Enzyme detection methods

Abstract

A novel enzyme detection method is provided that can efficiently detect a specific enzyme contained in a sample solution. [Solution] A method for detecting a specific enzyme contained in a sample liquid, comprising: an antibody conjugate preparation step of binding an antibody that binds to the specific enzyme to the surface of a carrier to prepare an antibody conjugate; an enzyme conjugate preparation step of contacting the antibody conjugate with the sample liquid and binding a specific enzyme contained in the sample liquid to the antibody of the antibody conjugate to prepare an enzyme conjugate; a sample liquid separation step of separating the enzyme conjugate from the sample liquid; and an enzyme detection step of detecting the specific enzyme bound to the enzyme conjugate after separating the sample liquid.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting a specific enzyme contained in a sample solution. [Background technology]

[0002] Humans have two arginase isoforms, of which arginase 1 is a urea cycle enzyme primarily localized in the liver, which converts L-arginine to L-ornithine and urea and plays an important role in removing toxic ammonia from the body.

[0003] As a method for measuring the enzymatic activity of such arginase, for example, a sample containing arginase is reacted in a buffer solution containing arginine, and the products obtained by a series of enzymatic reactions in which arginine is decomposed into ornithine and urea are quantified by the following methods 1) to 4) (see FIG. 5).

[0004] 1) A method for quantifying the amount of ornithine produced by HPLC (see, for example, Patent Document 1) 2) A method in which urea is decomposed into ammonia using urease and the amount of ammonia produced is quantified (see, for example, Non-Patent Document 1). 3) A method of quantifying the amount of urea produced by reacting urea with a color reagent (see, for example, Patent Document 2) 4) A method for quantifying urea production using radioisotope-labeled arginine as a substrate (see, for example, Non-Patent Document 2)

[0005] However, method 1) requires HPLC measurement equipment and is time-consuming to perform, making it unsuitable for analyzing multiple samples. Method 2) requires an additional enzymatic reaction with urease, making the procedure complicated. Furthermore, for samples such as plasma and serum, a process to remove endogenous urea is required to distinguish it from the urea generated. Method 3) also requires a process to remove endogenous urea to distinguish it from the urea generated. Method 4) can distinguish the urea generated from endogenous urea, but requires special care in handling because it uses a radioactively labeled compound. As described above, the above methods 1) to 4) have some problems, but in many studies, the method 3) using a color reagent is adopted.

[0006] Here, the method using a color reagent (3) mainly employs a dilution method using an ultrafiltration unit to remove endogenous urea. Specifically, the sample is diluted with an appropriate buffer solution, and the low-molecular-weight urea is eluted by ultrafiltration to concentrate the sample. The concentrated sample is then diluted again with a buffer solution and concentrated by centrifugation, and this procedure is repeated. This method can reduce the amount of urea in the sample to an unlimited extent (see Figure 6). However, this method requires a long time, making it difficult to simultaneously process a large number of samples, and there is a problem that the daily processing capacity is limited. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2015-516397 [Patent Document 2] International Publication No. 2017 / 086421 [Non-patent literature]

[0008] [Non-Patent Document 1] Cheryl L. Garganta, Judith S. Bond, Assay and kinetics of arginase, Analytical Biochemistry, Vol. 154(2), p. 388-394 (1986) [Non-patent document 2] Urs T. Rueegg,Anthony S. Russell,A rapid and sensitive assay for arginase,Analytical Biochemistry,Vol.102(1),p.206-212 (1980) Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a novel enzyme detection method that can efficiently detect a specific enzyme contained in a sample solution. [Means for solving the problem]

[0010] The present inventors discovered that a specific enzyme contained in a sample solution can be efficiently detected by using an antibody conjugate in which an antibody that binds to a specific enzyme is bound to the surface of a carrier, thereby separating the specific enzyme from other enzymes and impurities, and thus completed the present invention.

[0011] That is, the present invention is as follows. [1] A method for detecting a specific enzyme contained in a sample solution, comprising: an antibody conjugate preparation step of binding an antibody that binds to the specific enzyme to a carrier surface to prepare an antibody conjugate; an enzyme conjugate preparation step of contacting the antibody conjugate with the sample solution to allow a specific enzyme contained in the sample solution to bind to the antibody of the antibody conjugate, thereby preparing an enzyme conjugate; a sample liquid separating step of separating the enzyme conjugate from the sample liquid; an enzyme detection step of detecting a specific enzyme bound to the separated enzyme conjugate from the sample liquid; A method for detecting an enzyme, comprising:

[0012] [2] The method for detecting an enzyme according to [1] above, wherein the carrier is a magnetic material. [3] The method for detecting an enzyme according to [2] above, wherein the carrier is a particle having a particle size of 5 μm or less. [4] The enzyme conjugate preparation step is carried out in a well of a well plate; The sample liquid separating step is carried out by bringing a magnet close to or in contact with the backside of the well plate, and removing the sample liquid from the wells of the well plate while the oxygen binder is held in the wells of the well plate. The method for detecting an enzyme according to [2] or [3] above.

[0013] [5] The method for detecting an enzyme according to any one of [1] to [4] above, wherein the sample liquid is a biological fluid. [6] The method for detecting an enzyme according to any one of [1] to [5] above, wherein the specific enzyme is arginase. [7] The method for detecting an enzyme according to any one of [1] to [6] above, wherein the specific enzyme is arginase 1.

[0014] [8] A method for detecting an enzyme according to any one of [1] to [7] above, characterized in that the sample solution contains two or more isoforms of the enzyme, and one of the two or more isoforms of the enzyme is detected. [Effects of the Invention]

[0015] According to the present invention, a specific enzyme contained in a sample liquid can be efficiently detected. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic diagram illustrating a method for detecting an enzyme according to one embodiment of the present invention. [Figure 2]FIG. 2 is an explanatory diagram of an antibody conjugate preparation step and an enzyme conjugate preparation step in the enzyme detection method according to one embodiment of the present invention. [Figure 3] This is a calibration curve showing absorbance versus urea standard solution (urea amount). [Figure 4] 1 is a calibration curve showing absorbance versus enzyme activity of arginase 1 with known activity. [Figure 5] FIG. 1 is a diagram illustrating a method for measuring the enzyme activity of arginase. [Figure 6] FIG. 1 is a schematic diagram illustrating ultrafiltration. DETAILED DESCRIPTION OF THE INVENTION

[0017] The enzyme detection method of the present invention is a method for detecting a specific enzyme contained in a sample liquid, and is characterized by comprising an antibody conjugate preparation step of binding an antibody that binds to the specific enzyme to the surface of a carrier to prepare an antibody conjugate; an enzyme conjugate preparation step of contacting the antibody conjugate with the sample liquid and binding the specific enzyme contained in the sample liquid to the antibody of the antibody conjugate to prepare an enzyme conjugate; a sample liquid separation step of separating the enzyme conjugate from the sample liquid; and an enzyme detection step of detecting the specific enzyme bound to the enzyme conjugate separated from the sample liquid.

[0018] The enzyme detection method of the present invention may include other steps before, after, or between the sample solution preparation step, antibody conjugate preparation step, enzyme conjugate preparation step, sample solution separation step, and enzyme detection step. Specifically, for example, the method may include a sample solution preparation step of obtaining a serum sample from blood collected from a human body before the antibody conjugate preparation step.

[0019] The enzyme detection method of the present invention is a novel technique that uses an antibody conjugate in which an antibody that binds to this specific enzyme is bound to the surface of a carrier in order to detect a specific enzyme contained in a sample solution.For example, it can efficiently detect a specific enzyme contained in a sample solution without using expensive high-precision detection equipment, without using ultrafiltration, without using radioactive substances, and without being influenced by other enzymes or impurities other than the specific enzyme to be detected.

[0020] In the enzyme detection method of the present invention, the antibody conjugate is not particularly limited as long as it can bind to a specific enzyme contained in a sample solution, and examples thereof include a carrier having an antibody that binds to a specific enzyme bound to its surface (see Figure 2). Methods for binding the antibody to the carrier include, for example, a method in which the antibody is bound to the carrier surface via a binding substance previously provided on the carrier surface, and a method in which the antibody is bound to the carrier surface by a crosslinking reaction. Examples of binding substances include binding proteins such as protein G, protein A, and protein L.

[0021] Here, the carrier can be of various shapes, such as granular, flake, or rod-like, and its material can be magnetic, such as iron oxide, chromium oxide, cobalt, or ferrite, or non-magnetic, such as resin, plastic, or ceramic. Regarding the size of the carrier, for example, in the case of particles, the particle size is, for example, 1 mm or less, preferably 100 μm or less, more preferably 10 μm or less, and even more preferably 5 μm or less. Meanwhile, the lower limit is not particularly limited, but is, for example, about 1 μm.

[0022] Furthermore, the antibody is preferably one that does not inhibit the enzyme detection reaction in the enzyme detection step, and can be appropriately selected depending on the type of specific enzyme to be bound.

[0023] In the enzyme detection method of the present invention, the sample liquid is not particularly limited as long as it contains the specific enzyme to be detected, and may contain, for example, other enzymes and impurities other than the specific enzyme. Examples of sample liquids include biological fluids of humans, animals, or plants, solutions containing pharmaceuticals, foodstuffs, etc., and solutions containing the raw materials for these, and examples of human biological fluids include blood (plasma, serum), saliva, sweat, urine, etc.

[0024] When the sample solution contains two or more isoforms of an enzyme, the method of the present invention can detect one of the two or more isoforms of the enzyme.

[0025] Examples of specific enzymes to be detected include arginase, elastase, peroxidase (also referred to as MPO), etc., present in humans. In the present invention, one of the two isoforms of arginase present in the human body can be detected. Specifically, arginase has two isoforms, arginase 1 (hereinafter also referred to as ARG1) and arginase 2 (hereinafter also referred to as ARG2), and of these, for example, only arginase 1 can be selectively detected. While both arginase 1 and arginase 2 use arginine as a substrate, they have different functions, so it is very useful to distinguish between them and detect only one of the arginases.

[0026] Each step of the present invention will be described below. The enzyme detection method of the present invention comprises an antibody conjugate preparation step, an enzyme conjugate preparation step, a sample liquid separation step, and an enzyme detection step. Each step will be specifically described below.

[0027] (Antibody conjugate production process) The antibody conjugate preparation step is a step in which an antibody that binds to a specific enzyme is bound to the surface of a carrier to prepare an antibody conjugate (see Figure 2).

[0028] First, the carrier and the antibody are placed in a container containing a liquid and allowed to react. Here, for example, a microtube or the like can be used as the container. The liquid is preferably determined appropriately depending on the sample liquid. For example, when a human biological fluid is used as the sample liquid, phosphate buffered saline (PBS) or the like can be used.

[0029] Next, the carrier to which the antibody is bound (carrier conjugate) is separated from the liquid. The separation method is not particularly limited, but when the carrier is magnetic, separation can be performed using a magnet, for example. Specifically, for example, separation can be performed by removing the liquid while the carrier-bound substance is still contained in a container such as a microtube, or by transferring the carrier-bound substance and liquid to a container such as a well plate, and then using a magnet to hold the antibody-bound substance at the bottom of the container.

[0030] Furthermore, when the carrier is non-magnetic, separation can be performed using, for example, a filter. Specifically, for example, a container equipped with a mesh (filter) at the bottom and an openable lid is used, and the antibody conjugate is prepared with the lid closed, and then the lid is opened to discharge the liquid downward (outside the container) through the mesh. Separation can also be performed using ultrafiltration or centrifugation using centrifugal filtration or centrifugal sedimentation.

[0031] After separating the antibody conjugate from the liquid, it is preferable to wash the antibody conjugate one or more times with a washing solution, thereby removing unbound (unreacted) antibodies and other substances adhering to the surface of the antibody conjugate. In this washing treatment, as described above, for example, if the carrier is magnetic, a magnet can be used, or if the carrier is non-magnetic, a container with an openable lid and mesh at the bottom can be used to easily separate the antibody conjugate from the washing solution.

[0032] (Enzyme conjugate preparation process) The enzyme conjugate preparation step is a step of preparing an enzyme conjugate by contacting an antibody conjugate with a sample solution and allowing a specific enzyme contained in the sample solution to bind to the antibody in the antibody conjugate. Here, it is preferable to adjust the amount of the antibody conjugate and / or sample solution so that 30% or more, preferably 40% or more, and particularly preferably 50% of the enzyme contained in the sample solution binds to the antibody in the antibody conjugate.

[0033] First, the antibody conjugate and the sample solution are placed in a container and allowed to react. Here, the container may be, for example, a well plate, a microtube, etc. The container used for producing the antibody conjugate may also be used as it is with the antibody conjugate inside. The reaction temperature and reaction time of this sample solution can be changed depending on, for example, the type of specific enzyme, the binding state, etc. The reaction temperature is within a temperature range in which the enzyme is not inactivated, and is, for example, preferably about 3 to 40° C., and more preferably about 4 to 37° C. The reaction time is, for example, preferably 5 hours or more, more preferably 8 hours or more, and even more preferably 10 to 24 hours.

[0034] (Sample liquid separation process) The sample liquid separation step is a step of separating the enzyme conjugate from the sample liquid. This separation can be easily performed by using a magnet when the carrier is magnetic, as described above, or by using a container with an openable lid and mesh at the bottom when the carrier is non-magnetic, so that the antibody conjugate and the sample solution can be easily separated.

[0035] After separating the prepared enzyme conjugate from the sample solution, it is preferable to wash the enzyme conjugate once or multiple times with a washing solution such as a buffer solution, thereby removing unbound enzyme and impurities contained in the sample solution that are attached to the surface of the enzyme conjugate. In this washing treatment, for example, as described above, if the carrier is magnetic, a magnet can be used, and if the carrier is non-magnetic, a container with an openable lid and mesh at the bottom can be used to easily separate the enzyme conjugate from the washing solution.

[0036] (Enzyme detection process) The enzyme detection step is a step of detecting a specific enzyme bound to the separated enzyme conjugate in the sample liquid. Here, enzyme detection can take various forms depending on the purpose of detection, and is not particularly limited as long as a specific enzyme can be detected, and examples include a form in which the magnitude of enzyme activity is measured, a form in which the presence or absence of enzyme activity is simply detected, etc. Specific examples include a form in which the enzyme is quantitatively detected using a numerical value, and a form in which the presence or absence is detected non-quantitatively without using a numerical value.

[0037] Hereinafter, embodiments of the enzyme detection method of the present invention will be specifically explained with reference to the drawings, but the present invention is not limited to these embodiments.

[0038] 1 and 2, an enzyme detection method according to one embodiment of the present invention includes an antibody conjugate preparation step, an enzyme conjugate preparation step, a sample liquid separation step, and an enzyme detection step. Hereinafter, a case will be described in which a biological fluid containing two enzyme isoforms, arginase 1 (a specific enzyme) and arginase 2, is used as the sample liquid, and magnetic particles (hereinafter also referred to as magnetic beads) are used as the antibody conjugate.

[0039] (Antibody conjugate production process) As shown in Figures 1 and 2, the carrier and antibody are first introduced into a microtube (container) containing a buffer solution and allowed to react. The carrier is a magnetic bead with a particle size of 5 μm or less (approximately 2 to 4 μm) and a large number of immunoglobulin-binding proteins (binding substances) on its surface, and the antibody is an anti-arginase 1 antibody. This allows the antibody to react with the immunoglobulin-binding protein of the carrier, binding the antibody to the immunoglobulin-binding protein, and producing an antibody conjugate.

[0040] Next, as shown in Figure 1, the prepared antibody conjugate and buffer solution are dispensed into a well plate and allowed to stand, and a magnet is brought close to or into contact with the back of the plate to remove the buffer solution from the well while retaining the antibody conjugate in the well. Subsequently, the antibody conjugate in the well is washed multiple times with the buffer solution to remove unreacted antibody.

[0041] (Enzyme conjugate preparation process) As shown in Figure 1, the enzyme conjugate preparation process is carried out in the wells of a well plate. A sample solution is added to the well containing the prepared antibody conjugate and buffer solution. This allows the antibody conjugate to come into contact with the sample solution, and the arginase 1 contained in the sample solution binds to the antibody in the antibody conjugate through an antigen-antibody reaction to produce an enzyme conjugate. At this time, the arginase 2 in the sample solution remains in the sample solution without binding to the antibody in the antibody conjugate.

[0042] (Sample liquid separation process) As shown in Figure 1, a magnet is placed near or in contact with the back of a well plate, and while the oxygen conjugate is held in the well of the well plate, the sample solution containing impurities such as arginase 2 and endogenous urea is removed from the well of the well plate. The enzyme conjugate is then washed multiple times with a buffer solution in the well to remove any remaining impurities adhering to the enzyme conjugate. This allows the impurities to be removed without the need for processes such as ultrafiltration to remove endogenous urea, thereby improving both workability and efficiency.

[0043] (Enzyme detection process) First, a substrate solution containing arginine, which is a substrate for arginase, and a divalent cation such as manganese is added to each well of a well plate, and the mixture is incubated at 37°C for a certain period of time to allow the enzyme-substrate reaction to occur. Next, a urea color reagent is added and reacted for a certain period of time, and then the mixture is set in a measuring device and the absorbance is measured from the resulting color to detect arginase 1. Here, the color reagent is not particularly limited as long as it can detect urea. In addition, examples of measuring devices include spectrophotometers and microplate readers, and in particular, microplate readers that can measure multiple samples simultaneously are preferred. [Example]

[0044] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0045] [Comparative Example] A test to detect arginase was carried out using the conventional method described in the background art, "3) quantification of the amount of urea produced by reacting urea with a color reagent."

[0046] <Serum 1-3 Measurement> (Test Procedure 1: Removal of Endogenous Urea from Serum Samples) The serum samples (Serum 1 to 3) were loaded onto a column of an ultrafiltration unit (MWCO 10000; Merck) and concentrated by centrifugation. Subsequently, the sample was diluted with 20 mM Tris-HCl, pH 7.5, and the endogenous urea in the serum was removed by repeated centrifugation (see Figure 6). Finally, the serum in the column was recovered, and the volume was returned to the original volume by adding 20 mM Tris-HCl, pH 7.5. These were used as specimen samples (Sample Solutions 1 to 3).

[0047] (Test Procedure 2: Enzyme Detection Step (Enzyme-Substrate Reaction)) The sample was mixed with the buffer solution in a well plate to a total volume of 40 μL, and 10 μL of substrate solution (0.5 M arginine, pH 9.7, 10 mM MnCl2) was added using a multichannel pipettor (total volume: 50 μL). The mixture was then incubated at 37°C for 2 hours with shaking in a thermostatic bath to allow the enzyme-substrate reaction to proceed.

[0048] (Test Procedure 3: Enzyme Detection Step (Enzyme Inactivation and Urea Detection Reaction)) A urea detection reagent was prepared by mixing 16% by mass sulfuric acid with 400 mg / mL of O-phthalaldehyde, 300 mg / mL of primaquine diphosphate, 4 g / L of boric acid, and 0.3% of Brij-35. Next, 200 μL of the prepared urea detection reagent was added to the well plate using a multichannel pipettor. The mixture was then incubated at room temperature for 1 hour, and the absorbance of the sample was measured at 450 nm using a microplate reader. The absorbance was measured at 570 nm as a reference wavelength.

[0049] (Test Procedure 4: Enzyme Detection Step (Calculation of Arginase Activity)) A calibration curve (Figure 3) was created from the results of urea standard solutions of known concentrations. Based on this calibration curve, the amount of urea produced in the reaction solution of the sample was determined, and arginase activity was calculated from the amount of urea produced using the following formula. Arginase activity was expressed as 1 U, which is the reaction volume required to break down 1 μmol of arginine into ornithine and urea in 1 minute.

[0050] Arginase activity (mU / mL) = Amount of urea produced (nmol) ÷ Reaction time (min) ÷ Sample volume (mL)

[0051] For example, when 40 μL of sample is used and 60 nmol of urea is produced after a 2-hour reaction, the activity is calculated as follows: Arginase activity = 60 nmol ÷ 120 min ÷ 0.04 mL = 12.5 (nmol / min / mL, mU / mL)

[0052] The results are shown in Table 1. In Table 1, the unit of activity is mU / mL and it is shown as "mean value±standard deviation" (the same applies hereinafter).

[0053] [Table 1]

[0054] <Measurement of recombinant proteins> Using recombinant human arginase proteins (arginase 1, arginase 2), absorbance was measured according to the above test procedures 2 to 4, and their activity was calculated. Note that since the recombinant human arginase proteins were forcibly expressed and purified from heterologous cells, they do not contain urea, and therefore test procedure 1 above was not performed. The results are shown in Table 2.

[0055] [Table 2]

[0056] [Example] A test for detecting arginase 1 was carried out using the method of the present invention.

[0057] <Measurement of recombinant proteins> First, based on the results of the above comparative example, experiments were conducted using recombinant human arginase proteins (arginase 1, arginase 2) adjusted so that the activity of arginase 1 was 7.50 mU / mL and the activity of arginase 2 was 15.0 mU / mL.

[0058] (Test Procedure 1: Antibody Conjugate Preparation Step) For each sample, 2 μL of a magnetic carrier (Dynabeads Protein G, Thermo Fisher Scientific) and anti-arginase 1 antibody (Biovendor, clone 6G3) at a final concentration of 5 μg / mL were added to PBS in a microtube so that the total volume was 20 μL / sample, and the mixture was allowed to react at 4°C for 1 hour to produce an antibody conjugate.

[0059] The microtube was then placed on a magnetic stand to adsorb the antibody conjugates, and the supernatant was removed. The antibody conjugates were then washed three times with PBS to remove unreacted antibody, and resuspended in PBS containing 0.1% bovine serum albumin to a volume of 20 μL per sample.

[0060] (Test Procedure 2: Enzyme Conjugate Preparation Step and Sample Liquid Separation Step) The antibody conjugate was suspended in PBS containing protease inhibitors and 0.1% bovine serum albumin, and 80 μL of the suspension was added to a 96-well plate.

[0061] (Test Procedure 2.1) The sample was diluted with PBS containing 0.1% bovine serum albumin to prepare a sample solution. Subsequently, 20 μL of the sample solution was added to each well (total 100 μL), and the mixture was incubated overnight at 4° C. to allow the protein (arginase) to bind to the antibody of the antibody conjugate, thereby producing an enzyme conjugate.

[0062] (Test Procedure 2.2) After incubation, the enzyme conjugate was adsorbed to the bottom of the wells by placing the 96-well plate on a magnetic separator. The supernatant was then removed, and the plate was washed three times with PBS. After thoroughly removing the supernatant, 40 μL of PBS was added to each well.

[0063] (Test Procedure 3: Enzyme Detection Step) The substrate solution obtained in Test Procedure 2.2 was subjected to the above Comparative Test Procedures 3 and 4. The results are shown in Table 3.

[0064] [Table 3]

[0065] As shown in Table 3, when arginase 1 was adjusted to have the same level of activity as the comparative example, it showed the same level of absorbance. On the other hand, for arginase 2, even though the activity of the example was adjusted to be three times that of the comparative example, no absorbance was obtained. In other words, this example using anti-arginase 1 antibody showed absorbance similar to that of the conventional method, and it was revealed that arginase 1 can be detected with sufficient accuracy. Furthermore, since arginase 2 did not show absorbance (was not detected), and only arginase 1 was detected, it was revealed that the enzyme can be selectively detected.

[0066] <Serum 1-3 Measurement> For serums 1 to 3, the absorbance was measured according to the above test procedures 2 to 4, and the arginase 1 activity was calculated based on a calibration curve (Figure 4) created from the results of recombinant arginase 1 protein of known activity at multiple concentrations. The results are shown in Table 4.

[0067] [Table 4]

[0068] As shown in Table 4, arginase 1 activity was detected in all serum sample solutions, Serum 1 to Serum 3. The reason why the arginase activity is lower than that of the comparative example is thought to be because the comparative example shows the activity of both arginase 1 and arginase 2, while the example shows the activity of only arginase 1.

[0069] From the above, it has become clear that by using the enzyme detection method of the present invention, it is possible to detect a specific enzyme, and even when a sample solution contains two or more isoforms of an enzyme, it is possible to detect one specific enzyme. [Industrial Applicability]

[0070] The enzyme detection method of the present invention is industrially useful because it can detect a specific enzyme contained in a sample liquid such as a biological fluid.

Claims

1. A method for detecting a specific enzyme contained in a sample solution, comprising: an antibody conjugate preparation step of binding an antibody that binds to the specific enzyme to a carrier surface to prepare an antibody conjugate; an enzyme conjugate preparation step of contacting the antibody conjugate with the sample solution to allow a specific enzyme contained in the sample solution to bind to the antibody of the antibody conjugate, thereby preparing an enzyme conjugate; a sample liquid separating step of separating the enzyme conjugate from the sample liquid; an enzyme detection step of detecting a specific enzyme bound to the separated enzyme conjugate from the sample liquid; Equipped with The sample solution is serum containing arginase 1 and arginase 2, The specific enzyme is arginase 1, the antibody is an anti-arginase 1 antibody, In the enzyme detection step, arginine is added to the enzyme conjugate, and urea produced by the enzyme-substrate reaction is detected. A method for detecting an enzyme, comprising:

2. 2. The method for detecting an enzyme according to claim 1, wherein the carrier is a magnetic material.

3. 3. The method for detecting an enzyme according to claim 2, wherein the carrier is a particle having a particle size of 5 μm or less.

4. the enzyme conjugate preparation step is carried out in a well of a well plate; The sample liquid separating step is carried out by bringing a magnet close to or in contact with the backside of the well plate, and removing the sample liquid from the wells of the well plate while the oxygen binder is held in the wells of the well plate.

3. The method for detecting an enzyme according to claim 2.

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