JPH0329400B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for measuring biological components, and more particularly, the present invention relates to a method for measuring biological components, and more specifically, a method for measuring biological components, which is characterized in that the reaction interference of coexisting bilirubin is avoided when hydrogen peroxide produced by a reaction is colorimetrically determined in the presence of peroxidase. This invention relates to a method for measuring and a reagent composition used therefor. In recent years, advances in enzymatic analysis methods in clinical chemical analysis have been remarkable, and oxidative enzymes such as glucose oxidase, cholesterol oxidase, uricase, acyl coenzyme A oxidase, choline oxidase, and glycerol-3-phosphate oxidase are widely used. Hydrogen peroxide produced by the action of these oxidizing enzymes is usually determined colorimetrically using a peroxidase-hydrogen donor system. Reaction systems using peroxidase are susceptible to interference from various reducing substances, administered drugs, biological pigments, etc. present in biological samples. Among these interfering substances, interference by bilirubin is discussed, for example, in Clinical Chemistry, Vol. 8, 63-
72 pages, published in 1979. According to this, the mechanisms of interference by bilirubin are: (1) Direct effect due to absorption of bilirubin, (2) Bilirubin acts as a hydrogen donor for peroxidase, and (3) Direct effect on the generated color pigment. It is being considered that the The serum concentration of bilirubin in normal people is 1 mg/
dl or less, but pathologically it can reach 20 mg/dl, and its influence is a serious problem in clinical tests. Known techniques related to these problems include, for example, a method using ferrocyanide in a method for quantifying glucose using a glucose oxidase-peroxidase system (Japanese Patent Publication No. 55-25840 and Clinical Chemistry Vol. 26, 227-231).
Page, 1981), a method using ferrocyanide ions in a method for quantifying uric acid using the uricase-peroxidase system (Japanese Unexamined Patent Publication No. 138656/1982), and a method using ascorbic acid in a method for quantifying uric acid by reaction with picric acid. Method for inhibiting oxidation of bilirubin (Japanese Patent Application Laid-Open No. 55-29718), Method for correcting interference due to bilirubin using a multi-wavelength photometer (Japanese Patent Application Laid-open No. 104239/1982), Hydrogen peroxide-peroxidase system A method of adding an EDTA-iron complex in a method for measuring serum components (Japanese Patent Application Laid-open No. 71398/1983), and
Publication No. 151193 describes a method for degrading bilirubin using a bilirubin-specific fungal enzyme composition. The last mentioned bilirubin-specific fungal enzyme composition reacts with bilirubin and
produces fluorescent substances and hydrogen peroxide.
Therefore, the present enzyme composition is not an advantageous method for avoiding the interference of bilirubin with the hydrogen peroxide-peroxidase system, which is the object of the present invention. The present inventors conducted intensive studies on methods for avoiding interference caused by coexisting bilirubin when measuring biological components, and as a result, they discovered that bilirubin oxidase has such an effect. Bilirubin oxidase is an enzyme newly discovered by the present inventors, and does not generate hydrogen peroxide through reaction. The method of the present invention will be described in detail below. Two types of bilirubin oxidase, which is one embodiment of the present invention, can be supplied. One of them is disclosed in Japanese Patent Application No. 57-25613 as bilirubin oxidase NS-1 produced by a bacterium of the genus Myrocesium. This bilirubin oxidase NS
-1 oxidizes bilirubin in the presence of molecular oxygen, producing a green substance but not hydrogen peroxide. This enzyme acts on direct (glucuronide) bilirubin, but not on indirect bilirubin bound to albumin. To avoid interference with bilirubin, it is necessary to remove these total bilirubins. In order to solve this problem, we have found that it is effective to add one or more selected from the group consisting of surfactants, aromatic carboxylic acids, sulfur agents, and proteases. It is thought that a reaction promoter consisting of one or more of these (hereinafter simply referred to as a reaction promoter) has a function of promoting the reaction between bilirubin oxidase NS-1 and indirect bilirubin. Surfactants include cations, anions, non-ions,
Either type of amphoteric acid may have the above-mentioned effects, such as sucrose fatty acid ester, bile salt, dodecyl sulfate, p-toluenesulfonic acid, cetylpyridinium chloride, nonylphenol ethoxylate, polyoxyethylene-polyoxypropylene condensate. Examples include surfactants such as surfactants. Examples of aromatic carboxylic acids include salicylic acid and sulfosalicylic acid, sulfur drugs include sulfanilamide and sodium acetosulfamine, and examples of proteases include pronase (manufactured by Kaken Chemical Co., Ltd.). Specifically, a method for avoiding bilirubin interference using bilirubin oxidase NS-1 is to use bilirubin oxidase NS-1 in the reaction system when measuring biological components.
-1 and the reaction accelerator may be added. As a result, bilirubin is oxidized, the yellow color characteristic of bilirubin decreases and disappears, and it changes to biliverdin, which becomes a hydrogen donor for peroxidase and does not act on pigments. In addition, if the enzyme bilirubin oxidase NS-1 itself acts on the coloring system, a coloring reaction inhibitor may be added as necessary. These inhibitors inhibit the action of bilirubin oxidase on the coloring system, but must not inhibit the action on bilirubin. Examples of inhibitors that satisfy this requirement include sodium fluoride, hydroxylamine, and phenanthroline. Another type of bilirubin oxidase was discovered by the present inventors in a bacterium of the genus Coprinus as a result of screening. This enzyme, unlike bilirubin oxidase NS-1, also acts on indirect bilirubin. However, in order to make the reaction proceed more quickly, it is preferable to add a reaction promoter such as a surfactant. The method for avoiding interference by bilirubin using this enzyme can be carried out in the same manner as in the case of bilirubin oxidase NS-1. The reagent composition used in the method of the present invention will be explained. The amount of enzyme contained in the reagent composition is 0.01 mμ/ml to 20 μ/ml as bilirubin oxidase activity.
ml can be used. Most preferably, bilirubin oxidase is present at a concentration of 0.01 to 10 μ/ml in the reaction solution.
For example, in the case of sodium dodecyl sulfate, the reaction promoting substance is 0.02 to 0.2% in the reaction solution, preferably 0.05%.
~0.1%, also 0.1 for sodium cholate
~1.0%, preferably 0.5-0.7%. In the case of sodium fluoride, the reaction inhibitor is in the reaction solution.
The same applies to hydroxylamine and phenanthroline, 0.5 to 10 mM, preferably 1 to 7 mM. Other buffers, enzyme stabilizers, etc. may be added as necessary. The present invention will be explained in detail below using a method for measuring enzyme activity, test examples, and examples. Bilirubin oxidase activity measurement method 0.2M containing 1mM of ethylenediaminetetraacetic acid
Dissolve 5 mg of reagent bilirubin (manufactured by Wako Pure Chemical Industries, Ltd.) in 250 ml of Tris-HCl buffer (PH8.4), react this 2 ml with 0.2 ml of enzyme solution at 37°C, and measure the decrease in absorbance at 440 nm. One unit is the amount of enzyme that oxidizes 1 micromole of bilirubin per minute. Test Example 1 Preparation of bilirubin oxidase NS-1 Myrothecium bercariae
verrucaria) MT−1FERM−BP No.653(FERM
-P No. 5918) strain was cultured in a glucose/potato medium to obtain a culture solution containing bilirubin oxidase NS-1. The culture solution was sequentially subjected to ammonium sulfate salting out, dialysis,
Activated carbon treatment, QAE-Sephadex A-50 column chromatography, ultrafiltration, and Sephadex G-100 column chromatography were performed to obtain purified bilirubin oxidase NS-1. This specimen was unique in polyacrylamide gel disk electrophoresis. Test Example 2 Preparation of bilirubin oxidase produced by Coprinus genus Coprinus cinereus
IFO8371 strain was cultured in potato glucose medium,
A culture solution containing bilirubin oxidase activity was obtained. The culture solution was sequentially subjected to ammonium sulfate salting out, dialysis, and DEAE-
Cellulose column chromatography, DEAE−
Purified bilirubin oxidase was obtained by performing Sepharose column chromatography, ultrafiltration, and Sephadex G-100 column chromatography. Test Example 3 Preparation of Lactucase Produced by Polyporus Bacteria Polyporus versicolor (Polyporus versicolor)
versicolor) IFO9791 strain with glucose, L-asparagine, DL-phenylalanine, adenine,
The cells were cultured in a medium containing thiamine and inorganic salts to obtain a culture medium containing lactcase activity. The culture solution was sequentially subjected to ammonium sulfate salting out, dialysis, ultrafiltration, DEAE-Sephadex A-50 column chromatography, ultrafiltration, and Sephadex G-100 column chromatography to obtain a purified sample. This specimen was disk electrophoretically single. Test Example 4 Preparation of tyrosinase derived from pine room Using Tyrosinase (Grade) manufactured by Sigma, this was further added to DEAE-Sephadex A-50.
Column chromatography, hydroxyapatite column chromatography, and DEAE-Sephadex A-50 column chromatography were performed to obtain a purified sample. The purity of this specimen was approximately 90% by disk electrophoresis. Test Example 5 Preparation of Urushi laccase Sumac sap was salted out, dialyzed, ultrafiltered, and then subjected to Sephadex G-200 column chromatography to obtain a partially purified sample. Test Example 6 Substrate specificity of various enzymes In order to clarify the difference between bilirubin oxidase used in the method of the present invention and other similar enzymes, substrate specificity was investigated. The enzymes were bilirubin oxidase NS-1 (abbreviated as A, hereinafter the same) prepared in Test Examples 1 to 4, bilirubin oxidase (B) produced by Coprinus spp., latcase (C) produced by Polyporus spp. Tyrosinase (E), latcase derived from sumac (D), and ascorbic acid oxidase derived from Japanese cucumber (Glade, manufactured by Toyobo) (F) were used. Various substrates at a concentration of 7mM shown in Table 1 and a certain amount of the above enzyme were added.
The reaction was carried out at 37°C and the enzyme activity was measured. Results first
Shown in the table. In the same table, the enzyme showing the highest activity for each substrate is expressed as a relative activity, with the enzyme showing the highest activity as 100.

[Table] As shown in Table 1, bilirubin oxidase had the strongest effect on bilirubin, but weak reactivity was also observed with other enzymes. Test example 7 Effect of reaction accelerator A reaction solution with the following composition was reacted at 37°C, and 440nm
The decrease in absorption of was measured. Bilirubin Control (manufactured by Dade), which is albumin-bound bilirubin, was used as a substrate. 0.1M Tris-HCl buffer (PH8.4) 3ml Bilirubin control (manufactured by Dade, 20mg/
dl) 20μ Reaction promoter (10% aqueous solution) (Pronase P is a 0.1% aqueous solution) 50μ Bilirubin oxidase NS-1 (15u/ml) (prepared in Test Example 1) 20μ The results are shown in Table 2.

[Table] As can be seen from Table 2, particularly preferred substances for promoting the reaction between the enzyme and bilirubin used in the present invention were sodium cholate, sodium dodecyl sulfate, and salicylic acid. Test Example 8 Effect of color reaction inhibitor 3ml of 0.1M phosphate buffer (PH7.5) containing 0.4mM 4-aminoantipyrine and 15mM phenol
and bilirubin oxidase NS-1 (15u/ml)
30 μ and 200 μ of sodium fluoride or phenanthroline at various concentrations were reacted at 37° C. for 40 minutes, and the increase in absorbance at 505 nm was measured. The results are shown in Table 3.

[Table] From Table 3, bilirubin oxidase NS-1
It can be seen that the color development of the dye due to the influence of fluoride was inhibited by the addition of sodium fluoride or phenanthroline. Example 1 Glucose measurement reagent composition A 0.1M phosphate buffer (PH7.5) containing the following 4-aminoantipyrine 0.4mM Phenol 15mM Sodium fluoride 1mM Glucose oxidase (Amano Pharmaceutical) 17μ/ml Peroxidase (Boehringer) ) 0.3 μ/ml Sodium cholate 0.6% Bilirubin oxidase NS-1 (prepared in Test Example 1) 0.1 μ/ml Test solution B Glucose solution (including bilirubin controls at various concentrations) 200 mg/dl Reagent composition A 3 ml and test solution B20μ, 37
After reacting at ℃ for 20 minutes, absorbance at 505 nm was measured. As a control, a system excluding bilirubin oxidase NS-1 and/or bilirubin control was operated in the same manner. The results are shown in Figure 1. In the figure, -〇- represents the case where bilirubin oxidase NS-1 was added, and -●- represents the case when it was not added. It was found that by the method of the present invention, the influence of bilirubin coexisting in the test solution can be almost ignored. Example 2 Measurement of Glucose The same procedure as in Example 1 was carried out by varying the concentration of the glucose solution of test solution B described in Example 1 above.
The concentration of bilirubin control contained in test solution B is 20 mg/dl. The results are shown in Figure 2. -〇- in the figure is a calibration curve according to the method of the present invention,
Others were conducted as controls - without bilirubin and bilirubin oxidase NS-1 -△-, without bilirubin oxidase NS-1 -
●Represents − respectively. In the measured values obtained by the method of the present invention, almost no interference by bilirubin was observed. Example 3 Measurement of Glucose The same procedure as in Example 2 was carried out using bilirubin oxidase (3 mμ/ml) of the genus Coprinus prepared in Test Example 2 in place of the bilirubin oxidase NS-1 of Example 2. As a result, the calibration curve was the same as the result of Example 2. Example 4 Cholesterol measurement reagent composition A Cholesterol C-Test "Wako" (manufactured by Wako Pure Chemical Industries, Ltd., containing cholesterol esterase, cholesterol oxidase, and peroxidase), which is a kit for measuring total cholesterol, was dissolved in 75 ml of buffer solution, Into this were dissolved 1 mM sodium fluoride, 0.6% sodium cholate, and 0.1 u/ml of bilirubin oxidase NS-1 prepared in Test Example 1. Test solution B: The standard serum attached to the Cholesterol C-Test "Wako" was used, and various concentrations of bilirubin control were added thereto. After reacting 3ml of reagent composition A and 20μ of test solution B at 37°C for 20 minutes, the absorbance at 505nm was measured. Bilirubin oxidase NS-1 and/or
Alternatively, the same procedure was performed for a system excluding the bilirubin control. The results are shown in Figure 3. In the figure, -〇- represents the case where bilirubin oxidase NS-1 was added, and -●- represents the case when it was not added. It was found that by the method of the present invention, the influence of bilirubin coexisting in the test solution can be almost ignored. Example 5 Reagent composition for measuring neutral fat A commercially available kit for measuring neutral fat, TG Kit-GN (containing lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase, and peroxidase manufactured by Nippon Shoji Co., Ltd.) was used. Using. Solution A - One vial of enzyme reagent in the kit was dissolved with 15 ml of color reagent. Solution B - 1mM sodium fluoride in the coloring reagent,
0.6% sodium cholate and bilirubin oxidase NS-1 prepared in Test Example 1.
Each was dissolved to a concentration of 0.2 u/ml. Test Solution C Triolein solutions of various concentrations were prepared, and bilirubin control was dissolved therein to a concentration of 10 mg/dl. Mix 1.5ml of solution A and 20μ of test solution C, and incubate at 37℃ for 5 minutes.
Preheat for 37°C, then add 1.5ml of B solution.
After 15 minutes of reaction, absorbance at 505 nm was measured. The results are shown in Figure 4. In the figure, -〇- is the calibration curve obtained by the method of the present invention, and the others are -△-, bilirubin oxidase NS-1, which was conducted as a control without bilirubin and bilirubin oxidase NS-1.
If 1 is not included, -●- is respectively represented. In the measured values obtained by the method of the present invention, almost no interference by bilirubin was observed. Example 6 Measurement of neutral fat Sodium fluoride and sodium cholate were removed from solution B of Example 5, and bilirubin oxidase (0.2 The procedure was carried out in the same manner as in Example 5 using the following procedure.
As a result, all the calibration curves were the same as those of Example 5. Example 7 Measurement of Glucose Instead of the test solution B of Example 1, the same procedure as in Example 1 was performed using test solutions prepared by adding various concentrations of bilirubin control to human serum. The glucose concentration in the serum used was 96 mg/dl, and the bilirubin concentration was 0.6 mg/dl. The results are shown in Figure 5.
In the same figure, -〇- is bilirubin oxidase
When NS-1 is added, -●- represents the case where it is not added. The effect of the present invention is clear from FIG. Example 8 Measurement of Glucose in Serum The same procedure as in Example 1 was performed except that sodium fluoride was removed from the reagent composition of Example 1 and the serum sample was used as a test solution. The glucose concentration was determined from a calibration curve prepared in advance using a glucose standard solution and was found to be 96.1 mg/dl. As a control, when sodium cholate and bilirubin oxidase were omitted, the concentration was 90.2 mg/dl. Furthermore, when 1 mM of sodium fluoride was added to the above reagent and the operation was performed, the glucose concentration in the sample did not change, but the absorbance of the blank reaction solution was low, making the operation convenient. The total bilirubin concentration in the sample used was 12.5 mg/dl. Example 9 Measurement of Total Cholesterol in Serum The same procedure as in Example 4 was carried out using sodium dodecyl sulfate (0.07%) in place of the sodium cholate in the reagent composition of Example 4 and using the serum sample as the test solution. The total cholesterol concentration was determined from a calibration curve prepared in advance using a cholesterol standard solution and was found to be 163 mg/dl. As a control, when sodium fluoride, sodium dodecyl sulfate, and bilirubin oxidase were omitted, the concentration was 145 mg/dl. The total bilirubin concentration in the sample used was 15.2 mg/dl. Example 10 Measurement of neutral fats in serum In the reagent composition of Example 5, phenanthroline (2.5 mM) was used instead of sodium fluoride, and bilirubin oxidase produced by Coprinus sp. The same procedure as in Example 5 was carried out using the serum sample as the test solution. When the neutral fat concentration in the sample was determined from a calibration curve prepared in advance using triolein standard solution, it was found to be 78.0 as triolein.
It was mg/dl. phenanthroline as a control;
When sodium cholate and bilirubin oxidase were removed, the concentration was 68.6 mg/dl.
The total bilirubin concentration in the sample used was
It was 14.2 mg/dl. Example 11 Reagent composition for measuring free fatty acids in serum 0.1M phosphate buffer containing the following (PH6.75) Acyl coenzyme A synthetase (manufactured by Amano Pharmaceutical) 0.4u/ml Acyl coenzyme A oxidase (manufactured by Amano Pharmaceutical) 5u /ml Peroxidase (Boehringer) 5u/ml Adenosine triphosphate 4μM Coloring agent MMX (Kyowa Medics) 50μM Triton X-100 0.1% Bilirubin oxidase NS-1 0.1u/ml Sodium cholate 0.6% Sodium fluoride 2.5mM Mix 3 ml of the above reagent composition with 20 μ of serum sample, react at 37°C for 20 minutes, measure the absorbance at 660 nm, and calculate the free fatty acid concentration in the sample using a calibration curve prepared in advance using an oleic acid standard solution. When I calculated it, it was 210μEq/. For comparison, when the operation was performed without bilirubin oxidase, sodium cholate, and sodium fluoride, the amount was 182 μEq/. The total bilirubin concentration in the sample used was 10.8 mg/dl.
It was hot. Example 12 Measurement of serum phospholipids Determiner, a commercially available kit for measuring phospholipids
Using PL (manufactured by Kyowa Medics, containing 4-aminoantipyrine, phenol, phospholipase D, choline oxidase, and peroxidase),
Add 0.07% sodium dodecyl sulfate to the kit solution.
%, 2.5 mM of phenanthroline, and bilirubin oxidase produced by Coprinus bacteria prepared in Test Example 2 were dissolved to a concentration of 0.05 u/ml. Mix 3ml of the reagent solution with 20μ of serum sample, react at 37°C for 20 minutes, measure the absorbance at 500nm, and calculate the phospholipid concentration in the sample from a calibration curve prepared in advance using a choline chloride standard solution. When I calculated it, it was 277 mg/dl as choline chloride.
It was hot. Sodium dodecyl sulfate as a control;
When phenanthroline and bilirubin oxidase were removed, the concentration was 262 mg/dl. The total bilirubin concentration in the sample used was 14.1
It was mg/dl.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the interference of coexisting bilirubin when glucose is quantified by the method of the present invention. In the figure, -○- indicates the results obtained by the method of the present invention, and -●- indicates the results obtained by removing bilirubin oxidase NS-1 from the results. FIG. 2 is a diagram showing a calibration curve for glucose according to the method of the present invention. In the figure, -〇- represents the results obtained by the method of the present invention, -●- represents the results when bilirubin oxidase NS-1 was removed, and -△- represents the results when bilirubin oxidase NS-1 and bilirubin were removed. FIG. 3 is a diagram showing the interference of coexisting bilirubin when cholesterol is quantified by the method of the present invention. In the figure, -○- indicates the results obtained by the method of the present invention, and -●- indicates the results obtained by removing bilirubin oxidase NS-1 from the results. FIG. 4 is a diagram showing a neutral fat calibration curve obtained by the method of the present invention. In the same figure -
〇- is the result according to the method of the present invention, -●- is the result when bilirubin oxidase NS-1 is removed from it, -△- is the result when bilirubin oxidase NS-1 is removed.
and results excluding bilirubin. FIG. 5 is a diagram showing the interference of coexisting bilirubin when glucose in serum is determined by the method of the present invention. In the figure, -0- indicates the results obtained by the method of the present invention, and -●- indicates the results obtained by excluding bilirubin oxidase.

Claims (1)

[Scope of Claims] 1. A method for measuring biological components in human serum, in which bilirubin oxidase and one or more reaction promoters selected from the group consisting of surfactants, aromatic carboxylic acids, sulfur drugs, and proteases are used. A method for measuring biological components, which is characterized by adding a substance to avoid interference from bilirubin coexisting in a sample. 2. The method for measuring a biological component according to claim 1, wherein the bilirubin oxidase is an enzyme produced by a strain belonging to the genus Myrocesium or a strain belonging to the genus Coprinus. 3. The method for measuring biological components according to claim 1, wherein the method for measuring biological components includes a hydrogen peroxide-peroxidase-chromogenic hydrogen donor system. 4. In addition to bilirubin oxidase and one or more reaction promoters selected from the group consisting of surfactants, aromatic carboxylic acids, sulfur drugs, and proteases, a coloring reaction inhibitor may be added as necessary. The method for measuring biological components according to claim 1 or 3, wherein the action of the enzyme on the coloring system is inhibited. 5. The method for measuring a biological component according to claim 4, wherein the coloring reaction inhibitor has the property of not inhibiting the action of the enzyme on bilirubin, but inhibiting the action of the enzyme on the coloring system. 6. The method for measuring biological components according to claim 4 or 5, wherein the color reaction inhibiting substance is sodium fluoride, hydroxylamine, or phenanthroline. 7 Contains bilirubin oxidase and one or more reaction promoters selected from the group consisting of surfactants, aromatic carboxylic acids, sulfur drugs, and proteases to avoid interference of bilirubin in measuring biological components in human serum. A reagent composition having the property of 8. The reagent composition according to claim 7, wherein the bilirubin oxidase is an enzyme produced by a strain belonging to the genus Myrocesium or a strain belonging to the genus Coprinus. 9. The reagent composition according to claim 7, wherein the measurement of biological components comprises a hydrogen peroxide-peroxidase-chromogenic hydrogen donor system. 10 In addition to bilirubin oxidase and one or more reaction promoters selected from the group consisting of surfactants, aromatic carboxylic acids, sulfur drugs, and proteases, it also contains a coloring reaction inhibitor, if necessary, and 8. The reagent composition according to claim 7, which has a property of inhibiting the action of an enzyme on a coloring system. 11. The reagent composition according to claim 10, wherein the reaction inhibitor has the property of not inhibiting the reaction between the enzyme and bilirubin, but inhibiting the action of the enzyme on the coloring system. 12. The reagent composition according to claim 10 or 11, wherein the reaction inhibitor is sodium fluoride, hydroxylamine, or phenanthroline.