WO1999049076A1

WO1999049076A1 - Method for assaying trypsin inhibitor in urine and assay kit to be used therein

Info

Publication number: WO1999049076A1
Application number: PCT/JP1999/000972
Authority: WO
Inventors: Kazuhiro Okamoto; Satoshi Fukunaga
Original assignee: Arkray, Inc.
Priority date: 1998-03-20
Filing date: 1999-02-26
Publication date: 1999-09-30

Abstract

A method for assaying UTI which comprises inactivating α1-antitrypsin (α1-AT) in a specimen, mixing a trypsin solution with the specimen, then adding a substrate to thereby initiate an enzyme reaction, and determining UTI by measuring absorbance, etc. The α1-AT may be inactivated by adding a protease other than trypsin to the specimen and reacting the protease with α1-AT to thereby form a complex, or by adding an oxidizing agent to the specimen. As the protease described above, use may be made of elastase or subtilisin. As the oxidizing agent described above, use may be made of sodium iodate, iodine, copper sulfate and iron trichloride. As the graph in the figure shows, α1-AT in the specimen is inactivated by adding subtilisin and thus the amount of UTI can be accurately determined.

Description

Description ^: Urinary trypsin inhibitor assay method and assay kit used therefor

The present invention relates to a method for measuring urinary trypsin inhibitor and a measurement kit used for the method.

Background art

Recently, urinary trypsin inhibitor (UTI) has been attracting attention as an indicator of the state of a living body, and various studies have been conducted in the field of clinical medicine. UTIs are known to appear in urine, for example, when the body is exposed to internal and external stresses such as inflammation and surgery, or when it becomes infected (see “Urine trypsin”). Clinical significance of Yuichi Inhibi ", Shiro Kuwashima and others

JAPANESE JOURNAL OF INFLAMMATION REVIEW ARTICLE, VOL9, NO. 3, MAY 1989). UTI was first identified in urine, but subsequent studies have revealed that UTI is also present in other body fluids such as blood.

Since UTI inhibits trypsin activity depending on its amount, its measurement is performed by measuring the degree of inhibition of trypsin activity. Generally, the degree of inhibition of trypsin activity by UTI in a sample is measured by mixing a sample, a buffer solution, and an enzyme solution containing trypsin, adding a substrate solution thereto, and measuring the enzyme reaction. I do. Usually, in this measurement, calcium which is a trypsin activator is added to the buffer.

Disclosure of the invention

As mentioned above, UTI is an important indicator of the state of a living body. However, there is a problem that the disease specificity is low.

For example, the present inventors examined the correlation between the amount of UTI in urine and inflammation using C-reactive protein (CRP), which is a representative indicator of inflammation. Nevertheless, it was found that the measured value of UTI may be high. Further investigation of urine samples with high UTI measurements showed that in most of these samples albumin, an indicator of nephropathy, was positive. This means that UTI is increased in urine in the case of nephropathy as well as inflammation, which makes UTI a specific indicator of inflammation or a specific indicator of nephropathy. Can not be used.

In addition, according to the research results of the present inventors, it was also found that the conventional method for measuring UTI cannot accurately measure the amount of UTI present in urine.

Therefore, an object of the present invention is to provide a method for measuring UTI, which can enhance the clinical usefulness of UTI as an index indicating the state of a living body and accurately measure the amount of UTI, and a measurement kit used therefor. It is to be. In order to achieve the above object, a method for measuring UTI according to the present invention is a method for measuring the amount of urinary trypsin inhibitor in a sample, comprising the following steps (a) to (d): It is characterized by having.

(a) a step of inactivating a 1-antitrypsin (hereinafter referred to as “HI-AT”) in a sample;

(b) a step of mixing the sample and trypsin after the inactivation.

(c) a step of measuring the inhibition of the trypsin activity.

(d) determining the amount of urinary trypsin inhibitor in the sample from the inhibition.

The present inventors have further examined the above-mentioned findings and continued their research.As a result, in the case of nephropathy, the measured value of UTI by the conventional measurement method was increased. They found that the cause was the leakage of 1-AT from the blood into the urine. In nephropathy, prerenal proteins such as albumin appear in the urine because of a decrease in the barrier function of the glomerular basement membrane. Also, α AT-AT has a trypsin activity inhibitory function like UTI, is contained in large amounts in blood (occupies about 90% of trypsin inhibitor in blood), and has a molecular weight ゃ isoelectric point. Similar to albumin. Therefore, in the case of nephropathy, along with albumin, α1-AT in the blood should also leak into the urine. Therefore, in the conventional measurement method, in addition to the inhibition of UTI trypsin activity, the inhibition of trypsin activity of α1-AT leaked from blood into urine is measured. In addition, we found that in nephropathy, the UTI concentration in urine hardly increased. That is, if the effects of αΐ-AT are eliminated, not only can UTI be measured accurately, but also UTI in urine can be used as an index specific to inflammation. The present inventors have arrived at the measurement method of the present invention based on such research results and ideas.

In the measurement method of the present invention, as means for inactivating the above-mentioned AT, there are, for example, the following two methods.

One method (method A) is to inactivate α1-AT by adding a protease other than trypsin to the sample and reacting the protease with α11-AT to form a complex. How to

a 1—AT has only one protease inhibitory active site. On the other hand, UTI has two protease inhibitory sites, and the inhibitory protease is different at each inhibitory site. Therefore, when a protease other than trypsin is added to a sample to form a complex with the a1-AT in the sample and inactivate a1-AT, the UT coexisting in the sample is obtained. I also reacts with the protease to form a complex, There remains a trypsin activity inhibition site. Even if trypsin enzyme activity is measured in this state, UTI can inhibit trypsin activity, so that UTI can be measured without the influence of α1-AT. As the protease other than the trypsin, a protease that does not decompose a trypsin substrate is preferable, and a protease that does not inhibit trypsin is preferable.

Since α AT-AT is a serine protease inhibitor, the protease other than trypsin is preferably a serine protease, but the present invention is not limited to this. This is because αΐ-AT also reacts with cysteine protease and the like. Preferred specific examples of the protease other than the trypsin include eras yuichi, subtilisin, chymotrypsin, cathepsin, plasmin, thrombin, kallikrein, and perokinase. Among these proteases, more preferred are Erasase and Subtilisin, and particularly preferred is Subtilisin in terms of cost. As the protease other than trypsin, one kind may be used, or two or more kinds may be used in combination.

In the method A, the addition ratio of Protea Ichize other than the trypsin is preferably from ^{0. 3 3 X 1 0- 9 ~} 1 6. 6 X 1 0- 9 mo 1 to the sample lml, particularly preferably 3 . 3 X 1 0- ⁹ ~: in the range of ^{L 0. 0 X 1 0- 9 mo} l. When the content is less than ^{0. 3 3 X 1 0- 9 mo} l, it may not be completely inactivated the alpha 1-AT in the sample. In addition, in this method A, the addition amount of the protease other than trypsin is represented by mo 1 for the following reason. Usually, the amount of enzyme added is expressed in units based on its activity. However, as described above, in Method A, the number of the enzymes is a problem because the activity of the enzyme is not a problem and a complex of α1-AT and a protease other than trypsin is formed. Therefore, the amount of protease other than trypsin is preferably represented by “mo 1” representing the number of molecules. Good. It should be noted that if the molecular weight of the protease is known, it can be expressed in gram units (g). In practice, when this method A is carried out, it is preferable to use it in terms of gram units. In this case, the addition amount is preferably in the range of 100 to 500 g, particularly preferably in the range of 100 to 300 μg, per 1 ml of the sample.

When the subtilisin or elastase is used as a protease other than the trypsin, the addition ratio is preferably in the same range as the above-mentioned ratio. The same applies to the ratio of the amount added in grams. Next, another method (method B) is a method in which α1-AT in the sample is inactivated by adding an oxidizing agent to the sample. In α-AT, the reactive site with trypsin is the 358th methionine residue. Therefore, when this methionine residue is oxidized to methionine sulfoxide by an oxidizing agent, α 1 -AT is inactivated. In this method B, the oxidizing agent is preferably permanganic acid, permanganate, oxyacid, oxyacid salt, metal salts, oxides and peroxides, and particularly preferably sodium iodate, iodine, Copper sulfate and iron trichloride. One type of the oxidizing agent may be used, but it is preferable to use two or more types in combination since a sufficient effect can be obtained.

In the method B, the addition ratio of the oxidizing agent is preferably in the range of 0.05 to 0.5 mmo 1 with respect to the sample lml, and particularly preferably, in the range of 0.01 to 0.1 mmol. Range. If it is less than 0.05 mmol, α 1 -AT in the sample may not be completely inactivated. In the measurement method of the present invention, the trypsin is not particularly limited, and for example, trypsin derived from bovine gut, trypsin derived from grapevine, and the like can be used.

In the measurement method of the present invention, the substrate used is not particularly limited. Examples include natural proteins such as casein and albumin, as well as synthetic substrates. Examples of the synthetic substrate include N-benzoyl-arginine-p-nitroanilide (Β Α Ρ Ν Α), Να-benzoyl-lysine-1 ρ-nitro-2-lide, and t-butoxycarboyl arginine. 1-p-Nitroanilide, t-butoxycarboniluridine_p_Nitroanilide and the like can be used. These synthetic substrates produce chromogenic substances when decomposed by trypsin. Preferred among these substrates is BAPNA.

The buffer used in the measurement method of the present invention is not particularly limited, and examples thereof include a triethanolamine hydrochloride buffer, a Tris-HCl buffer, a phosphate buffer, and a good buffer.

Since the measurement method of the present invention enables UTI in urine to be used as an indicator of inflammation, urine is preferable as the sample to be measured. However, since UTI is also present in body fluids other than urine, the target sample for the measurement method of the present invention is not limited to urine, and may include blood, tracheal secretions, and the like.

Next, the UTI measurement kit of the present invention includes a measurement kit A and a measurement kit B as shown below, corresponding to the method A or the method B, respectively.

The measurement kit A includes trypsin, a substrate, and a protease other than trypsin. By using this measurement kit, the measurement method of the present invention by the method A can be easily and quickly performed.

In the measurement kit A, the protease other than the trypsin is preferably a serine protease, as in the method A, but is not limited thereto. Preferred specific examples of the protease other than the trypsin include Erasase, subtilisin, chymotrypsin, cathepsin, plasmin, thrombin, kallikrein and perokinase. This Among them, particularly preferred proteases are Erasase, subtilisin. As the protease other than trypsin, one kind may be used, or two or more kinds may be used in combination.

The measurement kit A includes a trypsin solution (R 1), a substrate solution (R 2), and a protease solution other than trypsin (R 3), and further has a buffer solution (R 4). May be. The preparation of these reagents (R1, R2, R3 and R4) can be carried out by the method described in the description of the measurement method of the present invention according to Method A. It is as described in the measurement method section.

In the measurement kit A, the reagents R1, R2, R3 and R4 may be independent of each other, or may be a combination thereof. Specifically, there are combinations of two-liquid systems and three-liquid systems as described below.

(1) Three-component system: a mixture of R 1 + R 2 + R 3 and R 4

(2) Two-component system: mixed solution of R 1 + R 2, R 3 and R 4

Next, the measurement kit B includes trypsin, a substrate, and an oxidizing agent. By using this measurement kit B, the measurement method of the present invention by the method B can be easily and quickly performed.

In the measurement kit B, the oxidizing agent is preferably permanganic acid, permanganate, oxyacid, oxyacid salt, metal salt, oxide, peroxide, or the like, as in Method B. Preferred are sodium iodate, iodine, copper sulfate and iron trichloride. Although one kind of the oxidizing agent may be used, it is preferable to use two or more kinds in combination.

The measurement kit B includes a trypsin solution (R 1), a substrate solution (R 2), and an oxidizing agent solution (R 5), and may further have a buffer solution (R 4) in addition thereto. . The preparation of these reagents (R1, R2, R5 and R4) can be carried out by the method described in the description of the measurement method of the present invention by Method B. The compositions and their ratios are as described in the section on the measuring method.

In the measurement kit B, the reagents R1, R2, R5 and R4 may be independent of each other, or may be a combination thereof. Specifically, there are three liquid combinations as shown below.

Three-part system: R 1 + R 5 + R 2 and R 4 mixture

In the measurement kits A and B of the present invention, the sample to be measured is preferably urine as in the above-described measurement method of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the results of measuring UTI by substituting α1-AT with subtilisin in one embodiment of the measuring method of the present invention according to method A.

FIG. 2 is a graph showing the results of measuring UTI in another example of the measurement method of the present invention according to Method A, by inactivating α11-AT using Erasase.

FIG. 3 is a graph showing the results of measuring UTI by changing the inactivation time of α1-AT with subtilisin in still another example of the measurement method of the present invention according to method A. .

FIG. 4 is a graph showing the results obtained by inactivating α1-AT using subtilisin and measuring UTI in still another example of the measurement method of the present invention according to method A.

FIG. 5 is a graph showing the results of measuring UTI by a conventional method in a comparative example.

FIG. 6 is a graph showing the relationship between protein concentration in urine and α1-AT concentration in Reference Example.

FIG. 7 shows the measurement method of the present invention using Method A and Method B together. FIG. 9 is a graph showing the results of measuring UTI by inactivating α1_AT using subtilisin and an oxidizing agent in another example.

BEST MODE FOR CARRYING OUT THE INVENTION

The measurement method of the present invention by the method A can be carried out using, for example, an enzyme solution containing trypsin (trypsin solution), a substrate solution, and a protease other than trypsin.

The protease other than trypsin may be used as it is, but is preferably used as a solution containing the protease from the viewpoint of easy operation. The solvent used for preparing the protease solution is usually a buffer, and the buffer is particularly preferable.

The buffer is prepared by a conventional method. The pH of the buffer is preferably in the range of pH 7 to 8, which is the optimal pH of trypsin. As a trypsin activator, calcium is usually added to the buffer. The compounding ratio is usually in the range of 0.01 to 0.5% by weight with respect to the buffer. The concentration of the trypsin solution is usually in the range of 13,000 to 10,000,000 UZ liters, preferably 130,000, relative to the total trypsin solution. It is in the range of 0 to 2, 000, 000 UZ little. The activity (U) is a value obtained when Nα-benzoyl_L-arginineethyl ester (ΒΑΕ) is used as a substrate in the measurement of trypsin activity. When the concentration of the trypsin solution is expressed in gram units, the concentration is appropriately determined depending on the specific activity and the like, but is usually in the range of 1 to 50 OmgZ liter with respect to the entire trypsin solution, and is preferably. Ranges from 10 to 10 Omg / litre. In addition, this trypsin solution may be adjusted to a pH range of 2.0 to 3.5 with, for example, hydrochloric acid, a buffer solution or the like in order to prevent autolysis of trypsin.

The concentration of the substrate solution is usually in the range of 1 to 10 gZ liter. The solvent used for preparing the substrate solution is usually water (purified water or the like), but may be the buffer. Further, since the synthetic substrate is hardly soluble in an aqueous solution or the like, it is preferable that the synthetic substrate be dissolved in an organic solvent such as dimethyl sulfoxide (DMSO) and then diluted with water or the like.

Next, the measurement method of the present invention according to Method A will be described below, taking urine as a sample as an example.

That is, first, a protease other than trypsin is mixed with a urine sample and inactivated. The protease may be directly added to the urine sample. However, in view of quickness and simplicity of operation, a buffer solution (protease solution) containing the protease is prepared as described above. And a urine sample are preferably mixed. The mixing ratio (volume ratio) varies depending on the concentration of the protease solution. However, when the concentration is 0.001 M, usually, the urine sample: the protease solution = 1: 5 to 1: 2 Set to 0 range. Thus, α1-AT in a urine sample is immediately inactivated at room temperature by the addition of the protease, but it is necessary to incubate at 25 to 37 ° C for 1 to 800 seconds. Is preferred.

Subsequently, a trypsin solution is added to the treated urine sample. This mixing ratio (volume ratio) is usually set in the range of the treated urine sample: the trypsin solution = 1: 1 to 5: 1. Typically, this mixture is incubated at 25-37 ° C for 1-5 minutes. Then, the substrate solution is added thereto, and the trypsin and the substrate are reacted. The mixing ratio of the substrate solution is usually in the range of 5 to 30% by volume based on the total reaction solution. The reaction conditions are usually at 25 to 37 ° C for 1 to 20 minutes. The pH of the reaction solution at this time is preferably in the range of pH 7 to 8, which is the optimum pH for trypsin. Then, the enzyme reaction of trypsin is detected by a predetermined method, and the enzyme activity is measured. In this reaction, α ΐ— A in the urine sample Since T is inactivated in advance, the enzymatic reaction of trypsin is inhibited according to the amount of UTI, and the amount of UTI can be measured accurately. As a method for detecting the enzymatic reaction of trypsin, for example, when a substrate that develops a color by an enzymatic reaction such as Β Β Ρ Ν 用い is used, a method of measuring the degree of the color development with a spectrophotometer or the like can be mentioned. . Alternatively, the enzyme activity can be measured by measuring the concentration of the enzyme reaction product.

In the measurement method of the present invention according to the method 2, it is sufficient that the ability of the sample to inhibit the trypsin activity of α11-AT before contacting the sample with the trypsin solution. For this reason, the protease treatment of the sample is not limited to the above method, and the protease solution, the sample, and the substrate solution can be mixed in any order. Then, a trypsin solution is added to the mixture to start a trypsin enzymatic reaction.

Next, the measurement method of the present invention by the method B can be carried out using, for example, an enzyme solution containing trypsin (trypsin solution), a substrate solution, and an oxidizing agent. Except for the oxidizing agent described below, unless otherwise specified, the same method as the measuring method according to the method A can be used, and it can be prepared in the same manner.

The oxidizing agent may be used as it is, but is preferably used as an oxidizing agent solution from the viewpoint of easy operation. The solvent used for preparing the oxidizing agent solution is preferably water in order to maintain its oxidizing power. Further, the oxidizing agent solution may contain other components such as ethylenediaminetetraacetic acid (EDTA). For example, the addition of EDTA forms a complex with the metal, thereby preventing the formation of a precipitate.

Next, the measurement method of the present invention according to the method B will be described below using urine as an example.

That is, first, an oxidizing agent is mixed with a urine sample and inactivated. Said The oxidizing agent may be added directly to the urine sample, but from the viewpoint of quickness and simplicity of operation, prepare the oxidizing agent solution as described above and mix it with the urine sample. Is preferred. The mixing ratio (volume ratio) varies depending on the concentration of the oxidizing agent solution. However, when the concentration is 0.1 mmo1 / 1, usually, the urine sample: the oxidizing agent solution = 1: 5 to 1 : Set to the range of 20. Thus, the addition of the oxidizing agent immediately inactivates α1 AT in the urine sample at room temperature, but incubates at 25 to 37 ° C for 10 to 600 seconds. Is preferred.

Subsequently, the treated urine sample, buffer solution and trypsin solution are mixed. The mixing ratio (volume ratio) is usually set in the range of the treated urine sample: the buffer solution: the trypsin solution = 1: 1 to 5: 1. After the mixing, a trypsin enzymatic reaction is carried out in the same manner as in the measurement method according to the above-mentioned method A, and this is detected to measure the enzymatic activity. In this reaction, α 1 -AT in the urine sample is previously inactivated by an oxidizing agent, so that the enzymatic reaction of trypsin is inhibited in accordance with the amount of UTI, so that an accurate amount of UTI can be measured.

In the measurement method of the present invention according to the method B, it is sufficient that the ability of the sample to inhibit the α1-AT trypsin activity is inactivated by the oxidizing agent before the sample comes into contact with the trypsin solution. For this reason, the treatment of the sample with the oxidizing agent is not limited to the above method, and the oxidizing agent solution, the sample, and the substrate solution can be mixed in any order. Then, a trypsin solution is added to the mixture to start a trypsin enzymatic reaction. However, the addition of the oxidizing agent is particularly preferably performed as a pretreatment of the sample, as described above, because of the simplicity of the operation.

Further, in the measurement method of the present invention, as described above, not only the method of inactivating α1-AT but also the method of 1-AT may be inactivated by a combination of the method and method B.

In this case, for example, the sample may be sequentially treated with a protease other than trypsin and an oxidizing agent, or the sample may be treated by simultaneously adding the two to the sample. When using Method A and Method B together,

: On the other hand, 0.33 X 10 16.6 X 10 mo

It is preferable to add the oxidizing agent in the range of 1 to 0.05 and 0.5 mmo1.

(Example)

Next, examples will be described together with comparative examples.

(Example A-1)

In Example A-1, subtilisin was used as a protease other than trypsin, and it was added to urine samples at various concentrations to inactivate α1-AT, and the degree of trypsin activity inhibition was determined as the amount of UTI. It is an example. The buffer, trypsin solution and substrate solution in Example A-1 were prepared by the methods described above. The composition and operation method of UTI measurement are shown below.

(Buffer: pH 8.1)

Triethanolamine hydrochloride 0.5 m o 1 Z liter

C a C 1 2 H O 150 mg Z liter

twenty two

Triton X-405 (manufactured by Nacalai Tesque) 2.0 gZ little (trypsin solution: pH 3.0)

Trypsin (14, 900 U / mg, Sigma) 50 mg / liter HC 11 mm o 1 Z liter

(Substrate solution)

It was prepared by dissolving L-BA PNA (manufactured by Peptide Research Laboratories) at a rate of 4 gZ liter in water heated to about 70 ° C. (Subtilisin)

Subtilisin derived from Bacillus subtilis (Boehringer Mannheim)

(1-AT)

Human serum-derived AT (ART)

(Sample 1, 2, 3)

Sample 1 is a sample (αΐ-AT-added urine) obtained by adding α 1 -AT to the urine of a healthy subject to a concentration of 14 mgZ 100 m 1. Sample 2 is a sample (serum-added urine) obtained by mixing serum of a urine protein-positive patient and urine of a healthy subject so that the volume ratio becomes serum: urine = 1: 7. The urine protein-positive patient is a patient whose serum CRP and serum amyloid A (SAA) are negative and whose urinary UTI is positive (by the conventional method) (hereinafter, referred to as “UTI”). the same). Sample 3 is healthy subject urine.

(Method of operation)

To the samples 1, 2, and 3, subtilisin was added at a predetermined concentration (O mgZ lOO ml, 0.4 mg / 100 m 0.8 mgZl 00 ml, 1.2 mgZl 00 ml, 1.6 mgZl 0 ml, 2 mg / lOOm 2.4 mg / 100 mU 3.2 mgZl 100 ml, 4 mg / 100 m1). Then, the urine sample 20/1, the buffer solution 201, and the trypsin solution 1001 after the subtilisin treatment were mixed and kept at 37 ° C. for 5 minutes. The substrate solution 1001 was added to the mixture to start the enzymatic reaction. Then, the reaction solution was kept at 37 ° C., and the change in absorbance (405 nm) of the reaction solution for 2 minutes was measured with a Hitachi 770 type automatic analyzer (manufactured by Hitachi, Ltd.). The relative absorbance (Δ〇.D.) Was determined. From this relative absorbance, the degree of trypsin activity inhibition was determined as the UTI amount using a calibration curve prepared in advance. Note that the calibration curve is a standard solution prepared by dissolving a UTI standard substance (Utinin, manufactured by Mech) in physiological saline. And the measurement was made in the same manner as described above. This UTI measurement was performed three times for each sample. The results are shown in the graph of FIG. '

As shown in FIG. 1, in the αΐ-AT-added urine (sample 1), as the amount of subtilisin added increased, the amount of UTI decreased, and subtilisin was added to the sample at 0.4 mg / 100m2. If (to the sample ^{lml 1 3 X 1 0- 9 mo} 1 0.) l is added to a above, UT I amount is constant, almost the same amount as UT I of healthy subjects urine (sample 3) Was. This indicates that α1-AT added to the urine of healthy subjects was completely inactivated. As for the above serum-supplemented urine Similarly, the addition of subtilisin so that the above ^{(0. 5 3 X 1 0- 9} mo 1 to the sample lml) 1. 6mg / 1 00m l to the sample It can be seen that α1 AT derived from the serum was inactivated. The reason that the UTI amount in the serum-added urine to which a sufficient amount of subtilisin was added was higher than the UTI amount in the healthy subject urine was considered to be due to the UTI present in the serum added. Can be

(Example A-2)

Example A-2 is an example in which elastase was added to a sample at various concentrations to inactivate α 1 -AT and the degree of trypsin activity inhibition was determined as the UTI amount. Unless otherwise indicated, the measurement operation of UTI is the same as in Example A-1.

(Eras Yuichi)

Erasase from human leukocytes (ART)

(Sample)

Urine from a urine protein-positive patient (urine protein concentration: 54 lmg / 100 ml) was used.

(Method of operation)

To the sample, add Eras Juice at a predetermined concentration (Omg / 100m1,2 mgZl 00 ml, 4 mg / 100 m 6 mg / 100 m 8 mg Zl 00 ml, 10 mg / 100 m 1). Thereafter, the UTI amount was determined in the same manner as in Example A-1. This UTI measurement was performed twice. The results are shown in the graph of FIG.

As shown in FIG. 2, the UTI amount decreased in proportion to the Elasic concentration. This sample is urine from a patient with non-inflamed nephropathy. Therefore, UTI showed a high value before the addition of Erasase, and the UTI value decreased as the concentration of Erasase was increased. It can be said that 1-AT is present, which inhibits trypsin activity. The a1-AT is inactivated by adding Erasase. From the results of Example A-2, it can be said that according to the measurement method of the present invention, UTI in urine can be used as an index specific to inflammation.

(Example A-3)

In Example A-3, the relationship between the treatment time of a sample with subtilisin and the amount of UTI was examined. Unless otherwise indicated, the used sample and the operation of measuring UTI were the same as those in Example A-1.

(Sample)

Sample 2 (urine with serum added) used in Example A-1 was used.

(Subtilisin solution)

The buffer was prepared by adding the same subtilisin as in Example A-1 to a concentration of 2 Omg / 100 ml.

(Method of operation)

The subtilisin solution 200 / X1 and the sample 20n1 were mixed and incubated for various times (25 seconds, 50 seconds, 425 seconds, 800 seconds). The temperature during this incubation is 37 ° C. Trip this solution The thin solution 1001 was added, and thereafter the UTI amount was measured in the same manner as in Example A-1. The absorbance was measured using a Roche COBAS MIRA automatic analyzer (manufactured by Roche Japan). The UTI was measured five times. The results are shown in the graph of FIG.

As shown in Fig. 3, there was no significant difference in the amount of UTI even when the inactivation time of 1-AT in the sample was changed by subtilisin. Α 1 -AT was inactivated.

(Example A-4, Comparative Example A-1)

In Example A-4 and Comparative Example A-1, the correlation between the UTI amount and inflammation was examined. Unless otherwise indicated, the measurement operation of UTI is the same as in Example A-1.

(Sample)

Patient urine (sample number: n = 77) was used as a sample for Example A-4 and Comparative Example A-1. Serum (sample number: n = 77) of the same patient was used as a control sample. used.

In Example A-4, the sample 201 was mixed with the subtilisin solution 2001 used in Example A-3, and then the trypsin solution 100a1 was added thereto. The UTI amount was measured in the same manner as in Example A-1. As a control, the serum (n = 77) was measured for CRP. This measurement was performed using a CRP measurement kit (manufactured by Den Rikiseiken Co., Ltd.) according to the method of use.

The results of Example A_4 and the control are shown in the graph of FIG. This graph is a graph showing the correlation between the amount of UTI in the urine sample after the subtilisin treatment and the CRP concentration in the serum sample. The equation in the figure is an equation representing the correlation, and R is the phase. The relation numbers are shown.

On the other hand, Comparative Example A-1 was treated with a protease other than trypsin. There is no conventional UTI measurement method. In Comparative Example A-1, the sample 20 was mixed with the buffer solution 200 ^ 1 and the trypsin solution 100 ^ 1, and the UTI amount was measured in the same manner as in Example A-1. This result and the result of the control are shown in the graph of FIG. This graph is a graph showing the correlation between the UTI amount in the urine sample not treated with the protease and the CRP concentration in the serum sample.The equation in the figure is an equation representing the correlation, and R is The correlation coefficient is shown.

As shown in the graphs of FIGS. 4 and 5, the correlation between the UTI amount and the CRP concentration in Example A-4 (R = 0.39) was the same as that in Comparative Example A-1 (R = 0.39). 24) was higher. From these results, it can be said that the measurement method of the present invention can enhance the specificity of UTI as an index for inflammation.

(Reference example)

This reference example is an example in which the relationship between urine protein concentration and α1-AT concentration in urine was examined.

(Sample)

Patient urine (sample size: n = 98)

The protein amount of each patient urine (n = 98) was measured using a protein measurement kit (manufactured by Wako Pure Chemical Industries, Ltd.) according to the method of use (pyrogallol red method). For this measurement, a Hitachi 770-type automatic analyzer (manufactured by Hitachi, Ltd.) was used. On the other hand, in each patient's urine (n = 98), the αΐ-AT concentration was measured by the ELISA method. These results are shown in the graph of FIG. This graph is a graph showing the correlation between the urine protein concentration and the above-mentioned urinary AT. The equation in the figure is an equation representing the correlation, and R is a correlation coefficient.

As shown in Fig. 6, the sample with high urine protein concentration is The T concentration also tends to be high, and the two have a high correlation. From this, it can be seen that in nephropathy in which urine protein concentration is increased, α1-AT having the same ability to inhibit trypsin activity as UTI is leaked into urine. In other words, in the urine of patients with nephropathy, since α1-AT leaked into the urine inhibits triscine activity, it can be said that the amount of UTI in the urine is apparently increased by the conventional method.

(Example B-1)

Example B-1 is an example in which various oxidizing agents were used, added to a urine sample to inactivate 11-AT, and the degree of trypsin activity inhibition was determined as the UTI amount. The same applies to the following Examples B-1 to B_5. The buffer, tribcine solution, substrate solution and oxidizing agent in Example B-1 were prepared by the methods described above. The composition and operation method of UTI measurement are shown below.

(Buffer: pH 8.3)

Triethanolamine hydrochloride 0.5 mol / liter

C a C 1 2 H O 150 mg Z liter

twenty two

Triton X—405 (manufactured by Nacalai Tesque) 2.0 g / liter

(Trypsin solution: pH 3.0)

Trypsin (14, 900 U / mg, Sigma) 50 mg Z liter HC 1 1 mm o 1 Z liter

(Substrate solution)

It was prepared by dissolving L-BAPNA (manufactured by Peptide Research Laboratories) at a rate of 4 gZ liter in water heated to about 70 ° C.

(Oxidizing agent solution)

Prepare 0.05 MNa IO (manufactured by Nacalai Tesque, Inc., the same applies hereinafter) and 0.05 M I 2 (manufactured by Wako Pure Chemical Industries, Ltd., the same applies hereinafter). Mixing was performed so that the product ratio was 1: 1. Water was used as a solvent for the preparation. In the preparation of each oxidizing agent solution described below, water was also used as a solvent.

(Sample)

Serum-containing urine was prepared by mixing normal human urine and serum of a urine protein-positive patient in a volume ratio of 7: 1. As a control sample, a mixture of physiological saline and the urine of a healthy subject so as to have a volume ratio of 1: 1 (diluted normal subject urine) was used.

(Method of operation)

First, the sample and the oxidant solution were mixed so that the volume ratio was 1: 1. Then, the urine sample 20 ^ 1, the buffer solution 20111 and the trypsin solution 1001 after the treatment with the oxidizing agent were mixed and kept at 37 ° C for 5 minutes. The substrate solution (100 × 1) was added to the mixture to start an enzyme reaction. Then, the reaction solution was kept at 37 ° C., and the change in absorbance (405 nm) of the reaction solution for 2 minutes was measured with a Hitachi 7770 automatic analyzer (manufactured by Hitachi, Ltd.). The relative absorbance (△ 〇.D.) Was determined. From this relative absorbance, the degree of trypsin activity inhibition was determined as the UTI amount using a calibration curve prepared in advance. The calibration curve was prepared by using a standard solution prepared by dissolving a UTI standard substance (Utinin, manufactured by Mekt) in physiological saline in the same manner as described above. As a control example, the diluted healthy urine 201, buffer solution 201, and trypsin solution 1001 were mixed, and the UTI amount was measured in the same manner as in Example B-1. . The results are shown in Table 1 below.

(Example B-2)

0.1 M N a I 〇 ₃ was prepared and mixed with the above-mentioned 0.05 MI so that the volume ratio was 1: 1. The UTI measurement operation was performed in the same manner as in Example B-1. The results are shown in Table 1 below.

(Example B-3)

0.05 M FeCl 3 · 6H 2 O (manufactured by Wako Pure Chemical Industries, Ltd .; same hereafter) was prepared. Same as Example B-1 except that this was used as an oxidizing agent solution.

The operation of UTI measurement was performed. The results are shown in Table 1 below.

(Example B-4)

In Example B-4, the degree of inhibition of trypsin activity when EDTA was added as another component to the oxidizing agent solution was determined as the UTI amount.

That is, 0.05M CuSO · 5HO (manufactured by Nacalai Tesque) and 0.05M EDTA—2Na · 2HO (manufactured by Nacalai Tesque, hereinafter the same) were prepared, and the volume ratio of these was adjusted. The mixture mixed at a ratio of 1: 1 was used as an oxidizing agent solution. This solution and the same sample as in Example B-1 were mixed, and the UTI amount was determined in the same manner as in Example B-1. The results are shown in Table 1 below.

(Example B-5)

The above-mentioned 0.05 M FeCl 3 '61 "120 and the above-mentioned 0.05 M EDTA

A mixture of 2Na · 2HO with a volume ratio of 1: 1

Two

An oxidizing agent solution was used. This solution and the same sample as in Example B-1 were mixed, and thereafter, the UTI amount was determined in the same manner as in Example B-1. The results are shown in Table 1 below.

(Comparative Example B-1)

Comparative Example B-1 is an example of a conventional UTI measurement method without treatment with an oxidizing agent. In Comparative Example B-1, first, the same sample (serum added urine) and physiological saline as in Example B-1 were mixed in a volume ratio of 1: 1. Then, the mixed solution 20 / X1, the buffer solution 201, and the trypsin solution 100 1 were mixed, and the UTI amount was measured in the same manner as in Example B-1. The results are shown in Table 1 below.

(table 1)

UT I amount a 1 -AT

(I UZ liter) Deactivation degree

Control 1 43

Example B 1 54

Example B 2 8 5

Example B 3 3 94 〇

Example B 4 3 2 5 〇

Example B 5 3 26 〇

Comparative Example B 1 18 26 X As shown in Table 1 above, the values of the UTI amount in Examples B-1 and B-2 are the values of the UTI amount in Comparative Example B-1 (conventional method). The value was significantly reduced as compared with, and a value almost similar to the value of the UTI amount in the urine of the diluted healthy control as a control was obtained. In addition, it was found that when two types of oxidizing agents were used in combination, the effect of inactivating a 1 -AT was sufficiently obtained. Also, the value of the UTI amount in Example B-3 was smaller than the value of the UTI amount in Comparative Example B-1 (conventional method), and the UTI amount in the diluted normal healthy urine as a control was also reduced. A value close to the value was obtained. Also, the values of UTI in Examples B-4 and B-5 to which EDTA was added were also smaller than the values of UTI in Comparative Example B-1 (conventional method). A value close to the value of UTI in human urine was obtained.

(Example C-11 and Comparative Example C-11)

Example C-11 is an example in which both a protease other than trypsin and an oxidizing agent were added to the sample, and the degree of trypsin inhibition was determined as the UTI amount. is there. In order to evaluate whether αΐ-AT was sufficiently inactivated when the protease and the oxidizing agent were used in combination, the amount of UTI when the sample was treated only with a protease other than trypsin was also determined. I asked.

(Buffer solution)

The same buffer as in Example A-1 was used.

(Subtilisin solution)

The same subtilisin as in Example A-1 was added to the buffer solution to a concentration of 0.02 mgZm1.

(Oxidizing agent solution)

0.2 M Cu S O 4 5 5 2Ο and 0.2 0 EDTA- 2 Na-

2 H ₂ 0 was prepared and mixed so that the volume ratio was 1: 1.

(Sample)

The serum of the patient was mixed with the urine of a healthy subject to a predetermined concentration (0% by volume, 20% by volume, 40% by volume, 60% by volume, 80% by volume, and 100% by volume). A sample was used.

(Method of operation)

The sample and the oxidant solution are mixed in a volume ratio of 2: 1 (0.05 mmo 1 of the oxidant per 1 m 1 of sample), and the mixed solution 7500 / and mixing the subtilisin solution 2 50 1 1 (subtilisin 1 3. to the sample ^{lm l 3 X 1 0- 9 mo} l). Thereafter, in the same manner as in Example A-1, the trypsin solution and the substrate solution were added, and the UTI amount was measured. The UTI amount was measured three times, and the average was determined.

On the other hand, the sample and physiological saline were mixed in a volume ratio of 2: 1 and the subtilisin solution 2501 (sample Fee 1 m 1 were mixed subtilisin ^{1 3. 3 X 1 0- 9 mo} 1) relative to measure the UT I amount in the same manner as described above.

Comparative Example C-11 is an example of a conventional UTI measurement method in which a sample is not treated with any protease or oxidizing agent other than trypsin. In this comparative example, the sample was mixed with physiological saline, and subtilisin was added. The UTI amount was measured in the same manner as described above except that the buffer was added instead of the solution. These results are shown in the graph of FIG. In the figure, ◊ indicates the amount of UTI when the sample was treated with subtilisin alone, the mouth indicates the amount of UTI when the sample was treated with subtilisin and an oxidizing agent, and 〇 indicates Comparative Example C where the sample was not treated. The UTI amount of 11 is shown.

As shown in the figure, Comparative Example C-1 showed a high UTI amount due to the influence of αΐ-AT, which exceeded the measurement range. On the other hand, when the sample was treated with both subtilisin and the oxidizing agent, and when the sample was treated with only subtilisin, the UTI amount was sufficiently reduced as compared with Comparative Example C-11, and both UTI amounts were reduced. Also showed almost the same values. From this, it was found that even when a protease other than trypsin and an oxidizing agent were used in combination, α1-AT in the sample could be sufficiently inactivated. It can be assumed that the increase in UTI value with increasing serum concentration (% by volume) in the sample is due to UTI contained in serum.

Industrial applicability

As described above, the method for measuring UTI according to the present invention is a method for measuring UTI in the sample by measuring trypsin activity in the presence of the sample, wherein the contact between the trypsin and the sample is measured. Prior to inactivating α AT-AT in a sample, the amount of UTI can be accurately measured and the usefulness of UTI as a clinical index can be enhanced. For example, if UTI in urine is measured by the measurement method of the present invention, UTI can be accurately measured even in urine of a nephropathy patient. Yes, this means that UTI can be used as an indicator of inflammation,

Claims

The scope of the claims

1. A method for measuring the amount of urinary trypsin inhibitor in a sample, the method comprising the following steps (a) to (d).

(a) a step of inactivating a 1 -antitrypsin in a sample;

(b) a step of mixing the sample and trypsin after the inactivation.

(c) a step of measuring the inhibition of the trypsin activity.

2. The means for inactivating a1-antitrypsin is a means for adding a protease other than trypsin to a sample and reacting the protease with a1-antitrypsin to form a complex. The measurement method described in 1.

3. The method according to claim 2, wherein the protease other than trypsin is a serine protease.

4. The method according to claim 2, wherein the protease other than trypsin is at least one protease selected from the group consisting of elasase, subtilisin, chymotrypsin, cathepsin, plasmin, thrombin, kallikrein, and perokinase.

5. Protea Ichize other than trypsin, claim 2-4 is added in a range of ^{0. 3 3 X 1 0- 9 ~1} 6. 6 X 1 0- 9 mo 1 to the sample 1 m 1 The measurement method according to claim 1.

6. Protea Ichize other than trypsin, or the preceding claims 2-4 is added in a range of 3. 3 X 1 0 one ^{9 ~ 1 0. 0 X 1 0-} 9 mo 1 to the sample 1 m 1 Measurement method described in 1.

7. The means to inactivate a 1 -antitrypsin in the sample 2. The method according to claim 1, which is a means for adding an agent.

8. The oxidizing agent according to claim 7, wherein the oxidizing agent is at least one selected from the group consisting of permanganic acid, permanganate, oxyacid, oxyacid salt, metal salt, oxide, and peroxide. Measurement method.

9. The method according to claim 7, wherein the oxidizing agent is at least one oxidizing agent selected from the group consisting of sodium iodate, iodine, copper sulfate, and iron trichloride.

10. The method according to claim 8 or 9, wherein two or more oxidizing agents are used in combination.

1. The method according to claim 7, wherein the oxidizing agent is added to the sample in an amount of 0.005 to 0.5 mmo 1 per 1 m 1.

12. The method according to any one of claims 7 to 9, wherein the oxidizing agent is added in a range of 0.01 to 0.1 lmmol per 1 ml of the sample.

1 3. The method according to claim 1, wherein the trypsin is at least one of trypsin derived from bovine kidney and trypsin derived from rat liver.

1 4. The substrates used for measurement of trypsin activity were N α-benzoyl-arginine-1 ρ-nitroanilide, Να-benzoyl-lysine-1 ρ-2 troanilide, and t-butoxycarbone-arginine-1. 2. The method according to claim 1, which is at least one synthetic substrate selected from the group consisting of p-nitro-2-lide and t-butoxycarboryl lysine-p-nitroanilide.

1 5. The method according to claim 1, wherein the sample is urine.

1 6. Urinary trypsin inhibitor kit containing trypsin, substrate and protease other than trypsin.

17. The measurement kit according to claim 16, wherein the protease other than trypsin is a serine protease.

1 8. Proteases other than trypsin are Erasu Yuichi, Subtilisin, 16. The measurement kit according to claim 16, which is at least one protease selected from the group consisting of chymotrypsin, cathepsin, plasmin, trombin, kallikrein, and perokinase.

1 9. Urine trypsin inhibitor assay kit with trypsin, substrate and oxidant.

20. The oxidizing agent is at least one selected from the group consisting of permanganic acid, permanganate, oxyacid, oxyacid salt, metal salts, oxide and peroxide. The measurement kit described.

21. The measurement kit according to claim 19, wherein the oxidizing agent is at least one oxidizing agent selected from the group consisting of sodium iodate, iodine, copper sulfate, and iron trichloride.

22. The measuring kit according to claim 20, wherein two or more oxidizing agents are used in combination.

23. The measurement kit according to claim 16 or 19, wherein the sample to be measured is urine.