JPH054079B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention provides a reagent composition for bilirubin containing a novel bilirubin oxidase, and a method using the same for quantifying bilirubin in a test fluid, such as a body fluid, or for eliminating the inhibitory effect of bilirubin. The present invention relates to methods for using the reagent compositions in analytical methods requiring such methods. Conventional technology Conventionally, a chemical measurement method called the diazo method using a diazonium salt such as diazosulfanilic acid has been mainly used to quantify bilirubin (Izumi Kanai and Masamitsu Kanai co-authored, Clinical Test Methods Proposal, No. 28
Edition, page XII-24, 1978, Kanehara Publishing). However, chemical measurement methods have drawbacks such as the specificity of the reaction, the complexity of the operation, and the irritant and corrosive nature of the reagents, and there are many problems in terms of installation, maintenance, management, and accurate measurement in automatic analyzers. One thing has been pointed out. Additionally, methods for quantifying bilirubin using enzymes are being developed recently. As an enzyme that reacts with bilirubin, R. Brodersen (R.
Brodersen) and P. Bortels (P.
Bortels first reported it [Europ.J.Biochem., Vol. 10, p. 465 (1969)].
They state that insoluble bilirubin oxidase isolated from guinea pig brain oxidizes bilirubin to biliverdin, but they do not investigate whether hydrogen peroxide is included in the reaction product. There is also a description that the enzyme composition of Agaricus bisporus has the effect of specifically reacting with bilirubin to cause a color change (Japanese Patent Publication No. 11194/1983). The reaction product between the enzyme composition and bilirubin has a wavelength of about 510 nm.
It is a substance that exhibits an absorption peak at , and emits fluorescence at about 525 nm when excited at an excitation wavelength of 450 nm.Hydrogen peroxide may or may not be produced depending on the method of adjusting the enzyme composition. A method has been proposed for quantifying bilirubin in body fluids using this bilirubin-specific fungal enzyme composition. It is very unclear whether it is catalyzed by the system. Furthermore, since the properties of the enzyme composition with respect to bilirubin have not been sufficiently described, it is difficult to say that it is an advantageous method for quantifying bilirubin. Furthermore, there is no description of the effect of this enzyme on albumin-bound and glucuronic acid-bound bilirubin, which are among the forms of bilirubin present in serum. Recently, when bilirubin oxidase produced by the genus Myrocesium is used to quantify bilirubin in body fluids, since the enzyme alone does not act on albumin-bound bilirubin, a surfactant or the like is added as a reaction accelerator. A method has been proposed to quantify the
No. 17999). On the other hand, when analyzing various components in body fluids such as serum and urine, one way to remove bilirubin from body fluids is to add a small amount of potassium ferrocyanide or potassium ferricyanide to the sample and use the redox potential to remove bilirubin and other components. A method of enzymatically quantifying glucose in body fluids by removing the interference of reducing substances (Special Publication No. 55-25840), a method of measuring body fluid components by reducing the reducing power of bilirubin using an oxidizing agent (Special Publication No. 55-25840), Japanese Patent Publication No. 107161/1982), a method for measuring target components in body fluids by decomposing bilirubin using the bilirubin-specific fungal enzyme composition mentioned above and removing its interference (Japanese Patent Publication No. 11194/1982), etc. There is. Among the bilirubin-specific fungal enzyme compositions used in the last method are compositions that produce hydrogen peroxide. The composition is not advantageous for assays using oxidase-peroxidase-hydrogen-donating chromogen reactions. Recently, a method for measuring bilirubin in body fluids has been known, which uses bilirubin oxidase, which oxidizes bilirubin but does not produce hydrogen peroxide, to remove interference from bilirubin coexisting in body fluids (Japanese Patent Application Laid-Open No. 1983-1993). −18000).
The feature of this measurement method is that a bilirubin oxidase reaction promoter (such as a surfactant) and a coloring reaction inhibitor (such as sodium fluoride) are added at the same time as bilirubin oxidase. However, bilirubin oxidase is a peroxidase used to detect hydrogen peroxide.
Chromogen in the hydrogen-donating chromogen coloring method, for example 4
- It has the effect of quantitatively oxidizing and condensing aminoantipyrine and phenol to develop a red color.
If a color reaction inhibitor is added to the reaction solution, bilirubin oxidase is also inhibited at the same time, so bilirubin cannot be removed sufficiently and it is difficult to obtain accurate measurements of body fluid components. The present inventors previously screened for bilirubin oxidase produced by Basidiomycetes, and found that Schizophyllum commune
found that bilirubin oxidase was produced in the culture solution (Japanese Patent Application No. 10149/1982). However, this enzyme was somewhat unstable and unsuitable for industrial production, and as a result of further intensive studies on bilirubin oxidase produced by Basidiomycetes, we found that Trachyderma tsunodae, which belongs to the genus Ebitake, was used in culture. We discovered that bilirubin oxidase, which has excellent properties, is produced in large quantities and clarified the enzymological properties of this enzyme. As a result, it was concluded that this enzyme is a new bilirubin oxidase different from the bilirubin oxidase that has already been reported, and a patent application was filed as Japanese Patent Application No. 58-218895 (see Japanese Patent Publication No. 3-24198). This enzyme is named novel bilirubin oxidase M-1. Purpose of the Invention The purpose of the present invention is to provide a reagent composition for bilirubin containing the novel bilirubin oxidase M-1, a method for quantifying bilirubin using the same, and a method for analyzing bilirubin that requires eliminating the interfering effect of bilirubin. An object of the present invention is to provide a method of using the composition. Structure of the Invention To summarize the present invention, the first invention of the present invention relates to a reagent composition for bilirubin, and is characterized in that the composition contains a novel bilirubin oxidase M-1. A second invention of the present invention is an invention relating to a method for quantifying bilirubin, in which a reagent composition containing the novel bilirubin oxidase M-1 is applied to a bilirubin-containing test solution, and a change in bilirubin occurs thereby. It is characterized by optically measuring. Furthermore, a third invention of the present invention relates to an analysis method, in which components other than bilirubin in a bilirubin-containing test solution in which bilirubin acts as an inhibitor of analysis are subjected to an oxidase-peroxidase-hydrogen-donating chromogen reaction. (a) a step of treating the test liquid with a reagent composition containing the novel bilirubin oxidase M-1 to eliminate the inhibitory effect of coexisting bilirubin; and (b) a step of eliminating the inhibitory effect of coexisting bilirubin. It is characterized by including the steps of analyzing the target component in the liquid. First, each property of the novel bilirubin oxidase M-1 in the present invention will be shown. (1) Enzyme-chemical and physicochemical properties of novel bilirubin oxidase M-1 (1) Action: This enzyme oxidizes bilirubin (albumin-bound type, glucuronic acid-bound type, free type) in the presence of oxygen, producing biliverdin. It has the effect of producing a pale purple substance and then an almost colorless substance, but it does not produce hydrogen peroxide. (2) Substrate specificity: Acts on bilirubin. It also acts on bilirubin, but at a rate of about 12% of the oxidation rate of bilirubin. It also acts on hydroquinone, pyrocatechol, para-phenylenediamine, pyrogallol, 4-aminoantipyrine and monophenols. It has no effect on hemin, vitamin _B12 and hemoglobin. (3) Optimal PH and PH stability: The optimal PH of this enzyme is around 5.5, and 37
It is stable between PH5 and PH9 when treated at each PH for 60 minutes at ℃. (4) Optimal temperature and thermostability: The optimal temperature of this enzyme is around 50℃, and the pH
7.0, it is stable up to 55°C when treated for 10 minutes at each temperature. (5) Molecular weight: The molecular weight of this enzyme is Cephacryl S-200.
(manufactured by Pharmacia), the gel filtration method uses approx.
It is 83000. (6) Uniformity: Disc electrophoresis was performed using 7.5% polyacrylamide gel (PH9.4), and protein staining and activity staining were performed. A single protein staining band was observed. Furthermore, since this enzyme has the effect of quantitatively developing a red color from 4-aminoantipyrine and phenol, when activity staining was performed, a red band was observed at the same position as the protein staining band. (7) Isoelectric point: The isoelectric point of this enzyme is 3.92±0.05 as determined by focusing electrophoresis using Pharmalite (PH3 to PH10, manufactured by Pharmacia). (8) Effects of metal ions, inhibitors, etc.: This enzyme is inhibited by L-ascorbic acid, sodium azide, dithiothreitol, potassium cyanide, L-cysteine, EDTA, Fe ²⁺ , etc. (Table 1) ).

【table】

[Table] (9) Visible absorption spectrum: When the visible absorption spectrum of this enzyme solution was measured, an absorption maximum was observed at 610 nm, indicating that it was blue protein. (10) Sugar content: Disc electrophoresis was performed using a 7.5% polyacrylamide gel (PH9.4), and PAS
Staining [Analytical Biochem. Vol. 30, p. 148 (1969)]
When this was performed, the same area as the protein staining position was stained. From this, this enzyme is a glycoprotein containing sugar. The sugar content is approximately 4.5% when measured by the phenol-sulfuric acid method. (11) Copper content: When the amount of copper in this enzyme was determined using an atomic absorption spectrometer, it was found that 4 mol of copper was contained per 1 mol of enzyme. (12) Enzyme activity measurement method: Enzyme activity was determined by decreasing the absorption of bilirubin at 460 nm. That is, 0.1 ml of omega high bilirubin control serum (manufactured by Highland, USA), 300 micromoles of phosphate buffer (PH7.0), and 0.1 ml of an appropriately diluted enzyme solution.
The reaction was carried out at 37° C. for 10 minutes in a reaction volume of 3.0 ml, and the decrease in absorption at 460 nm based on bilirubin was measured. Novel bilirubin oxidase M-1
One unit of is expressed as the amount of enzyme that oxidizes 1 micromole of bilirubin per minute in the above reaction system. Table 2 shows a comparison of the novel bilirubin oxidase M-1 used in the present invention with the conventional bilirubin oxidase.

【table】

[Table] Bilirubin oxidase M-1 according to the present invention
The reagent composition containing the above-mentioned novel bilirubin oxidase M-1 may contain conventional ingredients. In particular, addition of alkali metal and alkaline earth metal salts of deoxycholic acid, such as sodium deoxycholate, accelerates the reaction. The amount of enzyme contained in the reagent composition is at least 0.0001 unit, preferably 10 to
The unit is 0.001, and it is adjusted as appropriate depending on the measurement time and purpose. Sodium deoxycholate is
It is contained in an amount of 0.01 to 1.0%, preferably 0.05 to 0.5%.
Other known agents such as buffers and enzyme stabilizers may be added as necessary. The reagent composition can be provided by known methods such as solution, lyophilization, or impregnation into a carrier sheet. Enzymes can also be used by binding them to insoluble carriers according to known methods. (2) Bilirubin quantification method According to the present invention, the above-mentioned novel bilirubin
Utilizing the properties of oxidase M-1 [(1) action], bilirubin can be quantified in bilirubin-containing test fluids, such as body fluids such as serum and urine, or pretreated fluids thereof. The above-mentioned novel bilirubin oxidase M-1 used in the present invention oxidizes bilirubin in the presence of oxygen to produce biliverdin, and further produces a pale purple substance and then an almost colorless substance, so it can be used in body fluids such as serum and urine. The amount of bilirubin can be determined by the rate of decrease in absorbance at 460 nm after the novel bilirubin oxidase M-1 is applied. For example, add 0.1 to 2.0 units of novel bilirubin oxidase M-1 to 10 to 100μ of a sample such as serum with a buffer solution of pH 5.5 to 7.0 at 20 to 40℃, preferably 37℃.
React for 30 minutes, preferably 5-10 minutes, and add 460n before adding new bilirubin oxidase M-1.
The bilirubin concentration in the sample can be measured by obtaining the difference between the absorbance of m and the absorbance after reaction.
In addition, the novel bilirubin oxidase M-1 used in the present invention has a remarkable reaction promoting effect with sodium deoxycholate compared to sodium cholate, which is a previously reported reaction promoter for bilirubin oxidase (Table 3). . Table 3 Additives (final concentration 0.167%) Relative activity (%) No additives 100 Sodium cholate 120 Sodium deoxycholate 1650 The novel bilirubin oxidase M-1 used in the present invention is a The enzyme acts alone on all of the existing forms: albumin-bound, glucuronic acid-bound, and free. Enzymes with such properties have not been previously known. These properties make it superior to known enzymes in quantifying bilirubin and eliminating the bilirubin effect described below. (3) Method for eliminating the bilirubin effect in the analysis of body fluid components Next, when analyzing various components in body fluids such as serum and urine using the oxidase-peroxidase-hydrogen-donating chromogen reaction, coexisting bilirubin can be removed using a new bilirubin oxidase. The method for removing M-1 will be described. As described in the above property [(6) Uniformity], this enzyme is a chromogen in the peroxidase-hydrogen-donating chromogen coloring method used to detect hydrogen peroxide, such as 4-aminoantipyrine-phenol. It has the effect of quantitatively oxidizing the system and causing it to develop a red color. Therefore, for example, in methods for measuring various components such as glucose and total cholesterol, samples are prepared using the novel bilirubin oxidase M-1.
After removing the effect of bilirubin, which is a reducing substance, by adding an appropriate inhibitor, for example, sodium azide [Property (8) Effects of metal ions, inhibitors, etc. described above], new bilirubin can be produced. - Completely inhibits the action of oxidase M-1 on hydrogen-donating chromogens, and as a result, it is possible to obtain accurate measurements of various components such as glucose and total cholesterol. The reaction between the novel bilirubin oxidase M-1 and the specimen can be carried out under appropriate conditions depending on the quantitative method used for the body fluid component to be quantified, but usually a specimen sample such as serum or urine is used.
Novel bilirubin oxidase M at ~100μ
Inhibition of novel bilirubin oxidase M-1 after reacting 0.01 to 0.20 units of -1 with a buffer solution of pH 5.5 to 7.0 at 20 to 40°C, preferably 37°C, for 1 to 30 minutes, preferably for 10 to 20 minutes. The final concentration of the agent is 1-50mM, preferably 5mM of sodium azide.
M, and after that, usual methods for measuring various components can be carried out. Furthermore, in the above treatment method, the novel bilirubin oxidase M-1 inhibitor may be added to the reagents for measuring various components, and then added to the specimen treated with the novel bilirubin oxidase M-1. In addition, we tested the sample with novel bilirubin oxidase M-
1, the reaction accelerator sodium deoxycholate (final concentration 0.05-0.5
%), the amount of enzyme used for pretreatment can be reduced and the pretreatment time can also be shortened. DESCRIPTION OF EXAMPLES The method of using the novel bilirubin oxidase according to the present invention will be illustrated below with examples, but the present invention is not limited to the scope of the following examples. Example 1 Determination of bilirubin by absorbance reduction method at 460 nm 5 mg/dl, 10 mg/dl, 15 mg/dl, 20 mg/dl, 25
Prepare bilirubin solutions (albumin-binding type, manufactured by Daiichi Chemical Co., Ltd.) of mg/dl and 30 mg/dl, and add 0.3M potassium phosphate buffer (PH
7.0) 1.0ml, novel bilirubin oxidase M-
Add 0.5 units of 1 to make the total volume of each 3.0ml, 37
Incubate at ℃ for 10 minutes and measure absorbance at 460 nm (OD sample). Separately, as a control, the absorbance at 460 nm (OD blank) was measured for each bilirubin solution without the addition of novel bilirubin oxidase M-1, and ΔOD ₄₆₀ (OD blank - OD sample) was determined. The results are shown in Figure 1. That is, FIG. 1 is a graph showing the calibration curve as a relationship between bilirubin concentration (mg/dl) (horizontal axis) and absorbance difference (ΔOD ₄₆₀ ) (vertical axis). As is clear from FIG. 1, by using the novel bilirubin oxidase M-1 used in the present invention, it is possible to accurately measure bilirubin in body fluids from the change in absorbance at 460 nm. Example 2 Quantification of bilirubin by absorbance reduction method at 460 nm (effect of sodium deoxycholate) Each bilirubin solution was prepared in the same manner as in Example 1,
Add 0.1ml of this solution to 0.3M potassium phosphate buffer (PH
7.0) 1.0ml, 1.2% Sodium Deoxycholate 0.5
ml, 0.05 of novel bilirubin oxidase M-1
Add the units, make a total volume of 3.0 ml each, react at 37°C for 10 minutes, and measure the absorbance at 460 nm (OD sample). Separately, as a control, the absorbance at 460 nm (OD blank) was measured for each bilirubin solution without the addition of novel bilirubin oxidase M-1.
ΔOD ₄₆₀ (OD blank − OD sample) was determined.
The results are shown in Figure 2. As is clear from Figure 2, if sodium deoxycholate, which is a reaction accelerator of the novel bilirubin oxidase M-1, is added to quantify bilirubin, the amount of enzyme used will be greater than when sodium deoxycholate is not added. can be significantly reduced to about 1 in 10. Example 3 Removal of interference caused by bilirubin in serum total cholesterol measurement system (1) Coloring reagent: 167 mg of 4-aminoantipyrine,
1.32 g of phenol and 80 mg (100 units/mg) of horseradish peroxidase (manufactured by Sigma Chemical Co. (USA), type) were dissolved in 100 ml of 0.1 M potassium phosphate buffer (PH7.0). (2) Cholesterol esterase solution: Coriolus versicolor K-1213, FERM
- Deposited as P No. 4820), cottonseed oil, peptone,
The cells were cultured in a medium containing corn staple liquor to obtain a culture solution containing cholesterol esterase activity. The culture solution was sequentially subjected to Amberlite CG-50 column chromatography, ultrafiltration, DEAE-Sephadex column chromatography, SP-Sephadex column chromatography, and Sephadex G-150 column chromatography to obtain purified enzymes. Standard specimen (70 units/
mg) was obtained (according to the method described in the Japanese Society of Agricultural Chemistry, Abstracts of 1981 Conference, page 93). 5 mg of the sample was dissolved in 10 ml of 0.1M potassium phosphate buffer (PH7.0) (35 units/ml). (3) Cholesterol oxidase solution: Agrocybe pediades K-1, FERM
- Deposited as P No. 4343) was cultured in a medium containing soluble starch, soybean oil, peptone, etc. to obtain a culture solution containing cholesterol oxidase activity. The culture solution was sequentially added to Amberlight CG.
−50 column chromatography, ultrafiltration,
A purified enzyme preparation (15 units/mg) was obtained by performing DEAE-Sephadex column chromatography, SP-Sephadex column chromatography, and Sephadex G-100 column chromatography (Japan Agricultural Chemistry Society, Showa (According to the method described in the 1953 Conference Abstracts, page 99).
10 mg of this preparation was dissolved in 5 ml of 0.1M potassium phosphate buffer (PH7.0) (30 units/ml). (4) Procedure: In a test tube, add 0.1 ml of novel bilirubin oxidase M-1 (0.02 units), 1 ml of 0.3 M potassium phosphate buffer (PH7.0), 0.3 ml of 3% Triton X-100 solution, and control serum [ Hyland (USA) 0.02ml, 20mg/dl Albumin-bound bilirubin [Daiichi Chemical Co., Ltd.] 0.02
ml and reacted with shaking in a constant temperature bath at 37℃ for 20 minutes, then added 50mM sodium azide.
Add 0.3 ml, color reagent 0.3 ml, cholesterol esterase solution 0.1 ml, cholesterol oxidase solution 0.1 ml, and distilled water 0.76 ml,
After reacting for a minute, absorbance at 500 nm was measured. Separately, as a control, novel bilirubin oxidase M
-1 without treatment and without the addition of albumin-bound bilirubin, the absorbance was measured and the removal of bilirubin color development inhibition in a serum total cholesterol measurement system was investigated using this enzyme. The results are shown in Table 4.

[Table] As is clear from Table 4, by using the method of the present invention, it became possible to remove the interference of bilirubin and obtain accurate serum total cholesterol values. Example 4 Removal of interference caused by bilirubin in serum total cholesterol measurement system (effect of sodium deoxycholate) (1) Coloring reagent: The coloring reagent of Example 3 was used. (2) Cholesterol esterase solution and cholesterol oxidase solution: The enzyme solutions of Example 3 were used, respectively. (3) Procedure: In a test tube, add 0.1 ml of new bilirubin oxidase M-1 (0.002 units), 1.0 ml of 0.3 M potassium phosphate buffer (PH7.0), and control serum [manufactured by Highland Corporation (USA)]. 0.02ml, 20
Added 0.02 ml of mg/dl albumin-bound bilirubin [manufactured by Daiichi Chemical Co., Ltd.], 0.25 ml of 1.2% sodium deoxycholate, and 0.11 ml of distilled water, and reacted with shaking in a constant temperature bath at 37°C for 10 minutes. After, 3
Add 0.3 ml of % Triton Absorbance was measured. Separately, as a control, the absorbance was measured without treatment with novel bilirubin oxidase M-1 and without the addition of albumin-bound bilirubin, and the effect of sodium deoxycholate on removing bilirubin color inhibition in serum total cholesterol measurement system was investigated. investigated. The results are shown in Table 5.

[Table] As is clear from Table 5, by using sodium deoxycholate to remove the interference of bilirubin, it became possible to reduce the amount of enzyme used for pretreatment and to shorten the pretreatment time. Example 5 Removal of interference caused by bilirubin in serum glucose measuring system (1) Coloring reagent: The coloring reagent of Example 3 was used. (2) Glucose oxidase solution: Glucose oxidase [manufactured by Toyobo Co., Ltd., 100 units/mg]
20mg of 0.1M potassium phosphate buffer (PH7.0)2
ml (1000 units/ml). (3) Procedure: In a test tube, add 0.1 ml of new bilirubin oxidase M-1 (0.002 units), 1 ml of 0.3 M potassium phosphate buffer (PH7.0), and control serum [manufactured by Highland (USA)] 0.02 ml, 20
Add 0.02 ml of mg/dl albumin-bound bilirubin [manufactured by Daiichi Chemical Co., Ltd.] and store in a thermostat at 37°C for 20 minutes.
After reacting for a minute with shaking, 0.3 ml of 50 mM sodium azide, 0.3 ml of coloring reagent, 0.1 ml of glucose oxidase solution and 1.16 ml of distilled water were added, and after reacting for 10 minutes, the absorbance at 500 nm was measured. Separately, as a control, the absorbance was measured without treatment with the novel bilirubin oxidase M-1 and without the addition of albumin-bound bilirubin, and the removal of bilirubin color inhibition in a serum glucose measurement system was investigated using this enzyme.
The results are shown in Table 6.

[Table] As is clear from Table 6, by using the method of the present invention, it became possible to remove the interference of bilirubin and obtain accurate serum glucose values. Effects of the Invention In the reagent composition of the present invention, the novel bilirubin oxidase M-1 used oxidizes bilirubin in body fluids such as serum and urine, so bilirubin in body fluids can be easily and accurately quantified from the absorbance change at 460 nm. be able to. In addition, in the analysis of various components in body fluids using the oxidase-peroxidase-hydrogen-donating chromogen reaction, we investigated the interference effect of bilirubin coexisting in body fluids using a new bilirubin method.
It can also be removed with oxidase M-1, which is a remarkable effect.

[Brief explanation of the drawing]

FIG. 1 is a graph showing a calibration curve in Example 1 of the quantitative method of the present invention in terms of the relationship between bilirubin concentration and absorbance difference. FIG. 2 is a graph showing a calibration curve in Example 2 of the quantitative method of the present invention in terms of the relationship between bilirubin concentration and absorbance difference.

Claims (1)

[Scope of Claims] 1. A reagent composition for bilirubin characterized by containing the novel bilirubin oxidase M-1. 2. The reagent composition for bilirubin according to claim 1, wherein the composition contains deoxycholate. 3 Add new bilirubin to the bilirubin-containing test solution.
1. A method for quantifying bilirubin, which comprises applying a reagent composition for bilirubin containing oxidase M-1 and optically measuring the resulting change in bilirubin. 4. The quantitative determination method according to claim 3, wherein the composition contains deoxycholate. 5. In a method for analyzing components other than bilirubin in a bilirubin-containing test solution in which bilirubin is an inhibitor of analysis by an oxidase-peroxidase-hydrogen-donating chromogen reaction, (a) a novel bilirubin oxidase is added to the test solution; Includes the following steps: applying a reagent composition for bilirubin containing M-1 to eliminate the inhibitory effect of coexisting bilirubin; and (b) analyzing the target component in the remaining test liquid. An analysis method characterized by 6. The analytical method according to claim 5, wherein the novel bilirubin oxidase M-1 inhibitor is added after step (a), and then step (b) is performed. 7. The analytical method according to claim 5 or 6, wherein the composition contains deoxycholate.