JPH1118797A - Determination of direct type bilirubin and reagent therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and reagents for determination of direct type bilirubin, applicable to autoanlyzers and capable of completely avoiding interferance by indirect type bilirubin. SOLUTION: This method comprises making bilirubin oxidase act on samples at pH 5.0-6.0 in the coexistence of ferrous ions and determining direct type bilirubin in samples based on optical changes of the samples.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for determining direct bilirubin contained in a sample.

[0002]

2. Description of the Related Art Bilirubin is a metabolite of hemoglobin derived from aged erythrocytes and is a main component of bile pigment. In the blood, the fraction in which the propionic acid group in the side chain is enzymatically esterified mainly with glucuronic acid in the liver and becomes soluble in water (conjugated form), and the propionic acid group remains free and soluble The fraction with low affinity (free form) is mainly present. The former is called direct bilirubin because it easily reacts with the diazo reagent, and the latter is regarded as indirect bilirubin because it reacts with the diazo reagent for the first time in the presence of a reaction accelerator such as alcohol. Indirect bilirubin can be determined by subtracting direct bilirubin from total bilirubin obtained by diazo coloring of all bilirubins in the presence of a reaction accelerator. Differentiation and quantification of each of these conjugated (direct) and free (indirect) bilirubin concentrations enables the identification and diagnosis of jaundice due to various liver diseases and hemolytic diseases. It is an important item in clinical tests.

As described below, direct bilirubin quantification methods using a diazo reagent, bilirubin oxidase, high performance liquid chromatography, and a chemical oxidizing agent have been reported.

A) Conversion of direct bilirubin with diazo reagent
In the measurement method using a diazo reagent, bilirubin reacts with the diazo reagent to generate azobilirubin, and as a result, the maximum absorption of azobilirubin occurs in a longer wavelength region than the intrinsic maximum absorption wavelength of the bilirubin in the visible region. Is to determine bilirubin by the change in absorbance at Various of these have been reported depending on the type of indirect bilirubin reaction promoter, reaction termination conditions, and differences in the detection conditions for azobilirubin (Malloy, HT, Evelyn, KA; J. Bio).
l. Chem. 119 481 (1937); The determination of bi
lirubin with the photoelectric colorimeter. Jend
rassik, L., Grof, P .; Biochem. Z. 297 81 (1938); Ver.
einfachte Photometrische Methoden zur Bestimmung d
es Blutbilirubins .. Micha ёlsson, M; Scand JCli
n Lab Invest. 12 (Supp 56) 1-80 (1937); Bilirubi
n determination inserum and urine).

B) Direct form by bilirubin oxidase
The method of measuring bilirubin with bilirubin oxidase causes bilirubin oxidase to act on a sample containing bilirubin to oxidize bilirubin to bilirubin.At this time, the absorbance in the maximum absorption wavelength region of bilirubin disappears, so the amount of decrease in this absorbance Is determined by In this measurement method, various methods have been devised for the method of suppressing the reaction of indirect bilirubin, and many methods have been reported as follows.

B1) A method for directly measuring bilirubin, wherein bilirubin oxidase is allowed to act at a pH of 3.5 to 4.5 (JP-A-59-125899). B2) A method for measuring direct bilirubin characterized by allowing bilirubin oxidase to act in an acidic buffer solution containing an anionic surfactant at a pH of 5 to 6 (Shogo Otsu)
ji: Clin. Biochem. 21 33-38 (1988) and JP 6
0-152955). B3) A method for quantifying conjugated bilirubin, which comprises measuring the change in absorbance caused by the action of bilirubin oxidase in a buffer having a pH of 9 to 10 (Japanese Patent Application Laid-Open No. Sho 62-58999).
). B4) A method for quantifying direct bilirubin, characterized in that bilirubin oxidase is allowed to act in a buffer solution containing potassium ferrocyanide and / or potassium ferricyanide at a pH of 2.0 to 3.3, and the resulting change in absorbance is measured (JP-A 64- 5499). B5) A method for quantifying direct bilirubin, which comprises coexisting a fluorine compound or a reducing agent together with bilirubin oxidase (JP-A-5-276992). B6) A method for quantifying direct bilirubin, characterized by coexisting a tetrapyrrole ring compound with bilirubin oxidase (JP-A-7-231795).

C) High performance liquid chromatography (HPL)
Method for Measuring Direct Bilirubin by C) In the method of measuring direct bilirubin by HPLC, a gradient of an organic solvent is applied to a reversed-phase column to fractionate bilirubin fractions according to hydrophilic / hydrophobic order. HPLC
According to the results, bilirubin in serum is mainly composed of α, β, γ, and δ.
The α fraction is free bilirubin, and the β fraction is bilirubin (bilirubin) in which only one of the two side chain propionic acid groups in one molecule forms an ester bond with glucuronic acid. Monoglucuronide), the γ fraction is bilirubin in which both propionic acid groups have an ester bond with glucuronic acid (bilirubin diglucuronide), and the δ fraction is a covalent bond between albumin and bilirubin. Have been identified. It is also presumed that the δ fraction was formed as a result of the non-enzymatic reaction between the γ fraction and albumin (Toshio Yamamoto: Nichinai Jikkai 78 (11) 36-
41 (1989)). The α fraction in HPLC corresponds to indirect bilirubin in the method using the diazo reagent of the above A),
On the other hand, the β, γ, and δ fractions are considered to correspond to direct bilirubin (John J. Lauff: Clin. Chem. 28 (4) 629-
637 (1982)). The HPLC method has been improved while minimizing complicated sample pretreatment steps, and various reports have been made (Nakamura H .: Bunsekikagaku 36 352-
355 (1987). Yukihiko Adachi: Gastroenterologia Ja
ponica 23 (3) 268-272 (1988). Yuko Kato: Kinki University Medical Journal, Vol. 14, No. 1, 97-112 (1989). ).

D) Direct bilirubin with a chemical oxidizing agent
In the method using a chemical oxidizing agent, bilirubin is oxidized to biliverdin by using a low molecular weight oxidizing agent instead of bilirubin oxidase, and the amount is determined by the amount of decrease in absorbance based on bilirubin at this time. Various methods have been devised for suppressing the reaction of indirect bilirubin, and these are reported as follows. D1) A method for quantifying direct bilirubin, which comprises reacting a test solution with copper ions and thiourea or a derivative thereof (JP-A-63-118662). D2) A method for quantifying bilirubin, characterized in that vanadate ion or trivalent manganese ion is allowed to act as an oxidizing agent and the optical change of the sample is measured (JP-A-5-1897).
8). In order to measure direct bilirubin by this method, hydrazines, hydroxylamines, oximes, aliphatic polyamines, phenols, water-soluble polymers and HLB are used as indirect bilirubin reaction inhibitors.
One or more compounds selected from the group consisting of five or more nonionic surfactants are used. D3) A method for quantifying bilirubin (WO 96-17251), characterized by measuring the optical change of a sample by using nitrous acid as an oxidizing agent. In order to measure direct bilirubin by this method, polyoxyethylene (n-alkyl or iso-alkyl) ether having an HLB of 12 to 15, thiourea, hydrazine, polyvinylpyrrolidone, or the like is used as an indirect bilirubin reaction inhibitor. Use

Each of the above-mentioned measuring methods A) to D) has advantages and disadvantages, and at present, there is not always a satisfactory measuring method. The problems of each measurement method are listed below.

In the method A) using a diazo reagent, a reaction in the absence of a reaction accelerator is defined as a diazo direct reaction,
It is the origin of the name of direct bilirubin. However, there have been many reports that this direct diazo reaction can occur for some indirect bilirubins (eg, Killenberg PG: Gastroenterology 78 1011).
~ 1015 (1980). Blankaert N: J.Lab.Clin.Med. 96
198-212 (1980). Yukio Manabe: Analytical Chemistry 30 736 〜
740 (1981). Chan KM: Clin. Chem. 31 1560〜1563
(1985). Akira Takasaka: Inspection and Technology 14 971-975 (198
6). Yukihiko Adachi: Biological sample analysis 933-42 (1986)).
Therefore, the measured value of bilirubin defined by the diazo direct reaction cannot be said to accurately determine "direct bilirubin".

Method B) with bilirubin oxidase
Has developed a measurement system that can approximate the measured value of bilirubin defined by the diazo direct reaction. As a result, oxidation of some indirect bilirubin was observed. Is not quantified. Among them, a method of coexisting a fluorine compound with bilirubin oxidase (JP-A-5-276992) and a method of co-existing a tetrapyrrole ring compound (JP-A-7-231795)
The reaction to indirect bilirubin can be avoided. However, the use of a fluorine compound causes a problem of environmental pollution, and the presence of a tetrapyrrole ring compound in a reagent results in a lack of stability and a problem of long-term use in a solution state.

The method C) by high performance liquid chromatography has high analytical performance, but requires about one hour to process one sample, and is not suitable for processing a large number of samples. In addition, it requires expensive and special equipment and lacks versatility.

In the method D) using a chemical oxidizing agent, a measurement system was developed so as to approximate the measured value of direct bilirubin defined by the diazo direct reaction as in the case of bilirubin oxidase. Oxidation reaction was also observed in some parts, and it cannot be said that "direct bilirubin" was quantified accurately.

As described above, a stable and safe method for directly quantifying bilirubin which completely avoids the reaction with indirect bilirubin is not always present.
This development is awaited.

[0015]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and completely eliminates the interference of indirect bilirubin and is applicable to an automatic analyzer. The purpose is to provide.

[0016]

Means for Solving the Problems The present inventors diligently studied the reactivity of indirect bilirubin and direct bilirubin in the optimum pH range of bilirubin oxidase. 0 to
Under the conditions of 6.0 and containing divalent iron ions and, if desired, further potassium ions, the oxidation of indirect bilirubin is completely suppressed and the oxidation of direct bilirubin proceeds quantitatively. The inventors have found and completed the present invention.

That is, the present invention provides a method for quantifying direct bilirubin in a sample by causing bilirubin oxidase to act on the sample and optically changing the sample to obtain a bilirubin oxidase having a pH of 5.0 to 6 in the presence of divalent iron ions. A method for quantifying direct bilirubin, wherein bilirubin oxidase is allowed to act on the bilirubin oxidase at 2.0. In this case, it is preferable to coexist potassium ions in addition to divalent iron ions, because the reaction of indirect bilirubin is further suppressed.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a sample is not particularly limited as long as it contains direct bilirubin or indirect bilirubin. Usually, the sample is a biological fluid sample such as plasma, serum, urine or the like, or a model sample thereof.

In the method of the present invention, the divalent iron ion may be used in a free state or a chelate compound state, and may be ferrous chloride, sodium pentacyanoamine ferroate, potassium ferrocyanide, EDTA.
—Fe ^{2+ and the} like.

In the present invention, when bilirubin oxidase is allowed to act, the divalent iron ion present in the enzyme reaction solution is not sufficiently effective to suppress the reaction of indirect bilirubin when the concentration is too low. If the concentration is too high, the inhibitory effect of bilirubin oxidase is enhanced, and the oxidation reaction of direct bilirubin is hindered. Therefore, when bilirubin oxidase is allowed to act, in the enzyme reaction solution, divalent iron ions are preferably at a concentration of 1 to 1000 μl, more preferably 2 to 500 μl, and particularly preferably 4 to 250 μl.
1 is the concentration range.

On the other hand, as the potassium ion, potassium chloride, potassium bromide, potassium acetate, potassium citrate, potassium tartrate, potassium lactate, potassium phthalate, potassium sulfate and the like can be used without limitation.

In the method of the present invention, when potassium ions are allowed to coexist, the concentration of potassium ions in the final reaction solution is preferably from 100 mM to 800 mM, more preferably from 110 mM to 600 mM, and particularly preferably from 120 to 400 mM. If the potassium ion concentration does not exceed 100 mM, the reaction inhibitory effect of indirect bilirubin is weakened. 800mM potassium ion concentration
When the temperature exceeds the limit, the inhibitory effect of bilirubin oxidase is enhanced, and the oxidation reaction of direct bilirubin is easily hindered.

In the method of the present invention, the bilirubin oxidase is not particularly limited, but may be Myrothecium Verrucar.
ia-derived bilirubin oxidase (available from Amano Pharmaceutical Co., Ltd.), Trachyderma tsunodae-derived bilirubin oxidase (available from Takara Shuzo Co., Ltd.), or Pleurotus genus bilirubin oxidase (available from Morishin Co., Ltd.). But Pleurotu
Preferred is bilirubin oxidase from the s genus. The concentration of bilirubin oxidase in the final reaction solution is preferably 0.001 to 10 U / ml, more preferably 0.01 to 1 U / ml, and still more preferably 0.02 to 0.5 U / ml.

In the present invention, when bilirubin oxidase is allowed to act, the pH range is from 5.0 to 6.0. pH
If the pH is less than 5.0 or the pH exceeds 6.0, indirect bilirubin also tends to react. The buffer to be used is not particularly limited as long as it has a buffering ability in this pH range, but potassium hydrogen phthalate / sodium hydroxide buffer, sodium citrate / sodium hydroxide buffer, malic acid / sodium hydroxide buffer And those containing a liquid or the like. In particular, when a potassium salt such as potassium hydrogen phthalate is contained as a component of the buffer solution, potassium ions are preferred because they have an effect of suppressing indirect bilirubin reaction.

In the present invention, when bilirubin oxidase is allowed to act, a substance that suppresses the reaction between bilirubin oxidase and indirect bilirubin may be present in addition to divalent iron ions. Examples of such substances include thiocyanate ions, hydrazides, reduced nicotinamide amide adenine dinucleotide (NADH), and the like. Examples of the thiocyanate ion include an alkali metal thiocyanate, an alkaline earth metal thiocyanate, and ammonium thiocyanate, and sodium thiocyanate and potassium thiocyanate are preferable.

Examples of the hydrazides include acetylhydrazide, phthalhydrazide, isophthaloyldihydrazide, terephthalic dihydrazide, benzenesulfonylhydrazide and the like.

In the present invention, direct bilirubin in a sample can be quantified using, for example, a direct bilirubin quantification reagent containing bilirubin oxidase and divalent iron ion. If the reagent for quantification is a one-reagent system,
The pH of the reagent must be between 5.0 and 6.0.

Further, as a direct type bilirubin quantification reagent, a buffer solution having a pH of 5.0 to 6.0 containing divalent iron ions is used as a first reagent solution, and a solution containing bilirubin oxidase is used as a second reagent solution. A kit composed of these two reagents can also be used. As described above, examples of the composition of the buffer include those containing potassium hydrogen phthalate / sodium hydroxide buffer, sodium citrate / sodium hydroxide buffer, malic acid / sodium hydroxide buffer, and the like. In this case, the first reagent solution or the second reagent solution preferably further contains potassium ions.

The method of the present invention can be carried out, for example, using this kit as follows. A sample is mixed with the first reagent solution, and a specific wavelength in a wavelength range (430 to 460 nm) based on bilirubin in the mixed solution, preferably 450 nm
Is measured (absorbance 1). Next, a second reagent solution containing bilirubin oxidase is added to the obtained solution, and the bilirubin is oxidized at 25 to 40 ° C. for 3 to 15 minutes. Then, the absorbance at a specific wavelength based on bilirubin in the solution is again measured. Is measured (absorbance 2). After subjecting the obtained absorbance 1 and absorbance 2 values to liquid volume correction and the like,
The amount of change in absorbance before and after the oxidation reaction is determined. The concentration of direct bilirubin in the sample can be determined from this value and a calibration curve created based on the absorbance change amount obtained by the same operation as above using a standard solution with a known concentration in advance. A method for quantifying direct bilirubin using such a kit is as follows.
The present invention is applicable to general-purpose automatic analyzers such as Hitachi 7070 automatic analyzer. In addition, a sample is 0.005-1 ml.
Is preferred.

2 contained in the reagent for direct bilirubin determination
This reagent can also be used as a liquid reagent in the form of an aqueous solution, since valent iron or potassium ions are not particularly unstable in an aqueous solution. In addition, other reagents such as a preservative,
Any reagents such as chelating agents and surfactants that can be used in ordinary reagents and kits can be appropriately selected and used according to known methods. Further, the first reagent solution or the second
The reagent solution may contain, in addition to divalent iron ions, other substances that suppress the reaction of indirect bilirubin by bilirubin oxidase. As such a substance,
The above-mentioned thiocyanate ion, hydrazide, NADH and the like can be exemplified.

[0031]

【Example】

Example 1 and Comparative Example 1 Inhibitory effect of indirect bilirubin

When making bilirubin oxidase act,
The following experiment was conducted to observe whether or not the oxidation reaction of indirect bilirubin was suppressed under a condition in which the pH was 5.0 to 6.0 and contained divalent iron ions. In Comparative Example 1, the test was performed under the condition that divalent iron ions were not included. The reagents, samples, measurements and results are shown below.

(First reagent solution used in Comparative Example 1): phthalic acid 150 mM Triton X-100 0.05% pH 5.50 (adjusted with NaOH) (Second reagent solution used in Comparative Example 1): Tris (Hydroxymethyl) aminomethane 10 mM bilirubin oxidase 0.24 U / ml pH 10.2 (First reagent solution used in Example 1): potassium hydrogen phthalate 150 mM potassium ferrocyanide 12.5 μl Triton X-100 0.05% pH 5.50 (second reagent solution used in Example 1): tris (hydroxymethyl) aminomethane 10 mM bilirubin oxidase 0.24 U / ml pH 10.2

(Sample) The sample had an indirect bilirubin concentration of 5%.
0 mg / dl and human serum albumin concentration 6.0 g / l
Was used. The sample was prepared as follows. Weigh 5 mg of indirect bilirubin and disperse it in 0.4 ml of dimethyl sulfoxide. 0.4 to this dispersed liquid
Immediately after dissolving indirect bilirubin by adding 100 ml of 100 mM sodium carbonate solution, 100 ml containing human serum albumin
A sample was prepared by diluting with 9.2 ml of M Tris buffer (pH 7.00).

(Quantification in Comparative Example 1 and Example 1) In a Hitachi 7070 type automatic analyzer, a sample of 10 μl,
Under the conditions of 300 μl of the reagent solution and 75 μl of the second reagent solution, changes in absorbance at a main wavelength of 450 nm and a sub-wavelength of 546 nm were determined by a 2 Point End method. That is, after mixing the first reagent solution and the sample on an automatic analyzer and incubating at 37 ° C. for 5 minutes, the absorbance based on bilirubin in this solution is measured at a main wavelength of 450 nm and a sub wavelength of 546 nm (absorbance 1). ). Next, a second reagent solution containing bilirubin oxidase was added to the resulting solution, and the mixture was added at 37 ° C.
After performing the oxidation reaction of bilirubin for 5 minutes, the absorbance based on bilirubin in the solution is measured again at the above wavelength (absorbance 2). After subjecting the obtained absorbance 1 and absorbance 2 values to liquid volume correction and the like, the decrease in absorbance before and after the oxidation reaction is determined. These measurements and calculations are performed automatically by an automatic analyzer.

(Results of Comparative Example 1 and Example 1) Table 1 shows the results of the decrease in absorbance of indirect bilirubin in Comparative Example 1 and Example 1. FIG. 1 shows the course of reaction (reaction time course) on the automatic analyzer in Comparative Example 1 and Example 1.

[0037]

[Table 1]

As is clear from Table 1, in the method of the present invention (Example 1), the amount of decrease in absorbance due to oxidation of indirect bilirubin is about the measurement error of an automatic analyzer. Further, as is clear from FIG. 1, in the method of the present invention, a decrease in absorbance due to a change in the liquid volume is observed in the reaction time course, but a decrease in absorbance due to oxidation of indirect bilirubin is not observed. In contrast, in Comparative Example 1, a decrease in absorbance associated with oxidation of indirect bilirubin is clearly observed. This ensures that the pH is between 5.0 and 6.0 and 2
It was clarified that the reaction of indirect bilirubin by bilirubin oxidase was suppressed under the condition containing a multivalent iron ion.

Example 2 Direct bilirubin and indirect bilirubin
Determination of Direct Bilirubin in Samples Containing Rubin (Preparation of Sample) 5 mg / dl of ditaurobilirubin, a synthetic conjugated bilirubin, in 100 mM Tris buffer (pH 7.00)
(Equivalent concentration of bilirubin) and human serum albumin 6.0 g
The solution containing / l was prepared as a direct bilirubin solution.
Separately, a solution containing 5 mg / dl of unconjugated bilirubin and 6.0 g / l of human serum albumin in 100 mM Tris buffer (pH 7.00) was prepared as an indirect bilirubin solution. Next, the direct bilirubin solution was diluted with the indirect bilirubin solution so that the total bilirubin concentration was the same (5 m
g / dl) and different concentrations of direct bilirubin were prepared. The sample had a ratio of direct bilirubin to total bilirubin of 0.0, 0.2, 0.4, 0.6,
Six types of 0.8 and 1.0 were prepared.

(Quantitative conditions) Except for using this sample, the amount of decrease in absorbance was measured using the quantitative reagents and quantitative conditions described in Example 1 to obtain the data of Example 2.

(Results) The results are shown in FIG. In FIG. 2, the horizontal axis represents the ratio of direct bilirubin to total bilirubin, and the vertical axis represents the amount of decrease in absorbance. In the method of the present invention (Example 2), the amount of decrease in absorbance was increased in proportion to the amount of direct bilirubin, and good dilution linearity with regression to the origin was obtained. This indicates that under the conditions of pH 5.0 to 6.0 and containing divalent iron ions (Example 2), direct bilirubin was selectively quantified without interference of indirect bilirubin. I have.

Comparative Example 2 Direct Bilirubin by Conventional Method
Bilirubin in a sample containing and bilirubin
Determination contrast of using the sample used in Example 2 was quantified direct bilirubin in a conventional manner. The conventional method is a measurement method in which bilirubin oxidase is allowed to act under the condition of pH 3.5 to 4.5 (Japanese Patent Application Laid-Open No. 59-125891, Shogo Otsuji: Clin. Biochem.
38 (1988)). That is, it is based on the reagent conditions of the following composition.

(First Reagent Solution of Conventional Method) Trisodium citrate trihydrate 17.65 g / l Lactic acid 30.0 g / l Triton X-100 1.0 g / l EDTA · 2Na · 2H ₂ O 18.6 mg / l pH 3.70 (Second reagent solution of conventional method) Trisodium citrate trihydrate 3.0 g / l Lactic acid 160 mg / l Triton X-100 1.0 g / l CuSO _{4 · 5H 2 O 1.25 g /} l bilirubin oxidase 0.2 U / ml pH6.50

(Quantitative method) The quantitative conditions of the conventional method were the same as the quantitative conditions described in Example 1 except for the reagent solution.

(Results) The results are shown in FIG. In the conventional method (Comparative Example 2), it was found that linearity could not be obtained between the direct bilirubin concentration in the sample and the measured decrease in absorbance. In the conventional method, it is shown that direct bilirubin in the sample is not accurately determined due to the interference of indirect bilirubin in the sample.

Example 3 and Comparative Example 3 Determination Method of the Present Invention
Bilirubin in patient samples with high bilirubin levels under method conditions
As a sample (sample) in which non-reaction was confirmed by HPLC, a patient pool serum sample having a high bilirubin level was used. Serum samples were analyzed without treatment, without salting out with sodium sulfate. This is because the globulin in the sample is adsorbed to the column and the deterioration is accelerated, but the purpose is to prevent denaturation of the bilirubin fraction.

(Reagent) In Example 3, the reagent used in Example 1 was used. In Comparative Example 3, the reagent used in Comparative Example 2 was used.

(Oxidation reaction of bilirubin in a sample by bilirubin oxidase) To 16 µl of the sample, 480 µl of the first reagent solution was added and heated at 37 ° C for 5 minutes. Furthermore, 120 μl of the second reagent solution was added to the obtained solution,
After heating at 37 ° C. for 5 minutes, the reaction of bilirubin oxidase was stopped by adding 120 μl of a 2% aqueous solution of ascorbic acid.

(Analysis by HPLC) The HPLC analysis was performed according to the literature (John J. Lauff: Clin. Chem. 28 (4) 629-63).
7 (1982)), and the change in peak area based on indirect bilirubin before and after the reaction was examined. For the data before the reaction, a peak area based on indirect bilirubin was obtained by performing the same operation as the above-described oxidation reaction except that physiological saline was used as the second reagent solution. HPLC
Hitachi HPLC system (Column Oven L-7300, UV Detec
tor L-7400, PumpL-7100, Integrator D-7500) Lichrspher 100 RP-18 (1
0 μm). That is, the liquid obtained by the above oxidation reaction was filtered through a 0.45 μm membrane filter, and 150 μl of the filtrate was injected into an HPLC column to determine the peak area based on indirect bilirubin after the reaction. For elution, the bilirubin fraction was mixed with solution A: (purified water 95
PH at 0 volume / 2-methoxyethanol 50 volume / phosphoric acid
2.1) and solution B: (950 volumes of isopropanol /
2-methoxyethanol 50 volumes / phosphoric acid 2.5 volumes)
Performed by a linear gradient of isopropanol between the liquids,
Detection was at a wavelength of 450 nm.

(Results) The residual ratio of indirect bilirubin was determined by comparing the data before the reaction with 100 with the peak area data based on the indirect bilirubin after the reaction. Table 2 shows the results. The peak area based on indirect bilirubin hardly changed before and after the reaction,
It was confirmed that bilirubin oxidase and indirect bilirubin did not react under the conditions of 5.0 to 6.0 and containing divalent iron ions (Example 3). On the other hand, in the conventional method (Comparative Example 3), indirect bilirubin was reduced by 16.5%. This indicates that a part of indirect bilirubin reacted under the conditions of the conventional method.

[0051]

[Table 2]

[0052]

According to the present invention, direct bilirubin in a sample can be selectively quantified in a short time without being affected by indirect bilirubin. Further, the present invention can be applied to a general-purpose automatic analyzer. Therefore, it is useful in the field of clinical tests.

[Brief description of the drawings]

FIG. 1 shows a reaction time course of indirect bilirubin by bilirubin oxidase under a condition of pH 5.0 to 6.0 and containing divalent iron ions (Example 1). In addition, the reaction time course of indirect bilirubin by bilirubin oxidase when divalent iron ions are not present (Comparative Example 1) is shown. The horizontal axis indicates the photometry point (one point is about 20 seconds), and the vertical axis indicates absorbance × 10000.

FIG. 2: Reaction of direct bilirubin in a sample containing direct bilirubin and indirect bilirubin with bilirubin oxidase under conditions of pH 5.0 to 6.0 and containing divalent iron ions (Example 2) The quantified results are shown.
In addition, the result of quantifying the same sample by the conventional method (Comparative Example 2) is shown. The horizontal axis shows the ratio of direct bilirubin to total bilirubin, and the vertical axis shows the decrease in absorbance.

Claims (3)

[Claims] 1. A method for quantifying direct bilirubin in a sample by subjecting the sample to bilirubin oxidase by an optical change of the sample, wherein bilirubin is added at pH 5.0 to 6.0 in the presence of divalent iron ions. A method for quantifying direct bilirubin, which comprises reacting oxidase. 2. The method for quantifying direct bilirubin according to claim 1, wherein potassium ions are coexistent in addition to divalent iron ions. 3. Pleurotu as bilirubin oxidase
The method for quantifying direct bilirubin according to claim 1 or 2, wherein the method uses bilirubin oxidase derived from the genus s.