Methods, reagents, and in vitro diagnostic kits for detecting target substances

Abstract

To provide a simple, rapid, and highly sensitive detection method, reagents, and in vitro diagnostic kit for target substances related to the enzymatic reaction of transglutaminase in liquid samples. [Solution] A method for detecting a target substance related to the enzymatic reaction of transglutaminase in a liquid sample, comprising: a first step of forming a crosslinked structure between a plurality of the second substrates and the first substrates of the luminescent particles in a liquid sample comprising transglutaminase, luminescent particles having a first substrate of transglutaminase, and a second substrate of transglutaminase; a second step of obtaining a value relating to the polarization anisotropy of the liquid sample after the first step; and a third step of detecting a target substance related to the enzymatic reaction of transglutaminase in the liquid sample based on the value relating to the polarization anisotropy.

Description

[Technical Field]

[0001] The present disclosure relates to a method for detecting a target substance, a reagent, and an in vitro diagnostic kit. [Background Art]

[0002] Enzymes have the role of catalyzing chemical reactions in vivo, and measuring enzyme activity is extremely important not only in medical research on enzymes, but also in the fields of enzyme testing, clinical testing using enzymes as reagents, and substance production utilizing enzymes. Transglutaminase (hereinafter sometimes abbreviated as TG), a type of enzyme, uses a glutamine residue in a polypeptide as a substrate, and catalyzes an amide bond formation reaction between the carboxamide side chain of the glutamine residue and an amino group such as that contained in a lysine residue. The transglutaminase family consists of eight enzymes / enzyme precursors referred to as TG1 to TG7 and factor XIII. Transglutaminase is known to have physiological roles such as blood coagulation mediated by factor XIII and localized epidermal formation mediated by transglutaminase. In recent years, the pathological importance of transglutaminase in neurodegenerative diseases has also been demonstrated, and the development of highly sensitive measurement techniques for transglutaminase is expected for drug discovery, treatment, and diagnosis based on transglutaminase.

[0003] One method for measuring enzyme activity is fluorescence polarization. Fluorescence polarization, which utilizes fluorescence polarization, measures the rotational motion of a fluorescent substance. When a fluorescent substance is excited, its anisotropy (also called the degree of fluorescence polarization) changes according to its rotational motion. Specifically, when the fluorescent substance is not rotating, polarized emission is observed, whereas when the fluorescent substance rotates freely, fluorescence is emitted in all planes, resulting in the elimination of polarization. A key feature of fluorescence polarization is that, in the assay, the degree of fluorescence polarization is used as the indicator, rather than the emission intensity. Another feature is that it does not require separation or washing. In other words, a homogeneous assay is possible, and the sample can be added to the solution and measured directly, eliminating the need for complicated separation procedures and shortening the measurement time.

[0004] Methods for measuring transglutaminase activity using fluorescence have been disclosed before. Patent Document 1 investigates transglutaminase activity by measuring the fluorescence resonance energy transfer of fluorescent dyes after binding two types of substrate peptides labeled with different fluorescent dyes based on the transglutaminase activity. However, in the measurement method of Patent Document 1, there is one fluorescent molecule for each substrate peptide molecule, resulting in a small signal change per reaction. Therefore, it was sometimes difficult to measure enzyme activity when the enzyme concentration was very low. In addition, fluorescent dyes are easily affected by the surrounding environment, and in liquid samples containing many impurities such as blood, the substrate recognition ability of the peptide bound to the fluorescent dye decreases due to adsorption to proteins and lipids, which are impurities, making it difficult to perform high-sensitivity measurements.

[0005] Patent Document 2 discloses an immunoassay technique using fluorescence polarization. In Patent Document 2, a highly sensitive immunoassay is constructed by using luminescent particles, agglutinating the luminescent particles via an antigen-antibody reaction, and observing the change in fluorescence polarization before and after agglutination. Patent document 3 discloses a technique for producing hydrogels using transglutaminase. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2018 / 004014 [Patent Document 2] Japanese Patent Publication No. 2022-187791 [Patent Document 3] Special Publication No. 2013-544248 [Overview of the project] [Problems that the invention aims to solve]

[0007] However, the method described in the above-mentioned patent document made it difficult to easily and sensitively detect target substances related to the enzymatic reaction of transglutaminase in liquid samples. [Means for solving the problem]

[0008] As a result of diligent research to solve the above problems, the present inventors have found that by introducing a mechanism in which a cross-linked structure is formed in a liquid sample by the action of transglutaminase, thereby significantly reducing the mobility of luminescent particles, it becomes possible to obtain values ​​regarding the amount and polarization anisotropy of the activity of the enzyme or enzyme reaction-related substance in a short time and with high sensitivity, and have completed this disclosure.

[0009] In other words, according to one aspect of this disclosure, A method for detecting target substances related to the enzymatic reaction of transglutaminase in a liquid sample, A first step in which a plurality of the second substrates and the first substrates of the luminescent particles form a crosslinked structure in a liquid sample comprising transglutaminase, luminescent particles having a first substrate of transglutaminase, and a second substrate of transglutaminase, A second step involves obtaining a value relating to the polarization anisotropy of the liquid sample after the first step, A third step involves detecting a target substance related to the enzymatic reaction of the transglutaminase in the liquid sample based on the value relating to the polarization anisotropy, This is a method for detecting a target substance that has [certain properties].

[0010] Furthermore, another aspect of this disclosure is: A reagent for detecting transglutaminase using values ​​related to polarization anisotropy, comprising a luminescent particle having a first substrate of transglutaminase and a second substrate of a plurality of transglutaminases.

[0011] Furthermore, another aspect of this disclosure is: A reagent for detecting a third substrate of transglutaminase using values ​​related to polarization anisotropy, comprising transglutaminase, luminescent particles having a first substrate of transglutaminase, and a plurality of second substrates of transglutaminase.

[0012] Furthermore, another aspect of this disclosure is: A reagent for detecting transglutaminase activators or transglutaminase inhibitors using values ​​related to polarization anisotropy, comprising transglutaminase, luminescent particles having a first substrate of transglutaminase, and a second substrate of transglutaminase.

[0013] Furthermore, another aspect of this disclosure is: This is an in vitro diagnostic kit containing one of the aforementioned reagents. [Effects of the Invention]

[0014] The method for detecting target substances related to the enzymatic reaction of transglutaminase described herein makes it possible to measure enzymatic reaction-related substances simply and with high sensitivity. [Brief explanation of the drawing]

[0015] [Figure 1] This is a diagram illustrating the steps of a method for detecting a target substance according to one aspect of the present disclosure. [Figure 2] FIG. 1 is a schematic diagram illustrating a highly sensitive method for detecting transglutaminase according to one aspect of an embodiment of the present disclosure. [Figure 3] FIG. 2 is a schematic diagram illustrating a method for detecting a transglutaminase inhibitor according to one aspect of an embodiment of the present disclosure. [Figure 4] FIG. 3 is a schematic diagram illustrating a method for detecting a third substrate of transglutaminase according to one aspect of an embodiment of the present disclosure. MODES FOR CARRYING OUT THE INVENTION

[0016] Hereinafter, the present disclosure will be described in further detail. In the present disclosure, the term "detection" is used in the meaning that it includes all cases of: determining the presence or absence of a target substance, detecting a target substance, and qualitatively and quantitatively determining the amount and activity of a target substance.

[0017] (Value related to polarization anisotropy determined by fluorescence polarization method) The present disclosure provides a method for detecting the amount or activity of a transglutaminase enzyme reaction-related substance, such as transglutaminase, a third substrate of transglutaminase, a transglutaminase activator, or a transglutaminase inhibitor, by acquiring a value related to polarization anisotropy using luminescent particles.

[0018] Fluorescence polarization is used for analyzing the mobility of fluorescent molecules in a solution. The principle of fluorescence polarization will be described with reference to the case of the luminescent particles of the present disclosure. When luminescent particles in a liquid (particles containing luminescent molecules exhibiting polarization anisotropy) are excited by plane-polarized light, they emit fluorescence polarized in the same plane. However, if the luminescent particles rotate due to Brownian motion during the excited state, they emit fluorescence in a plane different from the excitation plane, and thus the polarization anisotropy is eliminated. That is, the value related to polarization anisotropy represents the degree of rotational motion of the luminescent particles between excitation and fluorescence emission.

[0019] When luminescent particles are dispersed individually in a liquid solution, they exhibit low polarization anisotropy due to their vigorous rotation caused by Brownian motion. On the other hand, if the viscosity of the dispersion medium for the luminescent particles increases, or if large cross-linking structures are bonded to the luminescent particles, the Brownian motion of the luminescent particles in the solution decreases, and the polarization anisotropy increases. Therefore, in fluorescence polarization, the change in polarization anisotropy is used as an indicator to analyze the mobility of luminescent particles in solution. Polarization anisotropy can be indicated by milli P (hereinafter abbreviated as mp), which represents the change in plane polarization.

[0020] (Method for detecting target substances) The method for detecting a target substance according to this disclosure is a method for detecting a target substance related to the enzymatic reaction of transglutaminase in a liquid sample, and includes the following steps as shown in Figure 1. (1) A first step (S1001) in which a liquid sample containing transglutaminase, luminescent particles having a first substrate of transglutaminase, and a second substrate of transglutaminase forms a crosslinked structure between a plurality of second substrates and the first substrates of the luminescent particles. (2) A second step (S1002) to obtain a value relating to the polarization anisotropy of the liquid sample after the first step. (3) A third step (S1003) in which a target substance related to the enzymatic reaction of transglutaminase in a liquid sample is detected based on the value of polarization anisotropy. According to this disclosure, a highly sensitive method for detecting target substances related to the enzymatic reaction of transglutaminase in a liquid sample is characterized by the following advantages: (1) unlike conventional low-molecular-weight substrates, the signal intensity per molecule is enhanced by the use of luminescent particles; (2) the enzyme rapidly forms a cross-linked structure consisting of the second substrate of transglutaminase (increasing the viscosity of the liquid sample solution); furthermore, the formed cross-linked structure also cross-links with the luminescent particles having the first substrate of transglutaminase, thereby significantly suppressing the mobility of the luminescent particles in a short time, and causing a large change in the value related to polarization anisotropy from the luminescent particles in a short time; and (3) since there is no need to separate and wash away impurities or unreacted substances by measuring the value related to polarization anisotropy, the method can detect the amount and activity of the target substance with high sensitivity and speed compared to existing methods, even when the activity of transglutaminase or its substrate as the target substance is low or the amount is small.

[0021] (Measurement principle 1: Measurement of transglutaminase activity) The principle of this disclosure will be explained using Figure 2, illustrating the method for measuring the activity of transglutaminase. The target substance, transglutaminase 40, can form a cross-linked structure with the transglutaminase's second substrate 30. This is because the activity of transglutaminase (catalyzing the reaction that forms an amide bond between a glutamine residue and an amino group in the polypeptide) causes cross-linking of the transglutaminase's second substrate 30 via glutamine and lysine contained in the transglutaminase's second substrate 30. Before the addition of transglutaminase 40, the luminescent particles 10 are dispersed in the liquid sample and have high mobility. In the presence of transglutaminase 40, depending on its activity, a cross-linked structure 50 of the transglutaminase's second substrate 30 is formed by cross-linking (also called aggregation) of the transglutaminase's second substrate 30. As a result, the viscosity of the liquid sample increases, and the mobility of the luminescent particles 10 decreases.

[0022] Furthermore, the target substance, transglutaminase 40, can crosslink the transglutaminase 10 with the crosslinked structure 50 of the transglutaminase's second substrate 30 via the first substrate 20 of transglutaminase present on the surface of the luminescent particle 10. Therefore, in the presence of transglutaminase 40, depending on its activity, the crosslinked structure 50 of the transglutaminase's second substrate 30 with the luminescent particle 10 results in reduced mobility of the luminescent particle 10.

[0023] The phenomenon of decreased mobility of the luminescent particles 10, which depends on transglutaminase activity, is observed as an increase in the polarization anisotropy of the luminescent particles 10. Therefore, by obtaining a value related to the polarization anisotropy of the luminescent particles 10, the activity of transglutaminase 40 in the sample can be determined. For example, a calibration curve of polarization anisotropy values ​​and activity can be obtained in advance using a transglutaminase with known activity, and the transglutaminase activity of the sample can be determined from the measurement results of the polarization anisotropy values ​​of the liquid sample. In this disclosure, the measurement may be of the quantity of transglutaminase, rather than its activity. That is, the activity can be kept constant, and the measurement can be performed by obtaining a value related to the polarization anisotropy of the liquid sample that depends on the amount of transglutaminase.

[0024] (Measurement principle 2: Measurement of transglutaminase inhibitors) This disclosure also shows that inhibitors, which are examples of substances involved in the enzymatic reaction of transglutaminase, can be measured in a similar manner. As shown in Figure 3, for example, the inhibitory efficiency of a transglutaminase inhibitor 60, which is the target substance, against a specified amount of transglutaminase 40 can be measured using a value related to the polarization anisotropy of the luminescent particles 10 as an indicator.

[0025] Specifically, the liquid sample contains a sufficiently large amount of transglutaminase 40 and / or transglutaminase 40 with sufficiently high activity. The target substance, the transglutaminase inhibitor 60, binds to transglutaminase 40 with arbitrary affinity and inhibits the activity of transglutaminase 40. That is, when transglutaminase 70 bound to the inhibitor 60 is produced, the activity of transglutaminase is lost. If the inhibition efficiency is 100%, a cross-linked structure will not be formed by cross-linking between the second substrates 30 of transglutaminase, and the viscosity of the liquid sample will not increase. In addition, the cross-linking reaction between the second substrate 30 of transglutaminase and the luminescent particles 10 will not occur. As a result, the mobility of the luminescent particles 10 will not change, and the polarization anisotropy of the luminescent particles 10 will not change. On the other hand, when the inhibition efficiency is 0%, the liquid sample becomes thicker due to the formation of a cross-linked structure 50 of the second substrate 30 of transglutaminase, as shown in Figure 2, and the luminescent particles 10 bind to the cross-linked structure. As a result, the polarization anisotropy of the luminescent particles 10 increases. That is, a change in the value of polarization anisotropy is observed in accordance with the inhibition efficiency of transglutaminase 40 by the inhibitor 60. Therefore, a large number of inhibitor candidates can be easily screened using the detection method disclosed herein.

[0026] (Measurement principle 3: Measurement of transglutaminase activators) In addition to inhibitors, the activation efficiency of transglutaminase activators can also be detected in a similar manner (not shown).

[0027] Specifically, the liquid sample contains a small amount of transglutaminase 40 and / or low-activity transglutaminase 40. The target substance, the activator, binds to the transglutaminase 40 with an arbitrary affinity, thereby increasing the activity of the transglutaminase 40. If the activation efficiency is 0%, no cross-linking structure is formed between the second substrates 30 of the transglutaminase, and the viscosity of the liquid sample does not increase. Also, no cross-linking reaction occurs between the second substrates 30 of the transglutaminase and the luminescent particles 10. As a result, the mobility of the luminescent particles 10 does not change, and the polarization anisotropy of the luminescent particles 10 does not change. On the other hand, if the activation efficiency is 100%, the liquid sample becomes thicker due to the formation of the cross-linking structure 50 of the second substrates 30 of the transglutaminase, as shown in Figure 2, and the luminescent particles 10 bind to the cross-linking structure. As a result, the polarization anisotropy of the luminescent particles 10 increases. In other words, a change in the value related to polarization anisotropy will be observed in accordance with the activation efficiency of transglutaminase 40 by the activator. Therefore, a large number of activator candidates can be easily screened using the detection method disclosed herein.

[0028] (Measurement principle 4: Measurement of the third substrate of transglutaminase) Furthermore, the third substrate of transglutaminase can also be measured in the same manner as the inhibition efficiency described in measurement principle 2. As shown in Figure 4, for example, the inhibitory efficiency of a third substrate 80 of the target transglutaminase against a specified amount of transglutaminase 40 can be measured using a value related to the polarization anisotropy of the luminescent particles 10 as an indicator. Here, "inhibition" means that the third substrate 80 competitively inhibits the reaction of transglutaminase 40 with the first substrate 20 and the second substrate 30.

[0029] Specifically, the liquid sample contains a sufficiently large amount of transglutaminase 40 and / or transglutaminase 40 with sufficiently high activity. The third substrate 80 of the target substance, transglutaminase, is assumed to preferentially form a crosslinked structure with respect to the activity of transglutaminase 40, over the first substrate 20 and the second substrate 30 (the third substrate 80 has higher substrate specificity than the first substrate 20 and the second substrate 30). That is, when the crosslinked structure 81 of the third substrate 80 is formed, the activity of transglutaminase 40 toward the first substrate 20 is inhibited. If the inhibition efficiency is 100%, no crosslinked structure will be formed by crosslinking between the second substrates 30 of transglutaminase, and the viscosity of the liquid sample will not increase. In addition, the crosslinking reaction between the second substrate 30 of transglutaminase and the luminescent particles 10 will not occur. As a result, the mobility of the luminescent particles 10 will not change, and the polarization anisotropy of the luminescent particles 10 will not change. On the other hand, when the inhibition efficiency is 0%, the liquid sample becomes thicker due to the formation of a cross-linked structure 50 of the second substrate 30 of the transglutaminase, as shown in Figure 2, and the luminescent particles 10 bind to the cross-linked structure. As a result, the polarization anisotropy of the luminescent particles 10 increases. That is, a change in the value of polarization anisotropy is observed in accordance with the inhibition efficiency of the transglutaminase 40 by the third substrate 80. Therefore, it is possible to easily screen a large number of transglutaminase substrate candidates using the detection method disclosed herein.

[0030] (Specific explanation of the detection method) An example of this disclosure includes the following steps: (1) A first step in which a plurality of second substrates and the first substrates of the luminescent particles form a crosslinked structure in a liquid sample containing transglutaminase, luminescent particles having a first substrate of transglutaminase, and a second substrate of transglutaminase. (2) A second step to obtain a value relating to the polarization anisotropy of the liquid sample after the first step. (3) A third step in which a target substance related to the enzymatic reaction of transglutaminase in a liquid sample is detected based on the value of polarization anisotropy. The following provides a more detailed explanation of each step.

[0031] (First step) In this disclosure, the first step is to form a crosslinked structure with a second substrate of multiple transglutaminases in a liquid sample, to which luminescent particles bind to the formed crosslinked structure, and further to which the viscosity of the liquid sample increases (also called thickening). Specifically, in the first step, it is preferable that a crosslinked structure consisting of a first substrate of transglutaminase and a second substrate of transglutaminase is crosslinked by transglutaminase. Furthermore, in the first step, it is preferable that the viscosity of the liquid sample increases due to the crosslinked structure consisting of the second substrate of transglutaminase. These processes may consist only of increased viscosity of the liquid sample, only binding of luminescent particles to the crosslinked structure, or both.

[0032] When the target substance is transglutaminase, the transglutaminase is reacted with luminescent particles having the first substrate of transglutaminase and the second substrate of transglutaminase to obtain a compound of the formed crosslinked structure and the luminescent particles. Alternatively, the viscosity of the liquid sample is increased. Here, the order of the reactions is not particularly limited. That is, the transglutaminase, the luminescent particles having the first substrate of transglutaminase and the second substrate of transglutaminase may be reacted simultaneously, or the transglutaminase and the second substrate of transglutaminase may be reacted first to form a crosslinked structure, and then the luminescent particles may be reacted with the crosslinked structure in an environment in which transglutaminase activity is maintained to obtain a compound of the luminescent particles and the crosslinked structure. Preferably, the transglutaminase, the luminescent particles having the first substrate of transglutaminase and the second substrate of transglutaminase are reacted simultaneously. This is because the crosslinked structure in the liquid sample may lead to a decrease in the dispersibility of the luminescent particles and a decrease in the reaction efficiency between the luminescent particles and the crosslinked structure. In this reaction, it is preferable to mix the liquid sample at a temperature (e.g., 30°C to 50°C) and pH (e.g., pH 5 to 8) at which transglutaminase is activated.

[0033] When the target substance is an inhibitor of transglutaminase, which is an example of an enzyme reaction-related substance, transglutaminase can be added to a liquid sample containing the inhibitor, luminescent particles having the first substrate of transglutaminase, and the second substrate of transglutaminase, and the reaction can be carried out. Here, the order of the reaction is not particularly limited, but since a crosslinking reaction via the substrate occurs with transglutaminase as a trigger, it is preferable to add the target substance, the inhibitor, before the transglutaminase. The liquid sample solution can also be divided into multiple solutions. That is, a solution A can be prepared by mixing transglutaminase and the target substance, the inhibitor, and then solution A can be mixed with a solution B containing luminescent particles having the first substrate of transglutaminase and the second substrate of transglutaminase to bind the luminescent particles to the crosslinking structure and thicken the liquid sample. In this case, even if the target substance is an activator of transglutaminase or a third substrate of transglutaminase, it can be detected by performing the same procedure as in the case of the inhibitor.

[0034] A preferred first step involves binding luminescent particles to a cross-linked structure formed by transglutaminase in a liquid sample, and simultaneously generating a cross-linked structure in the liquid sample independently of the luminescent particles, thereby increasing the viscosity of the liquid sample solution. To achieve this, it is preferable to use the same material for both the first and second substrates of the transglutaminase. For example, luminescent particles immobilized with casein as the first substrate and casein as the second substrate can be used. These actions significantly reduce the mobility of the luminescent particles, resulting in a significant increase in the polarization anisotropy of the luminescent particles. Obtaining a large change in the value of polarization anisotropy improves the detection sensitivity of the target substance.

[0035] (Liquid sample) The liquid sample that can be used in this disclosure is any liquid (also called a sample) that contains components for measuring the activity or concentration of the target substance and also contains the target substance. In this disclosure, the target substance is a substance related to the enzymatic reaction of transglutaminase. For example, samples can include bodily fluids such as blood, urine, and saliva containing the target substance, buffer solutions containing the target substance, culture media or tissue extracts of cells or microorganisms containing the target substance, drinking water containing the target substance, river water containing the target substance, or wastewater containing the target substance.

[0036] (Transglutaminase) An example of a target substance in this disclosure is the enzyme transglutaminase. Transglutaminases use glutamine residues in polypeptides as substrates and catalyze the formation of amide bonds between the carboxamide side chain of the glutamine residue and the amino group of the lysine residue. In this disclosure, eight enzymes / enzyme precursors, referred to as TG1-TG7 or factor XIII, can be used as transglutaminases.

[0037] (Enzyme reaction-related substances) Another example of a target substance in this disclosure, an enzyme reaction-related substance, is a substance related to the transglutaminase enzyme reaction, and is only required if its quantity and activity can be measured by the detection method of this disclosure. In other words, the enzyme reaction-related substance is only required if it is a substance that affects the activity of transglutaminase. For example, it could be a third substrate, activator, or inhibitor of transglutaminase. Depending on the quantity and activity of these enzyme reaction-related substances, the value of polarization anisotropy obtained by fluorescence polarization measurement in this disclosure can be changed. Preferred third substrates of transglutaminase include casein, gelatin, collagen, keratin, their denatured forms, and their partial peptides. In particular, it is preferable that the compound is catalytically activated by transglutaminase preferentially over the first and second substrates of transglutaminase. Preferred activators include calcium chloride, and preferred inhibitors include metal chelators and protein denaturants.

[0038] (Luminescent particles having the first substrate of transglutaminase) The luminescent particles having the first substrate of transglutaminase used in this disclosure are particles containing a luminescent substance, and any luminescent particles from which a value relating to the polarization anisotropy of their emission can be obtained by fluorescence polarization. Furthermore, the luminescent particles according to this disclosure have a substrate of transglutaminase (also called the first substrate of transglutaminase). Therefore, by the action of transglutaminase, a cross-linked structure can be formed between the first substrate of transglutaminase and a second substrate of transglutaminase present in the same solution. The cross-linking mode between these substrates can be any bond catalyzed by transglutaminase, but is preferably a chemical bond, and amide bonds, peptide bonds, isopeptide bonds, etc., are preferred. The first substrate of transglutaminase present in the luminescent particles can be in any state as long as it is recognized by transglutaminase present in the same solution, but is preferably on the surface of the luminescent particles.

[0039] For amide bond formation, it is preferable that the luminescent particles have at least one glutamine residue and at least one lysine residue on their surface as the first substrate for transglutaminase. Examples of the first substrate for transglutaminase immobilized on the luminescent particles include casein, gelatin, collagen, keratin, modified forms thereof, and partial peptides thereof. The method for immobilizing the first substrate for transglutaminase onto the luminescent particles can be any known method for immobilizing protein and peptide particles. For example, physical adsorption and chemical bonding methods can be used, and the first substrate for transglutaminase can be physically adsorbed onto luminescent particles having a hydrophobic surface. Alternatively, the first substrate for transglutaminase can be chemically bonded to luminescent particles having a reactive functional group. For example, an amide coupling method using water-soluble carbodiimide is preferred.

[0040] The luminescent particles of this disclosure have an average particle size, which is the average of the particle diameters, of 1 nm to 1000 nm, preferably 25 nm to 500 nm, and more preferably 50 nm to 300 nm. If the average particle size exceeds 500 nm, the polarization anisotropy increases before the reaction, i.e., when the luminescent particles exist in monodisperse, and the change in the value of polarization anisotropy after the enzymatic reaction, after bonding between the luminescent particles and the cross-linking structure, and after solvent thickening, may become small. Also, if the average particle size is less than 25 nm, the content of the luminescent substance in the particles decreases, the luminescence intensity per particle decreases, and as a result, the detection sensitivity and accuracy may decrease, so luminescent particles of 50 nm or more are preferred. The size of the luminescent particles can be determined by dynamic light scattering (DLS) measurement.

[0041] As the particle material (matrix material) for the luminescent particles, for example, nanoparticles of synthetic polymers such as polystyrene and polymethacrylate, inorganic nanoparticles such as silica, titanium dioxide, and iron oxide, and natural polymers such as dextran and casein can be used. Among these, synthetic polymers are preferred as the particle material for the luminescent particles according to this disclosure because they have a specific gravity that allows them to be dispersed in water, are stable, and have high productivity, and polystyrene particles are particularly preferred because they also have excellent particle size controllability.

[0042] Furthermore, it is preferable that the particle surface is hydrophilic. This is because hydrophilic particle surfaces can suppress nonspecific aggregation between luminescent particles and nonspecific adsorption between luminescent particles and the measurement container (plastic cuvette, quartz glass, etc.). In the detection method using fluorescence polarization described herein, changes in the mobility of luminescent particles, independent of enzyme activity, enzyme amount, and the concentration of enzyme reaction-related substances, can reduce the accuracy and precision of target substance detection. Therefore, it is necessary to avoid nonspecific aggregation between luminescent particles and nonspecific adsorption of luminescent particles to the container. For this purpose, it is preferable to coat the particle surface with a hydrophilic polymer. For example, polymers such as polyvinylpyrrolidone, polyethylene glycol, polyhydroxymethacrylate, and dextran are suitably used. As described above, by appropriately introducing crosslinkable functional groups to the surface of the luminescent particles, the first substrate of transglutaminase can be chemically bonded. For example, by having reactive functional groups such as thiol groups, amino groups, carboxyl groups, and maleimide groups on the surface of the luminescent particles, it is possible to bind the first substrate of a transglutaminase having thiol groups, carboxyl groups, amino groups, etc. Therefore, a preferred example of the surface of luminescent particles is one in which a hydrophilic polymer such as polyvinylpyrrolidone is coated, and a carboxyl group is introduced to chemically bind the first substrate of the transglutaminase.

[0043] The luminescent particles used in this disclosure are characterized by containing a luminescent substance. Here, the luminescent substance only needs to be something that can be contained in the particles and can be measured by fluorescence polarization. The luminescent substance may be contained in the particles on the surface or inside the particles, but is preferably inside the particles. This is because on the particle surface, interactions occur between the luminescent substance and impurities and water molecules in the solution, which can affect the acquisition of values ​​related to the dispersibility and polarization anisotropy of the luminescent particles. Examples of luminescent substances include fluorescent dyes such as fluorescein derivatives, rhodamine derivatives, and cyanine derivatives, and rare-earth luminescent complexes such as europium complexes and terbium complexes. Europium complexes are suitable as luminescent substances to be contained in the luminescent particles according to this disclosure because their emission wavelength and intensity are less affected by the surroundings, and their emission has a long lifetime.

[0044] Specific examples of europium complexes include [Tris(2-thenoyltrifluoroacetone)bis(triphenylphosphineoxide)europium(III)], [Tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfoxide)europium(III)], and [Tris(2-thenoyltrifluoroacetone)(phenanthroline)europium(III)].

[0045] A higher content of luminescent material in the luminescent particles is preferable. This is because it increases the luminescence intensity per particle, which in turn contributes to increased sensitivity or improved accuracy in the measurement of enzyme activity, enzyme quantity, and enzyme reaction-related substance concentrations as described in this disclosure. The content of rare earth luminescent complexes in the luminescent particles is preferably, for example, 0.001 g or more per gram of particle. With this content, a sufficiently high and stable luminescence intensity per particle can be obtained. Furthermore, the content of luminescent material can be measured by elemental analysis using the elemental characteristics of the luminescent material as an indicator. For example, the content of rare earth luminescent complexes can be calculated from the quantification of the rare earth luminescent complexes using inductively coupled plasma (ICP) emission spectroscopy.

[0046] (The second substrate of transglutaminase) In this disclosure, the second substrate of transglutaminase is a substrate molecule that serves as a raw material for forming a crosslinked structure. In this disclosure, it is preferable that the second substrate of transglutaminase plays the role of a precursor for the crosslinked structure. Therefore, the second substrate of transglutaminase is not particularly limited as long as it does not hinder the acquisition of values ​​related to the polarization anisotropy of the luminescent particles used in this disclosure, and it is sufficient that it is crosslinked with the second substrate present in the same solution by the action of transglutaminase. It is also preferable that it can crosslink with the first substrate of transglutaminase. The crosslinking mode between these substrates can be any bond catalyzed by transglutaminase, but it is preferably a chemical bond, and amide bonds, peptide bonds, isopeptide bonds, etc., are suitable. The second substrate of transglutaminase can be in any state as long as it is recognized by transglutaminase present in the same solution, but it is preferably water-soluble, and a carrier on which multiple second substrates of transglutaminase are immobilized may be used. For the amide bond to function, the second substrate of transglutaminase must have at least one glutamine residue and at least one lysine residue on its molecule. Examples of the second substrate of transglutaminase include casein, gelatin, collagen, keratin, their modified forms, and their partial peptides.

[0047] Regarding the second substrate for transglutaminase, the molecular weight of the substrate can be appropriately selected considering the required sensitivity and the handling of the reagent. In one example of the detection method disclosed herein, it is necessary to bind luminescent particles to the formed cross-linked structure and significantly reduce the mobility of the luminescent particles. Therefore, a larger molecular weight of the substrate is preferable. On the other hand, from the viewpoint of handling the solution before measuring enzyme activity, a smaller molecular weight is preferable. As the molecular weight increases, the viscosity of the solution increases, which can cause problems with dispensing accuracy. For these reasons, the molecular weight of the second substrate for transglutaminase is suitable to be in the range of approximately 1,000 to approximately 5,000,000, more preferably approximately 10,000 to 1,000,000. It is also desirable that it does not have inhibitory activity against the enzyme. Based on these requirements, casein, gelatin, modified forms thereof, and partial peptides thereof are more preferable as the second substrate for transglutaminase. The second substrate for transglutaminase may be the same substance as the first substrate for transglutaminase described above.

[0048] The size of the crosslinking structure that significantly changes the polarization anisotropy of the luminescent particles in response to enzyme activity can be observed by dynamic light scattering (DLS) measurements. For example, if the average particle size of the luminescent particles is about 100 nm, it is preferable that the crosslinking structure be about 1000 nm in size. When luminescent particles bind to a crosslinking structure that is about 10 times larger than their average particle size, the volume change increases by about 1000 times, which greatly reduces the mobility of the luminescent particles and makes it possible to obtain a large signal change using fluorescence polarization. The increase in average particle size is at least 1.5 times, preferably 5 times or more, and particularly preferably 10 times or more.

[0049] (Second step) In this disclosure, the second step is to obtain a value relating to the polarization anisotropy of the liquid sample after the first step. In this disclosure, as an example of a value relating to polarization anisotropy, a step of measuring the degree of fluorescence polarization or polarization anisotropy is shown. The fluorescence polarization method can be performed by known methods, and various commercially available measuring devices can be used for this purpose. The fluorescence polarization method is also called the fluorescence depolarization method, but in this specification, the fluorescence polarization method and the fluorescence depolarization method are synonymous.

[0050] In this disclosure, the target substance, such as an enzyme or enzyme reaction-related substance, can be measured by fluorescence polarization using the mobility of luminescent particles in a sample solution as an indicator. Specifically, depending on the amount and activity of the enzyme or enzyme reaction-related substance, a cross-linked structure is formed in the sample solution, and as a result of the luminescent particles binding to this cross-linked structure, the mobility of the luminescent particles decreases. Furthermore, the formation of the cross-linked structure in the solution increases the viscosity of the liquid sample, which in turn reduces the mobility of the luminescent particles. In this disclosure, the mobility of the luminescent particles is detected by fluorescence polarization. In fluorescence polarization, as mentioned above, for example, fluorescence polarization degree or polarization anisotropy can be used as an indicator, and here we show an example in which fluorescence polarization degree (mp) is used as an indicator.

[0051] A key feature of this disclosure is the use of luminescent particles as probes, which contain a europium complex exhibiting polarized emission as a luminescent material within the luminescent particles. Even slight changes in the rotational motion of these luminescent particles in a liquid sample can be detected as changes in their polarized emission properties. Specifically, when the luminescent particles are fixed to a cross-linked structure that is larger in size than the luminescent particles themselves, and the mobility of the luminescent particles is significantly reduced, the decrease in the rotational Brownian motion of the luminescent particles can be detected with high sensitivity as a change in the degree of fluorescence polarization.

[0052] The timing of the fluorescence polarization measurement can be set as appropriate. For example, the fluorescence polarization may be measured after the first step. Measuring the fluorescence polarization when the reaction is complete is a simple and preferred method. For example, the fluorescence polarization can be measured 5 minutes after adding the target substance to the solution.

[0053] Furthermore, in the first step, before reacting the target substance (enzyme or enzyme reaction-related substance), the fluorescence polarization degree of the luminescent particles may be measured and defined as the initial fluorescence polarization degree (mp(0)). Then, the reaction may proceed, and upon binding of the luminescent particles to the formed cross-linked structure, the fluorescence polarization degree (mp(t)) at a certain time point may be measured again, or it may be measured continuously at regular time intervals. That is, by measuring the change in fluorescence polarization degree over time, the change in fluorescence polarization degree (Δmp) may be determined from the difference between the fluorescence polarization degree (mp(t)) at a certain time point and the initial fluorescence polarization degree (mp(0)). The change in fluorescence polarization degree occurs rapidly, and for example, a sufficient Δmp can be obtained in about 1 to 10 minutes. Alternatively, the rate of change in fluorescence polarization degree (dmp / dt) may be determined.

[0054] The measurement conditions for fluorescence polarization are preferably, for example, in a liquid at a temperature of 1 to 50°C, with a viscosity of 0.5 to 50 mPa·s. If the luminescent particles are luminescent particles containing a europium complex, the concentration of the luminescent particles is not limited as long as the luminescence of the luminescent particles can be detected, and can be appropriately selected according to the type, amount, activity, etc. of the target substance. It is preferable to measure the fluorescence polarization at concentrations from 0.0001 mg / mL (equivalent to 0.00001 mass%) to 1.0 mg / mL (equivalent to 0.1 mass%), but in order to ensure the luminescence intensity from the luminescent particles and avoid the effects of scattering by the luminescent particles, it is preferable to measure the fluorescence polarization in the range of 0.001 mg / mL (equivalent to 0.0001 mass%) to 0.1 mg / mL (equivalent to 0.01 mass%).

[0055] (Third step) In this disclosure, the third step is to detect substances related to the enzymatic reaction in the liquid sample based on the polarization anisotropy value of the luminescent particles in the liquid sample obtained in the second step. More specifically, it is a step of relating the change in the polarization anisotropy value to the amount or activity of the enzyme or enzymatic reaction-related substance.

[0056] In the second step, the viscosity of the liquid sample and the degree of decrease in the mobility of the luminescent particles in response to their interaction with the cross-linked structure are detected as values ​​related to polarization anisotropy. The degree of decrease in the mobility of the luminescent particles depends on the amount and activity of transglutaminase or enzyme-related substances present in the liquid sample. Therefore, by relating the value related to polarization anisotropy obtained in the second step with the amount and activity of transglutaminase or enzyme-related substances in the liquid sample, the amount and activity of transglutaminase or enzyme-related substances in the liquid sample can be measured. For example, a high value related to polarization anisotropy obtained in the second step means that the liquid sample has thickened due to the formation of a cross-linked structure, or that the luminescent particles are strongly bound to the cross-linked structure, which can be associated with high transglutaminase activity.

[0057] Quantitative correlations can be established, for example, by using an enzyme solution with known enzyme activity to pre-determine a relationship between the amount or activity of the enzyme and the value related to polarization anisotropy. Using this relationship, the enzyme activity of the target substance can be determined from the value related to polarization anisotropy obtained by detecting the target substance. By performing such correlations, the enzyme activity in a liquid sample can be measured. The enzyme activity measured here can also be determined as enzyme concentration, mass, number of molecules, etc.

[0058] Furthermore, in the third step, the change in the value related to the polarization anisotropy of the luminescent particles can also be treated relatively. That is, the amount and activity of the enzyme or enzyme reaction-related substance may be related relatively, and for example, the value of fluorescence polarization degree (mp) obtained by fluorescence polarization measurement can be used. For example, in applications of screening highly active enzymes, screening may be performed simply based on the value of fluorescence polarization degree (mp) or the amount of change therein (Δmp) for a group of enzymes of the same concentration.

[0059] (Detection reagent) The present disclosure provides reagents for use in a method for detecting target substances related to the enzymatic reaction of transglutaminase, comprising luminescent particles having a first substrate of transglutaminase and a second substrate of transglutaminase.

[0060] One specific example is a reagent for detecting transglutaminase using values ​​related to polarization anisotropy, which comprises a luminescent particle having a first substrate of transglutaminase and a second substrate of multiple transglutaminases. Another example is a reagent for detecting a third substrate of transglutaminase using values ​​related to polarization anisotropy, comprising transglutaminase, luminescent particles having a first substrate of transglutaminase, and a plurality of second substrates of transglutaminase. Another example is a reagent for detecting transglutaminase activators or transglutaminase inhibitors using values ​​related to polarization anisotropy, comprising transglutaminase, luminescent particles having a first substrate of transglutaminase, and a second substrate of transglutaminase.

[0061] The reagent, comprising luminescent particles having a first substrate of transglutaminase, transglutaminase, a second substrate of transglutaminase, a third substrate of transglutaminase, a transglutaminase activator, or a transglutaminase inhibitor, is preferably provided in solution, but can also be provided in a dry state, frozen state, lyophilized state, etc. The dry reagent can be used for measurement by converting it to a solution state with the accompanying dissolving solution before measurement. Furthermore, the reagent of this disclosure may be a single reagent or may be divided into multiple reagents, for example, a first solution, a second solution, and a third solution.

[0062] The amount (concentration) of luminescent particles having the first substrate of transglutaminase contained in the reagent in this disclosure is preferably from 0.000001% by mass to 20% by mass, and more preferably from 0.0001% by mass to 1% by mass. In this disclosure, 1% by mass corresponds to 10 mg / mL. The reagent in this disclosure may also contain substances such as solvents and blocking agents in addition to the components described herein, to the extent that the objectives of this disclosure can be achieved. Two or more types of solvents and blocking agents may be included in combination. Examples of solvents used in this disclosure include various buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the solvents contained in the reagent in this disclosure are not limited to these.

[0063] (In vitro diagnostic kit) According to this disclosure, an in vitro diagnostic kit containing the above-mentioned reagents can also be provided. In addition to the above-mentioned reagents, the in vitro diagnostic kit may also include a standard solution, a positive control, a negative control, a serum diluent, etc. The standard solution is a solution of the target substance at a known concentration. As the medium for the positive control and negative control, serum, physiological saline, or a solvent that does not contain the detectable target substance may be used. [Examples]

[0064] The present disclosure will be described in further detail below with reference to examples. (Luminescent particle S1: Preparation of casein-immobilized luminescent particles) The luminescent particles used in the method for measuring the amount and activity of enzymes or enzyme reaction-related substances according to this disclosure are characterized by having a primary substrate of transglutaminase on their surface. Here, we show an example of the synthesis of luminescent particles having casein as the primary substrate of transglutaminase on their surface.

[0065] First, solvent A was prepared by dissolving polyvinylpyrrolidone (PVP-K30: manufactured by Tokyo Chemical Industry Co., Ltd.) in MES (2-morpholinoethanesulfonic acid) buffer at pH 7 (manufactured by Kishida Chemical Co., Ltd.). Next, reaction solution B was prepared by mixing the europium complex [Tris(2-thenoyltrifluoroacetone)bis(triphenylphosphineoxide)europium(III)] (manufactured by Central Techno Co., Ltd., hereinafter abbreviated as "Eu(TTA)3(TPPO)2"), styrene monomer (manufactured by Kishida Chemical Co., Ltd.), and 3-methacrylateoxypropyltrimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter abbreviated as "MPS"). Reaction solution B was added to a four-necked flask containing solvent A, and the mixture was stirred using a mechanical stirrer set to 300 rpm. After stirring for 15 minutes under nitrogen flow conditions, the temperature of the prepared oil bath was set to 70°C, and nitrogen flow was performed for another 15 minutes. After heating and stirring the mixture, an aqueous solution containing potassium persulfate (hereinafter abbreviated as "KPS") (manufactured by Aldrich) was added to the reaction solution, and emulsion polymerization was carried out for 20 hours. After the polymerization reaction, the obtained suspension was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 100K with approximately 4 L of deionized water to wash the product and obtain a dispersion of luminescent particles.

[0066] A dispersion of luminescent particles obtained by emulsion polymerization was taken and added to an aqueous solution containing 1% by mass of Tween20 (manufactured by Kishida Chemical Co., Ltd.). After stirring for 10 minutes, the silane coupling agent X12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added and stirred overnight (the mass ratio of the charged particles, pure water, and X12-1135 was 1:300:2). After stirring, the dispersion was centrifuged, the supernatant was removed, and the precipitate was redispersed in pure water. The centrifugation and redispersion process was repeated at least three times to wash the product. The precipitate after washing was redispersed in pure water. Luminescent particle 1 was obtained by the above procedure.

[0067] Next, 0.25 mL of the particle dispersion of luminescent particle 1 (particle concentration 1.2% by mass) was taken, and the solvent was replaced with 1.6 mL of MES buffer at pH 6.0. To the MES buffer containing the dispersed luminescent particles, 0.5% by mass of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and N-hydroxysulfosuccinimide sodium were added, and the mixture was reacted at 25°C for 1 hour. After the reaction, the dispersion was washed with phosphate-buffered saline (hereafter abbreviated as PBS) at pH 7.4, and then casein (derived from milk, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was reacted at 25°C for 2 hours to introduce casein to the particle surface. After introduction, the luminescent particles were washed with Tris buffer at pH 8. Subsequently, the luminescent particles were washed with PBS containing 0.01% by mass of Tween 20 (hereafter abbreviated as PBS-T) to obtain 1.0% by mass casein-immobilized luminescent particles (hereafter luminescent particle S1). The aqueous solution containing luminescent particles S1 is called the luminescent particle S1 solution. The particle concentration was adjusted by dilution with PBS-T.

[0068] The presence of casein on the particle surface was confirmed by adding BCA reagent (Protein Assay BCA Kit, Wako Pure Chemical Industries, Ltd.) to the luminescent particle S1 solution, centrifuging the solution, and measuring the absorbance (562 nm) of the supernatant. The increase in absorbance compared to before the addition of the BCA reagent confirmed the presence of casein on the particle surface. Dynamic light scattering (DLS) analysis of the size of luminescent particles in the aqueous solution revealed an average particle size of 143.1 nm (z-mean particle size). The polydispersity index was 0.019, indicating that monodisperse luminescent particles were obtained.

[0069] (Luminescent particle S2: Preparation of gelatin-immobilized luminescent particles) Except for replacing casein with gelatin (Type A, manufactured by Wako Pure Chemical Industries, Ltd.), luminescent particles having gelatin as the primary substrate for transglutaminase were synthesized in the same manner as the method for producing luminescent particle S1. The resulting gelatin-immobilized luminescent particle will hereafter be referred to as luminescent particle S2.

[0070] The presence of gelatin on the particle surface was confirmed by adding BCA reagent (Protein Assay BCA Kit, Wako Pure Chemical Industries, Ltd.) to the luminescent particle S2 solution, centrifuging the solution, and measuring the absorbance (562 nm) of the supernatant. The increase in absorbance compared to before the addition of the BCA reagent confirmed the presence of gelatin on the particle surface. Dynamic light scattering (DLS) analysis of the size of luminescent particle S2 in aqueous solution revealed an average particle size of 151.3 nm (z-mean particle size). The polydispersity index was 0.031, indicating monodisperse luminescent particles. The size was larger compared to luminescent particle S1 (casein-immobilized luminescent particle). This is thought to be due to the difference in molecular weight of the proteins (gelatin has a larger molecular weight than casein).

[0071] (Emitting particle NC: Fabrication of Tris-immobilized luminescent particles) Luminescent particles containing Tris were synthesized in the same manner as those used to produce luminescent particles S1, except that casein was replaced with tris(hydroxymethyl)aminomethane (Tris, manufactured by Wako Pure Chemical Industries, Ltd.). The resulting Tris-immobilized luminescent particles are hereafter referred to as luminescent particles NC. Luminescent particles NC do not contain a compound corresponding to the first substrate of transglutaminase and were used as control particles for comparative examples.

[0072] Dynamic light scattering (DLS) analysis of the size of luminescent particle NC in aqueous solution revealed an average particle size of 141.5 nm (z-mean particle size). The polydispersity index was 0.007, indicating monodisperse luminescent particles. NC was smaller in size than luminescent particles S1 and S2. This is thought to be due to the absence of protein immobilization.

[0073] (Example 1 and Comparative Example 1: Fluorescence polarization measurement of transglutaminase using casein-immobilized luminescent particles) PBS-T, luminescent particle S1 solution (particle concentration 0.1 mg / mL), and casein as a second substrate (final concentration 8% by mass) were mixed in 96-well microplates in the proportions shown in Table 1. To this aqueous solution, the target substance, transglutaminase (TG-S, Ajinomoto Co., Inc.), was added and thoroughly mixed (Example 1). In Table 1, it will be denoted as TG. Casein and transglutaminase were added after being dissolved in water. As a comparative example, a sample without the target substance was also prepared. In this case, 10 μL of PBS-T was added instead of the target substance (Comparative Example 1).

[0074] A microwell plate was placed in a fluorescence polarization analyzer (Nivo multimode microplate reader). The temperature inside the analyzer was adjusted to 37°C. Approximately 10 minutes after adding the target substance, the fluorescence polarization degree (mp) was measured. The conditions for the fluorescence polarization analyzer were as follows: Mode: FP Kinetics Excitation light: Center wavelength 355 nm / width 40 nm Light-emitting filter: (S) Center wavelength 615nm / width 8nm, (P) Center wavelength 615nm / width 8nm Dichroic mirror: D400 Measurement time: 1000ms Z-Focus: 5mm Measurement spot size: 2mm on the excitation side, 4mm on the emission side Flash energy: Low (10) PMT HV:1000

[0075] As shown in Table 1, the fluorescence polarization degree was 92.1 in the sample without transglutaminase (Comparative Example 1), but it increased to 105.6 in the sample containing transglutaminase (Example 1). This means that the mobility of the luminescent particles S1 (casein-immobilized luminescent particles) in the sample solution was reduced in Example 1, indicating that the transglutaminase activity caused the luminescent particles S1 to bind to the substrate cross-linking structure (the cross-linking structure of casein, the second substrate). From this example, it was found that transglutaminase can be detected simply and quickly by measuring the fluorescence polarization degree of the sample solution containing luminescent particles S1.

[0076] (Example 2 and Comparative Example 2: Fluorescence polarization measurement of transglutaminase using gelatin-immobilized luminescent particles) As shown in Table 1, PBS-T, luminescent particle S2 solution (particle concentration 0.1 mg / mL), and casein as a second substrate (final concentration 8 mass%) were mixed in a 96-well microplate, and fluorescence polarization measurements were performed in the same manner as in Example 1 and Comparative Example 1 (Example 2, Comparative Example 2). As shown in Table 1, the fluorescence polarization degree was 89.7 for the sample without transglutaminase (Comparative Example 2), but increased to 104.4 for the sample containing transglutaminase (Example 2). This indicates that the mobility of the luminescent particle S2 (gelatin-immobilized luminescent particle) in the sample solution was reduced in Example 2, and that binding to the substrate cross-linking structure of the luminescent particle S2 (the cross-linking structure of casein, the second substrate) occurred due to the activity of transglutaminase. From this example, it was found that transglutaminase can be detected simply and quickly by measuring the fluorescence polarization degree of a sample solution containing luminescent particle S2.

[0077] (Comparative Examples 3 and 4: Control tests using luminescent particles without the first substrate immobilized) PBS-T, luminescent particle NC solution (particle concentration 0.1 mg / mL), and casein as a second substrate (final concentration 8% by mass) were mixed in a 96-well microplate in the proportions shown in Table 1. Fluorescence polarization measurements were performed in the same manner as in Example 1 and Comparative Example 1. As shown in Table 1, the fluorescence polarization degree was 90.6 for the sample containing transglutaminase (Comparative Example 3) and 91.8 for the sample without transglutaminase (Comparative Example 4), indicating that the fluorescence polarization degrees were similar for both. Unlike Examples 1 and 2, no increase in fluorescence polarization degree was observed due to the presence of transglutaminase.

[0078] [Table 1]

[0079] Under the experimental conditions of Examples 1-2 and Comparative Examples 1-4 described above, the cross-linked structure formed in the solution (the cross-linked structure of casein, the second substrate) did not cause an increase in the viscosity of the solution that would alter the fluorescence polarization degree of the luminescent particles. From the results of Examples 1-2 and Comparative Examples 1-4 described above, it was found that by using a reagent for detecting the fluorescence polarization of transglutaminase, which contains luminescent particles having a first substrate of transglutaminase and a plurality of second substrates (casein) of transglutaminase as constituent components, it is possible to measure transglutaminase simply and rapidly.

[0080] (Example 3: Fluorescence polarization measurement of transglutaminase) PBS-T, luminescent particle S1 solution (particle concentration 0.1 mg / mL), and casein as a second substrate (final concentration 1% by mass) were mixed in 96-well microplates in the proportions shown in Table 2. To this aqueous solution, the target substance, transglutaminase (TG-S, manufactured by Ajinomoto Co., Inc.), was added and thoroughly mixed. In Table 2, it is denoted as TG. Fluorescence polarization measurements were performed in the same manner as in Example 1, except that the fluorescence polarization degree mp was measured at three points: before adding the target substance (fluorescence polarization degree before transglutaminase addition), approximately 3 minutes later (fluorescence polarization degree 3 minutes after transglutaminase addition), and approximately 45 minutes later (fluorescence polarization degree 45 minutes after transglutaminase addition).

[0081] (Example 4: Fluorescence polarization measurement of transglutaminase) As shown in Table 2, fluorescence polarization measurements were performed in the same manner as in Example 3, except that the final concentration of casein as the second substrate was 2% by mass.

[0082] (Example 5: Fluorescence polarization measurement of transglutaminase) As shown in Table 2, fluorescence polarization measurements were performed in the same manner as in Example 3, except that the final concentration of casein as the second substrate was 3% by mass.

[0083] (Example 6: Fluorescence polarization measurement of transglutaminase) As shown in Table 2, fluorescence polarization measurements were performed in the same manner as in Example 3, except that the final concentration of casein as the second substrate was 4% by mass.

[0084] (Example 7: Fluorescence polarization measurement of transglutaminase) As shown in Table 2, fluorescence polarization measurements were performed in the same manner as in Example 3, except that the final concentration of casein as the second substrate was 5% by mass.

[0085] (Example 8: Fluorescence polarization measurement of transglutaminase) As shown in Table 2, fluorescence polarization measurements were performed in the same manner as in Example 3, except that the final concentration of casein as the second substrate was 6% by mass.

[0086] As shown in Table 2, the fluorescence polarization degree after the addition of transglutaminase increased with increasing final concentration of casein as the second substrate. Furthermore, it was found that the fluorescence polarization degree increased over time. It is thought that transglutaminase formed cross-linked structures in the solution, and the luminescent particles bound to these cross-linked structures, thereby reducing the mobility of the luminescent particles over time.

[0087] The percentage increase in fluorescence polarization was calculated 3 minutes and 45 minutes after the addition of transglutaminase. Here, the percentage increase in fluorescence polarization was calculated by dividing the fluorescence polarization at 3 minutes or 45 minutes after the addition of transglutaminase by the fluorescence polarization before the addition of transglutaminase, and then multiplying the result by 100.

[0088] At both 3 minutes and 45 minutes after the addition of transglutaminase, the percentage increase in fluorescence polarization increased with increasing final concentration of casein as the second substrate. However, a peak was obtained in the sample with a final casein concentration of 5% by mass (Example 7). Under these conditions, it is considered that the binding of the luminescent particles containing the first substrate of transglutaminase to the cross-linked structure formed in the solution (the cross-linked structure of casein, the second substrate) occurs effectively.

[0089] [Table 2]

[0090] This embodiment includes the following configurations and methods. (Method 1) A method for detecting target substances related to the enzymatic reaction of transglutaminase in a liquid sample, A first step in which a plurality of the second substrates and the first substrates of the luminescent particles form a crosslinked structure in a liquid sample comprising transglutaminase, luminescent particles having a first substrate of transglutaminase, and a second substrate of transglutaminase, A second step involves obtaining a value relating to the polarization anisotropy of the liquid sample after the first step, A third step involves detecting a target substance related to the enzymatic reaction of the transglutaminase in the liquid sample based on the value relating to the polarization anisotropy, A method for detecting a target substance having [specific characteristics]. (Method 2) The method for detecting a target substance according to Method 1, wherein the luminescent particles are particles containing a rare earth luminescent complex. (Method 3) A method for detecting a target substance according to method 1 or 2, wherein the first step is to mix the liquid sample at a temperature of 30°C or higher and 50°C or lower and a pH of 5 or higher and 8 or lower. (Method 4) A method for detecting a target substance according to any one of methods 1 to 3, wherein the acquisition of the value relating to polarization anisotropy in the second step is also performed in the first step. (Method 5) A method for detecting a target substance according to any one of methods 1 to 4, wherein the target substance is transglutaminase. (Method 6) A method for detecting a target substance according to any one of methods 1 to 5, wherein the first substrate and the second substrate are each independently selected from the group consisting of casein, gelatin, collagen, keratin, modified forms thereof, and partial peptides thereof. (Method 7) A method for detecting a target substance according to any one of methods 1 to 6, wherein the first substrate and the second substrate are the same substance. (Method 8) A method for detecting a target substance according to any one of methods 1 to 7, wherein the target substance is a third substrate of transglutaminase. (Method 9) A method for detecting a target substance according to any one of methods 1 to 8, wherein the crosslinking structure is a bond selected from the group consisting of an amide bond, a peptide bond, and an isopeptide bond. (Method 10) The method for detecting a target substance according to Method 8, wherein the third substrate has higher substrate specificity than the first substrate and the second substrate. (Method 11) The method for detecting a target substance according to Method 8, wherein the first substrate, the second substrate, and the third substrate are each independently selected from the group consisting of casein, gelatin, collagen, keratin, modified forms thereof, and partial peptides thereof. (Method 12) A method for detecting a target substance according to any one of methods 1 to 11, wherein the target substance is an activator of transglutaminase. (Method 13) A method for detecting a target substance according to any one of methods 1 to 12, wherein the first step is a step of increasing the viscosity of the liquid sample. (Method 14) A method for detecting a target substance according to any one of methods 1 to 13, wherein the value relating to polarization anisotropy is either fluorescence polarization degree or polarization anisotropy. (Composition 15) A reagent for detecting transglutaminase using values ​​related to polarization anisotropy, comprising a luminescent particle having a first substrate of transglutaminase and a second substrate of a plurality of transglutaminases. (Composition 16) A reagent for detecting a third substrate of transglutaminase using a value relating to polarization anisotropy, comprising transglutaminase, luminescent particles having a first substrate of transglutaminase, and a plurality of second substrates of transglutaminase. (Composition 17) A reagent for detecting transglutaminase activators or transglutaminase inhibitors using values ​​related to polarization anisotropy, comprising transglutaminase, luminescent particles having a first substrate of transglutaminase, and a second substrate of transglutaminase. (Composition 18) The reagent according to any one of configurations 15 to 17, wherein the value relating to polarization anisotropy is either the degree of fluorescence polarization or polarization anisotropy. (Composition 19) The reagent according to configuration 16, wherein the third substrate has higher substrate specificity than the first substrate and the second substrate. (Composition 20) An in vitro diagnostic kit containing the reagent described in any one of components 15 to 17. [Explanation of Symbols]

[0091] 10 Luminescent particles 20 The primary substrate of transglutaminase 30. Second substrate of transglutaminase 40 Transglutaminase 50 Cross-linking structure of the second substrate of transglutaminase 60 Transglutaminase inhibitors 70 Transglutaminase bound to inhibitor 80. The third substrate of transglutaminase 81 Cross-linking structure of the third substrate

Claims

1. A method for detecting target substances related to the enzymatic reaction of transglutaminase in a liquid sample, A first step in which a plurality of the second substrates and the first substrates of the luminescent particles form a crosslinked structure in a liquid sample comprising transglutaminase, luminescent particles having a first substrate of transglutaminase, and a second substrate of transglutaminase, A second step involves obtaining a value relating to the polarization anisotropy of the liquid sample after the first step, A third step involves detecting a target substance related to the enzymatic reaction of the transglutaminase in the liquid sample based on the value relating to the polarization anisotropy, A method for detecting a target substance having [specific characteristics].

2. The method for detecting a target substance according to claim 1, wherein the luminescent particles are particles containing a rare earth luminescent complex.

3. The method for detecting a target substance according to claim 1 or 2, wherein the first step is to mix the liquid sample at a temperature of 30°C or higher and 50°C or lower and a pH of 5 or higher and 8 or lower.

4. The method for detecting a target substance according to claim 1 or 2, wherein the acquisition of the value relating to polarization anisotropy in the second step is also performed in the first step.

5. The method for detecting a target substance according to claim 1 or 2, wherein the target substance is transglutaminase.

6. The method for detecting a target substance according to claim 1 or 2, wherein the first substrate and the second substrate are each independently selected from the group consisting of casein, gelatin, collagen, keratin, modified forms thereof, and partial peptides thereof.

7. The method for detecting a target substance according to claim 1 or 2, wherein the first substrate and the second substrate are the same substance.

8. The method for detecting a target substance according to claim 1 or 2, wherein the target substance is a third substrate of transglutaminase.

9. The method for detecting a target substance according to claim 1 or 2, wherein the crosslinking structure is a bond selected from the group consisting of an amide bond, a peptide bond, and an isopeptide bond.

10. The method for detecting a target substance according to claim 8, wherein the third substrate has higher substrate specificity than the first substrate and the second substrate.

11. The method for detecting a target substance according to claim 8, wherein the first substrate, the second substrate, and the third substrate are each independently selected from the group consisting of casein, gelatin, collagen, keratin, modified forms thereof, and partial peptides thereof.

12. The method for detecting a target substance according to claim 1 or 2, wherein the target substance is an activator of transglutaminase.

13. The method for detecting a target substance according to claim 1 or 2, wherein the first step is a step of increasing the viscosity of the liquid sample.

14. The method for detecting a target substance according to claim 1 or 2, wherein the value relating to polarization anisotropy is the degree of fluorescence polarization or polarization anisotropy.

15. A reagent for detecting transglutaminase using values ​​related to polarization anisotropy, comprising a luminescent particle having a first substrate of transglutaminase and a second substrate of a plurality of transglutaminases.

16. A reagent for detecting a third substrate of transglutaminase using a value relating to polarization anisotropy, comprising transglutaminase, luminescent particles having a first substrate of transglutaminase, and a plurality of second substrates of transglutaminase.

17. A reagent for detecting transglutaminase activators or transglutaminase inhibitors using values ​​related to polarization anisotropy, comprising transglutaminase, luminescent particles having a first substrate of transglutaminase, and a second substrate of transglutaminase.

18. The reagent according to any one of claims 15 to 17, wherein the value relating to polarization anisotropy is the degree of fluorescence polarization or polarization anisotropy.

19. The reagent according to claim 16, wherein the third substrate has higher substrate specificity than the first substrate and the second substrate.

20. An in vitro diagnostic kit comprising the reagent described in any one of claims 15 to 17.

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