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 agglomerating the luminescent particles in a liquid sample containing transglutaminase and a plurality of luminescent particles having a first substrate of transglutaminase, by forming a crosslink structure between the first substrate of transglutaminase and the plurality of luminescent particles based on the activity of the 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 using enzymes. Transglutaminase (hereinafter sometimes abbreviated as TG), which is 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 transglutaminase measurement technology 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 previously. 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 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. However, Patent Document 2 does not disclose the measurement of enzyme activity, and furthermore, measuring enzyme activity in an antigen-antibody reaction is extremely difficult. [Prior art documents] [Patent Documents]

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

[0007] Therefore, 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 the action of transglutaminase in a liquid sample causes interparticle aggregation of luminescent particles and significantly reduces the mobility of the luminescent particles, it becomes possible to obtain values ​​related to the amount and polarization anisotropy 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, one aspect of this disclosure is: A method for detecting target substances related to the enzymatic reaction of transglutaminase in a liquid sample, A liquid sample comprising transglutaminase and a plurality of luminescent particles having a first substrate of transglutaminase, wherein a first step is to aggregate the luminescent particles by forming a crosslinking structure between the first substrate of transglutaminase present in the plurality of luminescent particles based on the activity of the 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 plurality of luminescent particles having a first substrate of transglutaminase.

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

[0012] Furthermore, another aspect of this disclosure is: A reagent for detecting a second substrate of transglutaminase using values ​​related to polarization anisotropy, comprising transglutaminase and a plurality of luminescent reagents having a first 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]It is a schematic diagram illustrating a high-sensitivity detection method for transglutaminase according to one aspect of an embodiment of the present disclosure. [Figure 3] It is a schematic diagram illustrating a detection method for inhibitors of transglutaminase according to one aspect of an embodiment of the present disclosure. [Figure 4] It is a schematic diagram illustrating a detection method for a second substrate of transglutaminase according to one aspect of an embodiment of the present disclosure. MODE 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 to include all cases: knowing the presence or absence of a target substance, detecting a target substance, and qualitatively and quantitatively knowing the amount or activity of a target substance.

[0017] (Value relating to polarization anisotropy determined by fluorescence polarization method) The present disclosure is a method for detecting the amount or activity of transglutaminase enzyme reaction-related substances, such as transglutaminase, a second substrate of transglutaminase, an activator of transglutaminase, and an inhibitor of transglutaminase, by using luminescent particles to obtain a value relating to the polarization anisotropy of said particles.

[0018] Fluorescence polarization is used to analyze the mobility of fluorescent molecules in a solution. The principle of fluorescence polarization will be described in 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 into a plane different from the excitation plane, whereby the polarization anisotropy is eliminated. That is, the value relating to polarization anisotropy represents the degree of rotational motion of the luminescent particles between excitation and fluorescence emission.

[0019] When luminescent particles in a liquid are dispersed individually in the solution, they exhibit low polarization anisotropy due to their vigorous rotation caused by Brownian motion. On the other hand, when luminescent particles aggregate, Brownian motion in the solution decreases, and 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 the 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, in a liquid sample containing transglutaminase and a plurality of luminescent particles having a first substrate of transglutaminase, the luminescent particles are aggregated by the formation of a crosslinking structure between the first substrate of transglutaminase present in the plurality of luminescent particles, based on the activity of the transglutaminase. (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: (1) unlike conventional low-molecular-weight substrates, the signal intensity per molecule is enhanced by using luminescent particles; (2) the mobility of the luminescent particles can be significantly suppressed in a short time by the action of the enzyme, so the value related to polarization anisotropy from the luminescent particles changes significantly in a short time; and (3) by measuring the value related to polarization anisotropy, there is no need to separate and wash away impurities or unreacted substances. Therefore, even when the activity of transglutaminase or its substrate as the target substance is low or the amount is small, the amount and activity of the target substance can be detected with high sensitivity and speed compared to existing methods.

[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 30, catalyzes the formation of crosslinks between luminescent particles 10 via the first substrate 20 (e.g., glutamine or lysine) of transglutaminase on the luminescent particles 10, due to its activity (catalyzing the formation of amide bonds between glutamine residues and amino groups in polypeptides). Before the addition of transglutaminase 30, the luminescent particles 10 are dispersed in solution and have high mobility. However, upon addition of transglutaminase 30, crosslinks (also called aggregation) are formed between the luminescent particles 10 depending on its activity, resulting in decreased mobility of the luminescent particles 10. This decrease in mobility 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 30 in the sample can be determined.

[0022] In reality, a large number of luminescent particles 10 are present in the solution, and the proportion of aggregated luminescent particles 10 increases depending on the activity of transglutaminase 30 in the sample. Here, since the value of polarization anisotropy obtained by fluorescence polarization is the average value for the entire solution, i.e., for all luminescent particles 10, the proportion of aggregated luminescent particles 10 correlates with the value of polarization anisotropy.

[0023] For example, by first obtaining calibration curves for polarization anisotropy and activity using a transglutaminase with known activity, the transglutaminase activity of a sample can be determined from the measurement results of the polarization anisotropy of the solution. 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 solution, which 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 40, which is the target substance, against a specified amount of transglutaminase 30 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 30 and / or transglutaminase 30 with sufficiently high activity. The target substance, the transglutaminase inhibitor 40, binds to transglutaminase 30 with arbitrary affinity and inhibits the activity of transglutaminase 30. That is, when transglutaminase 50 bound to the inhibitor 40 is produced, the activity of transglutaminase is lost. As a result, the degree of aggregation of luminescent particles 10 by transglutaminase 30 is reduced. When the inhibition efficiency is 100%, the luminescent particles 10 do not aggregate. 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, when the inhibition efficiency is 0%, the luminescent particles 10 aggregate. As a result, a cross-linking structure between the luminescent particles 10 occurs as shown in Figure 2, and 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 inhibition efficiency of transglutaminase 30 by the inhibitor 40. Therefore, a large number of candidate inhibitors 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 30 and / or low-activity transglutaminase 30. The target substance, the activator, binds to the transglutaminase 30 with an arbitrary affinity, thereby increasing the activity of the transglutaminase 30. As a result, the degree of aggregation of the luminescent particles 10 by the transglutaminase 30 can be increased. When the activation efficiency is 0%, the luminescent particles 10 do not aggregate. As a result, the mobility of the luminescent particles 10 does not change, and the polarization anisotropy of the luminescent particles 10 also does not change. On the other hand, when the activation efficiency is 100%, the luminescent particles 10 aggregate. As a result, a cross-linking structure between the luminescent particles 10 occurs, as shown in Figure 2, and the polarization anisotropy of the luminescent particles 10 increases. That is, a change in the value related to polarization anisotropy is observed in accordance with the activation efficiency of transglutaminase 30 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 second substrate of transglutaminase) Furthermore, the second 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 the second substrate 60 of the target transglutaminase against a specified amount of transglutaminase 30 can be measured using a value related to the polarization anisotropy of the luminescent particles 10 as an indicator. Here, "inhibition" means that the second substrate 60 competitively inhibits the reaction of transglutaminase 30 with the first substrate 20.

[0029] Specifically, the liquid sample contains a sufficiently large amount of transglutaminase 30 and / or transglutaminase 30 with sufficiently high activity. The second substrate 60 of the target substance, transglutaminase, is assumed to preferentially form a crosslink structure with respect to the activity of transglutaminase 30, rather than with respect to the first substrate 20 (the second substrate 60 has higher substrate specificity than the first substrate 20). That is, when the crosslink structure 61 of the second substrate 60 is formed, the activity of transglutaminase 30 with respect to the first substrate 20 is inhibited. As a result, the degree of aggregation of luminescent particles 10 by transglutaminase 30 is reduced. When the inhibition efficiency is 100%, the luminescent particles 10 do not aggregate. 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, when the inhibition efficiency is 0%, the luminescent particles 10 aggregate. As a result, the formation of crosslink structures between the luminescent particles 10, as shown in Figure 2, occurs, and the polarization anisotropy of the luminescent particles 10 increases. In other words, a change in the value related to polarization anisotropy is observed in accordance with the inhibition efficiency of transglutaminase 30 by the second substrate 60. 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) One example of the present invention comprises the following steps. (1) A first step in which, in a liquid sample containing transglutaminase and a plurality of luminescent particles having a first substrate of transglutaminase, the luminescent particles are aggregated by the formation of a crosslinking structure between the first substrate of transglutaminase present in the plurality of luminescent particles, based on the activity of the 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 cross-linking structure (also called aggregation) between multiple luminescent particles in a liquid sample using an enzyme. For example, if the target substance is transglutaminase, the luminescent particles can be aggregated by reacting it with the transglutaminase to form a cross-linking structure between the luminescent particles. 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.

[0032] Furthermore, if the target substance is an inhibitor of transglutaminase, which is an example of an enzyme reaction-related substance, transglutaminase can be added to a solution containing the inhibitor and luminescent particles, and the reaction can be carried out to detect the aggregation reaction of the luminescent particles. Here, the order of the reaction is not particularly limited, but since interparticle aggregation is generated with transglutaminase as a trigger, it is preferable to add the target substance, the inhibitor, before the transglutaminase. The reaction solution can also be divided into multiple parts. That is, a first solution may be prepared by mixing transglutaminase and the target substance, the inhibitor, and then the first solution may be mixed with a second solution containing luminescent particles to carry out the aggregation reaction of the luminescent particles. In this case, even if the target substance is an activator of transglutaminase or a second substrate of transglutaminase, it can be detected by performing the same procedure as in the case of the inhibitor.

[0033] (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.

[0034] (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.

[0035] (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 second 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 second 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 to the first substrate of transglutaminase. Preferred activators include calcium chloride, and preferred inhibitors include metal chelators and protein denaturants.

[0036] (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, since the luminescent particles according to this disclosure have the first substrate of transglutaminase, cross-linking structures can be formed between the luminescent particles by the action of transglutaminase. The cross-linking mode between the luminescent particles can be any type of bond catalyzed by transglutaminase, but is preferably a chemical bond, such as an amide bond, peptide bond, or isopeptide bond. The first substrate of 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.

[0037] 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.

[0038] 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 before crosslinking between luminescent particles increases, and the change in the value of polarization anisotropy after aggregation between luminescent particles may become small. Also, if the average particle size is less than 25 nm, the content of luminescent material in the particles decreases, resulting in a lower luminescence intensity per particle, which may reduce detection sensitivity and accuracy. Therefore, luminescent particles of 50 nm or larger are preferred. The size of the luminescent particles can be determined by dynamic light scattering (DLS) measurement.

[0039] As the particle material (matrix material) for the luminescent particles, for example, synthetic polymer particles such as polystyrene and polymethacrylate, inorganic particles 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.

[0040] 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.

[0041] 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.

[0042] 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)].

[0043] 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.

[0044] (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.

[0045] In this disclosure, the target substance's 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. That is, depending on the amount and activity of the enzyme or enzyme reaction-related substance, a cross-linking structure is formed between luminescent particles in the sample solution, resulting in a decrease in the mobility of the luminescent particles. This disclosure is characterized by detecting the mobility of luminescent particles using 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 using fluorescence polarization degree (mp) as an indicator.

[0046] A key feature of this disclosure is the use of luminescent particles as probes, which contain a europium complex as a luminescent material within the luminescent particles, exhibiting a long luminescence lifetime and polarized emission. Even slight changes in the rotational motion of these luminescent particles in a liquid sample can be detected as changes in their polarized emission characteristics. Specifically, when the mobility of the luminescent particles decreases significantly due to crosslinking between the particles, 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.

[0047] 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.

[0048] 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 the fluorescence polarization degree (mp(t)) at a certain time point may be measured again due to the crosslinking between the luminescent particles, 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.

[0049] 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 depending on 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%).

[0050] (Third step) In this disclosure, the third step is to detect enzyme reaction-related substances in a liquid sample based on values ​​relating to the polarization anisotropy of luminescent particles in the liquid sample obtained in the second step. More specifically, it is a step of relating changes in the values ​​relating to polarization anisotropy to the amount or activity of the enzyme or enzyme reaction-related substances.

[0051] In the second step, the degree of reduction in the mobility of the luminescent particles, depending on the cross-linking structure between the luminescent particles, is detected as a value related to polarization anisotropy. The degree of reduction 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, if the value related to polarization anisotropy obtained in the second step is high, it means that the luminescent particles are aggregated and the proportion of these aggregates is large, which can be associated with high transglutaminase activity.

[0052] 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.

[0053] 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.

[0054] (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.

[0055] One specific example is a reagent for detecting transglutaminase using values ​​related to polarization anisotropy, which is a reagent containing multiple luminescent particles having a first substrate of transglutaminase. Another example is a reagent for detecting either a transglutaminase activator or a transglutaminase inhibitor using values ​​related to polarization anisotropy, the reagent comprising transglutaminase and a plurality of luminescent particles having a first substrate of transglutaminase. Another example is a reagent for detecting a second substrate of transglutaminase using values ​​related to polarization anisotropy, the reagent comprising transglutaminase and a plurality of luminescent particles having a first substrate of transglutaminase.

[0056] The reagent, comprising luminescent particles having a first substrate of transglutaminase, transglutaminase, a second 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.

[0057] 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 buffer solutions 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. Furthermore, the reagent in this disclosure may contain a sensitizer that promotes the aggregation of luminescent particles. Examples of sensitizers include polyvinyl alcohol, polyvinylpyrrolidone, and polyalginic acid, but this disclosure is not limited to these.

[0058] (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]

[0059] 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 that allows for crosslinking between the luminescent particles. Here, we show an example of the synthesis of luminescent particles having casein as the primary substrate of transglutaminase on their surface.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] (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.

[0065] 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).

[0066] (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 are designed not to contain a compound corresponding to the first substrate of transglutaminase and not to cause aggregation between luminescent particles by transglutaminase. In this example, they were used as control particles for comparative examples.

[0067] 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.

[0068] (Example 1 and Comparative Example 1: Fluorescence polarization measurement of transglutaminase using casein-immobilized luminescent particles) Diluted PBS-T and luminescent particle S1 solution (particle concentration 0.1 mg / mL) were mixed in a 96-well microplate in the proportions shown in Table 1. To this aqueous solution, the target substance, transglutaminase solution (TG-S, 2.29 mg / mL, Ajinomoto Co., Inc.), was added and thoroughly mixed (Example 1). In Table 1, this is 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).

[0069] 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

[0070] As shown in Table 1, the fluorescence polarization degree was 60.7 for the sample without transglutaminase (Comparative Example 1), but it was 63.3 for the sample containing transglutaminase (Example 1), indicating a significant increase in fluorescence polarization degree. This means that in Example 1, the mobility of the luminescent particles S1 (casein-immobilized luminescent particles) in the sample solution was reduced, indicating that aggregation of luminescent particles S1 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 particles S1.

[0071] (Example 2 and Comparative Example 2: Fluorescence polarization measurement of transglutaminase using gelatin-immobilized luminescent particles) As shown in Table 1, PBS-T and luminescent particle S2 solution (particle concentration 0.1 mg / mL) 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 62.1 for the sample without transglutaminase (Comparative Example 2), but it was 64.3 for the sample containing transglutaminase (Example 2), indicating a significant increase in fluorescence polarization degree. This means that the mobility of luminescent particles S2 (gelatin-immobilized luminescent particles) in the sample solution was reduced in Example 2, indicating that aggregation of luminescent particles S2 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 particles S2.

[0072] Furthermore, when comparing Comparative Example 1, which does not contain transglutaminase, with Comparative Example 2, the fluorescence polarization degree was higher in Comparative Example 2. This is due to the difference in particle size of the luminescent particles containing the first substrate of transglutaminase. Specifically, because luminescent particle S2 (151.3 nm) has a larger particle size than luminescent particle S1 (143.1 nm), its rotational motion is slower.

[0073] (Comparative Examples 3 and 4: Control tests using luminescent particles without the first substrate immobilized) PBS-T and luminescent particle NC solution (particle concentration 0.1 mg / mL) 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 59.4 for the sample containing transglutaminase (Comparative Example 3) and 59.6 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.

[0074] [Table 1]

[0075] The results from the above examples show that by using a reagent for detecting the fluorescence polarization of transglutaminase, which contains multiple luminescent particles having the first substrate of transglutaminase as a component, it is possible to measure transglutaminase simply and rapidly.

[0076] 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 liquid sample comprising transglutaminase and a plurality of luminescent particles having a first substrate of transglutaminase, wherein a first step is to aggregate the luminescent particles by forming a crosslinking structure between the first substrate of transglutaminase present in the plurality of luminescent particles based on the activity of the 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 target substance is a second substrate of transglutaminase. (Method 7) The method for detecting a target substance according to method 6, wherein the second substrate has higher substrate specificity than the first substrate. (Method 8) A method for detecting a target substance according to any one of methods 1 to 7, wherein the target substance is an activator of transglutaminase. (Method 9) A method for detecting a target substance according to any one of methods 1 to 8, wherein the target substance is a transglutaminase inhibitor. (Method 10) A method for detecting a target substance according to any one of methods 1 to 9, wherein the first substrate of the transglutaminase is selected from the group consisting of casein, gelatin, collagen, keratin, modified forms thereof, and partial peptides thereof. (Method 11) A method for detecting a target substance according to any one of methods 1 to 10, wherein the value relating to polarization anisotropy is either fluorescence polarization degree or polarization anisotropy. (Composition 12) A reagent for detecting transglutaminase using values ​​related to polarization anisotropy, comprising a plurality of luminescent particles having a first substrate of transglutaminase. (Composition 13) A reagent for detecting either a transglutaminase activator or a transglutaminase inhibitor using values ​​related to polarization anisotropy, comprising transglutaminase and a plurality of luminescent particles having a first substrate of transglutaminase. (Composition 14) A reagent for detecting a second substrate of transglutaminase using values ​​related to polarization anisotropy, comprising transglutaminase and a plurality of luminescent particles having a first substrate of transglutaminase. (Composition 15) The reagent according to any one of configurations 12 to 14, wherein the value relating to polarization anisotropy is either the degree of fluorescence polarization or polarization anisotropy. (Composition 16) The reagent according to configuration 14, wherein the second substrate has higher substrate specificity than the first substrate. (Composition 17) An in vitro diagnostic kit containing the reagent described in any one of components 12 to 14. [Explanation of Symbols]

[0077] 10 Luminescent particles 20 The primary substrate of transglutaminase 30 Transglutaminase 40. Transglutaminase inhibitors 50 Transglutaminase bound to inhibitor 60 Second substrate of transglutaminase 61 Crosslinking structure of the second substrate

Claims

1. A method for detecting target substances related to the enzymatic reaction of transglutaminase in a liquid sample, A liquid sample comprising transglutaminase and a plurality of luminescent particles having a first substrate of transglutaminase, wherein a first step is to aggregate the luminescent particles by forming a crosslinking structure between the first substrate of transglutaminase present in the plurality of luminescent particles based on the activity of the 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 target substance is a second substrate of transglutaminase.

7. The method for detecting a target substance according to claim 6, wherein the second substrate has higher substrate specificity than the first substrate.

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

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

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

11. 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.

12. A reagent for detecting transglutaminase using values ​​related to polarization anisotropy, comprising a plurality of luminescent particles having a first substrate of transglutaminase.

13. A reagent for detecting either a transglutaminase activator or a transglutaminase inhibitor using values ​​related to polarization anisotropy, comprising transglutaminase and a plurality of luminescent particles having a first substrate of transglutaminase.

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

15. The reagent according to any one of claims 12 to 14, wherein the value relating to polarization anisotropy is the degree of fluorescence polarization or polarization anisotropy.

16. The reagent according to claim 14, wherein the second substrate has higher substrate specificity than the first substrate.

17. An in vitro diagnostic kit comprising the reagent described in any one of claims 12 to 14.

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