Compound, conjugate, light emission detection system, and measurement device

Abstract

The main purpose of the present invention is to provide a technology for, in relation to a compound in which a plurality of enzymes are substituted or indirectly bonded, suppressing steric hindrance between the plurality of enzymes and suppressing a decrease in activity of the plurality of enzymes. As a result of performing intensive studies, the present inventors have found that, by obtaining a compound which is provided with at least one repeating structure comprising a repeating unit having an arbitrarily defined chemical structure and in which the repeating unit has one or more side chains in which an enzyme is substituted, it is possible to maintain the distance between enzymes within a certain range and suppress steric hindrance.

Description

Compounds, conjugates, luminescence detection systems and measuring devices

[0001] The present technology relates to compounds, conjugates, luminescent detection systems and measurement devices.

[0002] Conventionally, a method has been known in which a conjugate having a plurality of enzymes is used in an enzyme chemiluminescence immunoassay or the like.

[0003] For example, Non-Patent Document 1 below discloses an example of using a conjugate in which an HRP (horseradish peroxidase) enzyme and an antibody are conjugated to the surface of gold nanoparticles in an enzyme chemiluminescence immunoassay, although this technology does not focus on suppressing steric hindrance between multiple enzymes in the conjugate.

[0004] Chemiluminescence immunoassay for the rapid and sensitive detection of antibody against porcine parvovirus by using Horseradish peroxidase / detection antibody-coated gold nanoparticles as nanoprobes, Luminescence 2014, 29(4): 338- 343

[0005] The main purpose of this technology is to provide a technology that suppresses steric hindrance between multiple enzymes and suppresses a decrease in the activity of multiple enzymes in relation to a compound to which the multiple enzymes are substituted or indirectly bound.

[0006] As a result of extensive research, the present inventors have found that by using a compound having one or more repeating structures made up of repeating units of any chemical structure, in which the repeating units have one or more enzyme-substituted side chains, the distance between the enzymes can be maintained within a certain range and steric hindrance can be suppressed.

[0007] That is, the present technology provides a compound having one or more repeating structures made of repeating units having any chemical structure, and the repeating units have one or more enzyme-substituted side chains. The compound of the present technology may have a structure represented by the following formula (1): (1) n is an integer of 1 or more, m is an integer of 1 or more, A is a repeating unit having any chemical structure, B is an enzyme unit, and when n is an integer of 2 or more, the two or more Bs may be the same or different from each other, L1 is a single bond or a divalent group, and when n is an integer of 2 or more, the two or more L1s may be the same or different from each other, L2, L3, or L4 is a single bond or a divalent group, Q is a reactive group capable of forming a bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q', and when there are two or more Qs, the two or more Qs may be the same or different from each other, R1 is H or a monovalent group, and R2 is H, a monovalent group, or Q. Furthermore, in the compound according to the present technology, A may be represented by the following formula (2): (2) l is an integer of 1 or more, L6 and L7 are single bonds or divalent groups, and L6 and L1, or L7 and L1, may be bonded to each other to form a ring, L8 is a single bond or a divalent group, and when l is an integer of 2 or more, two or more L8s may be the same or different from each other, R4 is H or a monovalent group, and two or more R4s may be the same or different from each other, R5 is H or a monovalent group, and two or more R5s may be the same or different from each other, and R6 is H or a monovalent group. In the compound according to the present technology, Q may be a reactive group capable of forming a bond with a complementary reactive group Q', and one of Q or Q' may be avidin or an avidin derivative, and the other may be biotin or a biotin derivative. In the compound according to the present technology, B may be ALP, HRP, β-gal, or α-araf. The compound according to the present technology may be used for identification labeling.

[0008] Next, the present technology provides a conjugate in which an antibody substituted with or indirectly bound to the complementary reactive group Q' is bound to one or more compounds of the present technology.The present technology also provides a luminescence detection system in which a conjugate having an antibody against the antigen and an enzyme unit is bound to an antigen directly or indirectly via antigen-antibody bond, and luminescence is detected when a luminescent substrate is decomposed by the enzyme unit, wherein the antibody is substituted with or indirectly bound to the complementary reactive group Q', and the antibody is bound to one or more compounds of the present technology to form the conjugate. Furthermore, the present technology provides a luminescence detection system that directly or indirectly binds an antibody against the antigen, the antibody being substituted with the complementary reactive group Q' or to which the complementary reactive group Q' can indirectly bind, via antigen-antibody binding, and then binds the antibody to one or more compounds of the present technology via the bond between Q and the complementary reactive group Q' to form a conjugate, and detects luminescence when a luminescent substrate is decomposed by the enzyme unit. In the luminescence detection system of the present technology, the antigen may be present as particles comprising the antigen, and the particles may be cells. Next, the present technology provides a measurement device that employs the luminescence detection system of the present technology.

[0009] FIG. 1 is an image showing an example of a compound according to the present technology. FIG. 2 is an image showing an example of a conjugate according to the present technology. FIG. 3 is an image showing an example of an antibody according to the prior art. FIG. 4 is an image showing the binding state between a conjugate and an antigen according to the present technology when luminescence detection is performed by a direct method using a luminescence detection system according to the present technology. FIG. 5 is an image showing the binding state between a conjugate and an antigen according to the present technology when luminescence detection is performed by an indirect method using a luminescence detection system according to the present technology. FIG. 6 is an image showing the process of forming a binding state between a conjugate and an antigen according to the present technology, for a luminescence detection system using a direct method in a sensitization method. FIG. 7 is an image showing the process of forming a binding state between a conjugate and an antigen according to the present technology, for a luminescence detection system using an indirect method in a sensitization method. FIG. 8 is a measurement result of ALP enzyme activity measured by luminescence measurement for a conjugate etc. of the present technology.

[0010] Preferred embodiments of the present technology will be described below. However, the embodiments shown below are examples of typical embodiments of the present technology, and the present technology is not limited to only the preferred embodiments below and can be freely modified within the scope of the present technology.

[0011] <Compound> The compound according to the present technology has one or more repeating structures made up of repeating units having any chemical structure, and the repeating units have one or more side chains substituted with an enzyme.

[0012] In the compounds of the present technology, the "repeating structure" refers to a structure in which the "repeating unit" is repeated two or more times. The number of repeating units is not particularly limited as long as it is two or more, and any number of repeating units can be selected, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.

[0013] There is no upper limit to the number of repeats of the repeating unit, and any number of repeats that can be synthesized can be used. For example, the number of repeats can be selected arbitrarily, such as 30 or less, 20 or less, 15 or less, or 10 or less.

[0014] In the compound of the present technology, the repeating unit has one or more side chains substituted with an enzyme. Any enzyme can be appropriately selected as the enzyme depending on the application of the present technology. For example, when the present technology is used for identification labeling in a luminescence detection system, any sensitized enzyme such as ALP (Alkaline Phosphatase), HRP (Horseradish Peroxidase), β-gal (β-Galactosidase), or α-arabinofuranosidase (α-Arabinofuranosidase) can be suitably used.

[0015] As used herein, the term "identification labeling application" refers to labeling performed to identify a specific analyte, such as applications for determining the presence, location, or amount of a specific analyte such as a biomolecule, and also includes immunohistochemical staining.

[0016] In the compound of the present technology, the repeating unit may have two or more enzyme-substituted side chains, in which case the two or more enzymes may be the same enzyme or different enzymes.

[0017] The compound of the present technology has a structure in which the side chains substituted with the enzymes are linked as repeating units, and therefore the distance between adjacent enzymes arranged on the side chains is maintained at a predetermined distance, which is expected to suppress so-called steric hindrance, which occurs when the enzymes are too closely packed together, blocking the active site of the enzyme and preventing it from reacting with the substrate, thereby reducing activity.

[0018] The compound of the present technology may have two or more of the repeating structures. In this case, the repeating structures may be the same structure or different structures.

[0019] The compounds of the present technology may further have reactive groups capable of forming bonds with complementary reactive groups.

[0020] An example of a combination of the complementary reactive group and a reactive group capable of forming a bond with the complementary reactive group is a combination of avidin and biotin.

[0021] The compound of the present technology further has a reactive group capable of forming a bond with a complementary reactive group, thereby forming a conjugate with an antibody or the like that substitutes or indirectly binds to the complementary reactive group. This allows, for example, detection of an antigen by directly or indirectly binding to the antigen through antigen-antibody binding.

[0022] An example of the compound of the present technology may have a structure of the following formula (1):

[0023] (1)

[0024] In formula (1), n ​​represents the number of side chains possessed by the repeating unit A. n is an integer of 1 or greater, and can be any integer such as 1, 2, 3, or 4. The two n's defined in formula (1) may be the same or different.

[0025] In formula (1), m represents the number of repetitions of the repeating unit A. m is an integer of 1 or more, and can be any integer selected within the range in which synthesis is possible, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. As mentioned above, there is no upper limit to m, but it can be any integer selected from, for example, 30 or less, 20 or less, 15 or less, 10 or less, etc.

[0026] In formula (1), B is an enzyme unit, and any enzyme can be appropriately selected depending on the application of the present technology. For example, when the present technology is used for identification labeling in a luminescence detection system, enzymes such as ALP, HRP, β-gal, and α-araf can be suitably used.

[0027] When n is an integer of 2 or more, two or more Bs may be the same, but depending on the purpose of use of the present technology, two or more enzymes that are different from each other may also be used.

[0028] In formula (1), L1 is a single bond or a divalent group. When n is an integer of 2 or more in formula (1), two or more L1 may be the same or different from each other.

[0029] In formula (1), L-B may, for example, have the following structure:

[0030] wherein L1a and L1b each independently represent a single bond or a divalent group.

[0031] Furthermore, in formula (1), L1-B may be, for example, the following structure:

[0032] wherein L1a and L1b each independently represent a single bond or a divalent group.

[0033] In some embodiments, L1a and L1b may, for example, independently have the following structures:

[0034]

[0035] The ion implantation device may have one of the following:

[0036] In formula (1), L2, L3, or L4 is a single bond or a divalent group, R1 is H or a monovalent group, and R2 is H, a monovalent group, or Q described below.

[0037] In formula (1), Q is a reactive group capable of forming a bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q'. These may further have one or more substituents, as needed, as long as the desired physical properties are not significantly impaired. Furthermore, when there are two or more Qs in formula (1), the two or more Qs may be the same or different from each other.

[0038] In formula (1), when Q is a moiety containing a reactive group capable of forming a bond with a complementary reactive group Q′, a conjugate in which the compound of the present technology is bound to, for example, an antibody can be formed by binding Q and Q′.

[0039] Here, a "reactive group" is a moiety that can react with a second reactive group (a "complementary reactive group") to form one or more bonds, for example, by substitution, oxidation, reduction, addition, or cycloaddition reaction, etc. Exemplary reactive groups are provided in Table 1 below and include, for example, nucleophilic groups, electrophilic groups, dienes, dienophiles, aldehydes, oximes, hydrazones, alkynes, amines, azides, acyl azides, acyl halides, nitriles, nitrones, sulfhydryls, disulfides, sulfonyl halides, isothiocyanates, imidoesters, activated esters, ketones, α,β-unsaturated carbonyls, alkenes, maleimides, α-haloimides, epoxides, aziridines, tetrazines, tetrazoles, phosphines, biotin, thiirane, and the like.

[0040] In the above case, Q and Q' are not particularly limited as long as they are a combination that can form a bond through interaction, and an example is a combination of biotin and avidin.

[0041] In the present technology, by making one of Q and Q' avidin or an avidin derivative and the other biotin or a biotin derivative, a conjugate between the compound of the present technology and an antibody or the like can be suitably formed by avidin-biotin bonding.

[0042] Examples of Q that can be used in formula (1) are shown in Table 1 below. Note that Table 1 shows examples of Q, and Q is not limited to these.

[0043]

[0044]

[0045]

[0046]

[0047] In formula (1), A is a repeating unit having any chemical structure. The chemical structure of the repeating unit is not particularly limited as long as it can maintain a predetermined distance between the enzyme units B. In the compound of the present technology, the presence of A is expected to suppress so-called steric hindrance, in which the active site of the enzyme related to B is blocked, making it unable to react with the substrate, thereby reducing activity. For example, the chemical structure shown in the following formula (2) can be used as A.

[0048] (2)

[0049] In formula (2), l is an integer of 1 or more. By adjusting the value of l, the distance between the Bs can be adjusted. The l is an integer of 1 or more, and any integer can be selected within a range that can be combined, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. There is no upper limit to l, but it can be selected arbitrarily from, for example, 30 or less, 20 or less, 15 or less, 10 or less, etc.

[0050] In formula (2), L6 and L7 are single bonds or divalent groups, L6 and L1 or L7 and L1 may be bonded to each other to form a ring, L8 is a single bond or divalent group, and when l is an integer of 2 or more, two or more L8 may be the same or different from each other.

[0051] In formula (2), when L6 and L1 or L7 and L1 are bonded to each other to form a ring, the structure of the formed ring is not particularly limited and may be a carbocyclic ring or a heterocyclic ring. For example, when L1 and L7 are bonded to each other to form a ring, the following structure is considered, but is not limited thereto.

[0052]

[0053] In formula (2), R4 is H or a monovalent group. Two or more R4s may be the same or different from each other. R5 is H or a monovalent group. Two or more R5s may be the same or different from each other. R6 is H or a monovalent group.

[0054] In the present technology, the "monovalent group" is not particularly limited as long as it is a group having one binding site, and suitable examples include monovalent groups such as OH, SH, alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether, monovalent carbocyclic groups such as aryl or cycloalkyl, and heterocyclic groups such as heteroaryl or heterocycloalkyl. These may further have one or more substituents as necessary, as long as the desired physical properties are not significantly impaired.

[0055] In the present technology, the "divalent group" is not particularly limited as long as it is a group having two binding sites, and for example, divalent groups such as alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker, arylene, heteroarylene, etc. can be suitably used. These may further have one or more substituents as necessary, as long as the desired physical properties are not significantly impaired.

[0056] In the present technology, the term "substituent" refers to an atom or atomic group other than a hydrogen atom that is introduced by replacing a hydrogen atom that constitutes a hydrocarbon having a target chemical structure.

[0057] "Alkyl" refers to a straight or branched hydrocarbon chain group consisting of carbon and hydrogen atoms, containing no unsaturation, e.g., 1 to 12 carbon atoms (C1-C12 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), or 1 to 6 carbon atoms (C1-C6 alkyl), attached to the remainder of the molecule by a single bond (e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, etc.). Unless otherwise specifically stated in the specification, the hydrogen atoms comprising an alkyl group may optionally be substituted.

[0058] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain, consisting of carbon and hydrogen, containing no unsaturation, and having 1 to 12 carbon atoms (e.g., methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc.), that connects the rest of the molecule to the radical group. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule or to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, alkylene can have optional substituents.

[0059] "Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain, consisting of carbon and hydrogen, containing at least one carbon-carbon double bond, e.g., 2 to 12 carbon atoms, that connects the rest of the molecule to a radical group (e.g., ethenylene, propenylene, n-butenylene, etc.). The alkenylene chain is attached to the rest of the molecule through a single bond and to the radical group through a double bond or a single bond. The points of attachment of the alkenylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, alkenylene is optionally substituted.

[0060] "Alkynylene" or "alkynylene chain" refers to a straight or branched divalent hydrocarbon chain, consisting of carbon and hydrogen, containing at least one carbon-carbon triple bond, e.g., 2 to 12 carbon atoms, (e.g., ethenylene, propenylene, n-butenylene, etc.), that connects the rest of the molecule to a radical group. The alkynylene chain is attached to the rest of the molecule through a single bond and to the radical group through a double bond or a single bond. The points of attachment of the alkynylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, alkynylene can optionally have substituents.

[0061] "Alkyl ether" refers to any alkyl group, as defined above, in which at least one carbon-carbon bond is replaced with a carbon-oxygen bond. The carbon-oxygen bond can be terminal (i.e., C-O-H) (as in an alkoxy group) or the carbon-oxygen bond can be internal (i.e., C-O-C). An alkyl ether contains at least one carbon-oxygen bond, but can contain more than one. For example, polyethylene glycol (PEG) is included within the meaning of an alkyl ether. Unless otherwise specifically stated herein, alkyl ether groups can optionally have substituents.

[0062] "Alkoxy" refers to a group of the formula -ORa, where Ra is an alkyl group as defined above, for example, containing 1 to 12 carbon atoms. Unless stated otherwise specifically in the specification, an alkoxy group may optionally bear a substituent.

[0063] "Alkoxyalkyl ether" refers to a group of the formula -ORaRb, where Ra is an alkylene group as defined above, for example, containing 1 to 12 carbon atoms, and Rb is an alkyl ether group as defined herein. Unless stated otherwise specifically in the specification, an alkoxyalkyl ether group may optionally bear a substituent.

[0064] "Heteroalkyl" refers to an alkyl group (as defined above) that includes at least one heteroatom (e.g., N, O, P, or S) within or at the terminus of the alkyl group. Unless otherwise specifically stated in the specification, heteroalkyl groups can have optional substituents.

[0065] "Heteroalkoxy" refers to a radical of the formula -ORa, where Ra is a heteroalkyl radical as defined above, e.g., containing 1 to 12 carbon atoms. Unless stated otherwise specifically in the specification, a heteroalkoxy radical may optionally bear a substituent.

[0066] "Heteroalkylene" refers to an alkylene group, as defined above, containing at least one heteroatom (e.g., N, O, P, or S) within the alkylene chain or at the terminus of the alkylene chain. Unless stated otherwise specifically in the specification, heteroalkylene groups can have optional substituents.

[0067] Exemplary heteroalkylene groups include ethylene oxide (e.g., polyethylene oxide) and the "C" linking groups illustrated below: Includes.

[0068] Multimers of the above C-linkers are included in the various embodiments of heteroalkylene linkers.

[0069] "Heteroalkenylene" is a heteroalkylene (as defined above) containing at least one carbon-carbon double bond. Unless stated otherwise specifically in the specification, heteroalkenylene groups may optionally bear substituents.

[0070] "Heteroalkynylene" means a heteroalkylene (as defined above) containing at least one carbon-carbon triple bond. Unless stated otherwise specifically in the specification, a heteroalkynylene group may optionally bear substituents.

[0071] A "heteroatom linker" refers to a linker group consisting of one or more heteroatoms. Exemplary heteroatom linkers contain one or more heteroatoms selected from the group consisting of O, N, P, and S (e.g., linkers having the formula -P(O-)(=O)O- or -OP(O-)(=O)O-, as well as multimers and combinations thereof). Unless otherwise specifically stated herein, heteroatom linkers may optionally have substituents.

[0072] "Phosphate" refers to the group -OP(=O)(Ra)Rb, where Ra is OH, O-, or ORc; and Rb is OH, O-, ORc, a thiophosphate group as described below, or a further phosphate group, where Rc is a counterion (e.g., Na+, etc.). Unless otherwise specifically stated herein, the phosphate group may have optional substituents.

[0073] "Phosphoalkyl" refers to the group -OP(=O)(Ra)Rb, where Ra is OH, O-, or ORc; and Rb is -Oalkyl, where Rc is a counterion (e.g., Na+, etc.). Unless otherwise specifically stated herein, phosphoalkyl groups may optionally have substituents.

[0074] "Phosphoalkyl ether" refers to the group -OP(=O)(Ra)Rb, where Ra is OH, O-, or ORc; and Rb is an -Oalkyl ether, where Rc is a counterion (e.g., Na+, etc.). Unless otherwise specifically stated herein, phosphoalkyl ether groups may optionally have substituents.

[0075] "Thiophosphate" refers to the group -OP(=Ra)(Rb)Rc, where Ra is O or S; Rb is OH, O-, S-, ORd, or SRd; and Rc is OH, SH, O-, S-, ORd, SRd, a phosphate group, or an additional thiophosphate group, where Rd is a counterion (e.g., Na+), and with the proviso that: i) Ra is S; ii) Rb is S- or SRd; iii) Rc is SH, S-, or SRd; or iv) a combination of i), ii), and / or iii). Unless specifically stated otherwise herein, thiophosphate groups can optionally bear substituents.

[0076] "Thiophosphoalkyl" refers to the group -OP(=Ra)(Rb)Rc, where Ra is O or S; Rb is OH, O-, S-, ORd, or SRd; and Rc is -Oalkyl, where Rd is a counterion (e.g., Na+), and with the proviso that: i) Ra is S; ii) Rb is S- or SRd; or iii) Ra is S and Rb is S- or SRd. Unless stated otherwise specifically in the specification, thiophosphoalkyl groups may bear optional substituents.

[0077] "Thiophosphoalkyl ether" refers to the group -OP(=Ra)(Rb)Rc, where Ra is O or S, Rb is OH, O-, S-, ORd, or SRd; and Rc is an -Oalkyl ether, where Rd is a counterion (e.g., Na+), and with the proviso that: i) Ra is S, ii) Rb is S- or SRd, or iii) Ra is S and Rb is S- or SRd. Unless otherwise specifically stated herein, thiophosphoalkyl ether groups may bear optional substituents.

[0078] "Carbocyclic" refers to a stable 3- to 18-membered aromatic or non-aromatic ring containing, for example, 3 to 18 carbon atoms. Unless stated otherwise specifically in the specification, a carbocyclic ring can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system which can include fused or bridged ring systems and can be partially or fully unsaturated. Non-aromatic carbocyclyl radicals include cycloalkyl, while aromatic carbocyclyl radicals include aryl. Unless stated otherwise specifically in the specification, a carbocyclic group can optionally bear substituents.

[0079] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic carbocyclic ring, e.g., having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, saturated or unsaturated, and attached to the remainder of the molecule by a single bond, which may include fused or bridged ring systems. Monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyls include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo-[2.2.1]heptanyl, and the like. Unless otherwise specifically stated in the specification, cycloalkyl groups may optionally bear substituents.

[0080] "Aryl" refers to a ring system containing at least one carbocyclic aromatic ring. In some embodiments, an aryl contains 6 to 18 carbon atoms. The aryl ring can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or bridged ring systems. Aryl includes, but is not limited to, aryl derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless stated otherwise specifically in the specification, aryl groups can optionally bear substituents.

[0081] "Heterocyclic" refers, for example, to a stable 3- to 18-membered aromatic or non-aromatic ring containing from 1 to 12 carbon atoms and from 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless otherwise specifically stated herein, the heterocyclic ring can include fused or bridged ring systems, the nitrogen, carbon, or sulfur atoms in the heterocyclic ring can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclic ring can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system which can be partially or fully unsaturated.

[0082] Examples of aromatic heterocyclic rings are listed below in the definition of heteroaryl below (ie, heteroaryl is a subset of heterocyclic). Examples of non-aromatic heterocyclic rings include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, a heterocyclic group may optionally have substituents.

[0083] "Heteroaryl" refers, for example, to a 5-14 membered ring system containing 1 to 13 carbon atoms, 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. For purposes of certain embodiments of the present invention, the heteroaryl radical can include fused or bridged ring systems; the nitrogen, carbon, or sulfur atoms in the heteroaryl radical can be optionally oxidized; and the nitrogen atom can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can be optionally quaternized.Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzoindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzo Furanyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, benzoxazolinonyl, benzimidazolethionyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizyl nyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyridinonyl, pyrazinyl, pyrimidinyl, pyri Unless stated otherwise specifically in the specification, heteroaryl groups may optionally bear substituents.

[0084] "Heterocycloalkyl" refers to a cycloalkyl group, as defined above, containing at least one heteroatom (e.g., N, O, P, or S) within the cycloalkyl chain or at the terminus of the cycloalkyl chain. Unless stated otherwise specifically in the specification, heterocycloalkyl groups may optionally bear substituents.

[0085] "Arylene" refers to a divalent ring system containing at least one carbocyclic aromatic ring that links the rest of the molecule to a radical group. Unless stated otherwise specifically in the specification, an arylene group can optionally bear substituents.

[0086] "Heteroarylene" refers to an arylene group, as defined above, containing at least one heteroatom (e.g., N, O, P, or S) within or at the terminus of the arylene group. Unless otherwise specifically stated in the specification, heteroarylene groups may optionally bear substituents.

[0087] "Fused" refers to a ring system containing at least two rings, wherein the two rings share at least one common ring atom, e.g., two common ring atoms. When the fused ring is a heterocyclyl ring or a heteroaryl ring, the common ring atom can be carbon or nitrogen. Fused rings include bicyclic, tricyclic, tetracyclic, etc.

[0088] "Conjugation" refers to the overlap of one p orbital with another p orbital across an intervening sigma bond. Conjugation can occur in cyclic or acyclic compounds. "Degree of conjugation" refers to the overlap of at least one p orbital with another p orbital across an intervening sigma bond. For example, 1,3-butadiene has a degree of conjugation of one, while benzene and other aromatic compounds typically have multiple degrees of conjugation. Fluorescent and colored compounds typically contain at least one degree of conjugation.

[0089] In the compound of formula (1), examples of analyte molecules that can be Q include "biomolecules."

[0090] "Biomolecule" refers to any of a variety of biological substances, including nucleic acids, carbohydrates, amino acids, polypeptides, glycoproteins, hormones, aptamers, and mixtures thereof. More specifically, the term is intended to include, but is not limited to, RNA, DNA, oligonucleotides, modified or derivatized nucleotides, enzymes, receptors, prions, receptor ligands (including hormones), and the like.

[0091] In the compounds of formula (1), the "solid support" which can be Q refers to any solid substrate known in the art for solid-phase support of molecules, including, for example, microparticles, cells, etc. Here, "microparticle" refers to any of a number of small particles useful for binding to the compounds of the invention, including, but not limited to, glass beads, magnetic beads, polymeric beads, non-polymeric beads, etc. In certain embodiments, the microparticle comprises a polystyrene bead.

[0092] "Solid support residue" refers to a functional group that remains attached to a molecule when the molecule is cleaved from the solid support. Solid support residues are known in the art and can be readily obtained based on the structure of the solid support and the group linking the molecule to the solid support.

[0093] In compounds according to Formula (1), a "targeting moiety," which can be Q, is a moiety that selectively binds to or associates with a particular target (e.g., an analyte molecule). "Selectively" binding or associating means that the targeting moiety preferentially associates with or binds to the desired target compared to other targets. In some embodiments, the compounds disclosed herein include a linkage to a targeting moiety for the purpose of selectively binding or associating the compound with the analyte of interest (i.e., the target of the targeting moiety), thereby enabling detection of the analyte. Exemplary targeting moieties include, but are not limited to, antibodies, antigens, nucleic acid sequences, enzymes, proteins, cell surface receptor antagonists, and the like. In some embodiments, the targeting moiety is a moiety, such as an antibody, that selectively binds to or associates with a target feature on or in a cell (e.g., a target feature on the cell membrane or other cellular structure), thereby enabling detection of the cell of interest. Small molecules that selectively bind to or associate with the desired analyte are also contemplated as targeting moieties in certain embodiments. Those of skill in the art will recognize other analytes and corresponding targeting moieties that are useful in various embodiments.

[0094] Examples of compounds having the structure of formula (1) are shown in Table 2 below. Note that Table 2 shows examples of compounds having the structure of formula (1), but the compounds having the structure of formula (1) are not limited to these.

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] <Synthesis of Compounds of the Present Technology> The following reaction scheme shows an exemplary method for preparing compounds having the structure of formula (1). Those skilled in the art can prepare these compounds by similar methods or by combining other methods known to those skilled in the art. Those skilled in the art can prepare compounds having the structure of formula (1) that are not specifically exemplified herein by using appropriate starting components in a manner similar to that described below and, if necessary, modifying the synthesis parameters.

[0103] Generally, the starting components can be obtained from sources such as Sigma-Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or can be synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (Wiley, December 2000)), or can be prepared as described herein.

[0104] Reaction Scheme I

[0105] Reaction Scheme I illustrates an exemplary method for preparing intermediates useful for the preparation of compounds having the structure of Formula (1). Referring to Reaction Scheme 1, compounds of structure a can be purchased or prepared by methods well known to those skilled in the art. Reaction with B-X (where X is a halogen, such as bromo) under art-known Suzuki coupling conditions yields compounds of structure b. Compounds of structure b can be used to prepare compounds having the structure of Formula (1) as described below.

[0106] In the above formula, Lc is a monovalent group having at least one double structure, and becomes a divalent group by reaction with B-X. Furthermore, Ld and Le are divalent groups, and Ra, Rb, and R6 are monovalent groups.

[0107] Reaction Scheme II

[0108] Reaction Scheme II illustrates an alternative method for preparing intermediates useful in preparing compounds having the structure of Formula (1). Referring to Reaction Scheme II, a compound of structure c, which can be purchased or prepared by well-known techniques, is reacted with B-G' to provide a compound of structure d, where G and G' represent functional groups with complementary reactivity (i.e., functional groups that react to form a covalent bond). G' can be pendant to B or can be part of the structural backbone of B. G and G' can be any number of functional groups described herein, such as an alkyne and azide, respectively, an amine and activated ester, respectively, or an amine and isothiocyanate, respectively.

[0109] In the above formula, Lf, Ld, and Le are divalent groups, and Ra, Rb, and R6 are monovalent groups.

[0110] Compounds having the structure of formula (1) can be synthesized by synthesis of the following structure (e): (hereinafter "click" reaction)

[0111] DNA synthesis methods are well known in the art. Briefly, two alcohol groups (e.g., R and R in intermediates b or d above) are functionalized with a dimethoxytrityl (DMT) group and a 2-cyanoethyl-N,N-diisopropylamino phosphoramidite group, respectively. The phosphoramidite group is typically coupled to the alcohol group in the presence of an activator such as tetrazole, followed by oxidation of the phosphorus atom with iodine. The dimethoxytrityl group can be removed with an acid (e.g., chloroacetic acid) to reveal a free alcohol, which can be reacted with the phosphoramidite group. The 2-cyanoethyl group can be removed after oligomerization by treatment with aqueous ammonia.

[0112] The preparation of phosphoramidites used in the above oligomerization methods is also well known in the art. For example, a primary alcohol (e.g., Rb) can be protected as a DMT group by reaction with DMT-Cl. A secondary alcohol (e.g., Ra) is then functionalized as a phosphoramidite by reaction with an appropriate reagent, such as 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. Methods for the preparation of phosphoramidites and their oligomerization are well known in the art and are described in more detail in the Examples.

[0113] Compounds having the structure of formula (1) are prepared by oligomerization of intermediates b or d and e according to the well-known phosphoramidite chemistry described above. The desired number of m and l repeat units are incorporated into the molecule by repeating the phosphoramidite coupling the desired number of times.

[0114] In various other embodiments, compounds useful for the preparation of compounds having the structure of formula (1) are provided. These compounds can be prepared as described above in the form of monomers, dimers, and / or oligomers, and then a B moiety can be covalently attached to the compound via any number of synthetic methodologies (e.g., the "click" reaction described above) to form a compound having the structure of formula (1).

[0115] Alternatively, a compound having the structure of formula (1) can be prepared by subjecting a compound of structure c to the above-mentioned "click" reaction to form an intermediate shown in the following formula, which can then be reacted with the above-mentioned B-G'. In the following formula relating to the intermediate, G and G' defined in structure C are functional groups having complementary reactivity (i.e., functional groups that react to form a covalent bond), and are otherwise the same as defined in formula (1).

[0116]

[0117] <Luminescent detection system> When Q of the compound of the present technology is a moiety containing a reactive group capable of forming a bond with a complementary reactive group Q′, a conjugate obtained by bonding the compound of the present technology to an antibody substituted with or indirectly bound to the complementary reactive group Q′ can be suitably used for identification labeling purposes in a luminescent detection system.

[0118] The above-mentioned antibody may be directly substituted with the complementary reactive group Q' by replacing a hydrogen atom, other atom, or atomic group in the antibody, or the antibody may be indirectly bound to the complementary reactive group Q' by binding to a molecule or multimer having two or more complementary reactive groups Q' substituted with the reactive group Q.

[0119] The above-mentioned "multimer" may be a homomultimer in which two or more molecules of the same type are bound by interaction, or a heteromultimer in which two or more different types of molecules are bound by interaction. An example of a multimer is avidin, which usually forms a tetramer.

[0120] For example, when the conjugate of the present technology is used for identification labeling purposes in a luminescence detection system, the conjugate of the present technology is bound to an antigen directly or indirectly through antigen-antibody binding, and as described above, the antigen can be visually detected by detecting luminescence generated when a luminescent substrate is decomposed by an enzyme unit contained in the compound of the present technology bound to the antibody.

[0121] Here, "visual" and "visually detectable" refer to the ability to be visualized and detected directly or indirectly using a detection device. The enzymatic reaction caused by the enzyme unit of the compound of the present technology can be detected by observation with the naked eye or with the aid of an optical-based detection device (including, but not limited to, an absorption spectrophotometer, a transmission light microscope, a digital camera, and a scanner). Visually detectable substances are not limited to those that emit and / or absorb light within the visible spectrum. Substances that emit and / or absorb light within the ultraviolet (UV) region (about 10 nm to about 400 nm), the infrared (IR) region (about 700 nm to about 1 mm), and substances that emit and / or absorb in other regions of the electromagnetic spectrum are also included within the scope of "visually detectable" substances.

[0122] In this specification, the term "luminescence detection system" is not limited to a method for detecting luminescence, but also includes a kit for realizing a method for detecting luminescence, an apparatus equipped with a system for detecting luminescence, and the like.

[0123] When the compounds of the present technology are used for identification labeling purposes in luminescence detection systems, they are useful in a wide variety of analytical applications (e.g., biochemical and biomedical applications) that require the determination of the presence, location, or amount of a specific analyte (e.g., a biomolecule).Therefore, in another aspect, the present invention provides a method for visually detecting a biomolecule, the method comprising: (a) providing a biological system with a biomolecule having a compound of the present technology linked to the biomolecule; (b) promoting an enzymatic reaction by the enzyme unit of the compound of the present technology; and (c) detecting the biomolecule based on its visual properties.

[0124] For purposes of the present invention, "detecting a biomolecule by its visual properties" means detecting the biomolecule of interest directly or indirectly using an optically based detection device, including but not limited to, an absorbance spectrophotometer, a transmitted light microscope, a digital camera, and a scanner.

[0125] A densitometer can be used to quantify the amount of visually detectable biomolecules present. For example, the relative amounts of the biomolecules in two samples can be determined by measuring their relative optical density. If the stoichiometry of dye molecules per biomolecule is known and the extinction coefficient of the dye molecule is known, the absolute concentration of the biomolecule can also be determined from the optical density measurement. As used herein, the term "biological system" refers to any solution or mixture containing one or more biomolecules in addition to visually detectable biomolecules. Non-limiting examples of such biological systems include cells, cell extracts, tissue samples, electrophoresis gels, assay mixtures, and hybridization reaction mixtures.

[0126] [Method for detecting an antigen using a pre-prepared conjugate] As an example of a luminescence detection system to which the present technology can be applied, a method for detecting an antigen using a pre-prepared conjugate will first be described. This method includes a direct method and an indirect method, which will be described later.

[0127] When the present technology is used in a luminescence detection system that detects an antigen using a pre-prepared conjugate, a conjugate of the present technology having an antibody against the target antigen and an enzyme unit that catalyzes the decomposition reaction of a luminescent substrate is prepared in advance. The conjugate is formed by binding an antibody substituted with or indirectly bound to a complementary reactive group Q' to a compound of the present technology through the interaction between the reactive group Q and the complementary reactive group Q'.

[0128] FIG. 1 is an image diagram showing an example of a compound according to the present technology. In this example, compound 10 has seven enzyme units 11 and a reactive group 12 in the molecule. In this example, biotin is used as the reactive group 12, but the reactive group possessed by the compound of the present technology is not limited thereto. Note that this example is an example of a compound according to the present technology, and the compound of the present technology is not limited to the compound of this example.

[0129] FIG. 2 is an image diagram showing an example of a conjugate according to the present technology. In this example, two compounds 10 shown in FIG. 1 are conjugated to an antibody 14 to form a conjugate 20. In this example, the antibody 14 also has biotin as a reactive group 12, and the compounds 10 and the antibody 14 are conjugated via a reactive group-complementary reactive group bond 13 via a multimer of avidin, which is a complementary reactive group. As a result, the conjugate according to the present technology shown in this example integrally comprises 14 enzyme units. Furthermore, since the present technology has a structure in which repeating units in which enzyme units are substituted are linked, the distance between adjacent enzyme units is maintained at a predetermined distance, and this can suppress so-called steric hindrance, which occurs when the enzyme units are too closely packed together and the active site of the enzyme in the enzyme unit is blocked, making it unable to react with the substrate and resulting in a decrease in activity.

[0130] 3 shows an image of an example of an antibody according to the prior art for comparison with the compound of the present technology. In the example of the present technology, the prior art antibody 20-2 has an enzyme unit at the position of reactive group 12. As a result, the entire antibody integrally comprises two enzyme units.

[0131] As described above, when comparing the conjugate of the present technology with the antibody of the prior art, it can be confirmed that the conjugate of the present technology can integrally comprise more enzyme units than the antibody of the prior art. Furthermore, the present technology can also comprise even more enzyme units by adjusting the number of repeating units.

[0132] In a luminescence detection system that detects an antigen using a pre-prepared conjugate, the conjugate is added to a medium containing the antigen, and the conjugate binds directly or indirectly to the antigen through antigen-antibody binding. A luminescent substrate is then added to the medium, and the enzyme unit contained in the conjugate causes a decomposition reaction of the luminescent substrate, resulting in luminescence. The luminescence can be detected by the aforementioned method or the like, allowing the location of the antigen to be detected to be visually detected.

[0133] The "medium" used in the luminescence detection system is not particularly limited, and a suitable medium can be used appropriately depending on the state of the antigen to be detected. For example, known media such as a solid phase, a liquid layer, a gel layer, immobilized cells, a cell extract, or a sliced ​​tissue sample can be suitably used.

[0134] As used herein, the term "antigen" refers to a substance that specifically reacts with a particular antibody in vivo or in vitro. An antigen may also become an antibody against a different antigen.

[0135] The direct method refers to a luminescence detection system in which the conjugate of the present technology directly binds to the antigen to be detected through an antigen-antibody reaction.

[0136] Specifically, as shown in Fig. 4, the conjugate 20 of the present technology binds to the antigen 16 through an antigen-antibody bond. In this state, by adding a luminescent substrate to a medium or the like, the enzyme unit 11 advances the decomposition reaction of the luminescent substrate, generating luminescence. By detecting the luminescence using the method described above or the like, the position of the antigen 16 to be detected can be visually detected.

[0137] On the other hand, in the indirect method, the antigen to which the conjugate of the present technology binds through an antigen-antibody reaction is not the antigen to be detected, but an antibody against a different antigen. That is, in the indirect method, the conjugate of the present technology binds to a primary antibody, and the primary antibody binds to the antigen to be detected, thereby indirectly binding to the antigen to be detected via the primary antibody. When the indirect method is adopted in a luminescence detection system, multiple conjugates of the present technology can be bound to the primary antibody, allowing for more sensitive antigen detection.

[0138] In addition, the indirect luminescence detection system can be designed so that the conjugate of this technology binds to the antigen to be detected not only via a primary antibody, but also via multiple multi-antibodies, such as a primary antibody that binds to the antigen to be detected and a secondary antibody that further binds to the primary antibody.

[0139] FIG. 5 shows an example of an indirect method of a luminescence detection system that detects an antigen using a pre-prepared conjugate. Specifically, as shown in FIG. 5 , a conjugate 20 of the present technology binds to a primary antibody 17, which then binds to the antigen 16, thereby indirectly binding to the target antigen via the primary antibody 17. In FIG. 5 , to easily understand the binding state between the antigen and the conjugate, an image of one conjugate bound to the primary antibody 17 is shown. However, multiple conjugates 20 may bind to the primary antibody 17. In this state, by adding a luminescent substrate to the medium or the like, the enzyme unit 11 advances the decomposition reaction of the luminescent substrate, generating luminescence. By detecting this luminescence using the method described above or the like, the location of the target antigen 16 can be detected with higher sensitivity.

[0140] [Method of forming a conjugate after binding of an antibody to an antigen (sensitization method)] Next, an antigen detection method in which a conjugate is formed after binding of an antibody to an antigen (hereinafter referred to as "sensitization method") will be described. Note that, like the method of detecting an antigen using a pre-prepared conjugate, sensitization methods can be divided into direct and indirect methods.

[0141] When this technology is used in a luminescence detection system for a sensitization method, before forming a conjugate, an antigen is bound to an antibody against the antigen that is substituted with a complementary reactive group or to which a complementary reactive group is indirectly bound.

[0142] The term "indirect binding of the complementary reactive group" refers to, for example, the case where the antibody is substituted with a reactive group and binds to a molecule or polymer having two or more complementary reactive groups, thereby indirectly binding to the complementary reactive group.

[0143] More specifically, the antibody is added to a medium containing the antigen to promote the binding by the antigen-antibody bond.

[0144] As mentioned above, the sensitization method can be a direct method or an indirect method. In the direct method, the antibody that forms the conjugate of the present technology directly binds to the antigen to be detected.

[0145] On the other hand, in the indirect method, the antibody that forms the conjugate of the present technology binds to a primary antibody (the antigen of the antibody that forms the conjugate of the present technology), and the primary antibody binds to the antigen to be detected, thereby indirectly binding to the antigen to be detected via the primary antibody.

[0146] In the indirect method of sensitization, as in the case of a method of detecting an antigen using a pre-prepared conjugate, the antibody that forms the conjugate of this technology can be designed to bind not only to the antigen to be detected via a primary antibody, but also to bind via multiple multi-antibodies, such as a primary antibody that binds to the antigen to be detected and a secondary antibody that further binds to the primary antibody.

[0147] In both the direct and indirect sensitization methods, after binding of the antigen and antibody, the compound of the present technology is further added to a medium or the like, whereby the antibody bound to the antigen forms a conjugate through binding of the reactive group and the complementary reactive group with the compound of the present technology while maintaining the bound state with the antigen.

[0148] Then, by adding a luminescent substrate to the medium, etc., the enzyme unit of the conjugate causes the decomposition reaction of the luminescent substrate to proceed, resulting in luminescence. By detecting the luminescence using the method described above, etc., the position of the antigen to be detected can be visually detected.

[0149] When the sensitization method is used in the luminescence detection system of the present technology, there is no need to prepare a different conjugate for each antibody in advance, and therefore the luminescence detection system of the present technology can be used by providing the compound of the present technology as a general-purpose reagent for antibodies designed against the target antigen. Therefore, while narrowing the types of reagents manufactured, sold, etc., the luminescence detection system of the present technology can be provided for various antibody libraries, etc.

[0150] FIG. 6 is an image diagram showing the process of forming a binding state between the conjugate and the antigen of the present technology in a luminescence detection system using the direct method in the sensitization method.

[0151] As shown in Fig. 6A, the antibody 14 is added to a medium or the like containing the antigen 16, thereby binding the antibody 14 to the antigen 16. In the example shown in Fig. 6, the antibody 14 has biotin as the reactive group 12. Note that, as with the other examples, the reactive group is not limited to biotin.

[0152] Then, a multimer 15 (avidin) having two or more complementary reactive groups is added to the above-mentioned medium, and as shown in Figure 6B, the antibody 14 bound to the antigen 16 forms an antibody to which the complementary reactive groups are indirectly bound while maintaining the binding state with the antigen 16.

[0153] Thereafter, by adding the compound 10 of the present technology to the above-mentioned medium or the like, the antibody 14 forms a conjugate 20 of the present technology through a bond 13 between the reactive group and the complementary reactive group (avidin-biotin bond) while maintaining a bonded state with the antigen 16, as shown in FIG. 6C .

[0154] In this state, by adding a luminescent substrate to the medium or the like, the decomposition reaction of the luminescent substrate proceeds due to the enzyme unit 11, and luminescence occurs. By detecting the luminescence using the method described above or the like, the position of the antigen 16 to be detected can be visually detected.

[0155] FIG. 7 is an image diagram showing the process of forming a binding state between the conjugate and the antigen of the present technology in a luminescence detection system using the indirect method in the sensitization method.

[0156] As shown in FIG. 7A , a primary antibody 17 is first added to a medium containing an antigen 16, causing the primary antibody 17 to bind to the antigen 16. Next, an antibody 14 is further added, causing the antibody 14 to bind to the primary antibody 17. As a result, the antibody 14, which forms a conjugate with the compound of the present technology, indirectly binds to the antigen 16 via the primary antibody 17. Note that in the example shown in FIG. 7 , the antibody 14 has biotin as the reactive group 12, but as in other examples, the reactive group is not limited to biotin. Also, in FIG. 7 , to make it easier to understand the binding state between the antigen 16 and the antibody 14, an image of one antibody 14 binding to the primary antibody 17 is shown; however, multiple antibodies 14 may bind to the primary antibody 17.

[0157] Then, a multimer 15 (avidin) having two or more complementary reactive groups is added to the above-mentioned medium, and as shown in Figure 7B, the antibody 14 indirectly bound to the antigen 16 via the primary antibody 17 forms an antibody indirectly bound to the complementary reactive group while maintaining the binding state with the antigen 16.

[0158] Thereafter, by adding the compound 10 of the present technology to the above-mentioned medium or the like, the antibody 14 forms a conjugate 20 of the present technology through a reactive group-complementary reactive group bond 13 (avidin-biotin bond) while maintaining a bonded state with the antigen 16, as shown in FIG. 7C .

[0159] In this state, by adding a luminescent substrate to the medium or the like, the decomposition reaction of the luminescent substrate proceeds due to the enzyme unit 11, and luminescence occurs. By detecting the luminescence using the method described above or the like, the position of the antigen 16 to be detected can be detected with higher sensitivity.

[0160] [Antigen] In the luminescence detection system of the present technology, the antigen to be detected may be present in any form, such as cells, cell extracts, tissue samples, electrophoresis gels, assay mixtures, and hybridization reaction mixtures, depending on the state and purpose of the measured object, or may be present as particles comprising the antigen, for example.

[0161] Here, examples of particles carrying antigens include biological samples such as cells (including free cells of microorganisms) and microparticles such as fluorescent beads.

[0162] Furthermore, the luminescence detection system of the present technology can also detect the luminescence of particles by flow cytometry, which measures and analyzes particles passing through a flow channel such as a flow cell.

[0163] As described above, the luminescence detection system according to the present technology includes a plurality of enzymes, and detects luminescence generated when a luminescent substrate is decomposed using a conjugate in which steric hindrance between adjacent enzymes is suppressed, thereby enabling highly sensitive measurement. For this reason, the luminescence detection system can be suitably employed in measurement devices such as microscopes and plate readers.

[0164] The present technology will be described in detail below based on specific examples, but the present technology is not limited to the contents of the examples shown below.

[0165] <Raw Materials> Seven enzymes can be substituted into the structure of oligofluoroside (#SND2346, 5'-Biotinylated SND1471, Sony), which is an intermediate for the compound having the structure of formula (1). TE buffer pH 8.0 (#AM9849, Invitrogen), streptavidin (#S4762, Sigma), DEMSO, Anhydrous (#D12345, Thermo), IgG antibody (anti-human CD3, #317301, Biolegend), UltraComp eBeads (#01-2222-42, Thermo), Alkaline Phosphatase Labeling Kit-NH2 (LK12, Dojindo), Biotin Labeling Kit-NH2 (LK03, Dojindo), Immun-Start AP substrate (#1705018, BioRad)

[0166] <Preparation of Compounds and Conjugates of the Present Technology> [ALP Modification of Oligofluorosides] Using the oligofluorosides described above as intermediates for compounds having the structure of formula (1), ALP modification was carried out according to the following procedure, thereby obtaining oligofluoroxides substituted with seven ALPs.

[0167] (1) Dissolve 5 mg of dried oligofluoroside in 0.1 mL of reaction buffer (included in the Alkaline Phosphatase Labeling Kit). (2) Add 50 μL of the above oligofluoroside solution to the NH2-Reactive Alkaline Phosphatase. (3) After thoroughly dissolving the NH2-Reactive ALP by pipetting, allow to react at 37°C for 1 hour. (4) Add 100 μL of washing buffer provided with the Alkaline Phosphatase Labeling Kit to the reaction solution, and transfer this solution to the filtration tube provided with the Alkaline Phosphatase Labeling Kit. (5) Centrifuge at 8,000 × g for 10 minutes. (6) Discard the filtrate and add 200 μL of washing buffer. (7) Centrifuge at 8,000 × g for 10 minutes. Discard the filtrate, add 200 μL of washing buffer again, and centrifuge at 8,000 × g for 10 minutes. (8) Add 465 μL of the storage buffer included in the Alkaline Phosphatase Labeling Kit and pipette about 10 times to recover the ALP-modified oligofluoroside. (9) Store in a microtube at 0-5°C. (1 mM KDB w / ALP solution)

[0168] [Biotin Modification of Oligofluoroside] The ALP-modified oligofluoroside obtained in the above step was modified with biotin according to the following procedure.

[0169] (1) Dispense 25 μL of the pre-adjusted 0.5 mg / mL oligofluoroside solution into the filtration tube provided with the Biotin Labeling Kit. (2) Next, dispense 100 μL of the WS buffer provided with the Biotin Labeling Kit into the filtration tube. (3) Mix gently by pipetting, then centrifuge at 8000 × g for 10 minutes. (4) Add 10 μL of DMSO to the NH2-Reactive Biotin and dissolve by pipetting. (5) Add 100 μL of the Reaction Buffer included with the Biotin Labeling Kit onto the membrane of the Filtration Tube, then add 8 μL of NH2 reactive biotin DMSO solution. (6) Mix thoroughly with the oligofluoroside on the membrane by pipetting, then allow to react at 37°C for 10 minutes. (7) Add 100 μL of WS buffer to the Filtration Tube and centrifuge at 8000 × g for 10 minutes. After centrifugation, discard the filtrate. (8) Add 200 μL of WS buffer to the Filtration Tube and centrifuge at 8000 × g for 10 minutes. After centrifugation, discard the filtrate. This procedure is repeated twice. (9) Add 200 μL of WS buffer to the filtration tube and pipette about 10 times to recover the biotin-modified oligofluoroside. (10) Store in a microtube at 0-5°C.

[0170] [Synthesis of Biotinylated Antibody] Next, using the IgG antibody described above, the IgG antibody was modified with biotin according to the following procedure.

[0171] (1) Dispense 25 μL of the pre-adjusted 0.5 mg / mL IgG solution into the filtration tube provided with the Biotin Labeling Kit. (2) Next, dispense 100 μL of the WS Buffer provided with the Biotin Labeling Kit into the filtration tube. (3) Mix gently by pipetting, then centrifuge at 8,000 × g for 10 minutes. (4) Add 10 μL of DMSO to the NH2-Reactive Biotin and dissolve by pipetting. (5) Add 100 μL of the Reaction Buffer included with the Biotin Labeling Kit onto the membrane of the Filtration Tube, then add 8 μL of NH2 reactive biotin DMSO solution. (6) Mix thoroughly with the IgG on the membrane by pipetting, then allow to react at 37°C for 10 minutes. (7) Add 100 μL of WS buffer to the Filtration Tube and centrifuge at 8000 × g for 10 minutes. After centrifugation, discard the filtrate. (8) Add 200 μL of WS buffer to the Filtration Tube and centrifuge at 8000 × g for 10 minutes. After centrifugation, discard the filtrate. This procedure is repeated twice. (9) Add 200 μL of WS buffer to the filtration tube and pipette about 10 times to recover the biotin-modified IgG antibody (IgG-Biotin). (10) Store in a microtube at 0-5°C.

[0172] [Conjugate Formation] Using the above-mentioned ALP and biotin-modified oligofluoroside (KDB-ALP) and the above-mentioned biotin-modified IgG antibody, a conjugate of the present technology was formed on the surface of beads according to the following procedure. Because the IgG antibody can be bound to two oligofluorooxides via biotin-avidin bonds, the resulting conjugate has 7 x 2 = 14 ALPs. Note that this procedure is an example of conjugate formation used in the luminescence detection system using the direct method with sensitization, as shown in Figure 6.

[0173] (1) Dispense 50 μL of well-mixed Ultra eCompBeads into a microtube. (2) Add 10 μL of IgG-Biotin and allow to react in the refrigerator for 30 minutes. (3) Add 50 μL of TE buffer, centrifuge at 2000 × g for 1 minute, and discard 50 μL of the supernatant. Repeat this procedure three times. (4) Add 20 μL of 0.06 mg / mL streptavidin solution prepared with TE buffer and pipette thoroughly. (5) Allow to react at room temperature for 30 minutes. (6) Add 50 μL of TE buffer, centrifuge at 2000 × g for 1 minute, and discard 50 μL of the supernatant. Repeat this procedure three times. (7) Add 20 μL of KDB-ALP and allow to react at room temperature for 30 minutes. (8) Add 100 μL of TE buffer and centrifuge at 2000×g for 1 minute, then discard 180 μL of the supernatant. Repeat this process five times. (9) Add 90 μL of TE buffer to complete the process.

[0174] <Preparation of ALP-modified antibody for comparison> As a comparison with this technology, an IgG antibody was directly modified with ALP, an example of an enzyme, to synthesize an ALP-modified IgG antibody (IgG-ALP) using the following procedure. Since this IgG antibody can bind to two ALPs, the IgG antibody obtained by this procedure has two ALPs. (See the image in Figure 3.)

[0175] (1) Dispense 25 μL of the pre-adjusted 0.5 mg / mL IgG solution into the filtration tube provided with the Alkaline Phosphatase Labeling Kit. (2) Dispense 100 μL of the WS buffer provided with the Alkaline Phosphatase Labeling Kit into the filtration tube. (3) Mix gently by pipetting, then centrifuge at 8,000 × g for 10 minutes. (4) Add 10 μL of the reaction buffer provided with the Alkaline Phosphatase Labeling Kit to the NH2-Reactive ALP and dissolve by pipetting. (5) Add the solution containing NH2-Reactive ALP onto the membrane of the filtration tube where the IgG is concentrated. (6) Mix thoroughly with the IgG on the membrane by pipetting, and then allow to react for 2 hours at 37°C. (7) Add 200 μL of the storage buffer included with the Alkaline Phosphatase Labeling Kit, and recover the labeled product by pipetting approximately 10 times. (8) Store in a microtube at 0-5°C.

[0176] Using the above-mentioned ALP-modified IgG antibody (IgG-ALP), IgG-ALP was immobilized on the surface of beads by the following procedure.

[0177] (1) Dispense 50 μL of well-mixed Ultra eCompBeads into a microtube. (2) Add 10 μL of IgG-ALP and allow to react in a refrigerator for 30 minutes. (3) Add 50 μL of TE buffer, centrifuge at 2000 × g for 1 minute, and discard 50 μL of the supernatant. Repeat this procedure three times. (4) Add 50 μL of TE buffer to complete the reaction.

[0178] <Luminescence measurement> Using beads onto which the conjugate of KDB-ALP according to the present technology and biotin-modified IgG antibody obtained above was immobilized and beads onto which IgG-ALP for comparison was immobilized, the enzymatic activity of each ALP was measured by luminescence measurement according to the following procedure.

[0179] (1) Prepare a black-walled 96-well plate. (2) Start the measurement device (Spark 10M, Tecan). (3) Dispense 25 μL of each of the following samples into the microwells: a) Negative control: beads only b) Beads immobilized with an antibody labeled with two ALPs (IgG-ALP for comparison) c) Beads immobilized with a conjugate of KDB-ALP and biotin-modified IgG antibody (IgG-KDB-ALPs) according to this technology (4) Add 25 μL of luminescent substrate. (5) Implement measurement (measurement conditions are as follows): Exposure time: 1 second Interval: 1 minute Measurement mode: Luminescence Attenuation: None

[0180] The results of the luminescence measurement are shown in Figure 9. As mentioned above, the comparative IgG-ALP has two ALPs, whereas the IgG-KDB-ALPs conjugate according to the present technology has 14 ALPs. The measurement results in Figure 9 suggest that the conjugate according to the present technology has a structure in which ALP-substituted side chains are linked as repeating units, and therefore the distance between adjacent ALPs can be maintained at a predetermined distance, thereby suppressing steric hindrance.

[0181] The present technology can have the following configurations: [1] A compound having one or more repeating structures made of repeating units having any chemical structure, wherein the repeating units have one or more enzyme-substituted side chains. [2] A compound having a structure of the following formula (1): (1) n is an integer of 1 or more, m is an integer of 1 or more, A is a repeating unit having any chemical structure, B is an enzyme unit, and when n is an integer of 2 or more, the two or more Bs may be the same or different from each other, L1 is a single bond or a divalent group, and when n is an integer of 2 or more, the two or more L1s may be the same or different from each other, L2, L3, or L4 is a single bond or a divalent group, Q is a reactive group capable of forming a bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q', and when there are two or more Qs, the two or more Qs may be the same or different from each other, R1 is H or a monovalent group, and R2 is H, a monovalent group, or Q. [3] The compound according to [2], wherein A is represented by the following formula (2): (2) l is an integer of 1 or more, L6 and L7 are single bonds or divalent groups, and L6 and L1, or L7 and L1, may be bonded to each other to form a ring, L8 is a single bond or a divalent group, and when l is an integer of 2 or more, two or more L8s may be the same or different from each other, R4 is H or a monovalent group, and two or more R4s may be the same or different from each other, R5 is H or a monovalent group, and two or more R5s may be the same or different from each other, and R6 is H or a monovalent group. [4] The compound according to [2] or [3], wherein Q is a moiety containing a reactive group capable of forming a bond with the complementary reactive group Q'. [5] The compound according to [4], wherein one of Q or Q' is avidin or an avidin derivative, and the other is biotin or a biotin derivative. [6] The compound according to any one of [2] to [5], wherein B is ALP, HRP, β-gal, or α-araf. [7] The compound according to any one of [1] to [6], which is for use as an identification label. [8] A conjugate comprising an antibody to which the complementary reactive group Q' is substituted or to which the complementary reactive group Q' is indirectly bound, and one or more of the compounds according to any one of [2] to [7]. [9] A luminescence detection system in which a conjugate having an antibody against the antigen and an enzyme unit is bound to an antigen directly or indirectly by antigen-antibody binding, and luminescence generated when a luminescent substrate is decomposed by the enzyme unit is detected, wherein the antibody is substituted with the complementary reactive group Q' or to which the complementary reactive group Q' is indirectly bound, and the antibody forms the conjugate by binding to one or more of the compounds according to any one of [2] to [7].

[10] A luminescence detection system comprising: an antibody against the antigen, the antibody being substituted with the complementary reactive group Q' or capable of indirectly binding to the complementary reactive group Q', and then binding the antibody to one or more of the compounds according to any one of [2] to [7] via the bond between Q and the complementary reactive group Q' to form a conjugate; and detecting luminescence produced when a luminescent substrate is decomposed by the enzyme unit.

[11] The luminescence detection system according to [9] or

[10] , wherein the antigen exists as a particle comprising the antigen.

[12] The luminescence detection system according to

[11] , wherein the particle is a cell.

[13] A measuring device employing the luminescence detection system according to any one of [9] to

[12] .

[0182] 10 Compound of the present technology 11 Enzyme unit 12 Reactive group (biotin) 13 Bond of reactive group-complementary reactive group (avidin-biotin bond) 14 Antibody 15 Multimer having complementary reactive group (avidin) 16 Antigen 17 Primary antibody 20 Bond of the present technology 20-2 Antibody of the prior art

Claims

1. A compound comprising one or more repeating structures made of repeating units having any chemical structure, said repeating units having one or more enzyme-substituted side chains.

2. A compound having the structure of the following formula (1): (1) n is an integer of 1 or more; m is an integer of 1 or more; A is a repeating unit having any chemical structure; B is an enzyme unit, and when n is an integer of 2 or more, two or more Bs may be the same or different from each other; L1 is a single bond or a divalent group, and when n is an integer of 2 or more, two or more L1s may be the same or different from each other; L2, L3, or L4 is a single bond or a divalent group; Q is a reactive group capable of forming a bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q', and when there are two or more Qs, the two or more Qs may be the same or different from each other; R1 is H or a monovalent group; and R2 is H, a monovalent group, or Q.

3. The compound according to claim 2, wherein A is represented by the following formula (2): (2) l is an integer of 1 or more; L6 and L7 are a single bond or a divalent group, and L6 and L1 or L7 and L1 may be bonded to each other to form a ring; L8 is a single bond or a divalent group, and when l is an integer of 2 or more, two or more L8s may be the same or different from each other; R4 is H or a monovalent group, and two or more R4s may be the same or different from each other; R5 is H or a monovalent group, and two or more R5s may be the same or different from each other; R6 is H or a monovalent group.

4. The compound of claim 2, wherein Q is a moiety that contains a reactive group capable of forming a bond with a complementary reactive group, Q'.

5. The compound according to claim 4, wherein one of Q or Q' is avidin or a derivative of avidin, and the other is biotin or a derivative of biotin.

6. The compound according to claim 2, wherein B is ALP, HRP, β-gal, or α-araf.

7. The compound according to claim 2, which is used as an identification label.

8. A conjugate comprising an antibody substituted with or indirectly attached to said complementary reactive group Q' and one or more compounds according to claim 2.

9. A luminescence detection system which comprises a conjugate having an antibody against the antigen and an enzyme unit, which is directly or indirectly bound to an antigen via antigen-antibody binding, and which detects luminescence when a luminescent substrate is decomposed by the enzyme unit, wherein the antibody is substituted with the complementary reactive group Q' or the complementary reactive group Q' is indirectly bound to the antibody, and the antibody forms the conjugate by binding to one or more compounds according to claim 2.

10. A luminescence detection system comprising: an antibody against the antigen, which is substituted with the complementary reactive group Q' or to which the complementary reactive group Q' can indirectly bind, and then binding the antibody to one or more compounds according to claim 2 via the bond between Q and the complementary reactive group Q' to form a conjugate; and detecting luminescence produced when a luminescent substrate is decomposed by the enzyme unit.

11. The luminescence detection system of claim 10, wherein the antigen is present as a particle comprising the antigen.

12. The luminescence detection system of claim 11, wherein the particle is a cell.

13. A measuring device employing the luminescence detection system according to claim 10.

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