JP2007202568A - Method for determination of direct bilirubin and reagent therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a reagent for determining indirect bilirubin, which little have risks of environmental pollution due to unnecessary waste liquid treatment and perfectly avoid interference of the indirect bilirubin. <P>SOLUTION: This method for determining the indirect bilirubin in a specimen, comprising making bilirubin oxidase to act on the specimen to optically change the specimen, is characterized by allowing 100 to 800 mM of potassium ion to coexist. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for measuring direct bilirubin contained in a sample and a measuring reagent used therefor.

Bilirubin is a metabolite of hemoglobin derived from aging erythrocytes and is the main component of bile pigment. In the blood, the fraction in which the side chain propionate group is enzymatically bound mainly with glucuronic acid in the liver to increase water solubility (conjugated), and the propionate group remains free and water soluble. There is mainly a fraction (free form) with low properties. The former is called direct bilirubin because it easily reacts with a diazo reagent, and the latter is regarded as indirect bilirubin because it reacts with a diazo reagent for the first time in the presence of a reaction accelerator such as alcohol. Indirect bilirubin can be obtained by subtracting direct bilirubin from the total bilirubin obtained by diazo coloration of all bilirubin in the presence of a reaction accelerator.
Because the bilirubin concentration of these conjugated (direct) and free (indirect) bilirubin can be separately quantified, it is possible to distinguish and diagnose jaundice due to various liver diseases, hemolytic diseases, etc. It is an important item in clinical examination.

  As a direct quantification method of bilirubin, as shown below, a method using a diazo reagent, a method using bilirubin oxidase, a method using high performance liquid chromatography, a method using a chemical oxidizing agent, and the like have been reported.

A) Method for measuring direct bilirubin using diazo reagent The method using diazo reagent is that bilirubin reacts with diazo reagent to produce azobilirubin, and as a result, azobilirubin has a longer wavelength than the maximum visible absorption wavelength of bilirubin. Since maximum absorption occurs, bilirubin is quantified by the change in absorbance at this wavelength. Various types of these have been reported depending on the type of reaction promoter for indirect bilirubin, reaction termination conditions, and detection conditions for azobilirubin (Non-patent Document 1, Non-patent Document 2 and Non-patent Document 3).

B) Method for measuring bilirubin directly using bilirubin oxidase In the method using bilirubin oxidase, bilirubin oxidase is allowed to act on a sample containing bilirubin to oxidize bilirubin to biliverdin. Therefore, it is determined by the amount of decrease in absorbance. In this measurement method, various contrivances have been made to the method for suppressing the reaction of indirect bilirubin, and many methods have been reported as follows.

B1) A method for measuring direct bilirubin, wherein bilirubin oxidase is allowed to act at pH 3.5 to 4.5 (Patent Document 1).
B2) A method for measuring direct bilirubin, wherein bilirubin oxidase is allowed to act in an acidic buffer solution having a pH of 5 to 6 containing an anionic surfactant (Non-patent Document 4 and Patent Document 2).
B3) A method for quantifying conjugated bilirubin, characterized by measuring a change in absorbance caused by the action of bilirubin oxidase in a pH 9-10 buffer (Patent Document 3).
B4) Direct bilirubin quantification method characterized by measuring the change in absorbance caused by the action of bilirubin oxidase in a buffer containing potassium ferrocyanide and / or potassium ferricyanide at pH 2.0 to 3.3 (Patent Document) 4).
B5) A method for quantifying direct bilirubin characterized by coexisting a fluorine compound or a reducing agent together with bilirubin oxidase (Patent Document 5) A direct bilirubin characterized by coexisting a tetrapyrrole ring compound together with bilirubin oxidase Quantitative method (Patent Document 6).

C) Method for measuring direct bilirubin by high performance liquid chromatography (HPLC) The method for measuring direct bilirubin by HPLC is a method in which a reverse phase column is subjected to a gradient of organic solvent and the fraction of bilirubin is classified according to the order of hydrophilicity / hydrophobicity. Fractionation. According to HPLC, bilirubin in serum is mainly separated into 4 fractions of α, β, γ, and δ. Each α fraction is free bilirubin, and β fraction is a side chain propion that is two in one molecule. Bilirubin (bilirubin monoglucuronide) in which only one of the acid groups has an ester bond with glucuronic acid, and γ fraction has a bilirubin (bilirubin diglucuronide) in which both propionic acid groups have an ester bond with glucuronic acid. Id), the δ fraction has been identified as a covalent bond between albumin and bilirubin. The δ fraction is presumed to have been generated as a result of non-enzymatic reaction of the γ fraction and albumin (Non-patent Document 5). The α fraction in HPLC corresponds to indirect bilirubin in the method using the diazo reagent of A) above, while the β, γ and δ fractions correspond to direct bilirubin (Non-patent Document 6). The HPLC method has been improved in such a way that a complicated sample pretreatment process is omitted as much as possible, and various reports have been made (Non-Patent Document 7, Non-Patent Document 8 and Non-Patent Document 9).

D) Method for measuring direct bilirubin with chemical oxidant In the case of using chemical oxidant, bilirubin is oxidized to biliverdin by using a low molecular weight oxidant instead of bilirubin oxidase, and this is based on bilirubin. It is determined by the amount of decrease in absorbance. These are also variously devised in the method of suppressing the reaction of indirect bilirubin, and are reported as follows.
D1) A direct bilirubin quantification method characterized by allowing copper ions and thiourea or a derivative thereof to act on a test solution (Patent Document 7).
D2) A method for quantifying bilirubin, characterized by measuring vanadate ions or trivalent manganese ions as oxidizing agents and measuring optical changes in the sample (Patent Document 8). In order to measure direct bilirubin by this method, hydrazines, hydroxylamines, oximes, aliphatic polyvalent amines, phenols, water-soluble polymers and HLB are used as reaction inhibitors for indirect bilirubin. One or more compounds selected from the group consisting of the above nonionic surfactants are used.
D3) A method for quantifying bilirubin, characterized in that nitrous acid is allowed to act as an oxidizing agent to measure an optical change of a sample (Patent Document 9). In order to measure direct bilirubin by this method, as an indirect bilirubin reaction inhibitor, polyoxyethylene (n-alkyl or iso-alkyl) ether having 12 to 15 HLB, thiourea, hydrazine, polyvinylpyrrolidone, etc. Is used.

  Each of the measurement methods A) to D) has advantages and disadvantages, and at present there is not always a satisfactory measurement 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, which is also the origin of the name of direct bilirubin. However, it has been reported in large numbers that this diazo direct reaction can occur even for a part of indirect bilirubin (for example, Non-Patent Document 10, Non-Patent Document 11, Non-Patent Document 12, Non-Patent Document 13, Non-patent document 14 and Non-patent document 15). Therefore, the bilirubin measurement value defined by the diazo direct reaction cannot be said to be accurately measuring “direct bilirubin”.

  Method B) with bilirubin oxidase has developed a measurement system that can approximate the measured bilirubin defined by the diazo direct reaction. As a result, oxidation of some indirect bilirubin is observed. Is not measuring “direct bilirubin”. Among them, the method of coexisting a fluorine compound with bilirubin oxidase (Patent Document 10) and the method of coexisting a tetrapyrrole ring compound (Patent Document 11) can avoid a reaction with indirect bilirubin. However, since the fluorine compound is used, there is a problem in environmental pollution, and since the tetrapyrrole ring compound is present in the reagent, the stability is insufficient, and there is a problem in using it in a solution state for a long time.

  Method C) by high performance liquid chromatography has high analytical performance, but it takes about one hour to process one sample, and is not suitable for processing a large number of samples. Moreover, an expensive and special apparatus is required and lacks versatility.

  Method D) with a chemical oxidant has developed a measurement system that can be approximated to a direct bilirubin measurement defined by a diazo direct reaction in the same way as with bilirubin oxidase. However, an oxidation reaction is observed, and it cannot be said that “direct bilirubin” is measured accurately.

As described above, there is currently no stable and safe method for measuring direct bilirubin that completely avoids the reaction to indirect bilirubin, and this development is awaited.
JP 59-125899 A JP-A-60-152955 Japanese Patent Laid-Open No. 62-58999 JP-A 64-5499 Japanese Patent Application Laid-Open No. 5-276922 Japanese Patent Laid-Open No. 7-231895 JP-A-63-118662 JP-A-5-18978 International Publication No. 96-17251 Pamphlet Japanese Patent Application Laid-Open No. 5-276922 Japanese Patent Laid-Open No. 7-231895 Malloy, H.M. T.A. , Evelyn, K .; A. J .; Biol. Chem. , 119, 481 (1937); The determination of bilirubin with the photoelectric colorimeter. Jendrasik, L.M. Grof, P .; Biochem. Z. , 297.81 (1938): Vereinfachte Photometriciche Method zur Best des des Blutbilibrubins Michael eleson, M.M. , Scand. J. et al. Clin. Lab. Invest. , 12 (Supp. 56), 1-8 (1937); Birubin determination in serum and urine) Shogo Otsuji: Clin. Biochem. , 21, 33-38 (1988) Toshio Yamamoto: Nichinaikai 78 (11), 36-41 (1989) John J. Lauff, Clin. Chem. , 28 (4) 629-637 (1982) Nakamura H. et al. : Bunseki kagaku, 36, 352-355 (1987) Yukihiko Adachi: Gastroenterologia Japan, 23 (3), 268-272 (1988) Yuko Kato: Kinki University Medical Journal Vol.14 No.1 97-112 (1989) Killenberg, P.M. G. , Gastroenterology, 78, 1011-1015 (1980). Blankaert, N.A. , J .; Lab. Clin. Med. 96, 198-212 (1980) Manabe Yukio: Analytical Chemistry, 30, 736-740 (1981) Chan, K.M. M.M. , Clin. Chem. 31, 1560-1563 (1985) Akira Takasaka: Inspection and Technology 14 971-975 (1986) Yukihiko Adachi: Biological Sample Analysis 9 33-42 (1986)

  The present invention has been made in view of the above-described situation, and its object is to provide a method for measuring direct bilirubin that completely avoids interference of indirect bilirubin and has no danger of environmental pollution due to waste liquid, and a reagent for measurement. To do.

  As a result of intensive studies on the reactivity of indirect bilirubin and direct bilirubin in the optimal pH range of bilirubin oxidase, the present inventors have found that when bilirubin oxidase is allowed to act on bilirubin, thiocyanate ions, hydrazides, reduced nicotine In the presence of amidoadenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), or potassium ion of 100 mM to 800 mM, indirect bilirubin oxidation is completely suppressed and direct bilirubin As a result, it was found that the oxidation of azobenzene progresses quantitatively and direct bilirubin can be accurately measured, and the present invention has been completed.

That is, the present invention relates to a method of measuring bilirubin oxidase, hydrazides, reduced nicotinamide adenine dinucleotide (NADH) in a method in which bilirubin oxidase is allowed to act on a sample and direct bilirubin in the sample is measured by optical change of the sample. , A direct bilirubin characterized by allowing bilirubin oxidase to act in the presence of at least one indirect bilirubin reaction inhibitor selected from reduced nicotinamide adenine dinucleotide phosphate (NADPH) and 100 mM to 800 mM potassium ion This is a measurement method.
Furthermore, the present invention is selected from i) bilirubin oxidase and ii) thiocyanate ions, hydrazides, reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate and 100 mM to 800 mM potassium ions as essential components. A reagent for measuring direct bilirubin, comprising at least one indirect bilirubin reaction inhibitor.

  According to the present invention, direct bilirubin in a sample can be selectively measured without the influence of indirect bilirubin, which is useful in the field of clinical examination.

In the present invention, the 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 a model sample thereof.
In the method of the present invention, the thiocyanate ion used as the indirect type bilirubin reaction inhibitor is not particularly limited, and examples thereof include alkali thiocyanate, alkaline earth metal thiocyanate, ammonium thiocyanate, and the like. , Potassium thiocyanate and the like are preferable.

  Examples of hydrazides include acetyl hydrazide, phthalhydrazide, isophthaloyl dihydrazide, terephthalic dihydrazide, and benzenesulfonyl hydrazide.

  Reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) may be directly present in the reaction reagent, or alcohol dehydrogenase, glycol-6-phosphate dehydrogenase, etc. The object of the present invention can also be achieved by using an enzyme derived from oxidized nicotinamide adenine dinucleotide.

  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 present invention, when bilirubin oxidase is allowed to act on bilirubin in a sample, the indirect bilirubin reaction inhibitor coexisting in the enzyme reaction solution is difficult to obtain the indirect bilirubin reaction inhibitory effect when the concentration is too low. If the concentration is too high, the inhibition of bilirubin oxidase is promoted, and the direct bilirubin oxidation reaction tends to be disturbed. Therefore, when using thiocyanate ions or hydrazides as the indirect bilirubin reaction inhibitor, the concentration in the enzyme reaction solution is preferably 0.1 mM to 100 mM, more preferably 0.2 mM to 50 mM.
When NADH or NADPH is used, the concentration range is 0.1 mM to 10 mM, preferably 0.2 mM to 5 mM.

  When potassium ions are used, the potassium ion concentration in the enzyme reaction solution is preferably 100 mM to 800 mM, more preferably 110 mM to 600 mM, and particularly preferably 120 to 400 mM. If the potassium ion concentration does not exceed 100 mM, the reaction suppressing effect of indirect bilirubin is weakened. Inhibition of bilirubin oxidase with a potassium ion concentration exceeding 800 mM is promoted, and the oxidation reaction of direct bilirubin tends to be disturbed.

  In the method of the present invention, two or more indirect bilirubin reaction inhibitors may be used. For example, two or more reaction inhibitors selected from thiocyanate ions, hydrazides, NADH and NADPH can be used in combination. In addition, one or more reaction inhibitors selected from thiocyanate ions, hydrazides, NADH and NADPH may be used in combination with potassium ions.

  In the method of the present invention, bilirubin oxidase is not particularly limited, but bilirubin oxidase derived from Myrothecerium verrucaria (available from Amano Pharmaceutical Co., Ltd.), bilirubin oxidase derived from Trachyderma tsunodae (available from Takara Shuzo Co., Ltd.) or Pleurotus genus. Derived from bilirubin oxidase (available from Shengshin Co., Ltd.) and the like, and the necessary amount thereof is preferably used at a concentration of 0.001 to 10 U / ml in the final reaction solution. The concentration range is more preferably 0.01 to 1 U / ml, and particularly preferably 0.02 to 0.5 U / ml.

  When bilirubin oxidase is allowed to act, the pH range is not limited as long as the enzyme activity can be expressed in an optimal state, but pH 4.5 to 6.5 is preferable, and pH 5.0 to 6.0 is particularly preferable. The buffer to be used is not particularly limited as long as it has a buffer capacity in this pH range, but potassium phthalate / sodium hydroxide buffer, sodium citrate / sodium hydroxide buffer, malic acid / sodium hydroxide buffer The thing containing a liquid etc. is mentioned. In particular, when a potassium salt such as potassium hydrogen phthalate is included as a component of the buffer solution, potassium ions are preferred because they have an indirect bilirubin reaction inhibitory effect.

  In the present invention, direct bilirubin in a sample can be measured using, for example, a reagent for measuring direct bilirubin containing one or more of bilirubin oxidase and an indirect bilirubin reaction inhibitor.

  As a reagent for direct bilirubin measurement, for example, a kit containing thiocyanate ion or hydrazide is used as a first reagent solution, and a solution containing bilirubin oxidase is used as a second reagent solution. It is preferable that The first reagent solution preferably further contains potassium ions.

Alternatively, for example, a buffer solution of pH 4.5 to 6.5, preferably a buffer solution of pH 5.0 to 6.0 is used as the first reagent solution, and a solution containing bilirubin oxidase and NADH, NADPH or potassium ions is used. It is preferable from the stability of NADH, NADPH or bilirubin oxidase to be a second reagent solution and a kit composed of these two reagents. When NADH or NADPH is used, the pH of the second reagent solution is preferably 9 or more, and more preferably 9 to 11.0, from the stability of NADH or NADPH in the solution state. Further, it preferably contains potassium ions.
When potassium ion is used alone as a reaction inhibitor for indirect bilirubin oxidase, the pH of the second reagent solution containing bilirubin oxidase is preferably 7 to 11 from the stability of bilirubin oxidase.
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 as described above.
The first reagent solution preferably further contains potassium ions.

  The method of the present invention can be carried out, for example, using the kit described above as follows. The sample and the first reagent solution are mixed, and the absorbance at a specific wavelength in the wavelength region (430 to 460 nm) based on bilirubin in the mixed solution, preferably at a wavelength of 450 nm is measured (absorbance 1). Next, after adding a second reagent solution containing bilirubin oxidase to the obtained solution and conducting an oxidation reaction of bilirubin at 25 to 40 ° C. for 3 to 15 minutes, the absorbance at a specific wavelength based on bilirubin in the solution again. Is measured (absorbance 2). The obtained absorbance 1 and absorbance 2 values are subjected to liquid volume correction and the like, and the amount of change in absorbance before and after the oxidation reaction is obtained. The direct bilirubin concentration in the sample can be obtained from this value and a calibration curve created based on the absorbance change amount obtained in the same manner as described above using a standard solution with a known concentration in advance. The direct bilirubin measurement method using such a kit can be applied to a general-purpose automatic analyzer such as a Hitachi 7070 automatic analyzer. The sample is preferably 0.005 to 2 ml.

  The thiocyanate ion, hydrazide and potassium ion contained in the reagent for direct bilirubin measurement are not particularly unstable in an aqueous solution, and NADH or NADPH and bilirubin oxidase are not particularly unstable in an aqueous solution at pH 9 or higher. This reagent can also be used as a liquid reagent in the form of an aqueous solution. The direct bilirubin measurement reagent is appropriately selected according to known methods as long as it can be used in other reagents, for example, conventional reagents and kits such as preservatives, chelating agents, and surfactants. Can be used.

Examples 1 to 6 and Comparative Example 1
Inhibitory effect of indirect bilirubin When bilirubin oxidase is allowed to act, 100 mM to 800 mM potassium ion (Example 1), thiocyanate ion (Example 2), hydrazides (Example 3 and Example 4), NADH (Example) 5) In order to observe whether or not the oxidation reaction of indirect bilirubin is suppressed when NADPH (Example 6) coexists, the following experiment was conducted. In Comparative Example 1, the test was carried out under conditions that did not contain these thiocyanate ions. Those reagents, samples, measurements, and results are shown below.

(1) First and second reagent solutions
First reagent solution used in Comparative Example 1 : phthalic acid 150 mM
Triton X-100 0.05%
pH 5.50 (adjusted with NaOH)
First reagent solution used in Example 1 : 150 mM phthalic acid
Potassium chloride 200 mM
Triton X-100 0.05%
pH 5.50 (adjusted with NaOH)
First reagent solution used in Example 2 : potassium hydrogen phthalate 150 mM
Sodium thiocyanate 10 mM
Triton X-100 0.05%
pH 5.50 (adjusted with NaOH)
First reagent solution used in Example 3 : potassium hydrogen phthalate 150 mM
Benzenesulfonyl hydrazide 10 mM
Triton X-100 0.05%
pH 5.50 (adjusted with NaOH)
First reagent solution used in Example 4 : potassium hydrogen phthalate 150 mM
Isophthaloyl dihydrazide 10 mM
Triton X-100 0.05%
pH 5.50 (adjusted with NaOH)
First reagent solution used in Example 5 and Example 6 : potassium hydrogen phthalate 150 mM
Triton X-100 0.05%
pH 5.50
Second reagent solution used in Comparative Example 1 and Examples 1-4 : Tris (hydroxymethyl) aminomethane
10 mM
Bilirubin oxidase 0.24U / ml
(Derived from the genus Pleurotus)
pH 10.2
Second reagent solution used in Example 5 : Tris (hydroxymethyl) aminomethane
10 mM
NADH 5 mM
Bilirubin oxidase 0.24U / ml
pH 10.2
Second reagent solution used in Example 6 : Tris (hydroxymethyl) aminomethane
10 mM
NADPH 5 mM
Bilirubin oxidase 0.24U / ml
pH 10.2

(2) A sample having an indirect bilirubin concentration of 50 mg / dl and a human serum albumin concentration of 6.0 g / l was used. The sample was prepared as follows.
Weigh 5 mg of indirect bilirubin and disperse in 0.4 ml of dimethyl sulfoxide. Immediately after 0.4 ml of 100 mM sodium carbonate solution was added to this dispersed solution to dissolve indirect bilirubin, a sample was prepared by diluting with 9.2 ml of 100 mM Tris buffer (pH 7.00) containing human serum albumin. did.

(3) Measurement in Comparative Example 1 and Examples 1-6 In the Hitachi 7070 type automatic analyzer, the absorbance at a primary wavelength of 450 nm and a secondary wavelength of 546 nm under the conditions of a sample of 10 μl, a first reagent solution of 300 μl, and a second reagent solution of 75 μl. The change was determined by the 2 Point End method.
That is, the first reagent solution and the sample are mixed on an automatic analyzer and incubated at 37 ° C. for 5 minutes, and then the absorbance based on bilirubin in this solution is measured at a main wavelength of 450 nm and a subwavelength of 546 nm (absorbance 1 ). Next, a second reagent solution containing bilirubin oxidase is added to the resulting solution, the bilirubin oxidation reaction is carried out at 37 ° C. for 5 minutes, and the absorbance based on the bilirubin in the solution is again measured at the above wavelength. (Absorbance 2). The obtained absorbance 1 and absorbance 2 values are subjected to liquid volume correction and the like, and the amount of decrease in absorbance before and after the oxidation reaction is determined. These measurements and calculations are automatically performed by an automatic analyzer.

(4) Results of Comparative Example 1 and Examples 1-6 Table 1 shows the results of the decrease in absorbance with indirect bilirubin in Comparative Example 1 and Examples 1-6. Moreover, the reaction progress process (reaction time course) on the automatic analyzer in the comparative example 1 and Examples 1-6 is shown in FIGS.

As is clear from Table 1 and FIGS. 1 to 6, in the methods based on the present invention (Examples 1 to 6), the amount of decrease in absorbance based on the oxidation of indirect bilirubin is about the measurement error of the automatic analyzer, There is no decrease in absorbance even in the reaction time course. On the other hand, in Comparative Example 1, a clear decrease in absorbance associated with indirect bilirubin oxidation is observed.
Thus, it has been clarified that the reaction of indirect bilirubin by bilirubin oxidase is suppressed in the presence of thiocyanate ions, hydrazides, 100 mM to 800 mM potassium ions, NADH, and NADPH.

Examples 7-12
Measurement of direct bilirubin in a sample containing direct and indirect bilirubin (1) Preparation of sample 5 mg / dl (corresponding to bilirubin) of ditaurobilirubin, which is a synthetic conjugated bilirubin, in 100 mM Tris buffer (pH 7.00) And a solution containing 6.0 g / l of human serum albumin was prepared as a direct bilirubin solution. Separately, a solution containing 5 mg / dl unconjugated bilirubin and 6.0 g / l 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, and various samples having the same total bilirubin concentration (5 mg / dl) and different direct bilirubin concentrations were prepared. In addition, six types of samples having a ratio of direct bilirubin to total bilirubin of 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 were prepared.

(2) Measurement conditions Except for using this sample, the amount of decrease in absorbance was measured using the measurement reagents and measurement conditions described in Examples 1 to 6, and Example 7 (using potassium ions) and Example 8 (thiocyanate) were used. Acid ion use), Example 9 (hydrazide use), Example 10 (hydrazide use), Example 11 (NADH), and Example 12 (NADPH).

(3) Results The results are shown in FIGS. 7 to 12, 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 (Examples 7 to 12), the absorbance increased in proportion to the amount of direct bilirubin, and good dilution linearity of origin regression was obtained. This indicates that in Examples 7 to 12, direct bilirubin is selectively measured without interference of indirect bilirubin.

Comparative Example 2
Measurement of direct bilirubin in a sample containing direct bilirubin and indirect bilirubin by a conventional method On the other hand, direct bilirubin was measured by a conventional method using the samples used in Examples 7-12. The conventional method was performed by a measurement method in which bilirubin oxidase was allowed to act under conditions of pH 3.5 to 4.5 (JP-A 59-125899; Shogo otsuji, Clin. Biochem., 21, 33-38 (1988)). That is, it is based on the reagent conditions of the following composition.

(1) First and second reagent solutions
Conventional first reagent solution 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
Conventional second reagent solution trisodium citrate trihydrate 3.05 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
pH 6.50

(2) Measurement method The measurement conditions of the conventional method were the same as those described in Example 1 except for the reagent solution.

(3) Results The results are shown in FIGS. In the conventional method (Comparative Example 2), it was found that no linearity was obtained between the concentration of the direct bilirubin in the sample and the measured absorbance. In the conventional method, it is shown that the direct bilirubin in the sample is not accurately measured due to the large interference of the indirect bilirubin in the sample.

Examples 13 to 18 and Comparative Example 3
Example in which indirect bilirubin in a patient sample with high bilirubin level does not react under the measurement method conditions of the present invention (1) A patient pool serum sample with high bilirubin level was used as a sample sample. Serum samples were analyzed without being subjected to salting out with sodium sulfate. This is because globulin in the sample is adsorbed on the column and accelerates the deterioration, but the purpose is to prevent denaturation of the bilirubin fraction.

(2) Reagent Examples 13 (using potassium ions), 14 (using thiocyanate ions), 15 (using hydrazides), 16 (using hydrazides), 17 (using NADH), In Example 18 (using NADPH), the reagents used in Examples 1 to 6 were used, respectively. In Comparative Example 3, the reagent used in Comparative Example 2 was used.

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

(4) Analysis by HPLC Analysis of HPLC is performed by the method described in the literature (John J. Lauff, Clin. Chem, 28 (4), 629-637 (1982)), and the peak area based on indirect bilirubin before and after the reaction. I examined the changes. 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 is a Hitachi HPLC system (Column Oven L-7300, UV Detector L-7400, Pump L-7100, Integrator D-7500) manufactured by Kanto Chemical Co., Ltd., reverse phase system Licherspher 100 RP-18 (10 μm). Connected and used.
That is, the liquid obtained by the above oxidation reaction was filtered through a 0.45 μm membrane filter, 150 μl of the filtrate was injected into an HPLC column, and the peak area based on indirect bilirubin after the reaction was determined. For elution, the bilirubin fraction was obtained by liquid A: (purified water 950 vol / 2-methoxyethanol 50 vol / pH adjusted to 2.1 with phosphoric acid) and liquid B: (isopropanol 950 vol / 2-methoxyethanol 50 vol / Phosphoric acid (2.5 vol) was detected with a linear gradient of isopropanol between two liquids, and detection was performed at a wavelength of 450 nm.

(5) Results The data before the reaction was set to 100, and the residual ratio of indirect bilirubin was determined by comparing the data with the peak area data based on the indirect bilirubin after the reaction. The results are shown in Table 2. The peak area based on indirect bilirubin hardly changed before and after the reaction, and it was confirmed that indirect bilirubin did not react under the measurement conditions of the present invention. On the other hand, in Comparative Example 3 (conventional method), indirect bilirubin was reduced by 16.5%. It shows that a part of indirect bilirubin reacted under the conditions of the conventional method.

  According to the present invention, direct bilirubin in a sample can be selectively measured without the influence of indirect bilirubin, which is useful in the field of clinical examination.

The reaction time course in Example 1 and Comparative Example 1 is shown. The abscissa indicates a photometric point (1 point is about 20 seconds), and the ordinate indicates absorbance × 10000. The reaction time course in Example 2 and Comparative Example 1 is shown. The abscissa indicates a photometric point (1 point is about 20 seconds), and the ordinate indicates absorbance × 10000. The reaction time course in Example 3 and Comparative Example 1 is shown. The abscissa indicates a photometric point (1 point is about 20 seconds), and the ordinate indicates absorbance × 10000. The reaction time course in Example 4 and Comparative Example 1 is shown. The abscissa indicates a photometric point (1 point is about 20 seconds), and the ordinate indicates absorbance × 10000. The reaction time course of indirect type bilirubin by bilirubin oxidase in Example 5 and Comparative Example 1 is shown. The abscissa indicates a photometric point (1 point is about 20 seconds), and the ordinate indicates absorbance × 10000. The reaction time course of indirect type bilirubin by bilirubin oxidase in Example 6 and Comparative Example 1 is shown. The abscissa indicates a photometric point (1 point is about 20 seconds), and the ordinate indicates absorbance × 10000. The result in Example 7 and Comparative Example 2 is shown. The horizontal axis shows the ratio of direct bilirubin to total bilirubin, and the vertical axis shows the amount of decrease in absorbance. The result in Example 8 and Comparative Example 2 is shown. The horizontal axis shows the ratio of direct bilirubin to total bilirubin, and the vertical axis shows the amount of decrease in absorbance. The result in Example 9 and Comparative Example 2 is shown. The horizontal axis shows the ratio of direct bilirubin to total bilirubin, and the vertical axis shows the amount of decrease in absorbance. The result in Example 10 and Comparative Example 2 is shown. The horizontal axis shows the ratio of direct bilirubin to total bilirubin, and the vertical axis shows the amount of decrease in absorbance. The result in Example 11 and Comparative Example 2 is shown. The horizontal axis shows the ratio of direct bilirubin to total bilirubin, and the vertical axis shows the amount of decrease in absorbance. The result in Example 12 and Comparative Example 2 is shown. The horizontal axis shows the ratio of direct bilirubin to total bilirubin, and the vertical axis shows the amount of decrease in absorbance.

Claims (1)

  A reagent for direct bilirubin measurement, comprising as essential components i) bilirubin oxidase and ii) an indirect bilirubin reaction inhibitor that is 100 mM to 800 mM potassium ion.

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