JP5170407B2 - Diabetes complication test reagent - Google Patents

Description

  The present invention relates to a reagent for testing diabetic complications, which is effective in testing diabetic complications, particularly diabetic nephropathy, and managing the degree of progression.

In recent years, markers for onset testing of diabetes and diabetic complications have been found.
For example, “hemoglobin with N-terminal carboxylation (Patent Document 1)” and “specific pyridinium compound (Patent Document 2)” are disclosed as effective markers for the diagnosis of diabetic complications. In addition, a method is also known in which the creatinine concentration in blood and urine is measured to examine the degree of decrease in renal function due to diabetic nephropathy.
Japanese Patent No. 3542245 Japanese Patent No. 3205899

However, the above conventional techniques have the following problems.
(1) All of the techniques disclosed in (Patent Document 1) and (Patent Document 2) and the inspection method based on the measurement of creatinine concentration use a specific substance such as carboxylated hemoglobin, a specific pyridinium compound, or creatinine. As an index, the degree of the presence of the specific substance in the body fluid is measured to determine a decrease in function of an organ such as a kidney due to diabetes. For this reason, there has been a problem that the onset and the degree of progression of diabetes and diabetic complications cannot be detected when a decrease in organ function due to diabetes does not appear as a change in the concentration of these specific substances.
(2) Diabetic nephropathy progresses into the first stage (early nephropathy), the second stage (early nephropathy), the third stage (apparent nephropathy), and the fourth stage (renal failure stage). The function is abolished and the dialysis therapy period is reached. The method of measuring the creatinine concentration in blood and urine is mainly used in the fourth stage test, and creatinine is a marker for determining whether or not to introduce dialysis. Therefore, in the method using creatinine, it is impossible to determine the degree of progression of the disease state from the first phase to the fourth phase only by determining whether or not to introduce dialysis. There was a problem that it was impossible to judge whether blood glucose control, blood pressure management and treatment performed during the fourth period were effective.
(3) When diabetic nephropathy progresses to the third stage, it becomes difficult to completely prevent the progression of nephropathy because the renal function rapidly decreases. Therefore, it is very important to manage the degree of progression of the first to third stage medical conditions. However, there was no technique for managing the progress of the disease state during this period.
(4) It has been well known that urine and plasma contain a substance exhibiting proteolytic enzyme activity (hereinafter referred to as an enzyme). As enzymes contained in urine, urokinase and urinaly kallikrein are known. It is also possible that an unknown enzyme leaks due to a decrease in renal function. However, since it is difficult to analyze individually the activity, abundance, stability, etc. of each enzyme, these enzymes could not be regarded as renal function markers.

  The present invention solves the above-mentioned conventional problems, and does not specify and track markers contained in body fluids of diabetic patients by a simple operation of measuring the activity by contacting body fluids such as urine and blood of a subject. However, it is possible to determine whether or not diabetic nephropathy has developed, and if so, whether the degree of progression is from the first stage to the fourth stage, and from the first stage to the fourth stage The purpose is to provide a diagnostic reagent for diabetes complications that is extremely effective for doctors' diagnosis support by refining the progress prediction of disease states such as examination of the effectiveness of treatment and when to introduce artificial dialysis And

In order to solve the above conventional problems, the reagent for testing for diabetic complications of the present invention has the following configuration.
In the reagent for testing for diabetic complications according to claim 1 of the present invention, four or more kinds of substrate peptides to which three amino acids are bound are combined, and four of the combinations of the substrate peptides are (a ) Leu-Val-Tyr, His-Glu-Lys, Glu-Lys-Lys, Thr-Arg-Ala, (b) Leu-Gly-Arg, Gly-Arg-Ile, Gly-Arg-Thr, Gly-Arg- Gly, (c) Asn-Pro-Arg, Asp-Gly-Arg, Arg-Arg-Gln, Pro-Leu-Gly or Ala-Lys-Ser, (d) Gln-Arg-Arg, His-Leu-Lys, Glu-Lys-Gly, Ala-Arg-Ser, (e) Val-Leu-Lys, Gly-Arg-Ile, Ala-Lys-Ser, Gly-Arg-Gly, (f) Val-Val-Arg, Val- Pro-Arg, Thr-Arg-Val, Asn-Lys-Tyr, (g) Gly-Arg-Ile, Gly-Arg-Thr, Phe-Lys-Ile, Ala-Lys-Ser, (h) Ala-Pro- Phe, Leu-Leu-Glu, Leu-Val-Tyr, Val-Leu-Lys, (i) Thr-Arg-Ala, Pro-Val-Arg, Gln-Arg-Ile, Leu-Met-Lys, (j) Val-Val-Arg, Pro-Val-Arg, Gln-Arg-Ile, Leu-Met-Lys, (k) Ser-Pro-Arg, Lys-Arg-Lys, Lys-Arg-Asp, Ser-Arg-Leu (l) His-Leu-Lys, Lys-Arg-Lys, Lys-Arg-Asp, Ser-Arg-Leu .
With this configuration, the following effects can be obtained.
(1) Various enzymes are secreted into body fluids such as urine and blood, and the amount and type of enzymes secreted into body fluids are constantly changing with the onset of diabetes and the progression of diabetic nephropathy. The present inventors use four or more kinds of substrate peptides to which three amino acids are bonded, collectively evaluate the whole enzyme activity of body fluid, and associate it with renal dysfunction, so that the marker can be identified. It was also found that the onset and progression of diabetic nephropathy can be determined regardless of the type of enzyme present in the body fluid, the amount of each enzyme present, and the intensity of activity. Accordingly, whether or not diabetic nephropathy has developed by a simple operation of measuring the activity by contacting a body fluid such as urine or blood of the subject, the degree of progression is from the first stage to It is possible to determine whether it is in the 4th stage, and to refine the prediction of the progress of the disease state, such as examination of the effectiveness of the 1st to 4th stage treatments and when to introduce artificial dialysis. Is possible.
(2) Since the enzyme secreted into urine and the enzyme secreted into blood are different from each other, the activity can be detected according to the type of body fluid by combining the substrate peptides, and the applicability is excellent.
(3) Since the substrate peptide can be easily produced using a peptide synthesizer or the like, it is excellent in productivity.
(4) (a) Leu-Val-Tyr, His-Glu-Lys, Glu-Lys-Lys, Thr-Arg-Ala, (b) Leu-Gly-Arg, Gly-Arg-Ile, Gly-Arg-Thr , Gly-Arg-Gly, (c) Asn-Pro-Arg, Asp-Gly-Arg, Arg-Arg-Gln, Pro-Leu-Gly or Ala-Lys-Ser, (d) Gln-Arg-Arg, His -Leu-Lys, Glu-Lys-Gly, Ala-Arg-Ser, (e) Val-Leu-Lys, Gly-Arg-Ile, Ala-Lys-Ser, Gly-Arg-Gly, (f) Val-Val -Arg, Val-Pro-Arg, Thr-Arg-Val, Asn-Lys-Tyr, (g) Gly-Arg-Ile, Gly-Arg-Thr, Phe-Lys-Ile, Ala-Lys-Ser, (h ) Ala-Pro-Phe, Leu-Leu-Glu, Leu-Val-Tyr, Val-Leu-Lys, (i) Thr-Arg-Ala, Pro-Val-Arg, Gln-Arg-Ile, Leu-Met- Lys, (j) Val-Val-Arg, Pro-Val-Arg, Gln-Arg-Ile, Leu-Met-Lys, (k) Ser-Pro-Arg, Lys-Arg-Lys, Lys-Arg-Asp, Ser-Arg-Leu, (l) His-Leu-Lys, Lys-Arg-Lys, Lys-Arg-Asp, Ser-Arg-Leu substrate peptide combination obtained from urine or plasma of diabetic patients It was confirmed that a positive discrimination rate of almost 100% was obtained by analyzing the measurement data obtained by contacting with the sample liquid and measuring the enzyme activity of the sample liquid. As a result, it was found that diabetic nephropathy samples can be accurately grouped. Therefore, the enzymatic activity of urine or plasma collected from an unknown subject is measured using these substrate peptides, and the degree of progression of diabetic nephropathy in the unknown subject is determined by applying a set discriminant. can do.

Here, as the amino acid, an L-amino acid usually found in a naturally occurring protein can be used.
The substrate peptide can be synthesized using a normal peptide synthesis method such as a solid phase method or a liquid phase method. Also, use a sequential extension method that sequentially extends from the C-terminal side to the N-terminal side of the target amino acid sequence, or a fragment condensation method that synthesizes a plurality of short peptide fragments and extends them by coupling between peptide fragments. Can do. Moreover, 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid, t-butyloxycarbonyl (Boc) amino acid, etc. can also be synthesize | combined using a peptide synthesizer. Furthermore, it can also be synthesized by generating peptide bonds using proteases or utilizing genetic engineering.

  When testing for diabetes, each substrate peptide is contacted with a body fluid such as urine or blood of the subject or a culture fluid of body fluid, and the enzyme activity is measured by reacting with the enzyme in the sample fluid. Do. As the sample liquid, a biological sample (blood or the like) can be used as it is, or a sample obtained by removing blood cell components or the like by a filter, centrifugation, or the like. Moreover, what performed the condition setting (pH adjustment, active agent introduction | transduction, etc.) that an enzyme expresses activity based on a biological sample can also be used. As a pH adjuster, a buffering agent such as Tris-HCl or Hepes-KOH can be added as a reaction buffer. In addition, salts and active protective agents necessary for the expression of enzyme activity can be added.

As a method for measuring activity, various known methods can be used. For example, a method for measuring the amount of decrease in substrate peptide by absorbance at a specific wavelength, a method for measuring substrate peptide or reaction product by coloring with chemical reagents, and other measurements using radioactivity, colorimetry, fluorescence, luminescence, etc. It is also possible to use a method of
In measuring the enzyme activity, it is preferable to trace the reaction time and obtain the initial reaction rate and the amount of change. This is because the reaction rate gradually decreases as the reaction time elapses.

Diabetes complications and progress management should be done first by sample fluid such as urine from patients who have diabetes but have not developed diabetic nephropathy, sample fluid from patients who have diabetic nephropathy Is measured by reacting with each of a plurality of substrate peptides, and the enzyme activity is measured. By a known method such as discriminant analysis, a criterion for discriminating between a group that develops diabetic nephropathy and a group that does not develop (discrimination Function) is set in advance. To obtain the discriminant function, either a linear method or a non-linear method can be used.
Next, the sample liquid of an unknown subject is reacted with each of the same types of substrate peptides as used when the discriminant function was set, and the enzyme activity is measured. It is possible to determine the onset and progression of diabetic nephropathy.
In addition, when the data is converted into a two-dimensional map by SOM (self-organizing map) analysis, it becomes easier to visualize and can be classified into a plurality of groups according to the degree of progression of diabetic nephropathy on the map. It is easy to grasp the degree of progression (1st to 4th stages), and it is possible to visually predict the progress of the disease state such as when artificial dialysis will be introduced.
The obtained enzyme activity data can be analyzed by various nonlinear classification methods and nonlinear factor analysis methods other than SOM, and diabetes complications can be examined and the degree of progress can be managed.

  As a combination of substrate peptides, 4 or more and 8 or less, preferably 4 or more and 6 or less are suitably used. This is because the enzyme activity can be measured and analyzed relatively easily and the discrimination accuracy is excellent. As the number of combinations decreases from four, the discrimination accuracy tends to decrease. If the number exceeds 6, the measurement and analysis of the enzyme activity tends to be complicated, and if the number exceeds 8, the tendency becomes remarkable.

As the reagent for testing for diabetic complications, it is possible to use four types of substrate peptides having the sequences (a) to (l) as separate compounds. In addition, one obtained by extending a substrate peptide to 1 to 3 kinds of compounds can also be used. It is preferable to use four types of substrate peptides as separate compounds. This is because the analysis of the measurement result is easy and the accuracy can be improved. When urine is used as the sample liquid, the substrate peptides (a), (h) to (l) are suitable. When blood (plasma) is used as the sample liquid, the substrate peptides (b) to (g) are suitable.
The substrate peptide can also bind any peptide or other compound or molecule that does not have enzyme specificity. Alternatively, one having one end bound to an insoluble carrier can be used.

The invention according to claim 2 of the present invention is the reagent for detecting diabetes according to claim 1 , and has a configuration including a first fluorescent group bonded to one end of the substrate peptide.
With this configuration, in addition to the operation obtained in the first aspect , the following operation can be obtained.
(1) When the substrate peptide is cleaved by an enzyme in urine or blood, the nature of the first fluorescent group changes before and after that, and the fluorescence wavelength and fluorescence intensity change. be able to.
(2) Since the enzyme activity can be detected simply by measuring the fluorescence intensity of the sample liquid after contact reaction with urine or blood sample liquid, the measurement time can be shortened and the workability improved. Measurement efficiency can be increased, and detection sensitivity and measurement accuracy can be increased.

Here, as the first fluorescent group, those in which the property of the fluorescent group changes before and after the substrate peptide is cleaved by the enzyme and the fluorescent wavelength and the fluorescence intensity change are used. When the substrate peptide is bound to an insoluble carrier, a fluorescent group that causes a change in fluorescence wavelength or fluorescence intensity due to concentration quenching is also used. The concentration quenching phenomenon means that the fluorescence wavelength and fluorescence intensity change when the fluorescent groups are close to each other on the carrier and when the fluorescent groups are separated in the urine or blood sample liquid and the distance between the fluorescent groups is increased. A phenomenon.
Examples of the first fluorescent group include 4-methylcoumaryl-7-amide (MCA), 7-amino-4-carboxymethylcoumarin (ACC), α-naphthylamide, α-naphthyl ester, fluorescein, rare earth complex Alternatively, derivatives thereof are used.
Thereby, since the fluorescence wavelength and fluorescence intensity of the first fluorescent group released from the carrier are different from those of the first fluorescent group before release, the amount of cleavage by the enzyme is measured using the fluorescence intensity in the specific wavelength region as an index. be able to.

  The first fluorescent group is preferably a fluorescein derivative such as fluorescein or fluorescein isothiocyanate (FITC). Since fluorescein emits fluorescence at around 525 nm, it can be distinguished from the fluorescence wavelength of fluorescent substances contained in medicines, etc., and the urine of a subject taking medicines or vitamins can be used as a sample liquid as it is. It is because it is excellent in property. In the urine of a subject who is taking drugs or vitamins, various fluorescent substances contained in the drug etc. appear, but since many of these fluorescent substances have a fluorescence wavelength of around 400 nm, urine is used as the sample liquid. When used as it is, the fluorescence of the fluorescent substance contained in the medicine or the like cannot be distinguished from the fluorescence of the first fluorescent group released into the sample liquid due to the enzyme activity.

  In addition to the method using a fluorescence spectrophotometer, the fluorescence measurement of the sample liquid is performed by irradiating the sample liquid with light from a light emitting element such as a light emitting diode through a filter that passes the excitation wavelength of the fluorescent group to detect the fluorescence of the sample liquid. It is also possible to use a method of measuring fluorescence intensity or the like with a light receiving element such as a CCD arranged at a position where it can be used.

The invention according to claim 3 of the present invention is the reagent for testing diabetic complications according to claim 1 , wherein the second fluorescent group bonded to one end of the substrate peptide and the other of the substrate peptide And a quenching group bonded to the terminal.
With this configuration, in addition to the operation of the first aspect , the following operation can be obtained.
(1) When the substrate peptide is cleaved by the enzyme, the fluorescence spectrum of the second fluorescent group changes due to the distance between the quenching group and the second fluorescent group, so this spectral change is used as a measurement index for enzyme activity. Thus, enzyme activity can be detected using changes in fluorescence intensity or the like as an index.
(2) Since the enzyme activity can be detected simply by measuring the fluorescence intensity of the sample liquid after contact reaction with urine or blood sample liquid, the measurement time can be shortened and the workability improved. Measurement efficiency can be increased, and detection sensitivity and measurement accuracy can be increased.

Here, as the quenching group, a substance that absorbs light corresponding to the fluorescence excitation wavelength of the second fluorescent group, a substance that forms a non-light emitting complex by loose bonding with the second fluorescent group, a second fluorescent group, A substance that causes fluorescence resonance energy transfer is used.
Fluorescence resonance energy transfer is when the excitation spectrum of the quenching group (acceptor) and the fluorescence spectrum of the second fluorescent group (donor) overlap when the second fluorescent group and the quenching group are located at close distances. When the energy of the excitation wavelength of the second fluorescent group is applied, the quenching group takes the excitation energy and the fluorescence of the second fluorescent group that should be observed is attenuated.

  As the second fluorescent group or quenching group, a combination of a donor and an acceptor in which fluorescence resonance energy transfer occurs can be used. For example, a quenching group that is an atomic group having an absorption band in a wavelength region that overlaps the fluorescence wavelength of the second fluorescent group is used. Specifically, the combination of (7-methoxycoumarin-4-yl) acetyl (MOAc), anthraniloylbenzyl (ABz), N-methylanthranilic acid (Nma) and the like and dinitrophenyl (Dnp), Dabsyl and EDANS ( 5- (2′-aminoethyl) aminonaphthalene-1-sulfonic acid), tryptophan (Trp) and 5-dimethylamino-1-naphthalenesulfonic acid (Dns), carboxydichlorofluorescein (CDCF) and carboxymethyl A combination of rhodamine (CTMR), a combination of carboxydichlorofluorescein (CDCF) and carboxy X-rhodamine (CXR), a combination of lucifer yellow (LY) and carboxymethyl rhodamine (CTMR), and the like are used.

The quenching group may be bound directly to the substrate peptide, or may be bound to the substrate peptide via an atomic group to which the peptide or other compound or molecule is bound.
For the bond between the substrate peptide and the atomic group, and the bond between the atomic group and the quenching group, an amide bond, an ester bond, an ether bond, a thioether bond, a urethane bond, or the like that is not cleaved by an enzyme is used. This is because even when the quenching group is cleaved by the enzyme and released from the substrate peptide, the fluorescence spectrum of the second fluorescent group changes, but the enzyme activity depending on the amino acid sequence of the substrate peptide cannot be detected.

  The length between the binding part of the second fluorescent group and the quenching group bonded to the substrate peptide is preferably 100 mm or less. As the distance between the binding parts of the second fluorescent group and the quenching group becomes longer, changes in fluorescence intensity and the like tend to become smaller, and when the distance exceeds 100 mm, this tendency becomes remarkably small and the change in fluorescence intensity becomes extremely small and sensitivity decreases. Because it does.

As described above, according to the reagent for testing for diabetic complications of the present invention, the following advantageous effects can be obtained.
According to the invention of claim 1,
(1) Since the activity of a plurality of enzymes secreted into a body fluid is measured using a plurality of substrates and the results are analyzed, the onset and progression of diabetic nephropathy can be determined. Whether or not diabetic nephropathy has developed in a simple operation of measuring the activity by contacting body fluids such as blood and blood, and if so, the degree of progression is any of the first to fourth stages Diabetic complications that can be used to assess the effectiveness of treatments in the first to fourth stages and to predict the progress of disease states such as when to introduce artificial dialysis A test reagent can be provided.
(2) Since the enzymes secreted in the urine and the enzymes secreted in the blood are different, the activity detection according to the type of body fluid can be detected by the combination of substrate peptides, and it has excellent applicability for testing diabetic complications Reagents can be provided.
(3) Since the substrate peptide can be easily produced using a peptide synthesizer or the like, a reagent for testing a diabetic complication having excellent productivity can be provided.
(4) Samples of diabetic nephropathy can be accurately grouped by contacting the sample solution obtained from urine or plasma of diabetic patients and analyzing the measurement data obtained by measuring the enzyme activity of the sample solution. Therefore, the enzyme activity of urine or plasma collected from an unknown subject is measured using these substrate peptides, and the progression of diabetic nephropathy in an unknown subject is determined by applying a set discriminant. A reagent for testing diabetic complications can be provided.

According to invention of Claim 2 , in addition to the effect of Claim 1 ,
(1) Since the enzyme activity can be detected by simply measuring the fluorescence intensity of the sample liquid after the urine or blood sample liquid is contacted, the measurement time can be shortened and the workability is improved. It is possible to provide a reagent for testing diabetic complications that can improve measurement efficiency and is excellent in detection sensitivity and measurement accuracy.

According to the invention of claim 3 , in addition to the effect of claim 1 ,
(2) Since the enzyme activity can be detected simply by measuring the fluorescence intensity of the sample liquid after contact reaction with urine or blood sample liquid, the measurement time can be shortened and the workability improved. It is possible to provide a reagent for testing diabetic complications that can improve measurement efficiency and is excellent in detection sensitivity and measurement accuracy.

Hereinafter, the best mode for carrying out the present invention will be described with reference to the drawings.
(Embodiment 1)
FIG. 1 is a schematic diagram showing the principle of detecting enzyme activity of a reagent for testing diabetic complications in Embodiment 1 of the present invention.
In the figure, 1 is a reagent for testing for diabetic complications in the first embodiment, 2 is a synthetic resin (polystyrene etc.) insoluble in solvents such as halogenated hydrocarbons, esters, etc. (eg, PEGA), glass, etc. carrier formed in a spherical shape or polyhedral shape or the like, X is a compound having no enzyme specificity conjugated to a carrier 2, 3 AA ₃ -AA ₂ -AA ₁ amino acid sequence to which the compound X is bonded to an amino acid AA ₁ 4-methylcoumalyl is a kind of fluorescent group that changes in fluorescence wavelength and fluorescence intensity before and after substrate peptide 3 binds to the N-terminus of substrate peptide 3 and is cleaved by enzyme 5 described later. First fluorescent group such as -7-amide (MCA), fluorescein isothiocyanate (FITC), 5 is a peptide bond between the first fluorescent group 4 and substrate peptide 3, and substrate peptide 3 Enzymes with substrate specificity of the test solution which selectively cleaved at the cleavage site of, 6 is the first fluorescent groups liberated fluorescence wavelength or the like is changed by the substrate peptide 3 was cleaved by the enzyme 5.

  The reagent 1 for testing a complication of diabetes composing as described above includes a normal peptide synthesis method such as a solid phase method in which the substrate peptide 3 is extended from the C-terminal, and the N-terminal from the C-terminal side of the target amino acid sequence. Sequential extension method that sequentially extends to the side, fragment condensation method that synthesizes multiple short peptide fragments and extends by coupling between peptide fragments, Fmoc method, Boc method etc. using peptide synthesizer It can be synthesized using a method or the like.

The detection principle of the enzyme activity of the reagent for testing for diabetic complications in Embodiment 1 configured as described above will be described below.
The first fluorescent group 4 such as 4-methylcoumalyl-7-amide (MCA), fluorescein isothiocyanate (FITC), etc. of the reagent 1 for testing for diabetic complications shown in FIG. 1 (a) is a non-fluorescent substance in a specific wavelength region, Further, when it exists on the surface of the carrier 2 at a high density, the fluorescence intensity is weak due to concentration quenching. When the reagent solution containing the enzyme 5 is brought into contact with and reacted with the reagent 1 for testing for complications of diabetes, the peptide bond between the first fluorescent group 4 and the substrate peptide 3 and the substrate peptide 3 are selectively selected at a specific cleavage site. Disconnect.
The first fluorescent group 6 released from the substrate peptide 3 becomes a fluorescent substance such as 7-amino-methylcoumarin (AMC), or the effect of concentration quenching is reduced by moving away from the carrier 2, and the fluorescence of the first fluorescent group 6 is reduced. Since the fluorescence intensity in the wavelength or the specific wavelength region is different from that of the first fluorescent group 4 bound to the substrate peptide 3, the enzyme activity can be detected by using a change in the fluorescence intensity of the sample liquid as an index (FIG. 1 ( b)).

Diabetes complications and progress management, first, body fluid such as blood of patients who have diabetes but have not developed diabetic nephropathy, body fluid of patients who have diabetic nephropathy, The enzyme activity is measured by reacting with each of the diabetic complication test reagents 1 combined with four or more kinds of substrate peptides 3 having different amino acid sequences, and the whole enzyme activity of the body fluid is collectively collected by a known method such as discriminant analysis. Thus, a criterion (discriminant function) for discriminating between a group that develops diabetic nephropathy and a group that does not develop diabetic nephropathy is set in advance by a nonlinear method based on the Mahalanobis distance. Each discriminant function for discriminating patients in the first to fourth stages of diabetic nephropathy can be set in a database in advance.
Next, the body fluid of the unknown subject is reacted with each of the diabetic complication testing reagents 1 combined with the same type of substrate peptide 3 as when the discriminant function is set, and the enzyme activity is measured. By determining which of the preset groups it belongs, it is possible to easily determine whether or not diabetic nephropathy has developed, the degree of complication, and the like.

As described above, since the reagent for testing for diabetic complications in the first embodiment is configured, the following effects can be obtained.
(1) It is possible to determine whether or not diabetic nephropathy has developed by a simple operation of measuring the activity by contacting the body fluid of the subject and to what extent the progression has progressed. In addition, it is possible to refine the prediction of the progress of disease states such as the examination of the effectiveness of treatment and when to introduce artificial dialysis.
(2) By measuring the enzyme activity by changing the combination of substrate peptides 3 according to the type of body fluid such as urine and blood, the activity can be detected according to the type of body fluid and the applicability is excellent.
(3) Since the substrate peptide can be easily produced using a peptide synthesizer or the like, it is excellent in productivity.

In the present embodiment, the reagent 1 for testing for diabetic complications in which the substrate peptide 3 is bound to the carrier 2 has been described. However, it is not always necessary to use the substrate 1 having the specific amino acid sequence. As long as the peptide is contained, a lyophilized powder or a solution supplied in a solution can be used.
In addition, the case where the reagent 1 for testing diabetic complications has one kind of substrate peptide 3 and four or more of them are combined, but the substrate peptide obtained by combining and extending two or more kinds of substrate peptides 3 in combination is extended. May be used. In this case as well, the enzyme activity is evaluated by fluorescence measurement of a reagent for testing for diabetic complications, and grouping by each discriminant function for discriminating patients in the first to fourth stages of diabetic nephropathy, The effect is obtained.

(Embodiment 2)
FIG. 2 is a schematic diagram showing the principle of detecting the enzyme activity of the reagent for testing diabetic complications in Embodiment 2 of the present invention. In addition, the same thing as Embodiment 1 attaches | subjects the same code | symbol, and abbreviate | omits description.
In the figure, 10 is a reagent for testing diabetic complications in the second embodiment, and 11 is a substrate peptide having an amino acid sequence of AA ₃ -AA ₂ -AA ₁ including the cleavage site of enzyme 14 in the sample liquid described later. Y and Z represent an atomic group such as an arbitrary amino acid residue. In this embodiment, the atomic group Y at the end is bonded to the carrier 2.
12 is a quenching group such as dinitrophenyl (Dnp) and 5-dimethylamino-1-naphthalenesulfonic acid (Dns) that binds to the atomic group Y bound to the substrate peptide 11, and 13 is quenched at the other end of the substrate peptide 11 This is a second fluorescent group such as (7-methoxycoumarin-4-yl) acetyl (MOAc), tryptophan (Trp), etc. in which fluorescence resonance energy transfer is observed with the group 12. The quenching group 12 and the second fluorescent group 13 are bonded together at a distance (100 Å or less) at which an interaction affecting the fluorescence is observed. Reference numeral 14 denotes an enzyme present in the sample solution, and reference numeral 15 denotes a second fluorescent group whose fluorescence wavelength or the like has changed due to the substrate peptide 11 being cleaved and released at a specific cleavage site by the substrate specificity of the enzyme 14.

The detection principle of the enzyme activity of the reagent for testing for diabetic complications in Embodiment 2 configured as described above will be described below.
Since the quenching group 12 and the second fluorescent group 13 of the reagent 10 for testing for diabetic complications shown in FIG. 2 (a) are bonded to each other at a distance at which an interaction affecting fluorescence is observed, absorption of the quenching group 12 is achieved. The spectrum and the fluorescence spectrum of the second fluorescent group 13 overlap each other, and when the energy of the excitation wavelength of the second fluorescent group 13 is applied, attenuation of the fluorescence of the second fluorescent group 13 that should originally be observed is observed.
When the sample liquid containing the enzyme 14 is brought into contact with the reagent 10 for testing for diabetic complications and reacted, the enzyme 14 having substrate specificity cleaves the substrate peptide 11.
When the second fluorescent group 13 is released from the carrier 2, no interaction affecting the fluorescence is observed between the second fluorescent group 13 and the quenching group 12, so that the sample liquid has an excitation wavelength of the second fluorescent group 13. When energy is applied, the fluorescence wavelength of the second fluorescent group 15 that has not been observed before contact with the sample liquid is observed, and is different from the fluorescence wavelength before the reaction of the enzyme 14, so that the fluorescence intensity, etc. The enzyme activity can be detected using the change as an index (see FIG. 2B).
In addition, since the method of a diabetic complication test | inspection and progress management is the same as that of what was demonstrated in Embodiment 1, description is abbreviate | omitted.

As described above, since the reagent for testing for diabetic complications in the second embodiment is configured, in addition to the operation described in the first embodiment, the following operation can be obtained.
(1) Since the fluorescence wavelength of the second fluorescent group 13 can be set in the visible region by selecting the quenching group 12 and the second fluorescent group 13, a visible light detection device such as a commercially available CCD camera This makes it possible to measure with a superior versatility.

Also in this embodiment, it is not always necessary to bind the substrate peptide 11 to the carrier 2, and if the substrate peptide 11 having a specific amino acid sequence is included, it is supplied in a lyophilized powder state or a solution state. Can also be used.
In addition, although the case where the reagent 10 for testing for diabetic complications has one kind of substrate peptide 11 and four or more of them are combined, the substrate peptide obtained by combining and extending two or more kinds of substrate peptides 11 in combination is extended. May be used. In this case as well, the enzyme activity is evaluated by fluorescence measurement of a reagent for testing for diabetic complications, and grouping by each discriminant function for discriminating patients in the first to fourth stages of diabetic nephropathy, The effect is obtained.

Hereinafter, the present invention will be specifically described by way of examples. The present invention is not limited to these examples.
The amino acids, peptides, protecting groups, solvents, and the like described in this example use the abbreviations commonly used in the technical field or the abbreviations adopted by the IUPAC-IUB naming committee. For example, the following abbreviations are used. For example, FITC: Fluorescein-4-isothiocyanate isomer-I (fluorescein isothiocyanate), DMF: N, N-dimethylformamide, DIEA: N, N-diisopropylethylamine, DCM: dichloromethane, i-PrOH: 2-propanol, MeOH: Methanol, Lys (Dnp): (2S) -2-amino-6- (2,4-dinitrophenylamino) hexanoic acid, TFA: trifluoroacetic acid.
Example 1
In Example 1, a peptidyl fluorescent group-bound spherical carrier as a reagent for testing diabetic complications was synthesized, and then the activity of a sample solution obtained from urine was measured. The method will be described below.
<Synthesis of peptidyl fluorescent group-bound spherical carrier>
As the carrier, spherical NH ₂ -PEGA-resin (manufactured by Watanabe Chemical Industries, particle size of about 0.1 mm) was used. Five amino acids of Lys (Dnp), AA ₁ , AA ₂ , AA ₃ , and βAla were sequentially introduced into NH ₂ -PEGA Resin (0.5 g, 25 μmol) using a peptide synthesizer (Model 433A, Applied Biosystems). AA ₁ , AA ₂ and AA ₃ are amino acids shown in Table 1. Thereafter, the carrier was placed in a plastic vessel, and DMF was added to swell the carrier. After removing DMF by suction, FITC (30 μmol, 12 mg), DMF (2 ml) and DIEA (25 μmol, 4.4 μl) dissolved in a small amount of DMF were added, and the mixture was shaken at room temperature for 3 hours. After removing the reaction solution by suction, DMF (2 ml, 2 times), DCM (2 ml, 2 times), i-PrOH (2 ml, 2 times), DMF (2 ml, 2 times), MeOH (2 ml , Twice) and ether (2 ml, twice) in this order. Thereafter, the mixture was reacted with a mixed solution of phenol (75 mg), 1,2-ethanedithiol (25 μl), thioanisole (50 μl), distilled water (50 μl) and TFA (2 ml) for 3 hours. After removing the reaction solution by suction, DCM (2 ml, 2 times), DMF (2 ml, 2 times), 20% piperidine / DMF (2 ml, 2 times), DMF (2 ml, 2 times), i-PrOH (2 ml, 2 times), DMF (2 ml, 2 times), distilled water (2 ml, 2 times), ether (2 ml, 1 time) were washed in this order and dried under reduced pressure. A substrate peptide consisting of βAla-AA ₃ -AA ₂ -AA ₁ -Lys in which a second fluorescent group is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end (Table 1) (No. 1 to No. 4) of diabetic complication test reagent (SEQ ID NO: 1 to 4 in the sequence listing).

<Measurement of enzyme activity>
Add 190 μl of 20 mM Tris-HCl buffer (pH 8.0, NaCl 100 mM, CaCl ₂ 50 mM) containing 0.01% Tween 20 to 1 mg of each of No1 to No4 diabetic complication test reagents. Add the same amount of sample solution obtained from the urine of 5 subjects (4th stage) (Group A) and 5 non-diabetic nephropathy subjects (Phase 1) (Group B), and sample each measurement time. 10 μl was added, 190 μl of buffer was added and transferred to a 96-well plate, and the fluorescence value was measured with a Wallac 1420 ARVO sx plate reader manufactured by Perkin Elmer (measurement time 0.1 second). Change amount of fluorescence value 10 minutes after adding sample solution (difference between fluorescence value before adding sample solution and fluorescence value 10 minutes after adding sample solution) (excitation wavelength 485 nm, fluorescence wavelength 535 nm) is shown in Table 2.

  First, the homogeneity of the variance-covariance matrix of Group A and Group B shown in Table 2 was analyzed, and it was found that the homogeneity was not considered. Therefore, analysis was performed using a nonlinear (secondary) discriminant analysis based on Mahalanobis distance. Table 3 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 3, a positive discrimination rate of 100% was obtained as shown in Table 4.

The above measurement data was analyzed to obtain a secondary discriminant function, and a discriminant was created.
As described above, the test for diabetic complications of this example was brought into contact with the sample liquid obtained from the urine of a diabetic patient, and the measurement value obtained by measuring the enzyme activity of the sample liquid was analyzed by discriminant analysis. As a result, it was confirmed that the correct discrimination rate was as extremely high as 100%. This indicates that the diabetic nephropathy specimens can be grouped very accurately by using the reagent for testing diabetic complications of this example.
Therefore, by measuring the enzyme activity of the urine sample liquid collected from an unknown subject using the reagent for testing diabetic complications of this example, and applying the set discriminant, the diabetic property of the unknown subject It is clear that the degree of progression of nephropathy can be distinguished.
In this example, it was shown that the samples of the first and fourth stages of diabetic nephropathy can be accurately discriminated, but by increasing the number of specimens in the first to fourth stages, the same method can be used. It is also possible to manage the progress of the first to fourth stages of diabetic nephropathy.

In this example, a diabetic complication having a substrate peptide in which a second fluorescent group composed of fluorescein isothiocyanate (FITC) is bonded to one end and a quenching group composed of dinitrophenyl (Dnp) is bonded to lysine at the other end. Although the case where the test reagent was synthesized and the fluorescence change of the sample liquid brought into contact with the reagent was measured was described, (7-methoxycoumarin-4-yl) acetyl (MOAc) or the like was used as the second fluorescent group. In some cases, it was confirmed that similar results were obtained.
In addition, when using a reagent for examination of diabetic complications that has a substrate peptide with a carrier such as PEGA resin at one end and a first fluorescent group such as FITC attached to the other end, the fluorescence change of the sample liquid brought into contact with this reagent It was confirmed that it can be used for the examination of the complication of diabetes and the progress management by measuring the enzyme activity.

(Example 2)
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end Reagents for testing diabetic complications (SEQ ID NOs: 5 to 8 in the sequence listing) having substrate peptides (No5 to No8 in Table 5) comprising _1- Lys were obtained.

<Measurement of enzyme activity>
Add 190 μl of 20 mM Tris-HCl buffer (pH 8.0, NaCl 100 mM, CaCl ₂ 50 mM) containing 0.01% Tween 20 to each 1 mg of No5 to No8 Diabetes Complication Test Reagent. Add the same amount of sample solution obtained from the blood (plasma) of 5 subjects (Phase 4) (Group A) and 5 non-diabetic nephropathy subjects (Phase 1) (Group B), and measure the time. 10 μl was taken from each sample, 190 μl of buffer was added and transferred to a 96-well plate, and the fluorescence value was measured with a Wallac 1420 ARVO sx plate reader manufactured by Perkin Elmer (measurement time 0.1 second). Change amount of fluorescence value 10 minutes after adding sample solution (difference between fluorescence value before adding sample solution and fluorescence value 10 minutes after adding sample solution) (excitation wavelength 485 nm, fluorescence wavelength 535 nm) is shown in Table 6.

  The variance-covariance matrices of Group A and Group B shown in Table 6 were analyzed using a non-linear (second-order) discriminant analysis method based on Mahalanobis distance, as in Example 1. Table 7 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 7, a positive discrimination rate of 80% or more was obtained as shown in Table 8.

The above measurement data was analyzed to obtain a secondary discriminant function, and a discriminant was created.
As described above, the reagent for testing diabetic complications of this example is brought into contact with the sample liquid obtained from the plasma of the diabetic patient, and by analyzing the measurement data obtained by measuring the enzyme activity of the sample liquid, It was confirmed that the correct discrimination rate was extremely high. If the number of specimens increases, the positive discrimination rate is expected to further increase. This indicates that the diabetic nephropathy specimens can be grouped very accurately by using the reagent for testing diabetic complications of this example.
Therefore, the enzyme activity of the plasma sample fluid collected from an unknown subject is measured using the reagent for testing diabetic complications of this example, and the diabetic property of the unknown subject is determined by applying the set discriminant. It is clear that the degree of progression of nephropathy can be distinguished.

(Example 3)
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end Reagents for testing diabetic complications (SEQ ID NOs: 9 to 12 in the sequence listing) having substrate peptides (No9 to No12 in Table 9) comprising _1- Lys were obtained.

<Measurement of enzyme activity>
Using 1 mg each of the No9 to No12 diabetic complication test reagents, in the same manner as in Example 2, the amount of change in fluorescence value when a sample solution obtained from blood (plasma) was added was measured. The results are shown in Table 10.

  The variance-covariance matrices of Group A and Group B shown in Table 10 were analyzed using a non-linear (second-order) discriminant analysis method based on Mahalanobis distance, as in Example 1. Table 11 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 11, a positive discrimination rate of 100% was obtained as shown in Table 12. As a result, it was confirmed that the diabetic nephropathy specimens could be grouped very accurately by using the diabetic complication test reagent of this example.

Example 4
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end Reagents for testing diabetic complications (SEQ ID NO: 9 to 11, SEQ ID NO: 13 in the sequence listing) having substrate peptides (No9 to No11, No13 in Table 13) comprising _1- Lys were obtained.

<Measurement of enzyme activity>
Using 1 mg of each of the reagents for testing for diabetic complications No. 9 to No. 11 and No. 13, in the same manner as in Example 2, the amount of change in the fluorescence value when the sample liquid obtained from blood (plasma) was added was measured. The results are shown in Table 14.

  The variance-covariance matrices of Group A and Group B shown in Table 14 were analyzed using a non-linear (secondary) discriminant analysis method based on Mahalanobis distance, as in Example 1. Table 15 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 15, a positive discrimination rate of 100% was obtained as shown in Table 16. As a result, it was confirmed that the diabetic nephropathy specimens could be grouped very accurately by using the diabetic complication test reagent of this example.

(Example 5)
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end Reagents for testing diabetic complications (SEQ ID NOs: 8 to 13 in the sequence listing) having substrate peptides (No8 to No13 in Table 17) consisting of _1- Lys were obtained.

<Measurement of enzyme activity>
Using 1 mg of each of the No8 to No13 diabetic complication test reagents, the amount of change in fluorescence value when a sample solution obtained from blood (plasma) was added was measured in the same manner as in Example 2. The results are shown in Table 18.

  For the variance-covariance matrices of Group A and Group B shown in Table 18, assuming that the variance-covariance matrices of both groups are homogeneous based on the Mahalanobis distance (excluding the quadratic terms), linear discrimination Led function. Table 19 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 19, a positive discrimination rate of 100% was obtained as shown in Table 20. In general, as the number of specimens increases, the similarity between both groups increases, and it is expected that linear analysis will be possible. It was confirmed that samples of diabetic nephropathy can be grouped very accurately by performing linear analysis using the reagent for testing diabetic complications of this example.

(Example 6)
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end _A reagent for testing diabetic complications (SEQ ID NO: 17, 19, 21, 23 in the sequence listing) having a substrate peptide consisting of _1- Lys (No. 17, 19, 21, 23 in Table 21) was obtained.

<Measurement of enzyme activity>
Measure the amount of change in fluorescence when adding sample liquid obtained from blood (plasma) in the same manner as in Example 2 using 1 mg each of the reagents for testing diabetic complications No17, 19, 21, 23 did. The results are shown in Table 22.

  The variance-covariance matrices of Group A and Group B shown in Table 22 were analyzed using a non-linear (second-order) discriminant analysis method based on Mahalanobis distance, as in Example 1. Table 23 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 23, a positive discrimination rate of 100% was obtained as shown in Table 24. As a result, it was confirmed that the diabetic nephropathy specimens could be grouped very accurately by using the diabetic complication test reagent of this example.

(Example 7)
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end _A reagent for testing diabetic complications (SEQ ID NO: 6, 8, 13, 14 in the sequence listing) having a substrate peptide consisting of _1- Lys (No. 6, 8, 13, 14 in Table 25) was obtained.

<Measurement of enzyme activity>
Measure the amount of change in fluorescence when adding a sample solution obtained from blood (plasma) in the same manner as in Example 2, using 1 mg each of No. did. The results are shown in Table 26.

  The variance-covariance matrices of Group A and Group B shown in Table 26 were analyzed using a non-linear (second-order) discriminant analysis method based on Mahalanobis distance, as in Example 1. Table 27 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 27, a positive discrimination rate of 100% was obtained as shown in Table 28. As a result, it was confirmed that the diabetic nephropathy specimens could be grouped very accurately by using the diabetic complication test reagent of this example.

(Example 8)
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end _A reagent for testing diabetic complications (SEQ ID NOs: 15, 16, 18, and 22 in the sequence listing) having a substrate peptide consisting of _1- Lys (No. 15, 16, 18, and 22 in Table 29) was obtained.

<Measurement of enzyme activity>
Measure the amount of change in fluorescence when adding a sample solution obtained from blood (plasma) in the same manner as in Example 2 using 1 mg each of No15, 16, 18, and 22 diabetes complication test reagents did. The results are shown in Table 30.

  The variance-covariance matrices of Group A and Group B shown in Table 30 were analyzed using a non-linear (secondary) discriminant analysis method based on Mahalanobis distance, as in Example 1. Table 31 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 31, a positive discrimination rate of 100% was obtained as shown in Table 32. As a result, it was confirmed that the diabetic nephropathy specimens could be grouped very accurately by using the diabetic complication test reagent of this example.

Example 9
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end _A reagent for testing diabetic complications (SEQ ID NO: 6, 7, 13, 20 in the sequence listing) having a substrate peptide consisting of _1- Lys (No. 6, 7, 13, 20 in Table 33) was obtained.

<Measurement of enzyme activity>
Measure the amount of change in fluorescence when adding a sample solution obtained from blood (plasma) in the same manner as in Example 2 using 1 mg each of the reagents for testing diabetic complications No. 6, 7, 13, and 20. did. The results are shown in Table 34.

  The variance-covariance matrix of Group A and Group B shown in Table 34 was analyzed using a non-linear (secondary) discriminant analysis method based on Mahalanobis distance, as in Example 1. Table 35 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 35, a positive discrimination rate of 100% was obtained as shown in Table 36. As a result, it was confirmed that the diabetic nephropathy specimens could be grouped very accurately by using the diabetic complication test reagent of this example.

(Example 10)
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end _A reagent for testing diabetic complications (SEQ ID NO: 24, 25, 1, 14 in the sequence listing) having a substrate peptide consisting of _1- Lys (No 24, 25, ₁ , 14 in Table 37) was obtained.

<Measurement of enzyme activity>
Using 1 mg each of the reagents for testing for diabetic complications No. 24, 25, 1, and 14 in the same manner as in Example 1, the amount of change in the fluorescence value when the sample solution obtained from urine was added was measured. The results are shown in Table 38.

  The variance-covariance matrices of Group A and Group B shown in Table 38 were analyzed using a non-linear (second order) discriminant analysis method based on Mahalanobis distance, as in Example 1. Table 39 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 39, a positive discrimination rate of 100% was obtained as shown in Table 40. As a result, it was confirmed that the diabetic nephropathy specimens could be grouped very accurately by using the diabetic complication test reagent of this example.

(Example 11)
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end _A reagent for testing diabetic complications (SEQ ID NO: 4, 26, 27, 28 in the sequence listing) having a substrate peptide consisting of _1- Lys (No. 4, 26, 27, 28 in Table 41) was obtained.

<Measurement of enzyme activity>
Using 1 mg of each of No. 4, 26, 27, and 28 for diabetic complication testing reagent, the amount of change in fluorescence value when a sample solution obtained from urine was added was measured in the same manner as in Example 1. The results are shown in Table 42.

  The variance-covariance matrices of Group A and Group B shown in Table 42 were analyzed using a non-linear (second order) discriminant analysis method based on Mahalanobis distance, as in Example 1. Table 43 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 43, a positive discrimination rate of 100% was obtained as shown in Table 44. As a result, it was confirmed that the diabetic nephropathy specimens could be grouped very accurately by using the diabetic complication test reagent of this example.

(Example 12)
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end _A reagent for testing diabetic complications (SEQ ID NO: 15, 26, 27, 28 in the sequence listing) having a substrate peptide consisting of _1- Lys (No 15, 26, 27, 28 in Table 45) was obtained.

<Measurement of enzyme activity>
Using 1 mg of each of No15, 26, 27, and 28, a reagent for testing diabetic complications, the amount of change in fluorescence value when a sample solution obtained from urine was added was measured in the same manner as in Example 1. The results are shown in Table 46.

  The variance-covariance matrices of Group A and Group B shown in Table 46 were analyzed using a non-linear (second order) discriminant analysis method based on Mahalanobis distance, as in Example 1. Table 47 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 47, a positive discrimination rate of 100% was obtained as shown in Table 48. As a result, it was confirmed that the diabetic nephropathy specimens could be grouped very accurately by using the diabetic complication test reagent of this example.

(Example 13)
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end _A reagent for testing diabetic complications (SEQ ID NO: 29, 30, 31, 32 in the sequence listing) having a substrate peptide consisting of _1- Lys (No. 29, 30, 31, 32 in Table 49) was obtained.

<Measurement of enzyme activity>
Using 1 mg of each of the reagents for testing for diabetic complications No. 29, 30, 31, and 32, the amount of change in fluorescence value when a sample solution obtained from urine was added was measured in the same manner as in Example 1. The results are shown in Table 50.

  The variance-covariance matrices of Group A and Group B shown in Table 50 were analyzed using a non-linear (second-order) discriminant analysis method based on Mahalanobis distance, as in Example 1. Table 51 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 51, a positive discrimination rate of 100% was obtained as shown in Table 52. As a result, it was confirmed that the diabetic nephropathy specimens could be grouped very accurately by using the diabetic complication test reagent of this example.

(Example 14)
In the same manner as in Example 1, βAla-AA ₃ -AA ₂ -AA in which a second fluorescent group consisting of FITC is bound to β-alanine at one end and a quenching group consisting of dinitrophenyl (Dnp) is bound to lysine at the other end _A reagent for testing diabetic complications (SEQ ID NO: 19, 30, 31, 32 in the sequence listing) having a substrate peptide consisting of _1- Lys (No 19, 30, 31, 32 in Table 53) was obtained.

<Measurement of enzyme activity>
Using 1 mg each of No19, 30, 31, and 32 reagents for testing diabetic complications, the amount of change in fluorescence value when a sample liquid obtained from urine was added was measured in the same manner as in Example 1. The results are shown in Table 54.

  The variance-covariance matrices of Group A and Group B shown in Table 54 were analyzed using a non-linear (second order) discriminant analysis method based on Mahalanobis distance, as in Example 1. Table 55 shows the discrimination scores obtained from the analysis results.

  From the discrimination score shown in Table 55, a positive discrimination rate of 100% was obtained as shown in Table 56. As a result, it was confirmed that the diabetic nephropathy specimens could be grouped very accurately by using the diabetic complication test reagent of this example.

  The present invention relates to a reagent for testing diabetic complications, in particular diabetic nephropathy, and a diagnostic test for diabetic complications, which is effective for measuring activity by contacting a body fluid such as urine or blood of a subject. With the simple operation, without identifying or following the markers contained in the body fluid of diabetic patients, whether or not diabetic nephropathy has developed, and if so, the degree of progression is from 1st to 4th And can refine the prediction of the progression of the disease state, such as the examination of the effectiveness of the first to fourth treatments and when to introduce artificial dialysis. It is possible to provide a reagent for testing for diabetic complications that is extremely effective in supporting diabetes diagnosis by the use of the above.

Schematic diagram showing the principle of enzyme activity detection of the reagent for testing for diabetic complications in the first embodiment Schematic diagram showing the principle of detecting enzyme activity of a reagent for testing for diabetic complications in Embodiment 2

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Reagent for diabetic complication test 2 Carrier 3 Substrate peptide 4,6 First fluorescent group 5 Enzyme 10 Diabetes complication test reagent 11 Substrate peptide 12 Quenching group 13,15 Second fluorescent group 14 Enzyme

Claims (3)

Four or more kinds of substrate peptides to which three amino acids are bonded are combined, and four of the above-mentioned combinations of substrate peptides are (a) Leu-Val-Tyr, His-Glu-Lys, Glu-Lys- Lys, Thr-Arg-Ala, (b) Leu-Gly-Arg, Gly-Arg-Ile, Gly-Arg-Thr, Gly-Arg-Gly, (c) Asn-Pro-Arg, Asp-Gly-Arg, Arg-Arg-Gln, Pro-Leu-Gly or Ala-Lys-Ser, (d) Gln-Arg-Arg, His-Leu-Lys, Glu-Lys-Gly, Ala-Arg-Ser, (e) Val- Leu-Lys, Gly-Arg-Ile, Ala-Lys-Ser, Gly-Arg-Gly, (f) Val-Val-Arg, Val-Pro-Arg, Thr-Arg-Val, Asn-Lys-Tyr, ( g) Gly-Arg-Ile, Gly-Arg-Thr, Phe-Lys-Ile, Ala-Lys-Ser, (h) Ala-Pro-Phe, Leu-Leu-Glu, Leu-Val-Tyr, Val-Leu -Lys, (i) Thr-Arg-Ala, Pro-Val-Arg, Gln-Arg-Ile, Leu-Met-Lys, (j) Val-Val-Arg, Pro-Val-Arg, Gln-Arg-Ile , Leu-Met-Lys, (k) Ser-Pro-Arg, Lys-Arg-Lys, Lys-Arg-Asp, Ser-Arg-Leu, (l) His-Leu-Lys, Lys-Arg-Lys, Lys -A diagnostic reagent for diabetic complications characterized by being either Arg-Asp or Ser-Arg-Leu . The diagnostic reagent for diabetes complication according to claim 1 , further comprising a first fluorescent group bonded to one terminal of the substrate peptide. The diabetic complication test according to claim 1 , further comprising: a second fluorescent group bonded to one end of the substrate peptide; and a quenching group bonded to the other end of the substrate peptide. reagent.

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