Method for analyzing trace substance, and trace substance analysis kit

Abstract

A method for analyzing a trace substance, the method comprising: a step A for binding, to the trace substance, an enzyme-labeled antibody comprising an antibody specific to the trace substance and an enzyme α, or an enzyme-labeled nucleic acid probe comprising a nucleic acid probe specific to the trace substance and the enzyme α; a step B for allowing the enzyme α in the enzyme-labeled antibody or the enzyme α in the enzyme-labeled nucleic acid probe to act on a compound β that is a substrate for the enzyme α to produce a compound γ that is a substrate for a dehydrogenase; a step C for producing a polymerized thio-NAD(P)H through an enzymatic cycling reaction using the produced compound γ, a dehydrogenase, NAD(P)H, and the polymerized thio-NAD(P); and a step D for detecting the trace substance with the polymerized thio-NAD(P)H generated.

Description

Method for analyzing trace substances and kit for analyzing trace substances

[0001] The present invention relates to a method for analyzing a trace substance and a kit for analyzing a trace substance.

[0002] Highly sensitive methods for analyzing biopolymers such as proteins and nucleic acids include enzyme immunoassays and nucleic acid probe assays such as Southern blotting, Northern blotting, and Western blotting. Improved detection sensitivity is required for these analytical methods, and various studies have been conducted.

[0003] In Patent Document 1, an attempt was made to improve detection sensitivity by combining enzyme immunoassay or nucleic acid probe assay with enzyme cycling to exponentially amplify thioNAD(P)H as a signal substance.

[0004] Patent Document 1 discloses an enzyme measurement method using an antibody-enzyme complex, in which the reaction products of the antibody-enzyme complex are quantified by generating thioNADH and / or thioNADPH through an enzyme cycling reaction using NADH and / or NADPH, thioNAD and / or thioNADP, and dehydrogenase (DH), and measuring the amount of the generated thioNADH and / or thioNADPH or by measuring a color change caused by the generated thioNADH and / or thioNADPH. Furthermore, Patent Document 1 discloses a method for measuring a nucleic acid probe using an enzyme-labeled nucleic acid probe, in which the enzymatic reaction products of the enzyme-labeled nucleic acid probe are quantified by generating thioNADH and / or thioNADPH through an enzymatic cycling reaction using NADH and / or NADPH, thioNAD and / or thioNADP, and dehydrogenase (DH), and measuring the amount of the generated thioNADH and / or thioNADPH, or by measuring a color change due to the generated thioNADH and / or thioNADPH.

[0005] WO 2008 / 117816

[0006] Lindberg M, Larsson PO, Mosbach K. A new immobilized NAD+ analogue, its application in affinity chromatography and as a functioning coenzyme. Eur J Biochem. 1973 Dec 3;40(1):187-93. Muramatsu M, Urabe I, Yamada Y, Okada H. Synthesis and kinetic properties of a new NAD+ derivative carrying a vinyl group. Eur J Biochem. 1977 Oct 17;80(1):111-7.

[0007] In the measurement method described in Patent Document 1, there is a demand for shortening the time required for detecting thioNAD(P)H produced in the enzyme cycling reaction.

[0008] The present invention provides an analytical method that can shorten the time required for detecting thioNAD(P)H produced in an enzyme cycling reaction.

[0009] That is, the present invention provides the following methods for analyzing trace substances and kits for analyzing trace substances: 1. A method for analyzing trace substances, comprising: Step A of binding, to the trace substance, an enzyme-labeled antibody consisting of an antibody specific to the trace substance and enzyme α, or an enzyme-labeled nucleic acid probe consisting of a nucleic acid probe specific to the trace substance and enzyme α; Step B of allowing the enzyme α of the enzyme-labeled antibody or the enzyme α of the enzyme-labeled nucleic acid probe to act on compound β, which is a substrate of enzyme α, to produce compound γ, which is a substrate for dehydrogenase; Step C of producing polymerized thioNAD(P)H by an enzyme cycling reaction using the produced compound γ, dehydrogenase, NAD(P)H, and polymerized thioNAD(P); and Step D of detecting the trace substance using the produced polymerized thioNAD(P)H. 2. The method for analyzing a trace substance according to 1., wherein the trace substance is an antigen, and in step A, the enzyme-labeled antibody is bound to the antigen. 3. The method for analyzing a trace substance according to 2., wherein the analysis is carried out on a carrier carrying an antibody specific to the antigen. 4. The method for analyzing a trace substance according to 2., wherein the analysis is carried out on a biological tissue section in which the antigen is present. 5. The method for analyzing a trace substance according to 2., wherein the analysis is carried out on a membrane in which the antigen is present. 6. The method for analyzing a trace substance according to any of 2. to 5., wherein the antigen is a protein antigen. 7. The method for analyzing a trace substance according to 1., wherein the trace substance is a nucleic acid, and in step A, the enzyme-labeled nucleic acid probe is bound to the nucleic acid. 8. The method for analyzing a trace substance according to any of 1. to 7., wherein in step D, the produced polymerized thioNAD(P)H is quantitatively analyzed. 9. The method for analyzing a trace substance according to 8., wherein the polymerized thioNAD(P)H produced in step D is quantitatively analyzed by absorbance. 10. The method for analyzing a trace substance according to any one of 1. to 7., wherein the polymerized thioNAD(P)H produced in step D is qualitatively analyzed. 11. The method for analyzing a trace substance according to 10., wherein the polymerized thioNAD(P)H produced in step D is qualitatively analyzed by visual observation. 12. The polymerized thioNAD(P) is N６ The method for analyzing a trace substance according to any one of 1. to 11., wherein the polymerized thioNAD (P) contains a structure derived from -carboxymethyl-thioNAD. 13. The method for analyzing a trace substance according to any one of 1. to 12., wherein the polymerized thioNAD (P) contains a structure derived from acrylamide. 14. The method for analyzing a trace substance according to any one of 1. to 13., wherein the enzyme α contains one or more enzymes selected from the group consisting of transferases represented by EC number 2.-, hydrolases represented by EC number 3.-, elimination-addition enzymes represented by EC number 4.-, and isomerases represented by EC number 5.-. 15. The method for analyzing a trace substance according to 14., wherein the enzyme α contains alkaline phosphatase. 16. The method for analyzing a trace substance according to any one of 1. to 15., wherein the compound β contains a compound having an androsterone skeleton. 17. The method for analyzing a trace substance according to any one of 1. to 16., wherein the dehydrogenase contains hydroxysteroid dehydrogenase. 18. 19. The method for analyzing a trace substance according to any one of 1. to 17., wherein the compound γ includes a compound having an androsterone skeleton. 19. The polymerized thioNAD(P) has a number average molecular weight of 1.0 × 10 ４ Above 1.0 x 10 ９A method for analyzing a trace substance according to any one of 1. to 18., which is as follows: 20. A kit for analyzing a trace substance, comprising: an enzyme-labeled antibody consisting of an antibody specific to a trace substance to be measured and an enzyme α, or an enzyme-labeled nucleic acid probe consisting of a nucleic acid probe specific to the trace substance and an enzyme α; a compound β that is a substrate of the enzyme α; a dehydrogenase; NAD(P)H; and polymerized thioNAD(P). 21. The kit for analyzing a trace substance according to 20., wherein the trace substance is an antigen and the kit comprises the enzyme-labeled antibody. 22. The kit for analyzing a trace substance according to 21., which is used on a carrier carrying an antibody specific to the antigen. 23. The kit for analyzing a trace substance according to 21., which is used on a biological tissue section in which the antigen is present. 24. The kit for analyzing a trace substance according to 21., which is used on a membrane in which the antigen is present. 25. The kit for analyzing a trace substance according to any one of 21. to 24., wherein the antigen is a protein antigen. 26. The kit for analyzing a trace substance according to 20., wherein the trace substance is a nucleic acid, and the kit includes the enzyme-labeled nucleic acid probe. 27. The polymerized thioNAD(P) is N ６ The kit for analyzing trace substances according to any one of 20. to 26., wherein the polymerized thioNAD (P) contains a structure derived from -carboxymethyl-thioNAD. 28. The kit for analyzing trace substances according to any one of 20. to 27., wherein the polymerized thioNAD (P) contains a structure derived from acrylamide. 29. The kit for analyzing trace substances according to any one of 20. to 28., wherein the enzyme α contains one or more enzymes selected from the group consisting of transferases represented by EC number 2.-, hydrolases represented by EC number 3.-, elimination-addition enzymes represented by EC number 4.-, and isomerases represented by EC number 5.-. 30. The kit for analyzing trace substances according to 29., wherein the enzyme α contains alkaline phosphatase. 31. The kit for analyzing trace substances according to any one of 20. to 30., wherein the compound β contains a compound having an androsterone skeleton. 32. The kit for analyzing trace substances according to 20. to 31., wherein the dehydrogenase contains hydroxysteroid dehydrogenase. 33. The kit for analyzing a trace substance according to any one of the preceding claims, wherein the number average molecular weight of the polymerized thioNAD(P) is 1.0 × 10 ４ Above 1.0 x 10 ９The kit for analyzing a trace substance according to any one of 20. to 32. below.

[0010] According to the present invention, an analytical method can be provided that can shorten the time required for detecting thioNAD(P)H produced in an enzyme cycling reaction.

[0011] 1 is a schematic diagram showing step A when the trace substance to be analyzed is an antigen; FIG. 2 is a schematic diagram showing step A when the trace substance to be analyzed is a nucleic acid; FIG. 3 is a schematic diagram showing step B; FIG. 4 is a schematic diagram showing step C; and FIG. 5 is a graph showing the change in absorbance over time in Example 1 and Comparative Example 1.

[0012] The present invention will be described below based on embodiments.

[0013] In the present embodiment, when a numerical range is described in stages, the upper and lower limits of each numerical range can be combined in any way.

[0014] In this embodiment, NAD(P) means NAD and / or NADP, and NAD(P)H means NADH and / or NADPH.

[0015] In this embodiment, thioNAD(P) means thioNAD and / or thioNADP, and thioNAD(P)H means thioNADH and / or thioNADPH.

[0016] In this embodiment, polymerized thioNAD(P) means a polymer containing a unit derived from thioNAD(P) represented by the following general formula (1) (hereinafter referred to as a thioNAD(P) unit), and polymerized thioNAD(P)H means a polymer containing a unit derived from thioNAD(P)H represented by the following general formula (2) (hereinafter referred to as a thioNAD(P)H unit).

[0017]

[0018] In general formulas (1) and (2), R １ is a hydroxy group or a phosphate group. １ When R is a hydroxy group, general formula (1) represents a thio NAD unit.１ When R is a phosphate group, general formula (1) represents a thio-NADP unit. １ When R is a hydroxy group, general formula (2) represents a thio-NADH unit. １ is a phosphate group, general formula (2) represents a thio-NADPH unit.

[0019] [Trace Substance Analysis Method] Hereinafter, the trace substance analysis method of this embodiment will be described.

[0020] The method for analyzing a trace substance of this embodiment includes the following steps: Step A: binding, to the trace substance, an enzyme-labeled antibody consisting of an antibody specific to the trace substance and enzyme α, or an enzyme-labeled nucleic acid probe consisting of a nucleic acid probe specific to the trace substance and enzyme α; Step B: allowing the enzyme α of the enzyme-labeled antibody or the enzyme α of the enzyme-labeled nucleic acid probe to act on compound β, which is a substrate of enzyme α, to produce compound γ, which is a substrate of dehydrogenase; Step C: producing polymerized thioNAD(P)H by an enzyme cycling reaction using the produced compound γ, dehydrogenase, NAD(P)H, and polymerized thioNAD(P); and Step D: detecting the trace substance using the produced polymerized thioNAD(P)H.

[0021] First, each step of the method for analyzing a trace substance according to this embodiment will be described.

[0022] Step A will be described separately for the case where the trace substance to be analyzed is an antigen and the case where the trace substance to be analyzed is a nucleic acid. When the trace substance to be analyzed is an antigen, an enzyme-labeled antibody consisting of an antibody that specifically binds to the antigen to be analyzed and enzyme α is prepared, and the antibody of the enzyme-labeled antibody is allowed to bind to the antigen to be analyzed, as shown in Figure 1. When the trace substance to be analyzed is a nucleic acid, an enzyme-labeled nucleic acid probe consisting of a nucleic acid probe that specifically binds to the nucleic acid to be analyzed and enzyme α is prepared, and the nucleic acid probe of the enzyme-labeled nucleic acid probe is allowed to bind to the nucleic acid to be analyzed, as shown in Figure 2.

[0023] In step B, as shown in FIG. 3, enzyme α of the enzyme-labeled antibody or enzyme α of the enzyme-labeled nucleic acid probe is allowed to act on compound β, which is a substrate of enzyme α, to produce compound γ, which is a substrate of dehydrogenase.

[0024] In step C, as shown in FIG. 4 , polymerized thioNAD(P)H is produced by an enzymatic cycling reaction using the produced compound γ, dehydrogenase, NAD(P)H, and polymerized thioNAD(P). Specifically, when reduced compound γ serves as the starting point of the enzymatic cycling reaction, reduced compound γ is first oxidized to produce oxidized compound γ. At this time, polymerized thioNAD(P)H is produced from polymerized thioNAD(P). Then, oxidized compound γ is reduced by dehydrogenase to produce reduced compound γ. At this time, NAD(P) is produced from NAD(P)H. This completes one cycle of enzymatic cycling. As long as polymerized thioNAD(P) and NAD(P)H are supplied, this cycle is repeated, and polymerized thioNAD(P)H is amplified.

[0025] In step D, the trace substance to be analyzed is detected by the polymerized thioNAD(P)H amplified in step C. Since the polymerized thioNAD(P)H is amplified in step C, highly sensitive analysis can be performed even if the amount of the substance to be analyzed is small.

[0026] According to the method for analyzing trace substances of this embodiment, the time required for detecting thio-NAD(P)H produced in the enzyme cycling reaction can be shortened. Although the mechanism for this time reduction is unclear, it is presumed that the time reduction is possible due to the following mechanism. First, regarding the detection of trace substances using thio-NAD(P)H, when detecting trace substances using thio-NAD(P)H, detection is not possible unless the thio-NAD(P)H concentration reaches a certain level or higher. Therefore, if thio-NAD(P)H is prone to diffusion on a plate or in biological tissue, it takes time for the thio-NAD(P)H concentration at the detection point to reach a certain level or higher. The method for analyzing trace substances of this embodiment uses polymerized thio-NAD(P). Therefore, it is believed that diffusion on a plate or in biological tissue is suppressed, and the time required for the thio-NAD(P)H concentration to reach a certain level or higher is shortened. This is believed to shorten the time required for the trace substance to be detected.

[0027] The method for analyzing trace substances according to this embodiment, in which an enzyme-labeled antibody is used, will be described below.

[0028] When an enzyme-labeled antibody is used, as shown in FIG. 1, the antibody of the enzyme-labeled antibody is bound to an antigen, which is a trace substance to be analyzed.

[0029] The location where trace substances are analyzed is not particularly limited, and for example, the analysis is performed on a carrier carrying an antigen-specific antibody. An example of an analytical method performed on a carrier carrying an antigen-specific antibody is enzyme-linked immunosorbent assay (ELISA). The ELISA method is a technique in which a sample solution is assayed on a carrier carrying an antibody, and the antigen in the sample solution is captured by the antibody, thereby detecting and quantifying the antigen. The carrier on which the antibody is carried is not particularly limited, and for example, a microplate, a plastic tube, or magnetic beads can be used.

[0030] The location where trace substances are analyzed is not particularly limited, and may be, for example, performed on a biological tissue section where an antigen is present. Examples of analytical methods performed on biological tissue sections where antigens are present include immunohistochemical staining (IHC). The IHC method is a technique for visualizing antigens in situ on biological sections. The IHC method has the advantage of being able to analyze biological tissues while retaining anatomical and structural information, and can analyze the compartment in which an antigen is localized in the biological tissue.

[0031] The location where trace substances are analyzed is not particularly limited, and for example, the analysis is performed on a membrane where an antigen is present. An example of an analysis method performed on a membrane where an antigen is present is Western blotting. Western blotting is a technique in which proteins separated by electrophoresis are transferred to a membrane, and the proteins are detected and quantified using antibodies specific to the proteins to be analyzed.

[0032] The type of antigen is not particularly limited as long as it can bind to the enzyme-labeled antibody, and may be, for example, a protein, sugar, lipid, peptide, or low-molecular-weight compound that induces an immune response in the body, preferably a protein. Here, proteins and sugars include proteins and sugars bound to lipids and / or nucleic acids.

[0033] The method for analyzing trace substances according to this embodiment will be described below in connection with the case where an enzyme-labeled nucleic acid probe is used.

[0034] When an enzyme-labeled nucleic acid probe is used, as shown in FIG. 2, the nucleic acid probe of the enzyme-labeled nucleic acid probe is bound to a nucleic acid, which is a trace substance to be analyzed.

[0035] Examples of analytical methods in which an enzyme-labeled nucleic acid probe binds to a nucleic acid, which is a trace substance to be analyzed, include Southern blotting and Northern blotting. Southern blotting and Northern blotting are techniques in which nucleic acids separated by electrophoresis are transferred to a membrane, and the nucleic acid is detected and quantified using a nucleic acid probe specific to the nucleic acid to be analyzed. The analysis target of Southern blotting is DNA, and the analysis target of Northern blotting is RNA.

[0036] The produced polymerized thioNAD(P)H is detected in step D. The detection method is not particularly limited, and for example, the produced polymerized thioNAD(P)H may be quantitatively analyzed.

[0037] The method for quantitatively analyzing polymerized thioNAD(P)H is not particularly limited, and for example, quantitative analysis can be performed based on absorbance. Note that since polymerized thioNAD(P)H exhibits absorption in the visible range (maximum absorption wavelength: 400 nm, molar extinction coefficient = 11,900), it can be measured using, for example, a commonly used absorptiometer or a colorimetric microplate reader.

[0038] The produced polymerized thioNAD(P)H is detected in step D. The detection method is not particularly limited, and for example, the produced polymerized thioNAD(P)H may be subjected to qualitative analysis.

[0039] The method for qualitative analysis of polymerized thioNAD(P)H is not particularly limited, and for example, the produced polymerized thioNAD(P)H can be qualitatively analyzed visually. As described above, polymerized thioNAD(P)H exhibits absorption in the visible range, and therefore visual qualitative analysis is possible.

[0040] The structure of the polymerized thioNAD(P) is not particularly limited, but the polymerized thioNAD(P) is N represented by the following general formula (3): ６ It is preferable that the NAD-containing compound contains a structure derived from -carboxymethyl-thioNAD, and the NAD-containing compound is represented by the following general formula (4): ６ It is more preferred that the compound contains a structure derived from -[N-(N-acryloyl-1-methoxycarbonyl-5-aminopentyl)-propioamide].

[0041]

[0042] In general formulas (3) and (4), R １ is a hydroxy group or a phosphate group.

[0043] The structure of the polymerized thioNAD(P) is not particularly limited, but it is preferable that the polymerized thioNAD(P) contains a structure derived from acrylamide.

[0044] The method for preparing polymerized thioNAD(P) is not particularly limited, but it can be prepared by referring to the methods described in Non-Patent Documents 1 and 2.

[0045] The enzyme α of the enzyme-labeled antibody or enzyme-labeled nucleic acid probe is not particularly limited, but preferably includes one or more enzymes selected from the group consisting of transferases represented by EC numbers 2.-, hydrolases represented by EC numbers 3.-, elimination-addition enzymes represented by EC numbers 4.-, and isomerases represented by EC numbers 5.-, and more preferably includes alkaline phosphatase (ALP).

[0046] The compound β is not particularly limited as long as it can be a substrate for the enzyme α. However, from the viewpoint of combination with a dehydrogenase described later, the compound β preferably contains a compound having an androsterone skeleton, and more preferably contains androsterone 3-phosphate (A3P).

[0047] The dehydrogenase used in the enzymatic cycling reaction of step C is not particularly limited and may include, for example, one or more dehydrogenases selected from group X consisting of malate dehydrogenase, lactate dehydrogenase, glucose dehydrogenase, galactose dehydrogenase, 3α-hydroxysteroid dehydrogenase (abbreviated as 3α-HSD), galactose dehydrogenase, glucose dehydrogenase, testosterone-17β-dehydrogenase, and glycerol-3-phosphate dehydrogenase. From the viewpoint of improving the reaction rate, it is preferable to include a hydroxysteroid dehydrogenase, and it is more preferable to include a 3α-HSD.

[0048] The combination of dehydrogenase and compound β is not particularly limited, but from the perspective of improving the reaction rate, a combination of 3α-HSD and its substrate, a compound having an androsterone skeleton, is preferred. To explain the improvement in reaction rate using a specific example, while the reaction rate of many dehydrogenases when thioNAD is used as a coenzyme is within a few percent of that when NAD is used, in the dehydrogenation reaction of androsterone by 3α-HSD, the reaction rate with thioNAD is approximately 59% of the reaction rate with NAD, making this a preferable property for the enzyme in the cycling reaction according to this embodiment. Specific examples of compounds having an androsterone skeleton include androsterone 3-phosphate (A3P), androsterone, 11β-hydroxyandrosterone, 11-oxoandrosterone, 11α-hydroxyandrosterone, etiocholanolone, tetrahydrocortisol, pregnanediol, lithocholic acid, deoxycholate, chenodeoxycholic acid, and cholic acid. Among the combinations of 3α-HSD and compounds having an androsterone skeleton, the combination of A3P and 3α-HSD is preferred from the viewpoint that it can be combined with ALP, which is a general-purpose enzyme used in enzyme-labeled antibodies or enzyme-labeled nucleic acid probes.

[0049] Compound γ is not particularly limited as long as it can be a substrate for the enzymatic cycling reaction using dehydrogenase, and therefore may be either a reduced substrate or an oxidized substrate.

[0050] In the method for analyzing trace substances of this embodiment, the properties of the enzyme α, compound β, and dehydrogenase are not particularly limited, but preferably have the following properties: The turnover number of the labeled enzyme is large. Commercially available and widely used labeled enzymes can be used. The turnover number of the enzyme cycling is large. The dehydrogenase used in the enzyme cycling reaction is free from contamination of the labeled enzyme and does not have the same activity as the labeled enzyme. The substrate of the labeled enzyme is a commercially available compound or can be easily synthesized and is highly stable.

[0051] In the method for analyzing trace substances of this embodiment, the concentration of the enzyme-labeled antibody or enzyme-labeled nucleic acid probe in the reaction system is not particularly limited, but is, for example, 1 nM or more, preferably 10 nM or more, more preferably 20 nM or more, and is, for example, 100 nM or less, preferably 50 nM or less, more preferably 40 nM or less.

[0052] In the method for analyzing a trace substance of this embodiment, the concentration of compound β in the reaction system is not particularly limited, but is, for example, 0.001 mM or more, preferably 0.01 mM or more, more preferably 0.05 mM or more, and even more preferably 0.1 mM or more, and is, for example, 500 mM or less, preferably 100 mM or less, more preferably 50 mM or less, even more preferably 10 mM or less, even more preferably 5 mM or less, even more preferably 1 mM or less, and even more preferably 0.5 mM or less.

[0053] In the method for analyzing trace substances of this embodiment, the concentration of NAD(P)H in the reaction system is not particularly limited, but is, for example, 0.001 mM or more, preferably 0.01 mM or more, more preferably 0.05 mM or more, even more preferably 0.1 mM or more, even more preferably 0.5 mM or more, even more preferably 1.0 mM or more, and is, for example, 500 mM or less, preferably 100 mM or less, more preferably 50 mM or less, even more preferably 10 mM or less, even more preferably 5 mM or less.

[0054] In the method for analyzing trace substances of this embodiment, the concentration of dehydrogenase in the reaction system is not particularly limited, but is, for example, 0.01 U / mL to 5000 U / mL. From the viewpoint of improving the S / N ratio (signal / noise ratio), the concentration is preferably 0.1 U / mL to 500 U / mL, more preferably 1 U / mL to 100 U / mL, even more preferably 2 U / mL to 80 U / mL, even more preferably 5 U / mL to 60 U / mL, even more preferably 10 U / mL to 50 U / mL, and even more preferably 20 U / mL to 40 U / mL.

[0055] In the method for analyzing trace substances of this embodiment, the amount of thioNAD(P) units in the reaction system is not particularly limited. However, from the viewpoint of improving the S / N ratio (signal / noise ratio), for example, 1.00 × 10 thioNAD(P) units are used for 100 μL of 30 U / mL dehydrogenase solution, i.e., 3 U of dehydrogenase. ３ mer or more 1.00 x 10 ５ mer or less, preferably 2.00 x 10 ３ mer or above 8.00 x 10 ４ mer or less, more preferably 5.00 x 10 ３ mer or above 5.00 x 10 ４ mer or less, more preferably 7.00 x 10 ３ mer or above 3.00 x 10 ４ mer or less, more preferably 1.50 × 10 ４ Mer or more 2.80 x 10 ４ mer or less, more preferably 2.00 × 10 ４ Mer or more 2.40 x 10 ４ In this embodiment, the amount of thioNAD(P) units in the reaction system is represented by mer. Mer is a unit representing the number of thioNAD(P) units in the reaction system. For example, when the amount of thioNAD(P) units in the reaction system is 100 mer, this means that the number of thioNAD(P) units in the reaction system is 100.

[0056] In the method for analyzing trace substances of this embodiment, the reaction temperature is not particularly limited, but is, for example, 0°C or higher, preferably 10°C or higher, more preferably 20°C or higher, and is, for example, 100°C or lower, preferably 80°C or lower, more preferably 60°C or lower, and even more preferably 40°C or lower.

[0057] In the method for analyzing a trace substance of this embodiment, the number-average molecular weight of the polymerized thioNAD(P) is not particularly limited. However, from the viewpoint of further shortening the time required for detecting thioNAD(P)H produced in the enzyme cycling reaction, it is preferably 1.0 × 10 ４ Above 1.0 x 10 ９ or less, more preferably 5.0 × 10 ４ Above 5.0 x 10８ More preferably, 1.0 × 10 ５ Above 1.0 x 10 ８ or less, more preferably 6.5 × 10 ５ Above 7.0 x 10 ７ More preferably, 1.0 × 10 ６ Above 5.0 x 10 ７ More preferably, 5.0 × 10 ６ Above 3.0 x 10 ７ More preferably, 1.0 × 10 ７ Above 2.0 x 10 ７ The number average molecular weight of the polymerized thioNAD(P) can be analyzed by, for example, gel permeation chromatography (GPC).

[0058] [Trace Substance Analysis Kit] Hereinafter, the trace substance analysis kit of this embodiment will be described.

[0059] The trace substance analysis kit of this embodiment includes an enzyme-labeled antibody consisting of an antibody specific to the trace substance to be measured and an enzyme α, or an enzyme-labeled nucleic acid probe consisting of a nucleic acid probe specific to the trace substance and an enzyme α, a compound β that is a substrate of the enzyme α, a dehydrogenase, NAD(P)H, and polymerized thioNAD(P).

[0060] Hereinafter, the case where the trace substance analysis kit of this embodiment includes an enzyme-labeled antibody will be described.

[0061] When the trace substance analysis kit of this embodiment contains an enzyme-labeled antibody, the trace substance to be analyzed is an antigen.

[0062] The use of the trace substance analysis kit of this embodiment is not particularly limited, and for example, it is used on a carrier carrying an antibody specific to an antigen. Specifically, it is used in an ELISA method, etc. The ELISA method is as described above.

[0063] The use of the trace substance analysis kit of this embodiment is not particularly limited, and it is used, for example, on a biological tissue section in which an antigen is present. Specifically, it is used in an IHC method, etc. The IHC method is as described above.

[0064] The use of the trace substance analysis kit of this embodiment is not particularly limited, and it is used, for example, on a membrane where an antigen is present. Specifically, it is used in Western blotting, etc. The Western blotting method is as described above.

[0065] The type of antigen is not particularly limited as long as it can bind to the enzyme-labeled antibody, and may be, for example, a protein, sugar, lipid, peptide, or low-molecular-weight compound that induces an immune response in the body, preferably a protein. Here, proteins and sugars include proteins and sugars bound to lipids and / or nucleic acids.

[0066] Hereinafter, the case where the trace substance analysis kit of this embodiment includes an enzyme-labeled nucleic acid probe will be described.

[0067] When the trace substance analysis kit of this embodiment includes an enzyme-labeled nucleic acid probe, the trace substance to be analyzed is a nucleic acid. The use of the trace substance analysis kit is not particularly limited, and it can be used, for example, in Southern blotting and Northern blotting. Southern blotting and Northern blotting are as described above.

[0068] The structure of the polymerized thioNAD(P) is not particularly limited, but the polymerized thioNAD(P) is N represented by the above general formula (3). ６ It is preferable that the NAD-N has a structure derived from -carboxymethyl-thioNAD, and the NAD-N is represented by the general formula (4) above. ６ It is more preferred that the compound contains a structure derived from -[N-(N-acryloyl-1-methoxycarbonyl-5-aminopentyl)-propioamide].

[0069] The structure of the polymerized thioNAD(P) is not particularly limited, but it is preferable that the polymerized thioNAD(P) contains a structure derived from acrylamide.

[0070] The enzyme α of the enzyme-labeled antibody or enzyme-labeled nucleic acid probe is not particularly limited, but preferably includes one or more enzymes selected from the group consisting of transferases represented by EC numbers 2.-, hydrolases represented by EC numbers 3.-, elimination-addition enzymes represented by EC numbers 4.-, and isomerases represented by EC numbers 5.-, and more preferably includes alkaline phosphatase (ALP).

[0071] The compound β is not particularly limited as long as it can be a substrate for the enzyme α. However, from the viewpoint of combination with the above-mentioned dehydrogenase, the compound β preferably contains a compound having an androsterone skeleton, and more preferably contains androsterone 3-phosphate (A3P).

[0072] The dehydrogenase contained in the trace substance analysis kit of this embodiment is not particularly limited, and may include, for example, one or more types selected from the above-mentioned Group X. From the viewpoint of improving the reaction rate described above, it is preferable that the kit include hydroxysteroid dehydrogenase, and it is more preferable that the kit include 3α-hydroxysteroid dehydrogenase (3α-HSD).

[0073] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations may be adopted. Furthermore, the present invention is not limited to the above-described embodiments, and modifications and improvements within the scope of achieving the object of the present invention are included in the present invention.

[0074] The usefulness of the present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples.

[0075] [Preparation of Polymerized ThioNAD] Polymerized thioNAD was obtained by the following procedure.

[0076] <N ６ Preparation of carboxymethyl-thio NAD> (1) Thio NAD (2.0 μg, 1.8 × 10 thiomethyl NAD manufactured by Oriental Yeast Co., Ltd.) was added to an aqueous solution of iodoacetic acid (2.0 g) neutralized with 2 M LiOH. １５mer) was added and the pH was adjusted to 6.5. In this example, the amount of thioNAD and compounds synthesized from thioNAD is expressed in mer. Mer is a unit that represents the number of thioNAD and compounds synthesized from thioNAD. In other words, 1.8 × 10 １５ The addition of mer thio-NAD means that the number of thio-NAD added to the iodoacetic acid solution is 1.8 x 10 １５ (2) The solution obtained in (1) above is diluted with the aqueous iodoacetic acid solution, and the amount of thio-NAD in the solution is 2.16 × 10 ４ mer. 2.16 x 10 ４ To obtain mer, the solution was diluted 1000-fold twice, 100-fold once, and 833-fold once. (3) The obtained solution was left standing in the dark at room temperature for 10 days. The pH of the solution was checked as appropriate and adjusted with 2 M LiOH. (4) The pH of the solution was adjusted to 3.5 with 3 M HCl and cooled on ice. (5) The cooled solution was poured into 10 volumes of acetone (temperature: -5°C). (6) The precipitate was collected, washed with ether, and dissolved in 50 mL of water. (7) The obtained solution was diluted with sodium dithionite solution (1 g / 100 mL, 1% NaHCO ３ ) and allowed to stand at 100°C for 5 minutes. (8) The solution was cooled to room temperature and oxygenated for 15 minutes. (9) The pH of the solution was adjusted to 11.3 with 1 M NaOH and allowed to stand at 75°C for 1 hour. (10) To react the resulting solution with alcohol dehydrogenase, the solution was first cooled to room temperature and Tris buffer was added to a Tris concentration of 0.1 M. (11) The pH of the solution was adjusted to 7.5 with 3 M HCl, and 1 mL of acetaldehyde and 1000 U of alcohol dehydrogenase were added. (12) When the absorbance at 340 nm ceased to fluctuate, dehydrogenation (oxidation) was considered complete, and the resulting solution was poured into a 10-fold excess of cold ether (-5°C). (13) After standing overnight at 3°C, the oily precipitate was collected, washed with acetone and ether, dissolved in 1 L of water, and adjusted to pH 7.5 with 0.1 M NaOH. (14) The resulting solution was washed with a column and then filtered with N ６ A solution of N-carboxymethyl-thio NAD was obtained.６ In the preparation of 1,4-carboxymethyl-thioNAD, almost no unreacted products, by-products, or losses due to purification remained. ４ mer) and the same amount of N ６ -carboxymethyl-thio NAD was considered to have been obtained.

[0077] <Preparation of N(ε)-acryloyl-L-lysine methyl ester hydrochloride> (15) L-lysine hydrochloride (27.6 g) was dissolved in hot water (70 mL), and CuCO ３ Cu(OH) ２ (15 g) was added. (16) 4 M NaOH (37.5 mL) was added to the obtained solution, and dibutylhydroxytoluene (50 mg) was further added, followed by cooling to -5°C. (17) The obtained solution was mixed with acryloyl chloride (13.7 g) and 4 M NaOH (37.5 mL) under stirring at -5°C for 1.5 hours. (18) After mixing, the pH of the solution was adjusted to 6.0 using 5 M HCl. (19) The obtained precipitate was recovered by filtration and washed with ultrapure water. (20) The washed precipitate was suspended in ultrapure water (150 mL), and the pH was lowered to 1.5 to dissolve the precipitate. (21) The remaining precipitate was removed by filtration, and Na ２ CO ３ The pH of the solution was returned to 6.0 by filtration and precipitation was carried out again. (22) The precipitate was collected and suspended in ultrapure water (150 mL), and 5 M HCl was added to dissolve it. (23) Hydrogen sulfide was introduced into the solution, and the resulting copper sulfide was removed by filtration. (24) The filtrate was evaporated under reduced pressure at 30°C, and then ethanol was added to the resulting residue and evaporated to obtain crystals. (25) The obtained crystals were treated with 21% methanolic hydrogen chloride (200 mL) at room temperature for 48 hours to obtain N(ε)-acryloyl-L-lysine methyl ester hydrochloride.

[0078] <ThioNAD-N ６-[Preparation of N-(N-acryloyl-1-methoxycarbonyl-5-aminopentyl)-propioamide]> (26) N(ε)-acryloyl-L-lysine methyl ester hydrochloride (5.0 g) obtained in (25) above was dissolved in ultrapure water (10 mL), and the pH was adjusted to 4.5 using 5 M NaOH. ６ The total volume of the solution of -carboxymethyl-thio NAD (2.16 × 10 ４ mer), hydroquinone (10 mg), and an aqueous solution (1 mL) of 1.34 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride were added and mixed. (27) After stirring at room temperature for 20 hours while maintaining the pH of the solution with 1 M HCl, the solution was poured into cold acetone / ethanol (1:1). (28) The precipitate was collected by centrifugation, washed with acetone, dissolved in water (5 mL), and desalted through a column to obtain thioNAD-N. ６ -[N-(N-acryloyl-1-methoxycarbonyl-5-aminopentyl)-propionamide] was obtained. ６ In the preparation of N-[N-(N-acryloyl-1-methoxycarbonyl-5-aminopentyl)-propioamide], almost no unreacted products, by-products, or losses due to purification remained. ６ -Carboxymethyl-thio NAD (2.16 × 10 ４ mer) and the same amount of thioNAD-N ６ It was assumed that N-[N-(N-acryloyl-1-methoxycarbonyl-5-aminopentyl)-propioamide] was obtained.

[0079] <Preparation of polymerized thioNAD> (29) ThioNAD-N obtained in (28) above ６ The total amount of [N-(N-acryloyl-1-methoxycarbonyl-5-aminopentyl)-propionamide] (2.16 × 10 ４A solution was prepared by mixing 300 mM acrylamide, 50 mM 3-(dimethylamino)propionitrile, and 4.4 mM ammonium persulfate. (30) Nitrogen gas was bubbled into the prepared solution for 1 minute, and the solution was left standing in ice for 1 hour, followed by dialysis against water to obtain polymerized thioNAD. In the preparation of the polymerized thioNAD described above, almost no unreacted products, by-products, or losses due to purification remained. From this, it was confirmed that thioNAD-N ６ -[N-(N-acryloyl-1-methoxycarbonyl-5-aminopentyl)-propionamide] (2.16 × 10 ４ It was considered that polymerized thioNAD containing the same amount of thioNAD as mer was obtained.

[0080] <Analysis of polymerized thioNAD> The polymerized thioNAD obtained by the above method was analyzed by gel permeation chromatography (GPC). As a result, the number average molecular weight was 1.5 × 10 ７ The GPC measurement conditions were as follows: Measurement apparatus: 1260 Infinity II Bio-SEC system (manufactured by Agilent) Column: PLgel 20 μm MIXED-A (size: 300 mm × 7.5 mm I.D., manufactured by Agilent) × 4 Column temperature: 25°C Mobile phase: dimethylformamide Flow rate: 1.0 mL / min Detector: RI (differential refractive index detector) Standard substance: Polystyrene D-H2-10 kit (manufactured by Agilent)

[0081] [Enzyme-linked immunosorbent assay (ELISA)] Example 1 The following analyte was analyzed using the following solution A by the following analytical method.

[0082] (Analysis target) Glucose-Regulated Protein 78 kDa solution (hereinafter referred to as GRP78 solution) (BSA: 0.1%, Tween 20: 0.02% in PBS) (Solution A) 100 μL of Solution A at pH 9.0 containing the following components was obtained: Tris-HCl (100 mM) Polymerized thioNAD (total amount obtained in (30) above, amount of thioNAD unit: 2.16 × 10 ４mer) (The amount of thioNAD units was determined in Preliminary Experiment 2 described below.) NADH (1.0 mM) Androsterone 3-phosphate (A3P) (0.1 mM) 3α-Hydroxysteroid dehydrogenase (3α-HSD) (30 U / mL) (The concentration of 3α-HSD was determined in Preliminary Experiment 1 described below.) (Analytical Method) A GRP78 solution was added to a microplate on which anti-GRP78 antibody had been immobilized, and the mixture was incubated at 37°C for 120 minutes. Next, the solution in the wells was aspirated and washed with saline. Then, 100 μL of alkaline phosphatase (ALP)-labeled anti-GRP78 antibody solution (BSA: 0.1%, Tween 20: 0.02% in PBS) was added and incubated at 37°C for 60 minutes. Next, the solution in the wells was aspirated and washed with saline. The entire volume of Solution A (100 μL) was added to the wells. While heating at 37°C, absorbance was measured every 5 minutes after the addition of Solution A using a microplate reader (manufactured by Corona Electric Co., Ltd., model name: SH-1000, equipped with a 405 nm filter). The results are shown in Figure 5. (Preliminary Experiment 1) The 3α-HSD concentrations were adjusted to 1, 10, 30, 50, or 100 U / mL. The absorbance was measured 5 minutes after the addition of Solution A using the above-described measurement method, and the S / N ratio (signal / noise ratio) at each concentration was compared. As a result, the S / N ratio was greatest at 30 U / mL, so the concentration of 3α-HSD was set to 30 U / mL. (Preliminary Experiment 2) The amount of thioNAD units in polymerized thioNAD was set to 7.20 × 10 ３ , 1.44 × 10 ４ , 2.16 × 10 ４ or 2.88 x 10 ４ The absorbance was measured 5 minutes after adding Solution A using the above measurement method, and the S / N ratio (signal / noise ratio) at each concentration was compared. ４ Since the S / N ratio was the largest when mer was used, the amount of thioNAD units was set to 2.16 × 10 ４ It was mer.

[0083] Comparative Example 1: Instead of polymerized thioNAD, 2.16 × 10４ Except for using mer thioNAD, ELISA was performed in the same manner as in Example 1. The results are shown in Figure 5.

[0084] 5, the rate of increase in absorbance was faster in Example 1 than in Comparative Example 1. This shows that the analysis method of the present embodiment can shorten the time required for thioNAD(P)H produced in the enzyme cycling reaction to reach a detectable concentration, i.e., the time required for detection.

[0085] Furthermore, the S / N ratio of Example 1 was 2.8, and the S / N ratio of Comparative Example 1 was 2.2. This shows that the analysis method of this embodiment can improve the S / N ratio.

[0086] [Preliminary Experiment for Application to Immunohistochemical Staining (IHC)] A preliminary experiment was conducted to apply the analytical method according to the present embodiment to the IHC method. Specifically, the detection limits of the analytical method according to the present embodiment were compared with those of an analytical method using horseradish peroxidase (HRP), which is commonly used in conventional IHC methods.

[0087] Reference Example 1 The detection limit was determined by the following method using the following solution B. (Solution B) 100 μL of solution B at pH 9.0 containing the following components was obtained: Tris-HCl (100 mM) Polymerized thioNAD (total amount obtained in (30) above, amount of thioNAD unit: 2.16×10 ４ mer), NADH (1.0 mM), Androsterone 3-phosphate (A3P) (0.1 mM), 3α-Hydroxysteroid dehydrogenase (3α-HSD) (30 U / mL). (Method) A pH 9.0 ALP solution (ALP: 5.0 × 10 －１７ , 1.0×10 －１７ , 5.0×10 －１８ or 1.0 x 10 －１８ / 100 μL, Tris-HCl: 100 mM) was added to the wells. Next, the entire amount of solution B (100 μL) was added to the wells, and while heating at 37°C, the absorbance was measured 5 minutes after the addition of solution B using a microplate reader (manufactured by Corona Electric Co., Ltd., model name: SH-1000, equipped with a 405 nm filter) to obtain a measured value A. If there was a significant difference between measured value A and blank value B, it was judged to be detectable, and the ALP concentration at the lowest ALP concentration among the detectable values ​​was taken as the detection limit. The presence or absence of a significant difference was determined by the following method. First, the average value B of blank value B was calculated. ＡＶＥ and standard deviation B ＳＤ Next, the reference value B was calculated using the following formula (1). Ｘ Next, the reference value B Ｘ and measurement value A, and if the values ​​were different, it was determined that there was a significant difference. Ｘ =B ＡＶＥ +B ＳＤ × 3 (1) The detection limit thus obtained is 2.24 × 10 －２０ mol / 100 μL.

[0088] <Reference Comparative Example 1> The detection limit was obtained by the following method using the following solution C. (Solution C) 100 μL of TMB substrate solution (manufactured by Thermodix) was prepared. (Method) A pH 9.0 HRP solution (HRP: 1.0 × 10 －８ , 0.5×10 －８ , 1.0×10 －９ or 0.5 x 10 －９100 μL of a 100 mM Tris-HCl (100 μL / 100 μL, Tris-HCl: 100 mM) solution was then added. Next, the entire amount of Solution C (100 μL) was added to the wells, and while heating at 37°C, the absorbance was measured 10 minutes after the addition of Solution C using a microplate reader (manufactured by Corona Electric Co., Ltd., model name: SH-1000, equipped with a 405 nm filter) to obtain a measurement value A. If there was a significant difference between Measurement Value A and Blank Value B, it was determined that the HRP was detectable, and the HRP concentration at the lowest among the detectable values ​​was taken as the detection limit. The presence or absence of a significant difference was determined by the method described above. The detection limit value obtained in this manner was 4.31 × 10 －１２ mol / 100 μL.

[0089] The detection limit value of Reference Example 1 is approximately 10 times lower than the detection limit value of Reference Comparative Example 1, which is a general HRP-based analytical method in conventional IHC methods. ８ From this, it is believed that the detection sensitivity can be improved by applying the analysis method according to this embodiment to the IHC method.

[0090] This application claims priority based on Japanese Patent Application No. 2023-108447, filed on June 30, 2023, the disclosure of which is incorporated herein by reference in its entirety.

Claims

1. A method for analyzing a trace substance, comprising: step A of binding, to the trace substance, an enzyme-labeled antibody consisting of an antibody specific to the trace substance and enzyme α, or an enzyme-labeled nucleic acid probe consisting of a nucleic acid probe specific to the trace substance and enzyme α; step B of allowing the enzyme α of the enzyme-labeled antibody or the enzyme α of the enzyme-labeled nucleic acid probe to act on a compound β, which is a substrate of the enzyme α, to generate a compound γ, which is a substrate of a dehydrogenase; step C of generating polymerized thioNAD(P)H by an enzyme cycling reaction using the generated compound γ, dehydrogenase, NAD(P)H, and polymerized thioNAD(P); and step D of detecting the trace substance using the generated polymerized thioNAD(P)H.

2. The method for analyzing a trace substance according to claim 1, wherein the trace substance is an antigen, and in step A, the enzyme-labeled antibody is bound to the antigen.

3. The method for analyzing a trace substance according to claim 2, which is carried out on a carrier carrying an antibody specific to the antigen.

4. The method for analyzing a trace substance according to claim 2, which is carried out on a biological tissue section in which the antigen is present.

5. The method for analyzing a trace substance according to claim 2, which is carried out on a membrane in which the antigen is present.

6. The method for analyzing a trace substance according to any one of claims 2 to 5, wherein the antigen is a protein antigen.

7. The method for analyzing a trace substance according to claim 1, wherein the trace substance is a nucleic acid, and in step A, the enzyme-labeled nucleic acid probe is bound to the nucleic acid.

8. A method for analyzing a trace substance according to any one of claims 1 to 7, wherein in step D, the polymerized thioNAD(P)H produced is quantitatively analyzed.

9. The method for analyzing a trace substance according to claim 8, wherein in step D, the produced polymerized thioNAD(P)H is quantitatively analyzed by absorbance.

10. A method for analyzing a trace substance according to any one of claims 1 to 7, wherein in step D, the polymerized thioNAD(P)H produced is subjected to qualitative analysis.

11. The method for analyzing a trace substance according to claim 10, wherein in step D, the polymerized thioNAD(P)H produced is qualitatively analyzed by visual inspection.

12. The polymerized thioNAD(P) is N ６ The method for analyzing a trace substance according to any one of claims 1 to 11, comprising a structure derived from -carboxymethyl-thioNAD.

13. The method for analyzing a trace substance according to any one of claims 1 to 12, wherein the polymerized thioNAD (P) contains a structure derived from acrylamide.

14. The method for analyzing a trace substance according to any one of claims 1 to 13, wherein the enzyme α comprises one or more enzymes selected from the group consisting of transferases represented by EC number 2.-, hydrolases represented by EC number 3.-, elimination-addition enzymes represented by EC number 4.-, and isomerases represented by EC number 5.-.

15. The method for analyzing a trace substance according to claim 14, wherein the enzyme α includes alkaline phosphatase.

16. The method for analyzing a trace substance according to any one of claims 1 to 15, wherein the compound β includes a compound having an androsterone skeleton.

17. The method for analyzing a trace substance according to any one of claims 1 to 16, wherein the dehydrogenase comprises a hydroxysteroid dehydrogenase.

18. The method for analyzing a trace substance according to any one of claims 1 to 17, wherein the compound γ includes a compound having an androsterone skeleton.

19. The number average molecular weight of the polymerized thioNAD (P) is 1.0×10 ４ Above 1.0 x 10 ９ The method for analyzing a trace substance according to any one of claims 1 to 18, wherein:

20. A kit for analyzing a trace substance comprising: an enzyme-labeled antibody consisting of an antibody specific to a trace substance to be measured and an enzyme α, or an enzyme-labeled nucleic acid probe consisting of a nucleic acid probe specific to the trace substance and an enzyme α; compound β which is a substrate for the enzyme α; a dehydrogenase; NAD(P)H; and polymerized thioNAD(P).

21. The kit for analyzing a trace substance according to claim 20, wherein the trace substance is an antigen, and the kit comprises the enzyme-labeled antibody.

22. The kit for analyzing a trace substance according to claim 21, which is used on a carrier carrying an antibody specific to the antigen.

23. The trace substance analysis kit according to claim 21, which is used on a biological tissue section in which the antigen is present.

24. The kit for analyzing a trace substance according to claim 21, which is used on a membrane on which the antigen is present.

25. The kit for analyzing a trace substance according to any one of claims 21 to 24, wherein the antigen is a protein antigen.

26. The kit for analyzing a trace substance according to claim 20, wherein the trace substance is a nucleic acid, and the kit comprises the enzyme-labeled nucleic acid probe.

27. The polymerized thioNAD(P) is N ６ The kit for analyzing a trace substance according to any one of claims 20 to 26, comprising a structure derived from -carboxymethyl-thioNAD.

28. The kit for analyzing trace substances described in any one of claims 20 to 27, wherein the polymerized thioNAD (P) contains a structure derived from acrylamide.

29. The kit for analyzing a trace substance according to any one of claims 20 to 28, wherein the enzyme α comprises one or more enzymes selected from the group consisting of transferases represented by EC number 2.-, hydrolases represented by EC number 3.-, elimination-addition enzymes represented by EC number 4.-, and isomerases represented by EC number 5.-.

30. The kit for analyzing a trace substance according to claim 29, wherein the enzyme α comprises alkaline phosphatase.

31. The kit for analyzing a trace substance according to any one of claims 20 to 30, wherein the compound β includes a compound having an androsterone skeleton.

32. The kit for analyzing a trace substance according to any one of claims 20 to 31, wherein the dehydrogenase comprises a hydroxysteroid dehydrogenase.

33. The number average molecular weight of the polymerized thioNAD (P) is 1.0×10 ４ Above 1.0 x 10 ９ The kit for analyzing a trace substance according to any one of claims 20 to 32, wherein:

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