Creatinine measurement method and creatinine measurement reagent

Abstract

Suppresses measurement errors. [Solution] In the creatinine measurement reagent according to the present invention, the R1 reagent converts intermediate substances (creatine, sarcosine, etc.) contained in the sample into hydrogen peroxide, and the converted hydrogen peroxide is converted into a colorless quinone. Subsequently, the R2 reagent converts the CRE contained in the sample into hydrogen peroxide via the intermediate substances, and the converted hydrogen peroxide is converted into a quinone dye. This makes it possible to suppress the situation in which intermediate substances contained in the sample affect the measurement value, and thus it is possible to suppress the error in the measurement value. In particular, the inclusion of multiple types of Trinder reagents in the R1 reagent makes it possible to reduce the error in the measurement value.

Description

Technical Field

[0001] The present invention relates to a creatinine measurement method and a creatinine measurement reagent.

Background Art

[0002] Conventionally, as a creatinine measurement method, creatinine contained in a sample is converted into hydrogen peroxide via intermediate substances such as creatine and sarcosine, and in the coexistence of peroxidase, a dye generated by oxidative condensation of a coupler and a Trinder reagent which is a hydrogen donor compound is colorimetrically quantified (see Patent Document 1). However, in this method, when an intermediate substance is contained in the sample, there is a risk of an error in the measured value. Here, as a method for removing hydrogen peroxide contained in a sample, a method of reacting a hydrogen donor compound with hydrogen peroxide using peroxidase to convert it into colorless quinone is known (see Patent Document 2).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, in the conventional creatinine measurement method using peroxidase, there is a risk that the error in the measured value becomes large. In particular, when selecting a Trinder reagent, it is necessary to consider the influence of coexisting substances including creatine and various performances. Therefore, in reagent development, it is more advantageous to be able to use various Trinder reagents rather than being able to use only a specific Trinder reagent. An object of the present invention is to suppress an error in a measured value.

Means for Solving the Problems

[0005] To achieve the above objective, the creatinine measurement method according to the first invention is a creatinine measurement method that converts creatinine contained in a sample to hydrogen peroxide via an intermediate substance, and then colorimetrically quantifies the pigment generated by oxidative condensation of a coupler and a Trinder reagent in the presence of peroxidase, characterized in that the hydrogen peroxide converted from the intermediate substance contained in the sample is pretreated with a reagent containing peroxidase and a plurality of types of Trinder reagents. In the creatinine measurement method according to the first invention, during the pretreatment, intermediate substances contained in the sample are converted to hydrogen peroxide, and the converted hydrogen peroxide is then converted to a colorless quinone by peroxidase and multiple types of Trinder reagents. At this time, multiple Trinder reagents, each having a different degree of influence from the intermediate substances contained in the sample, cancel each other out. This makes it possible to suppress the situation in which intermediate substances contained in the sample affect the measurement value, and thus to reduce the error in the measurement value. In particular, by using multiple types of Trinder reagents during the pretreatment, it is possible to reduce the error in the measurement value.

[0006] The creatinine measuring reagent according to the second invention is characterized by comprising a first reagent containing creatinase, sarcosine oxidase, peroxidase, and multiple types of Trinder reagents, and a second reagent containing creatininase and a coupler. In the creatinine measurement reagent according to the second invention, the first reagent converts intermediate substances (creatine, sarcosine, etc.) contained in the sample into hydrogen peroxide, and the converted hydrogen peroxide is then converted into a colorless quinone. At this time, multiple Trinder reagents, each having a different degree of influence from the intermediate substances in the sample, cancel each other out. Subsequently, the second reagent converts the creatinine contained in the sample into hydrogen peroxide via the intermediate substances, and the converted hydrogen peroxide is then converted into a quinone dye. This makes it possible to suppress the influence of intermediate substances in the sample on the measurement value and to reduce the error in the measurement value. In particular, the inclusion of multiple types of Trinder reagents in the first reagent makes it possible to reduce the error in the measurement value. [Effects of the Invention]

[0007] According to the present invention, it is possible to suppress errors in measurement values. [Brief explanation of the drawing]

[0008] [Figure 1] This figure shows an example of Trinder's reagent. [Figure 2] This figure shows the reaction equation during the pretreatment process. [Figure 3] This figure shows the reaction equations during the post-treatment process. [Figure 4] This graph shows the error rate when measuring the concentration of CRE contained in a sample using the creatinine measurement reagents described in Comparative Examples 1 to 7. [Figure 5] This figure shows the error rate when measuring the concentration of CRE contained in a sample using the creatinine measurement reagents according to Examples 1 to 8. [Figure 6] This figure shows the error rate when measuring the concentration of CRE contained in a sample using the creatinine measurement reagents according to Examples 9 to 15. [Figure 7] This figure shows the relationship between the combination of Trinder reagents and the error rate. [Figure 8]This figure shows the error rate when measuring the concentration of CRE contained in a sample using the creatinine measurement reagents described in Examples 16 to 18. [Figure 9] This figure shows the error rate when measuring the concentration of CRE contained in a sample using the creatinine measurement reagent according to Example 19. [Figure 10] This figure shows the error rate when measuring the concentration of CRE contained in a sample using the creatinine measurement reagent according to Example 20. [Modes for carrying out the invention]

[0009] Embodiments of the present invention will be described below with reference to the drawings. The creatinine measurement method and creatinine measurement reagent according to the embodiment of the present invention are used to measure the concentration of creatinine (hereinafter referred to as "CRE") contained in a sample. As a "sample," anything that may contain CRE and needs to be measured can be used. For example, samples can include biological samples, meat, vegetables, grains, fruits, marine products, processed foods, beverages, drinking water, well water, river water, lake water, seawater, soil, air, pharmaceuticals, etc. Examples of "biological samples" include human or animal blood, serum, plasma, urine, feces, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, organs such as the brain, tissues such as hair, skin, nails, muscles or nerves, cells, etc. Here, the "sample" is preferably a liquid. However, if the sample containing CRE is not a liquid, pretreatment such as extraction or solubilization may be performed according to known methods to make the CRE contained in the liquid. Biological samples are preferred as "samples," and blood, serum, plasma, or urine are particularly preferred. The creatinine measurement method and creatinine measurement reagent according to the embodiment of the present invention can be used to calculate the estimated glomerular filtration rate (eGFR), which is a criterion for evaluating renal function.

[0010] (Composition of creatinine measurement reagent) First, a creatinine measurement reagent according to an embodiment of the present invention will be described. Figure 1 shows an example of Trinder's reagent. The creatinine measurement reagent according to an embodiment of the present invention comprises an R1 reagent used for pretreatment and an R2 reagent used for posttreatment. The R1 reagent consists of creatinase (hereinafter referred to as "CR"), sarcosine oxidase (hereinafter referred to as "SOD"), peroxidase (hereinafter referred to as "POD"), and several types of Trinder reagents. Trinder reagents are a general term for compounds that exhibit a characteristic color when oxidatively condensed with a coupler (such as 4-AAP) as a hydrogen donor, and can be either phenol derivatives or aniline derivatives. Furthermore, the R1 reagent may contain, as appropriate, buffers, chelating agents, enzyme stabilizers (salts), agents to prevent interference from coexisting substances, preservatives, surfactants, antifoaming agents, etc. Here, it is preferable to adjust the pH of the sample after adding reagent R1 to within the range of pH 7 to pH 8.5. As a buffer for adjusting to this pH range, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (hereinafter referred to as "TAPS"), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (hereinafter referred to as "TES"), etc., can be used.

[0011] As shown in Figure 1, the Trinder reagents include 3-hydroxy-2,4,6-triiodobenzoic acid (hereinafter referred to as "HTIB"), N-sulfopropylaniline (hereinafter referred to as "HALPS"), N-sulfopropyl-3,5-dimethoxyaniline (hereinafter referred to as "HDAPS"), N-sulfopropyl-3-methoxy-5-methylaniline (hereinafter referred to as "HMMPS"), N-(2-carboxyethyl)-N-ethyl-m-toluidine (hereinafter referred to as "CEMB"), N-(2-carboxyethyl)-N-ethyl-3-methoxyaniline (hereinafter referred to as "CEMO"), N-(2-carboxyethyl)-N-ethyl-3,5-dimethoxyaniline (hereinafter referred to as "CEDB"), and N-(2-hydroxy-3-sulfopropyl)-3,5- Dimethoxyaniline (hereinafter referred to as "HDAOS"), N,N-bis(4-sulfobutyl)-3-methylaniline (hereinafter referred to as "TODB"), N,N-bis(4-sulfobutyl)-3,5-dimethylaniline (hereinafter referred to as "MADB"), N-ethyl-N-sulfopropylaniline (hereinafter referred to as "ALPS"), N-ethyl-N-sulfopropyl-m-anisidine (hereinafter referred to as "ADPS"), N-ethyl-N-sulfopropyl-3-methylaniline (hereinafter referred to as "TOPS (ESPMT)"), N-ethyl-N-sulfopropyl-3,5-dimethylaniline (hereinafter referred to as "MAPS"), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (hereinafter referred to as "DAPS"), N-ethyl-N-sulfopropyl-3,5-Dimethoxy-4-fluoroaniline (hereinafter referred to as "FDAPS"), N-ethyl-N-sulfobutyl-m-toluidine (hereinafter referred to as "ESBMT"), N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine (hereinafter referred to as "EMAE"), N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine (hereinafter referred to as "EMSE"), N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (hereinafter referred to as "ALOS"), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (hereinafter referred to as "TOOS(EHSPT)"), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (hereinafter referred to as "ADOS"), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (hereinafter referred to as "MAOS"), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (hereinafter referred to as "DAOS"), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline (hereinafter referred to as "FDAOS"), etc. can be used.,

[0012] In particular, the R1 reagent contains multiple types (two or more types) of Trinder reagents as exemplified above. At this time, it is preferable that the R1 reagent contains a Trinder reagent that causes a positive error in the measured value and a Trinder reagent that causes a negative error in the measured value. In particular, it is preferable that the R1 reagent contains a specific Trinder reagent. That is, it is preferable that the R1 reagent contains a specific Trinder reagent and a Trinder reagent having a different structure (basic skeleton) from the specific Trinder reagent. Here, the "specific Trinder reagent" is a Trinder reagent having a structure in which N-(2-hydroxy-3-sulfopropyl) aniline is used as the basic skeleton and at least one methoxy group is bonded to the meta position. That is, the specific Trinder reagent is a Trinder reagent composed of N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline. Examples of the specific Trinder reagent include ADOS, DAOS, FDAOS, HDAOS, etc. Here, the "Trinder reagent that causes a positive error in the measured value" means that, for the R1 reagent, when the concentration of creatinine contained in a sample containing creatinine as a coexisting substance is measured using the R1 reagent and the R2 reagent with the structure in which the Trinder reagent is contained alone, the error of the measured value with respect to the true value (theoretical value) is a positive (+) Trinder reagent. On the other hand, the "Trinder reagent that causes a negative error in the measured value" means that, for the R1 reagent, when the concentration of creatinine contained in a sample containing creatinine as a coexisting substance is measured using the R1 reagent and the R2 reagent with the structure in which the Trinder reagent is contained alone, the error of the measured value with respect to the true value (theoretical value) is a negative (-) Trinder reagent.

[0013] The R2 reagent is composed of creatinine kinase (hereinafter referred to as "CRN") and a coupler. In addition, buffers (buffer solutions), coexisting substance influence avoiders (salts), enzyme stabilizers (salts), surfactants, defoamers, preservatives, etc. are appropriately added to the R2 reagent. 4-aminoantipyrine (hereinafter referred to as "4-AAP") can be used as a coupler. Here, it is preferable to adjust the pH of the sample after adding the R2 reagent to within the range of pH 7 to pH 8.5. Buffers such as TAPS and TES can be used to adjust the pH to this range.

[0014] (Creatinine measurement method) Next, a creatinine measurement method according to an embodiment of the present invention will be described. Figure 2 shows the reaction equation for the pretreatment. Figure 3 shows the reaction equation for the posttreatment. A creatinine measurement method according to an embodiment of the present invention comprises a pretreatment, a posttreatment, and a measurement process. The creatinine measurement method according to an embodiment of the present invention first involves pretreatment. In the pretreatment, reagent R1 is added to the sample and stirred. As shown in Figure 2, if creatine is present in the sample, it is decomposed into sarcosine and urea by the action of CR. Next, the sarcosine present in the sample and the sarcosine produced by the decomposition are decomposed into glycine, formaldehyde (HCHO), and hydrogen peroxide (H2O2) by the action of SOD. Then, the hydrogen peroxide produced by the decomposition is converted into a colorless quinone by the action of POD in the presence of multiple types of Trinder reagents. At this time, multiple Trinder reagents, each having a different degree of influence depending on the intermediate substances contained in the sample, cancel each other out. Note that in the pretreatment, since reagent R1 does not contain CRN, the CRE contained in the sample does not change (react). As a result, it becomes possible to convert the intermediate substance (creatine sarcosine) contained in the sample into a colorless quinone, thereby suppressing the situation in which hydrogen peroxide generated from the intermediate substance affects the measurement values. After the pretreatment is complete, posttreatment is performed. In posttreatment, reagent R2 is added to the sample after reagent R1 has been added, and the mixture is stirred. As shown in Figure 3, if CRE is present in the sample, it undergoes ring opening by the action of CRN, and creatine is produced. The produced creatine is then decomposed into sarcosine and urea by the action of CR. The sarcosine produced by the decomposition is then decomposed into glycine, formaldehyde, and hydrogen peroxide by the action of SOD. The hydrogen peroxide produced by the decomposition then undergoes oxidative condensation with 4-AAP and several types of Trinder reagents in the presence of POD, producing quinone dyes. Once post-processing is complete, the measurement process is performed. In this process, a known automated analyzer is used to measure the absorbance of the quinone dye generated during post-processing at wavelengths that include the absorption wavelength range of the quinone dye. This makes it possible to quantify the concentration (amount) of CRE present in the sample.

[0015] (Operation of the Embodiment of the Present Invention) In the creatinine measurement reagent according to the embodiment of the present invention, the R1 reagent converts intermediate substances (creatine, sarcosine, etc.) contained in the sample into hydrogen peroxide, and the converted hydrogen peroxide is converted into a colorless quinone. At this time, multiple Trinder reagents, each having a different degree of influence from the intermediate substances contained in the sample, cancel each other out. Subsequently, the R2 reagent converts the CRE contained in the sample into hydrogen peroxide via the intermediate substances, and the converted hydrogen peroxide is converted into a quinone dye. This makes it possible to suppress the influence of intermediate substances contained in the sample on the measurement value and to reduce the error in the measurement value. In particular, the inclusion of multiple types of Trinder reagents in the R1 reagent makes it possible to reduce the error in the measurement value. Furthermore, in the creatinine measurement method according to the embodiment of the present invention, during the pretreatment, intermediate substances contained in the sample are converted to hydrogen peroxide, and the converted hydrogen peroxide is converted to a colorless quinone by POD and multiple types of Trinder reagents. At this time, multiple Trinder reagents, each having a different degree of influence from the intermediate substances contained in the sample, cancel each other out. This makes it possible to suppress situations in which intermediate substances contained in the sample affect the measurement value, and thus to suppress errors in the measurement value. In particular, by using multiple types of Trinder reagents during the pretreatment, it is possible to reduce errors in the measurement value.

[0016] (Examples) Next, embodiments of the present invention will be described. Figure 4 is a graph showing the error rate when the concentration of CRE contained in a sample is measured using the creatinine measuring reagents according to Comparative Examples 1 to 7. Figure 5 is a diagram showing the error rate when the concentration of CRE contained in a sample is measured using the creatinine measuring reagents according to Examples 1 to 8. Figure 6 is a diagram showing the error rate when the concentration of CRE contained in a sample is measured using the creatinine measuring reagents according to Examples 9 to 15. Figure 7 is a diagram showing the relationship between the combination of Trinder reagents and the error rate. Figure 8 is a diagram showing the error rate when the concentration of CRE contained in a sample is measured using the creatinine measuring reagents according to Examples 16 to 18. Figure 9 is a diagram showing the error rate when the concentration of CRE contained in a sample is measured using the creatinine measuring reagent according to Example 19. Figure 10 is a diagram showing the error rate when the concentration of CRE contained in a sample is measured using the creatinine measuring reagent according to Example 20.

[0017] First, as a comparative example, a creatinine measurement reagent consisting of reagent R1 containing one type of Trinder reagent and reagent R2 was prepared. The composition of reagent R1 (pH 8.0) in the comparative example is as follows: TAPS = 50 mM, NaCl = 10 mM, SOD = 15 U / mL, CR = 50 U / mL, POD = 10 U / mL, ascorbate oxidase (hereinafter referred to as "ASO") = 10 U / mL, and Trinder's reagent = 10 mM. Furthermore, the composition of the R2 reagent (pH 8.0) in the comparative example is as follows: TAPS = 100 mM, potassium ferrocyanide = 0.15 mM, 4-AAP = 3 mM, MgCl2 = 2 mM, and CRN = 450 U / mL. As comparative examples, we prepared the following creatinine measurement reagents: one containing HTIB in the R1 reagent (Comparative Example 1), one containing ADOS in the R1 reagent (Comparative Example 2), one containing TODB in the R1 reagent (Comparative Example 3), one containing TOOS in the R1 reagent (Comparative Example 4), one containing DAOS in the R1 reagent (Comparative Example 5), one containing MADB in the R1 reagent (Comparative Example 6), and one containing MAOS in the R1 reagent (Comparative Example 7). As samples, we prepared a base sample consisting of pooled serum without added creatine, sample 1 with 10 mg / dL of creatine added to the pooled serum, sample 2 with 20 mg / dL of creatine added to the pooled serum, sample 3 with 30 mg / dL of creatine added to the pooled serum, sample 4 with 40 mg / dL of creatine added to the pooled serum, and sample 5 with 50 mg / dL of creatine added to the pooled serum. The sample volume was 6 μL, the sample dilution ratio was 5 times, the amount of reagent R1 was 60 μL, and the amount of reagent R2 was 20 μL. The creatinine concentration in each sample was measured using the creatinine measurement reagents described in Comparative Examples 1 to 7. The ratio of the measured values ​​of each sample (Samples 1 to 5) to the measured value of the base sample was then calculated as the error rate.

[0018] As a result, as shown in Figure 4, it was confirmed that the error rates for the creatinine measurement reagent related to Comparative Example 1 were 118% for sample 1, 112% for sample 2, 106% for sample 3, 106% for sample 4, and 110% for sample 5. On the other hand, regarding the creatinine measurement reagent related to Comparative Example 2, it was confirmed that the error rates were 96% for sample 1, 92% for sample 2, 91% for sample 3, 90% for sample 4, and 92% for sample 5. On the other hand, regarding the creatinine measurement reagent in Comparative Example 3, it was confirmed that the error rates were 118% for sample 1, 120% for sample 2, 122% for sample 3, 123% for sample 4, and 126% for sample 5. On the other hand, regarding the creatinine measurement reagent in Comparative Example 4, it was confirmed that the error rates were 104% for sample 1, 110% for sample 2, 112% for sample 3, 114% for sample 4, and 115% for sample 5. On the other hand, regarding the creatinine measurement reagent for Comparative Example 5, it was confirmed that the error rates were 94% for sample 1, 101% for sample 2, 110% for sample 3, 119% for sample 4, and 127% for sample 5. On the other hand, regarding the creatinine measurement reagent in Comparative Example 6, it was confirmed that the error rates were 114% for sample 1, 117% for sample 2, 119% for sample 3, 120% for sample 4, and 122% for sample 5. On the other hand, regarding the creatinine measurement reagent in Comparative Example 7, it was confirmed that the error rates were 117% for sample 1, 130% for sample 2, 135% for sample 3, 140% for sample 4, and 146% for sample 5. Based on the above, it was confirmed that the creatinine measurement reagents used in Comparative Examples 1 to 7 were greatly affected by the presence of creatine in the sample, resulting in a large error rate. Furthermore, it was confirmed that there are two types of Trinder reagents: those that produce a positive error relative to the measured value, and those that produce a negative error relative to the measured value. Specifically, HTIB, TODB, TOOS, ALPS, HDAOS, DAOS, MADB, and MAOS all have a positive (+) error rate, indicating that they are Trinder reagents that produce a positive error relative to the measured value. On the other hand, ADOS has a negative (-) error rate, indicating that it is a Trinder reagent that produces a negative error relative to the measured value.

[0019] Next, as an example of the present invention, a creatinine measurement reagent consisting of reagent R1 containing two types of Trinder reagents and reagent R2 was prepared. The composition of reagent R1 (pH 8.0) in the example is as follows: TAPS = 50 mM, NaCl = 10 mM, SOD = 15 U / mL, CR = 50 U / mL, POD = 10 U / mL, ASO = 10 U / mL, Trinder's reagent 1 = 5 mM, Trinder's reagent 2 = 5 mM. Furthermore, the composition of the R2 reagent (pH 8.0) used in the example is as follows: TAPS = 100 mM, potassium ferrocyanide = 0.15 mM, 4-AAP = 3 mM, MgCl2 = 2 mM, and CRN = 450 U / mL. Furthermore, as examples, we have a creatinine measurement reagent containing HTIB·ADOS in the R1 reagent (Example 1), a creatinine measurement reagent containing ADOS·TOOS in the R1 reagent (Example 2), a creatinine measurement reagent containing ADOS·ALPS in the R1 reagent (Example 3), a creatinine measurement reagent containing ADOS·HDAOS in the R1 reagent (Example 4), a creatinine measurement reagent containing TODB·HDAOS in the R1 reagent (Example 5), a creatinine measurement reagent containing TODB·DAOS in the R1 reagent (Example 6), a creatinine measurement reagent containing TOOS·HDAOS in the R1 reagent (Example 7), and a creatinine measurement reagent containing TOOS·DAOS in the R1 reagent. We prepared the following creatinine measurement reagents: one without creatinine (Example 8), one containing ALPS·HDAOS in the R1 reagent (Example 9), one containing ALPS·DAOS in the R1 reagent (Example 10), one containing ALPS·MAOS in the R1 reagent (Example 11), one containing HDAOS·MADB in the R1 reagent (Example 12), one containing HDAOS·MAOS in the R1 reagent (Example 13), one containing DAOS·MADB in the R1 reagent (Example 14), and one containing DAOS·MAOS in the R1 reagent (Example 15). As samples, the base sample described above, and samples 1 to 5 were prepared. The sample volume was 6 μL, the sample dilution ratio was 5 times, the amount of reagent R1 was 60 μL, and the amount of reagent R2 was 20 μL. The creatinine concentration in each sample was measured using the creatinine measurement reagents described in Examples 1 to 15. The ratio of the measured values ​​of each sample (Sample 1 to Sample 5) to the measured value of the base sample was then calculated as the error rate.

[0020] As a result, as shown in Figure 5(a), it was confirmed that the error rates for the creatinine measurement reagent according to Example 1 were 97% for sample 1, 96% for sample 2, 97% for sample 3, 97% for sample 4, and 99% for sample 5. In particular, it was confirmed that the measurement error was smaller for HTIB and ADOS compared to when they were used individually. On the other hand, as shown in Figure 5(b), it was confirmed that the error rates for the creatinine measurement reagents according to Example 2 were 99% for sample 1, 99% for sample 2, 99% for sample 3, 98% for sample 4, and 99% for sample 5. In particular, it was confirmed that the measurement error was smaller for ADOS and TOOS compared to when each was used individually. On the other hand, as shown in Figure 5(c), the error rates for the creatinine measurement reagents according to Example 3 were confirmed to be 102% for sample 1, 102% for sample 2, 102% for sample 3, 100% for sample 4, and 97% for sample 5. In particular, it was confirmed that the measurement error was smaller for ADOS and ALPS compared to when each was used individually. On the other hand, as shown in Figure 5(d), it was confirmed that the error rates for the creatinine measurement reagents according to Example 4 were 93% for sample 1, 92% for sample 2, 92% for sample 3, 92% for sample 4, and 93% for sample 5. In particular, it was confirmed that the measurement error was smaller for ADOS and HDAOS compared to when each was used alone. On the other hand, as shown in Figure 5(e), the error rates for the creatinine measurement reagents in Example 5 were confirmed to be 100% for sample 1, 104% for sample 2, 107% for sample 3, 109% for sample 4, and 113% for sample 5. In particular, it was confirmed that the error in the measured values ​​was smaller for TODB and HDAOS compared to when they were used individually. On the other hand, as shown in Figure 5(f), the error rates for the creatinine measurement reagents according to Example 6 were confirmed to be 89% for sample 1, 96% for sample 2, 101% for sample 3, 105% for sample 4, and 108% for sample 5. In particular, it was confirmed that the error in the measured values ​​was smaller for TODB and DAOS compared to when they were used individually. On the other hand, as shown in Figure 5(g), the error rates for the creatinine measurement reagents according to Example 7 were confirmed to be 95% for sample 1, 95% for sample 2, 94% for sample 3, 96% for sample 4, and 96% for sample 5. In particular, it was confirmed that the error in the measured values ​​was smaller for TOOS and HDAOS compared to when they were used individually. On the other hand, as shown in Figure 5(h), the error rates for the creatinine measurement reagents in Example 8 were confirmed to be 97% for sample 1, 98% for sample 2, 103% for sample 3, 108% for sample 4, and 112% for sample 5. In particular, it was confirmed that the error in the measured values ​​was smaller for TOOS and DAOS compared to when they were used individually.

[0021] On the other hand, as shown in Figure 6(a), the error rates for the creatinine measurement reagents according to Example 9 were confirmed to be 100% for sample 1, 102% for sample 2, 105% for sample 3, 108% for sample 4, and 111% for sample 5. In particular, it was confirmed that the measurement error was smaller for ALPS and HDAOS compared to when each was used alone. On the other hand, as shown in Figure 6(b), the error rates for the creatinine measurement reagents in Example 10 were confirmed to be 92% for sample 1, 101% for sample 2, 107% for sample 3, 111% for sample 4, and 116% for sample 5. In particular, it was confirmed that the error in the measured values ​​was smaller for ALPS and DAOS compared to when they were used individually. On the other hand, as shown in Figure 6(c), the error rates for the creatinine measurement reagents in Example 11 were confirmed to be 118% for sample 1, 128% for sample 2, 135% for sample 3, 139% for sample 4, and 144% for sample 5. In particular, it was confirmed that the measurement error was smaller for ALPS and MAOS compared to when each was used individually. On the other hand, as shown in Figure 6(d), the error rates for the creatinine measurement reagents in Example 12 were confirmed to be 99% for sample 1, 100% for sample 2, 102% for sample 3, 103% for sample 4, and 104% for sample 5. In particular, it was confirmed that the measurement error was smaller for HDAOS and MADB compared to when each was used individually. On the other hand, as shown in Figure 6(e), the error rates for the creatinine measurement reagents in Example 13 were confirmed to be 101% for sample 1, 104% for sample 2, 107% for sample 3, 109% for sample 4, and 111% for sample 5. In particular, it was confirmed that the measurement error was smaller for HDAOS and MAOS compared to when they were used individually. On the other hand, as shown in Figure 6(f), the error rates for the creatinine measurement reagents in Example 14 were confirmed to be 90% for sample 1, 90% for sample 2, 94% for sample 3, 99% for sample 4, and 103% for sample 5. In particular, it was confirmed that the error in the measured values ​​was smaller for DAOS and MADB compared to when each was used individually. On the other hand, as shown in Figure 6(g), the error rates for the creatinine measurement reagents in Example 15 were confirmed to be 82% for sample 1, 85% for sample 2, 87% for sample 3, 90% for sample 4, and 92% for sample 5. In particular, it was confirmed that the error in the measured values ​​was smaller for DAOS and MAOS compared to when they were used individually.

[0022] Based on the above, for example, when looking at the error rate of sample 5, as shown in Figure 7, it can be seen that the error in the measured value is smaller when using the R1 reagent with two types of Trinder reagents than when using the R1 reagent with Trinder reagent alone. In particular, it can be seen that the error in the measured values ​​is reduced when using R1 reagents that contain Trinder reagents that produce a positive error in the measured values ​​(TODB, TOOS, ALPS, HDAOS) and Trinder reagents that produce a negative error in the measured values ​​(ADOS). Furthermore, it can be seen that the error in the measured values ​​is reduced when using R1 reagents that contain specific Trinder reagents (ADOS, DAOS, HDAOS) and Trinder reagents that have a different composition (basic structure) from the specific Trinder reagents. Furthermore, it can be seen that the error in the measured values ​​is smaller when using R1 reagents containing HTIB and ADOS, R1 reagents containing ADOS and TODB, R reagents containing ADOS and TOOS, R1 reagents containing ADOS and ALPS, R1 reagents containing ADOS and HDAOS, R1 reagents containing TODB and DAOS, R1 reagents containing TOOS and HDAOS, R1 reagents containing HDAOS and MADB, R1 reagents containing DAOS and MADB, and R1 reagents containing DAOS and MAOS.

[0023] Next, as an example of the present invention, a creatinine measurement reagent consisting of reagent R1 containing two types of Trinder reagents and reagent R2 was prepared. The composition of reagent R1 (pH 7.3) in the example is as follows: TES = 50 mM, NaCl = 1%, SOD = 12 U / mL, CR = 135 U / mL, POD = 80 U / mL, ASO = 2 U / mL, and Trinder's reagent 1 + Trinder's reagent 2 = 10 mM. Furthermore, the composition of the R2 reagent (pH 8.3) used in the example is as follows: TAPS = 100 mM, potassium ferrocyanide = 0.075 mM, 4-AAP = 3 mM, MgCl2 = 2 mM, and CRN = 200 U / mL. As examples, the following creatinine measurement reagents were prepared: Example 16a, containing ADOS and MAOS in a 10:0 ratio in reagent R1; Example 16b, containing ADOS and MAOS in an 8:2 ratio in reagent R1; Example 16c, containing ADOS and MAOS in a 6:4 ratio in reagent R1; Example 16d, containing ADOS and MAOS in a 5:5 ratio in reagent R1; Example 16e, containing ADOS and MAOS in a 4:6 ratio in reagent R1; Example 16f, containing ADOS and MAOS in a 2:8 ratio in reagent R1; and Example 16g, containing ADOS and MAOS in a 0:10 ratio in reagent R1. Furthermore, as examples, the following creatinine measurement reagents were prepared: one containing ADOS·TOOS in a ratio of 10:0 in the R1 reagent (Example 17a), one containing ADOS·TOOS in a ratio of 8:2 in the R1 reagent (Example 17b), one containing ADOS·TOOS in a ratio of 6:4 in the R1 reagent (Example 17c), one containing ADOS·TOOS in a ratio of 4:6 in the R1 reagent (Example 17d), one containing ADOS·TOOS in a ratio of 2:8 in the R1 reagent (Example 17e), and one containing ADOS·TOOS in a ratio of 0:10 in the R1 reagent (Example 17af). Furthermore, as examples, the following creatinine measurement reagents were prepared: one containing ADOS·TODB in a ratio of 10:0 in reagent R1 (Example 18a), one containing ADOS·TODB in a ratio of 8:2 in reagent R1 (Example 18b), one containing ADOS·TODB in a ratio of 6:4 in reagent R1 (Example 18c), one containing ADOS·TODB in a ratio of 4:6 in reagent R1 (Example 18d), one containing ADOS·TODB in a ratio of 2:8 in reagent R1 (Example 18e), and one containing ADOS·TODB in a ratio of 0:10 in reagent R1 (Example 18f). As samples, the base sample described above, and samples 1 to 5 were prepared. The sample volume was 12 μL, the sample dilution ratio was 5 times, the amount of reagent R1 was 60 μL, and the amount of reagent R2 was 20 μL. The CRE concentration in each sample was measured using the creatinine measurement reagents from Examples 16a to 16g. The CRE concentration in each sample was also measured using the creatinine measurement reagents from Examples 17a to 17f. Furthermore, the CRE concentration in each sample was measured using the creatinine measurement reagents from Examples 18a to 18f. The error rate was calculated as the ratio of the measured values ​​of each sample (Sample 1 to Sample 5) to the measured value of the base sample.

[0024] As a result, as shown in Figure 8(a), it was confirmed that, regardless of the ratio of ADOS to MAOS, the error in the measurement values ​​was smaller for the creatinine measurement reagents related to Examples 16a to 16g compared to when ADOS and MAOS were used individually. In particular, for reagent R1, it was confirmed that the error in the measurement values ​​was smaller when the ADOS content was higher than when the MAOS content was lower. Furthermore, as shown in Figure 8(b), it was confirmed that, regardless of the ratio of ADOS to TOOS, the error in the measured values ​​was smaller for the creatinine measurement reagents used in Examples 17a to 17f compared to when ADOS and TOOS were used individually. In particular, for reagent R1, it was confirmed that the error in the measured values ​​was smaller when the ADOS content was lower than when the TOOS content was higher. Furthermore, regarding the creatinine measurement reagents used in Examples 18a to 18f, it was confirmed that the measurement error was smaller compared to when ADOS and TODB were used individually, regardless of the ratio of ADOS to TODB. In particular, with the R1 reagent, it was confirmed that the measurement error was smaller when the ADOS content was higher than when the TODB content was lower. As described above, by selecting two types of Trinder reagents to be used in mixture, and then changing the mixing ratio of each Trinder reagent, it is possible to reduce the error in the measurement.

[0025] Next, as an example of the present invention, a creatinine measurement reagent consisting of reagent R1 containing three types of Trinder reagents and reagent R2 was prepared. The composition of reagent R1 (pH 8.0) in the example is as follows: TAPS = 50 mM, NaCl = 10 mM, SOD = 15 U / mL, CR = 50 U / mL, POD = 10 U / mL, ASO = 10 U / mL, and Trinder's reagent 1 + Trinder's reagent 2 + Trinder's reagent 3 = 10 mM. Furthermore, the composition of the R2 reagent (pH 8.0) used in the example is as follows: TAPS = 100 mM, potassium ferrocyanide = 0.15 mM, 4-AAP = 3 mM, MgCl2 = 2 mM, and CRN = 450 U / mL. As an example, a creatinine measurement reagent (Example 19) was prepared using reagent R1 containing HTIB, ADOS, and TOOS in a ratio of 2:5:3. As samples, the base sample described above, and samples 1 to 5 were prepared. The sample volume was 6 μL, the sample dilution ratio was 5 times, the amount of reagent R1 was 60 μL, and the amount of reagent R2 was 20 μL. The creatinine concentration in each sample was measured using the creatinine measurement reagent described in Example 19. The ratio of the measured values ​​of each sample (Sample 1 to Sample 5) to the measured value of the base sample was then calculated as the error rate. As a result, as shown in Figure 9, it was confirmed that the error in the measurement values ​​was smaller for HTIB, ADOS, and TOOS when using the creatinine measurement reagents in Example 19 compared to when each was used individually. This shows that it is possible to reduce the error in measurement values ​​by selecting three types of Trinder reagents to be used in mixture and then adjusting the mixing ratio of each Trinder reagent, indicating that the number of Trinder reagents used in mixture is not limited to two.

[0026] Next, as an example of the present invention, a creatinine measurement reagent consisting of reagent R1 containing four types of Trinder reagents and reagent R2 was prepared. The composition of reagent R1 (pH 8.0) in the example is as follows: TAPS = 50 mM, NaCl = 10 mM, SOD = 15 U / mL, CR = 50 U / mL, POD = 10 U / mL, ASO = 10 U / mL, and Trinder's reagent 1 + Trinder's reagent 2 + Trinder's reagent 3 + Trinder's reagent 4 = 10 mM. Furthermore, the composition of the R2 reagent (pH 8.0) used in the example is as follows: TAPS = 100 mM, potassium ferrocyanide = 0.15 mM, 4-AAP = 3 mM, MgCl2 = 2 mM, and CRN = 450 U / mL. As an example, a creatinine measurement reagent (Example 20) was prepared in which ADOS, TODB, TOOS, and HDAOS were present in a ratio of 3:2:2:3 in the R1 reagent. As samples, the base sample described above, and samples 1 to 5 were prepared. The sample volume was 6 μL, the sample dilution ratio was 5 times, the amount of reagent R1 was 60 μL, and the amount of reagent R2 was 20 μL. The creatinine concentration in each sample was measured using the creatinine measurement reagent described in Example 20. The ratio of the measured values ​​of each sample (Sample 1 to Sample 5) to the measured value of the base sample was then calculated as the error rate. As a result, as shown in Figure 10, it was confirmed that the error in measurement values ​​was smaller for the creatinine measurement reagents in Example 20 compared to when ADOS, TODB, TOOS, and HDAOS were used individually. This shows that it is possible to reduce the error in measurement values ​​by selecting four types of TRINDER reagents to be used in mixture and then adjusting the mixing ratio of each TRINDER reagent, indicating that the number of TRINDER reagents used in mixture is not limited to two or three types.

Claims

1. A method for measuring creatinine, which involves converting creatinine contained in a sample to hydrogen peroxide via an intermediate substance, and then colorimetrically quantifying the pigment generated by oxidative condensation of a coupler and Trinder's reagent in the presence of peroxidase, A method for measuring creatinine, characterized by pretreatment of hydrogen peroxide converted from the intermediate substance contained in the sample with a reagent containing peroxidase and several types of Trinder reagents.

2. A first reagent comprising creatinase, sarcosine oxidase, peroxidase, and several types of Trinder reagents, A creatinine measuring reagent characterized by comprising a second reagent containing creatininase and a coupler.

Images