/29/2006
• Published: 10/30/2008
• Est. Priority Date: 12/29/2003
• Status: Abandoned Application

• Current Assignee: Universal Bio Research Co., Ltd (Precision System Science Co., Ltd)
• Original Assignee: Universal Bio Research Co., Ltd (Precision System Science Co., Ltd)
• Inventors: Tajima, Hideji
• Application Number:
  US11/477,836
• Publication Number:
  US 20080268545A1
• Time in Patent Office: Days
• US Class Current: 436/94
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/113 based on agglutination/precipitation
• CPC Class Codes: G01N 33/54333 Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

In order to separate a complex of a first probe, a second probe and a target substance from a reaction mixture obtained by reacting the first probe having first particle (such as labeled particle) and a first binding substance (such as a first oligonucleotide) immobilized on the first particle, the second probe having second particle (such as magnetic particle) and a second binding substance (such as a second oligonucleotide) immobilized on the second particle, and the target substance (such as a target nucleic acid) under mild conditions which do not cause destruction or degradation of the complex, the reaction mixture is mixed with a liquid having a specific gravity greater than that of the first particle, less than that of the second particle and less than that of a particle complex composed of the first particle and the second particle, or a liquid having a specific gravity less than that of the first particle, greater than that of the second particle and greater than that of a particle complex composed of the first particle and the second particle, and then allowed to stand undisturbed until a precipitate and a floating substance are formed.

Claims

1. A method for separating a complex of a first probe, a second probe and a target substance from a reaction mixture obtained by reacting the first probe having a first particle and a first binding substance immobilized on the first particle, the second probe having a second particle having a different specific gravity from the first particle and a second binding substance immobilized on the second particle, and the target substance having a first portion able to be bound by the first binding substance and a second portion able to be bound by the second binding substance, the method comprising:

a step in which, after mixing the reaction mixture with a liquid having a specific gravity greater than that of the first particle, less than that of the second particle and less than that of a particle complex composed of the first particle and the second particle, or a liquid having a specific gravity less than that of the first particle, greater than that of the second particle and greater than that of a particle complex composed of the first particle and the second particle, the mixture is allowed to stand undisturbed until a precipitate and a floating substance are formed.

2. The method according to claim 1, wherein the first particle and/or second particle is a magnetic particle, and

the reaction mixture is obtained by reacting the first probe, the second probe and the target substance in a reaction solvent housed in a container, followed by removing the reaction solvent in a state in which a magnet is arranged in the vicinity of the outer wall of the container.

3. The method according to claim 2, wherein the first particle is a non-magnetic particle, and the second particle is a magnetic particle.

4. A method for reacting a first probe having a first particle and a first binding substance immobilized on the first particle, a second probe having a second particle and a second binding substance immobilized on the second particle, and a target substance having a first portion able to be bound by the first binding substance and a second portion able to be bound by the second binding substance, wherein

the first particle and/or second particle is a magnetic particle, and the method comprises:

a first step in which the first probe, the second probe, the target substance and a reaction solvent are housed in a container,

a second step in which a magnet is allowed to act on the magnetic particle within the container through a predetermined region of the outer wall of the container,

a third step in which the action of the magnet is canceled, and

a fourth step in which the second and third steps are repeated while changing the location of the predetermined region.

5. The method according to claim 4, wherein the magnet is allowed to act by allowing the predetermined region and the magnet to approach each other in the second step,

the action of the magnet is canceled by separating the predetermined region from the magnet in the third step, and

the predetermined region and magnet are repeatedly allowed to approach and separate from each other while changing the location of the predetermined region in the fourth step.

6. The method according to claim 4 or claim 5, wherein the location of the predetermined region is changed in the circumferential direction or lengthwise direction of the container.

11. The method according to claim 7 or claim 8, wherein a magnetic particle is selected for the first particle and/or second particle in the first step, and

the first probe, the second probe and the target substance are reacted by the method according to any of claims 4 to 6.

12. The method according to claim 11, wherein the reaction mixture is obtained by reacting the first probe, the second probe and the target substance, followed by removing the reaction solvent in the state in which a magnet is arranged in the vicinity of the outer wall of the container in the second step.

13. The method according to claim 12, wherein a non-magnetic particle is selected for the first particle, and a magnetic particle is selected for the second particle in the first step.

7. A method for detecting a target substance having a first portion able to be bound by a first binding substance and a second portion able to be bound by a second binding substance, using a first probe having a first particle and the first binding substance immobilized on the first particle, and a second probe having a second particle and the second binding substance immobilized on the second particle, the method comprising:

a first step in which a labeled particle is selected for the first particle and a particle having a specific gravity greater than that of the first particle is selected for the second particle,

a second step in which the first probe, the second probe and the target substance are reacted to obtain a reaction mixture,

a third step in which the reaction mixture is mixed with a liquid having a specific gravity greater than that of the first particle, less than that of the second particle, and less than that of a particle complex composed of the first particle and the second particle, followed by allowing to stand undisturbed until a precipitate and a floating substance are formed, and

a fourth step in which a complex of the first probe, the second probe and the target substance contained in the precipitate is detected based on the label of the first particle.

9. The method according to claim 7 or claim 8, wherein a magnetic particle is selected for the first particle and/or second particle in the first step, and

the reaction mixture is obtained by reacting the first probe, the second probe and the target substance in a reaction solvent housed in a container, followed by removing the reaction solvent in a state in which a magnet is arranged in the vicinity of the outer wall of the container in the second step.

10. The method according to claim 9, wherein a non-magnetic particle is selected for the first particle, and a magnetic particle is selected for the second particle in the first step.

14. The method according to claim 7 or claim 8, wherein a magnetic particle is selected for the second particle in the first step, and

the first probe is reacted with a complex of the second probe and the target substance obtained by mixing a sample containing the target substance and the second probe, and recovering the complex of the second probe and the target substance by controlling magnetic force in the second step.

8. A method for detecting a target substance having a first portion able to be bound by a first binding substance and a second portion able to be bound by a second binding substance, using a first probe having a first particle and the first binding substance immobilized on the first particle, and a second probe having a second particle and the second binding substance immobilized on the second particle, the method comprising:

a first step in which a labeled particle is selected for the first particle and a particle having a specific gravity less than that of the first particle is selected for the second particle,

a second step in which the first probe, the second probe and the target substance are reacted to obtain a reaction mixture,

a third step in which the reaction mixture is mixed with a liquid having a specific gravity less than that of the first particle, greater than that of the second particle, and greater than that of a particle complex composed of the first particle and the second particle, followed by allowing to stand undisturbed until a precipitate and a floating substance are formed, and

a fourth step in which a complex of the first probe, the second probe and the target substance contained in the floating substance is detected based on the label of the first particle.