US-20260126435-A1 - TARGET SUBSTANCE DETECTION METHOD AND REAGENT

US20260126435A1US 20260126435 A1US20260126435 A1US 20260126435A1US-20260126435-A1

Abstract

A technique that can detect a target substance in a sample with higher sensitivity and at lower cost than a conventional technique is provided, which is a target substance detection method including: in a first reaction field, forming a complex by sandwiching a target substance between a first trapping substance immobilized on a solid phase and a second trapping substance labeled with the labeling substance, and separating a moiety including the labeling substance from the complex and moving it through a liquid filled in a tubular channel by a centrifugal force; and detecting the moiety including the labeling substance by light scattered by the moiety including the labeling substance.

Inventors

Rikiya Takeuchi
Junichi Ando
Toshiya Ota
Hirokazu Ohta
Saaya Shiraki
Ayami Mori
Taisuke Yamauchi
Yoshiatsu Yamamoto
Takeaki Chichibu
Shion Nagashima

Assignees

LABORATORIES, INC.

Dates

Publication Date: 20260507
Application Date: 20230421
Priority Date: 20220422

Claims (20)

1 . A target substance detection method, comprising: forming a complex by sandwiching a target substance between a first trapping substance bound to a labeling substance and a second trapping substance immobilized on a solid phase, and separating a moiety including the labeling substance from the formed complex; moving the separated moiety including the labeling substance through a liquid filled in a tubular channel by a centrifugal force; and detecting the moiety including the labeling substance by light scattered by the moiety including the labeling substance from light irradiated into the tubular channel.
2 . The target substance detection method according to claim 1 , comprising: implementing a washing treatment of washing the complex in a state in which the complex is prevented from flowing into the tubular channel, before the moiety including the labeling substance is separated from the complex.
3 . The target substance detection method according to claim 2 , wherein when the washing treatment is implemented, the tubular channel is filled with a washing liquid used for the washing.
4 . The target substance detection method according to claim 2 , wherein a washing liquid used for the washing treatment accumulates downstream from the tubular channel.
5 . The target substance detection method according to claim 2 , wherein the complex is retained in a predetermined chamber, whereby the complex is prevented from flowing into the tubular channel.
6 . The target substance detection method according to claim 5 , comprising: arranging a first reservoir that stores the washing liquid, the chamber, and a second reservoir that stores a liquid flowing out from the chamber, in a device formed to be rotatable, in this order from a rotation center side of the device toward an outer side; communicating the first reservoir and the chamber with each other through a self-opening and closing flow path, and communicating the chamber and the second reservoir with each other through the tubular channel; rotating the device, thereby supplying the washing liquid from the first reservoir to the chamber and then discharging the washing liquid to the second reservoir via the tubular channel so as to implement the washing treatment.
7 . The target substance detection method according to claim 6 , wherein the first reservoir and the chamber are communicated with each other through an openable flow path.
8 . The target substance detection method according to claim 6 , wherein the washing treatment is performed until the second reservoir and the tubular channel are filled with the washing liquid.
9 . The target substance detection method according to claim 8 , comprising: arranging a third reservoir that stores a liquid containing a reagent for separating the moiety including the labeling substance from the complex, in a side closer to the center of rotation than the chamber of the device while the third reservoir and the chamber communicate with each other through a self-opening and closing flow path; and rotating the device, thereby supplying a liquid containing the reagent to the chamber after the washing treatment, whereby the moiety including the labeling substance is separated and then the separated moiety including the labeling substance is moved into the tubular channel.
10 . The target substance detection method according to claim 9 , wherein the third reservoir and the chamber are communicated with each other through an openable flow path.
11 . The target substance detection method according to claim 1 , further comprising: detecting scattered light that is intermittently generated from the tubular channel within a predetermined time period.
12 . The target substance detection method according to claim 1 , wherein the labeling substance is a substance having a specific gravity larger than that of the liquid filled in the tubular channel.
13 . The target substance detection method according to claim 12 , wherein the labeling substance is a fine particle that contains one selected from the group consisting of a metal, a ceramic, glass and a resin, as a main constituent component.
14 . The target substance detection method according to claim 13 , wherein the labeling substance is a metal colloidal particle.
15 . The target substance detection method according to claim 1 , wherein the solid phase is at least one granular body selected from the group consisting of glass particles, ceramic particles, magnetic particles and resin particles.
16 . The target substance detection method according to claim 1 , wherein the solid phase is a structure of an inside of a container that is used for forming the complex.
17 . The target substance detection method according to claim 1 , wherein the moiety including the labeling substance is separated from the complex, by at least one treatment selected from the group consisting of heat treatment, pH adjustment treatment, denaturation treatment, oxidation treatment, reduction treatment, enzyme treatment and competitive reaction treatment.
18 . The target substance detection method according to claim 1 , wherein the first trapping substance is at least one selected from the group consisting of an antibody, an antibody fragment, a modified antibody, an antigen, an aptamer and a nucleic acid, each of which specifically binds to the target substance.
19 . The target substance detection method according to claim 1 , wherein the second trapping substance is at least one selected from the group consisting of an antibody, an antibody fragment, a modified antibody, an antigen, an aptamer and a nucleic acid, each of which specifically binds to the target substance.
20 . A reagent for use in the target substance detection method according to claim 1 , the reagent having an action of separating the moiety including the labeling substance from the complex.

Description

TECHNICAL FIELD The present invention relates to a detection method and a reagent for detecting a target substance in a sample, and in particular, to a detection method and a reagent that can detect the target substance with high sensitivity and at low cost. BACKGROUND ART An immunoassay has heretofore been known which discovers a disease or qualitatively or quantitatively analyzes therapeutic effects or the like by detecting a particular antigen or antibody associated with the disease as a biomarker. In recent years, there has been a growing demand for highly sensitive detection to detect a trace amount of a biomarker for the purpose of detecting a more effective biomarker. An enzyme-linked immunosorbent assay (also referred to as “ELISA” in the present specification), which is one of the immunoassays, is a method of detecting a target antigen, antibody or nucleic acid contained in a sample solution through the use of enzyme reaction while trapping the target with a specific antibody, an antigen that binds to the target antibody, or a complementary nucleic acid, and it widely prevalent because of its advantages such as a cost. Although ELISA is an assay capable of quantitatively detecting a target substance, most of the currently prevalent ELISA techniques are operated on a 96-well microplate, have a large number of steps, and require a complicated operation. Furthermore, such ELISA undergoes a plurality of reaction steps and therefore requires a great deal of labor and time for obtaining measurement results. Hence, it is desired to shorten an operating time or a reaction time. Moreover, more highly sensitive detection is demanded. In order to achieve a highly sensitive detection, it is considered that the reactivity between an enzyme and a substrate is improved. For this purpose, for example, a technique has been proposed in which an immune complex (labeled compound), to which a reporter is added, is liberated from a solid phase and is allowed to react in a free liquid medium (Patent Literature 1). In this Patent Literature 1, components of the immune complex are designed such that a biotin or a functionalized azo dye and an avidin are introduced in the immune complex. For example, an antibody bound to a reporter group via streptavidin-biotin binding is prepared as a labeled antibody. When the immune complex is formed on a solid phase, the reporter group is introduced into the immune complex via streptavidin-biotin binding. Then, an excess of streptavidin is added thereto so that streptavidin in the complex is replaced with the added streptavidin to liberate the reporter molecule from the immune complex. Hence, in this technique, the reporter molecule is detected in a free liquid medium and can thereby be associated with an analyte concentration in a specimen. Further, in order to detect an immune complex at high sensitivity, a microreactor where reaction is performed in a minute container of a μm scale has been developed by microfabrication technology. If a microreactor having a minute space serving as a reaction field is used, it can be expected that a reaction rate is improved. The realization of an experimental operation such as extraction or separation on a microreactor using a minute channel or reaction chamber is often called “lab on a chip”. It has been proposed that a biological material is isolated by forming a microreactor in a disc having a flat circular shape and dispensing a sample solution by centrifugal force caused by rotation (Patent Literature 2). It has also been proposed that immunoassay procedures, which have been manually performed in wells, are implemented in a microreactor by forming a plurality of chambers, channels, reservoirs and the like in a disc having a flat circular shape and carrying out the liquid feed of a reagent and waste liquid disposal by rotation of the disc (Patent Literature 3). A method using a labeling bead in an immunoassay in a microreactor has been proposed for the purpose of detecting a target substance having a low concentration (Patent Literature 4). For example, Patent Literature 4 discloses a disc having a flat circular shape, which is provided with a bead filled portion which is filled with a labeling bead of which surface is modified with an antibody, as well as a detection region. An antigen in a sample solution injected to the disc is trapped by the antibody on the labeling bead, and a labeling bead complex bound by the antigen on the labeling bead is sent to the detection region by the rotation of the disc and trapped by an antibody immobilized on a detection area. It is disclosed that the number of the labeling bead complexes in a state of thus being fixed in the detection area are counted by an optical reading means of an optical disk device, thereby detecting the target substance in the sample. On the other hand, an analysis method using a disk has been proposed in which antigen-carrying beads, a fluorescent IgG antibody-labeling reagent and a s