CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of priority to Japanese Patent Application No. JP2019-114031, filed Jun. 19, 2019, the entire content of which is incorporated herein by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (sequence_listing.txt; Date of Creation: Jun. 17, 2020; and Size: 988 bytes) is herein incorporated by reference in its entirety.

BACKGROUND

1. Field

The present disclosure relates to a target substance detection method, a target substance detection kit and a target substance detection system in which trapping substances are used.

2. Description of Related Art

An immuno chromatographic method, an ELISA method, a flow-through method, a surface plasmon resonance (SPR) method, a beads assay method, and the like are known as methods for detecting a target substance. For example, in an immuno chromatographic detection method, a protein such as an antigen or an antibody is used to trap a detection-target substance (target substance) in a sample (JP 2012-533064A). For the purpose of enhancing sensitivity, the development of an immuno chromatographic detection method in which nucleic-acid aptamers are used has been started (JP H10-512751A).

As a method for immobilizing a nucleic-acid aptamer, an end of a nucleic-acid aptamer that specifically binds to a detection-target substance is modified with NH2, biotin, digoxigenin, or the like, and the nucleic-acid aptamer is immobilized on a nitrocellulose membrane or coupled to a detecting substance via the modified end (Bruno, Pathogens. 2014 June; 3(2): 341-355). A UV irradiation method is employed to directly couple a nucleic-acid aptamer to an immobilization carrier. Streptavidin is employed for indirect coupling of a biotin-modified aptamer. An anti-digoxigenin antibody is employed for a digoxigenin-modified aptamer. In order to indirectly couple a nucleic-acid aptamer to an immobilization carrier without using a protein, a method can also be used in which single-stranded DNA or single-stranded RNA having a sequence complementary to the nucleic-acid aptamer is coupled to an immobilization carrier, and the nucleic-acid aptamer is coupled to the immobilization carrier through complementary coupling (JP H10-48216A).

SUMMARY

In a conventional method for detecting a target substance (the so-called sandwich method), a trapping substance is directly immobilized on at least one of the immobilization side (the support substance side) and the detection side (the reporter side) before a sample containing a target substance is brought into contact therewith. Accordingly, when the target substance is changed to another one, it is necessary to change the immobilized trapping substance to a trapping substance corresponding to the new target substance. In order to make this change, it is necessary to redesign the trapping substance together with the support and/or the reporter substance on which the trapping substance is immobilized, and thus the immobilization of the trapping substance needs to be examined again.

However, in the conventional detection method in which a trapping substance is directly immobilized, when a chemical reaction or the like for immobilizing the trapping substance is performed, the trapping substance itself is also exposed to the reaction. This causes a problem in that the ability of the trapping substance to recognize a target substance is lost or impaired, for example. Accordingly, it is necessary to examine the immobilization reaction conditions and search for the optimum conditions. A lot of samples are required for the examination of the conditions, and time for the examination is required. Furthermore, in the early stages of a search for a trapping substance, there is a problem in that the conditions need to be examined regarding many candidate trapping substances, and it takes a lot of time.

Examples of a method for immobilizing a nucleic-acid aptamer on a carrier include immobilization methods in which chemical formation of a covalent bond, formation of a covalent bond with physical energy caused by UV irradiation, and the like are utilized (JP H10-48216A). However, these immobilization processes cause damage to the nucleic-acid aptamer itself, which causes impairment of the ability of the nucleic-acid aptamer to bind to a target substance, or loss of the functions of the nucleic-acid aptamer.

To address this, an aspect of the present disclosure provides a target substance detection method in which an immobilization side (support substance side) and a trapping substance can be indirectly coupled to each other and a detection side (reporter side) and the trapping substance can be indirectly coupled to each other.

Regarding a conventional immuno chromatographic method, when a sandwich method in which a target substance is trapped using two types of binding substances (e.g., antibodies or haptens) is performed, the two types of binding substances are designed such that one specifically binds to the immobilization side (support side) and the other specifically binds to the detection side (reporter substance side). For example, regarding a sandwich method in which a target substance is trapped using two different types of monoclonal antibodies, it is known that, according to the types of antibodies, some are preferably used on the immobilization side (support side) and others are preferably used on the detection side (reporter substance side).

One example of the detection target of the immuno chromatography is an influenza virus. In order to identify subtypes and drug-resistant strains of an influenza virus, trapping substances such as antibodies and nucleic-acid aptamers targeting hemagglutinin (HA), neuraminidases, and RNA polymerases are needed in addition to those targeting nucleoproteins. However, these virus proteins are likely to undergo mutation, and the change of the trapping substance is imperative to maintain the sensitivity. This is not limited to the influenza virus and also applies to viruses, bacteria, fungi, and the like that may undergo mutation.

When an aptamer is applied to immuno chromatography, or an aptamer obtained for a new target substance or a mutated target substance is applied to immuno chromatography, it is necessary to design two types of aptamer probes, namely aptamer probes for the immobilization side (support side) and the detection side (reporter substance side), relative to one type of aptamer in the conventional method.

If one of the two types of aptamer probes that bind to a target is limited to being coupled to the immobilization side (support side) and the other is limited to being coupled to the detection side (reporter substance side), it is conceivable that the reaction efficiency of binding to the support and the reporter (target substance trapping efficiency) will decrease.

Accordingly, another aspect of the present disclosure provides an immuno chromatographic detection method in which an aptamer probe that can indirectly bind to both the immobilization side (support side) and the detection side (reporter substance side) is used.

An aspect of the present disclosure relates to a method for detecting a target substance in a sample, and the method includes: a reaction step of reacting the sample, a support including a first binding portion, a reporter substance including a second binding portion, a first trapping substance that includes a first binding partner portion capable of binding to the first binding portion and the second binding portion and that can bind to the target substance, and a second trapping substance that includes a second binding partner portion capable of binding to the first binding portion and the second binding portion and that can bind to the target substance to form a complex of “support—first trapping substance—target substance—second trapping substance—reporter substance” or a complex of “support—second trapping substance—target substance—first trapping substance—reporter substance”; and a detection step of detecting a signal from the reporter substance in the complex. This method is also referred to as a “first detection method according to the present disclosure” hereinafter.

Another aspect of the present disclosure relates to a target substance detection system for performing the above-mentioned method for detecting a target substance in a sample. This system is also referred to as a “first detection system according to the present disclosure” hereinafter.

Another aspect of the present disclosure relates to a method for detecting a target substance in a sample using an immuno chromatographic method, and the method includes: using an aptamer probe including an aptamer portion that can bind to the target substance, and a binding partner portion that can bind to both a binding portion immobilized on (and forming part of) a support and a binding portion immobilized on (and forming part of) a reporter substance to trap the target substance and the reporter substance on the support in a binding state represented as “support—aptamer probe—target substance—aptamer probe—reporter substance”; and detecting a signal from the reporter substance. The above method uses one or more aptamer probe. This method is also referred to as a “second detection method according to the present disclosure” hereinafter.

Another aspect of the present disclosure relates to a target substance detection system in which an immuno chromatographic method is used, and the system includes:

an immuno chromatographic test piece including a detection region on which a binding portion is immobilized;

a reporter substance on which a binding portion is immobilized; and

an aptamer probe including a binding partner portion that can bind to both the binding portion of the detection region and the binding portion of the reporter substance, and an aptamer portion that can bind to the target substance. This system is also referred to as a “second detection system according to the present disclosure” hereinafter.

Another aspect of the present disclosure relates to a kit for performing the second detection method according to the present disclosure, and the kit includes:

an immuno chromatographic test piece including a detection region on which a binding portion is immobilized;

a reporter substance on which a binding portion is immobilized; and

an aptamer probe including a binding partner portion that can bind to both the binding portion of the detection region and the binding portion of the reporter substance, and an aptamer portion that can bind to the target substance. This kit is also referred to as a “second detection kit according to the present disclosure” hereinafter.

With the first aspect according to the present disclosure (including the first detection method and the first detection system according to the present disclosure), a target substance detection method in which a trapping substance that can indirectly bind to both the immobilization side (support side) and the detection side (reporter substance side) is used can be provided, for example.

As a result, even if there are many candidate trapping substances, defining the first binding portion and the second binding portion in advance makes it possible to perform an assay in which many candidate trapping substances are utilized as they are without examining the conditions for immobilizing the candidate trapping substances. This leads to a reduction in the amounts of examination samples and thus a significant reduction in the examination time. In particular, when many trapping substances are subjected to screening in a trapping substance selecting step of the SELEX method or the like, employing the present disclosure makes it possible to significantly reduce the screening time and reduce the cost.

Moreover, since processing for immobilizing the trapping substances on a support or a reporter substance is not performed, damage to the trapping substance due to the immobilization processing can be minimized, thus making it possible to perform an assay while maintaining the function of recognizing a target substance.

Furthermore, immobilizing the trapping substance via the first binding portion on the immobilization side makes it possible to maintain the orientation of the trapping substance. Thus, for example, the trapping efficiency of the target substance by the trapping substance can be improved. The immobilization of the trapping substance via the first binding portion on the immobilization side can be performed, for example, by providing a binding partner portion at a position of the trapping substance that is different from the target substance recognition portion (portion that binds the target substance) and binding the binding partner portion to the first binding portion on the immobilization side. Thus, the trapping substance can be indirectly immobilized on the immobilization side. Thus, since the target substance recognition portion of the trapping substance is not used for binding to the support, the trapping substance is immobilized on the support in a state in which the trapping substance can bind to the target substance (that is, a state in which the target substance recognition portion of the trapping substance is exposed and the orientation of trapping substance is retained).

With the second aspect according to the present disclosure (including the second detection method, the second detection system, and the second detection kit according to the present disclosure), an immuno chromatographic detection method in which an aptamer probe that can bind to both the immobilization side (support side) and the detection side (reporter substance side) is used can be performed.

In one or more embodiments that are not limited, when a plurality of aptamers can bind to a target substance, preparing a single type of aptamer makes it possible to perform the immuno chromatographic detection method.

