JP2911696B2 - Reagents for direct or total bilirubin measurement - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent for measuring direct bilirubin or total bilirubin in a biological fluid.

[0002]

2. Description of the Related Art Bilirubin is a yellow pigment belonging to tetrapyrroles, is a physiological metabolite of heme, and is most frequently present in bile. In addition, bilirubin in the biological fluid is a direct form bilirubin conjugated with glucuronic acid,
It is broadly classified into unconjugated indirect bilirubin.

[0003] The measurement of bilirubin is an important test item that serves as an index for diagnosis of various diseases, and is routinely measured in the field of clinical tests. For example, it is known that the amount of indirect type bilirubin increases in conditions such as hemolytic anemia and hemolytic jaundice, and the amount of direct type bilirubin increases in conditions such as obstructive jaundice. It occupies an important position in such places. Conventionally, bilirubin measured in clinical tests and the like is total bilirubin and direct bilirubin, and accurate quantification of these bilirubins is indispensable in clinical tests and the like.

[0004] Known methods for measuring bilirubin include a method using a diazo reagent, a method using high performance liquid chromatography, and a method using an enzyme such as bilirubin oxidase. Of these, the method using a diazo reagent depends on the type of the diazotization reaction accelerator used and the difference in the method of quantifying the azobilirubin produced.
Various things have been reported. For example, a typical example is the reagent by Malloy & Evelyn [Journal of Biological Chemistry, 119
Vol., Pp. 481 (1937)].

[0005] The method of fractionation and quantification by high performance liquid chromatography includes a method of Lauff et al., In which a reverse-phase HPLC column is used to elute and analyze various bilirubins with a gradient of a phosphate buffer and isopropanol [Journal. Of chromatography (Journal
of Chromatography), Vol. 226, p. 391 (198
1 year)].

[0006] In the method using an enzyme, bilirubin is oxidized directly or indirectly by the action of an enzyme such as oxidase, the yellow color of the bilirubin itself at around 450 nm is eliminated, and bilirubin is converted from the change in absorbance before and after the reaction. It is to be measured. In this case, various bilirubins can be specifically oxidized or all bilirubins can be oxidized by changing reaction conditions in various ways. As a reagent used in this method, for example, a reagent using bilirubin oxidase [Clinical chemistry]
(Clinical Chemistry), vol. 20, p. 783 (197
4 years)], reagents using oxidases such as laccase, tyrosinase and ascorbate oxidase (JP-A-59-17999), anionic surfactants such as cholic acid and aromatic carboxylic acids as reaction accelerators. The reagent for measuring total bilirubin used (Japanese Patent Application Laid-Open No. 60-249060)
Publication). Further, a reagent for measuring direct or conjugated bilirubin in which the conditions under which bilirubin oxidase acts only on direct or conjugated bilirubin by selecting a pH, a buffer, and a surfactant has been proposed.

As a reagent for measuring bilirubin, for example, p
A reagent for quantifying conjugated bilirubin by allowing bilirubin oxidase to act in a buffer solution in the range of H9 to H11 (Japanese Patent Application Laid-Open No. 62-58999), an acidic buffer solution having a pH of 5 to 6 containing an anionic surfactant Reagent for quantifying direct bilirubin by the action of bilirubin oxidase in the medium (Japanese Patent Application Laid-Open No. 60-152950), and quantifying the direct bilirubin by the action of bilirubin oxidase in a buffer having a pH of 3.5 to 4.5 Reagents (JP-B-61-44000).

[0008]

In particular, in a method using an oxidizing agent such as a diazo reagent or an enzyme for oxidizing bilirubin,
The reagent is generally in the form of a two-part system consisting of a first reagent and a second reagent. In the method, first, a first reagent and a sample are mixed, absorbance after preheating is measured, and various bilirubin concentrations are measured by comparing with the absorbance after adding a second reagent that oxidizes various bilirubins. . However, since bilirubin is a relatively unstable substance, the various measurement methods used in the past have caused the measured values of various bilirubins to be practical because the bilirubin in the sample gradually decomposes after mixing the sample with the first reagent. There is a problem that the value becomes lower.

The present inventors have conducted intensive studies to solve these problems in bilirubin measurement using an oxidizing agent such as a diazo reagent or an enzyme. As a result, it was found that by adding ascorbic acid to the first reagent of the bilirubin measurement reagent, the decomposition of bilirubin before adding the second and subsequent reagents was prevented, and it was found that bilirubin could be measured with high accuracy. By including ascorbate oxidase in the reagent, it was found that the effect of ascorbic acid added to the first reagent on the measurement was prevented, and it was found that bilirubin could be measured with high accuracy, and the present invention was completed.

[0010]

That is, a first aspect of the present invention relates to a reagent for measuring direct bilirubin or total bilirubin, which optically measures a change in bilirubin by causing an oxidizing agent to act on bilirubin, which contains ascorbic acid. The gist of the present invention is a reagent for measuring direct bilirubin or total bilirubin, which is characterized by being obtained by the method. Further, the second invention divides a direct bilirubin or total bilirubin measuring reagent for measuring the change of bilirubin by causing an oxidizing agent to act on bilirubin into at least two reagents, and ascorbic acid as the first reagent, The present invention is directed to a reagent for measuring direct bilirubin or total bilirubin, characterized in that the second reagent contains ascorbate oxidase.

The reagent of the present invention is usually composed of a solution containing ascorbic acid (first reagent) and a solution containing oxidizing agents (for example, bilirubin oxidase and diazo reagent) for quantifying various bilirubins as main components (first reagent). (2 reagents), and ascorbate oxidase that specifically oxidizes ascorbic acid contained in the first reagent may be added to the second reagent. Further, the second reagent may be used as an oxidizing agent for quantifying direct bilirubin, and the third reagent may be used as an oxidizing agent for measuring indirect bilirubin.
In this case, there is an advantage that both direct bilirubin and indirect bilirubin can be measured at once in a series of measurements.

As described above, the reagent of the present invention can be applied not only to a reagent for measuring direct bilirubin but also to a reagent for measuring total bilirubin by changing the final pH and composition of the reagent. When various kinds of bilirubin are measured using this reagent, for example, the reaction temperature is 25 to 45 ° C, preferably 30 to 40 ° C, and the reaction time is 1 to 30 minutes, preferably 3 to 15 minutes.

The amount of ascorbic acid of the first reagent is 0.00
1 to 100 μg / ml, preferably 0.01 to 10 μg / m
l. If the amount is less than 0.001 μg / ml, the decomposition of bilirubin cannot be sufficiently prevented, and if it exceeds 100 μg / ml, the oxidation of bilirubin after the addition of the second reagent is hindered, which is not preferable.

Examples of ascorbate oxidase which can be added to the second reagent include enzymes derived from plants such as cucumber and enzymes obtained from microorganisms such as strains belonging to the genus Acremonium. It is 0.01 to 200 units / ml, preferably 0.1 to 20 units / ml.

Examples of the oxidizing agent used in the second reagent include oxidases such as bilirubin oxidase and the like, and oxidases such as glucose oxidase that generate hydrogen peroxide and the like, a mixture of their oxidases and peroxidase, and bilirubin oxidizing agents. Diazo reagents such as monovalent copper ions or salts thereof (for example, peroxides such as copper sulfate, copper chloride, and calcium persulfate), a hydrochloric acid solution of sulfanilic acid and sulfurous acid, and a mixed solution of 2,4-dichloroanilic acid. These can be used alone or in combination.

These oxidizing agents are 0.01 in the case of an enzyme.
~ 30 units / ml, preferably 0.05-20 units / ml,
When using peroxide, 0.01 to 50 mM, preferably 0.05 to 30 mM, and when using metal ions and their salts, 0.01 to 20 mM, preferably 0.05 to 5 mM.
When M or diazo reagent is used, it can be used in a concentration range of 0.05 to 5 mM, preferably 0.1 to 2 mM. When the amount of these components is outside the above range, the reaction for oxidizing bilirubin takes a long time, the deterioration of the reagent becomes remarkable, or a precipitate may be formed in the reagent, which is preferable. Absent. The source of the oxidase is not limited, and various sources may be used.
For example, as a laccase, an enzyme derived from a microorganism such as urushi or polyporous genus, as bilirubin oxidase, an enzyme obtained from a strain belonging to the genus Milosesium or Trachiderma, and as glucose oxidase, an enzyme derived from Aspergillus can be used.

The reagent for measuring bilirubin of the present invention contains, in addition to the above essential components, an aromatic carboxylic acid as a reaction accelerator, a surfactant, a buffer, a chelating agent, a commonly used activator and salts. be able to.

As the aromatic carboxylic acid, for example, p-
Toluenesulfonic acid, benzoic acid, etc., and 1 to 10
It can be used in a concentration range of 0 mM.

As the surfactant, any of anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants can be used. For example, sodium cholate, polyoxyethylene glycol, Triton X-
100, twin 20, lauryl trimethyl ammonium chloride, sodium dodecyl sulfate, N-lauryl myristyl-β-aminopropionic acid and the like. These surfactants can be used in a commonly used concentration range of 0.001 to 10%.

As the buffer for the reagent, a buffer having a buffering capacity at a pH suitable for the type of bilirubin to be measured may be used. For example, phthalate buffer, malate buffer,
Citrate buffer, phosphate buffer, Tris buffer, HEP
There are ES buffer, MOPS buffer and the like. The concentration of the buffer is 10 mM to 2M, preferably 20 mM to 1.5M.

Examples of the chelating agent include polyaminocarboxylic acids such as ethylenediaminetetraacetic acid and oxycarboxylic acids such as citric acid, which can be used in a concentration range of 0.01 to 100 mM, preferably 0.02 to 50 mM.

As the activator, polyethylene glycol, albumin, saccharides and the like can be used. 0.02 to 5% when using polyethylene glycol, 0.001 to 1% when using albumin, 5 when using saccharides.
Use in the range of １００100 mM.

Further, as the salts, for example, sodium chloride, ammonium chloride, potassium sulfate and the like are 1 to 50.
It can be used in the range of 0 mM.

The specific method of the present invention for measuring various kinds of bilirubin using a two-part reagent for measuring the change in the absorbance of bilirubin by allowing an enzyme to act on the various kinds of bilirubin thus obtained, for example, is described below. Show. First, an appropriate amount of a sample (such as serum) containing various bilirubins is added to the above-mentioned first reagent (0.6 ml) for measuring bilirubin, and after preheating in a cell room of a spectrophotometer, the absorbance at 460 nm is measured. To this, the above-mentioned second reagent for measuring bilirubin (0.15 ml) was added, and reacted for 1 to 10 minutes to oxidize the bilirubin to be measured. Then, the absorbance at 460 nm was measured. The amount of change in absorbance (A) is determined from the corrected liquid amount of the absorbance. Next, a standard substance containing known concentrations of various bilirubins is measured in the same manner to determine the amount of change in absorbance (B). From the amount of change in absorbance determined by the action of the sample and the amount of change in absorbance determined by the action of the standard substance, the content of various bilirubins in the sample can be determined by the following equation. Various bilirubin concentrations in the specimen (mg / dl) = A / B × various bilirubin concentrations in the standard substance (mg / dl) The amount of the specimen is preferably 0.005 to 0.1 ml. The measurement wavelength is not limited to 460 nm, and any wavelength between 400 and 480 nm can be selected. Further, an arbitrary wavelength is set as an auxiliary wavelength in addition to an arbitrary wavelength of 400 to 480 nm (hereinafter, referred to as a main wavelength), and bilirubin is measured using a change in the difference between the absorbance at the main wavelength and the absorbance at the auxiliary wavelength. be able to. In addition, the liquid amounts of the first reagent, the second reagent, and the sample can be appropriately changed.

[0025]

Next, the present invention will be described more specifically with reference to examples and comparative examples. In Examples 1 and 2 and Comparative Examples 1 and 2 , the composition of the direct bilirubin measurement reagent was 60 mM potassium hydrogen phthalate (pH 5.0), 2.0 mM N-acetylcysteine, 0.001 mg / ml ascorbic acid. Was prepared as described above. Further, potassium hydrogen phthalate 60 mM (pH 5.0), bilirubin oxidase derived from a strain of the genus Trachiderma (purchased from Takara Shuzo) 5.0 units / m 2
1. Reagent 2 was prepared so that the concentration of ascorbate oxidase derived from microorganisms (purchased from Asahi Kasei Kogyo) was 2.0 units / ml. Tricine buffer 20 mM (pH 8.5), bilirubin oxidase derived from a strain of the genus Myrocesium (purchased from Amano Pharmaceutical) 6.0 units / ml, ascorbate oxidase derived from microorganisms 2.0 units / ml (purchased from Asahi Chemical Industry) Reagent 3 was prepared so that Further, reagent 4 was prepared by removing ascorbic acid from reagent 1.

Human serum (0.02 ml) was added to reagent 1 (0.4 ml), and the mixture was heated at 37 ° C. for 5 minutes. Then, reagent 2 (0.1 ml) was added and reacted at 37 ° C. for 5 minutes. In this case, 450 nm and 57
The time course of the difference in absorbance at 0 nm was recorded. The above operation was performed using a Hitachi 7050 automatic analyzer. The results are shown in FIG. 1 (Example 1). In addition, 450 nm and 570 nm
The change over time in the difference in absorbance at nm was measured (Comparative Example 1), and these are also shown in FIG. further,
Reagent 3 instead of Reagent 1 and Reagent 2 in Example 1
Was used to measure the change over time in the difference in absorbance at 450 nm and 570 nm, and the results are shown in FIG. 2 (Example 2). Further, the change with time of the difference in absorbance at 450 nm and 570 nm was measured using the reagent 4 instead of the reagent 1 in Example 2 (Comparative Example 2), and this is also shown in FIG. From these results, in the direct bilirubin measuring reagent of the comparative example, human serum was added to the first liquid, and bilirubin was gradually decomposed during heating at 37 ° C. for 5 minutes, and a decrease in absorbance was observed. However, in the direct bilirubin reagent of the present invention, no phenomenon of a decrease in bilirubin absorption was observed.

Example 3 and Comparative Example 3 In the reagent for measuring total bilirubin, the composition was HEP.
ES buffer 100 mM (pH 7.8), Triton X-100
Reagent 5 was prepared so as to be 0.01%, sodium cholate 0.1%, and ascorbic acid 0.005 mg / ml. In addition, HEPES buffer 100 mM (pH 7.8), Triton X
-100 0.01%, bilirubin oxidase from Myrocesium sp. (Purchased from Amano Pharmaceutical) 4.0 units / m
1. Reagent 6 was prepared so as to have a cucumber-derived ascorbate oxidase (purchased from Boehringer Mannheim Yamanouchi) 10.0 units / ml. Further, Reagent 7 obtained by removing ascorbate oxidase from Preparation 5 and Reagent 8 obtained by removing ascorbate oxidase from Reagent 6 were prepared.

Human serum (0.02 ml) was added to reagent 5 (0.4 ml), and the mixture was heated at 37 ° C. for 5 minutes. Then, reagent 6 (0.1 ml) was added and reacted at 37 ° C. for 5 minutes. In this case, 450 nm and 57
The change with time of the difference in absorbance at 0 nm was measured. The above operation was performed using a Hitachi 7050 automatic analyzer. The result is shown in FIG. 3 (Example 3). Further, with the use of the reagents 7 and 8 in place of the reagents 5 and 6 in Example 3, the change with time in the difference in absorbance at 450 nm and 570 nm was measured (Comparative Example 3). It was shown to. From these results, in the reagent for measuring total bilirubin of the comparative example, human serum was added to the first liquid,
While heating at 37 ° C. for 5 minutes, the absorption of bilirubin gradually decreased, but no decrease in the absorption of bilirubin was observed in the total bilirubin reagent of the present invention.

[0029]

As described above, according to the present invention, it is possible to accurately measure bilirubin, which is known to increase remarkably in biological fluids of patients with hemolytic anemia, jaundice, etc. Useful in laboratory tests and others.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing a change over time in absorbance when human serum is used as a sample in a direct bilirubin measurement reagent using bilirubin oxidase derived from a genus Trachyderma as a bilirubin oxidase.

FIG. 2 is a graph showing the time-dependent change in absorbance when human serum is used as a sample in a direct bilirubin measurement reagent when a bilirubin oxidase derived from a strain of the genus Myrocesium is used as the bilirubin oxidase.

FIG. 3 is a graph showing a change over time in absorbance when human serum is used as a sample in a reagent for measuring total bilirubin using an enzyme derived from a strain of the genus Myrocesium as bilirubin oxidase.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hitoshi Kondo 23 Uji Kozakura, Uji-city, Kyoto Unitika Unit Research Institute, Inc. (72) Inventor Masami Kojima 1460-6 Mitodai, Mitodai, Tako-cho, Katori-gun, Chiba Pref. (56) References US Patent 4,563,429 (US, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12Q 1/00-3/00 G01N 33/72 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A direct type bilirubin or total bilirubin measuring reagent for optically measuring a change in bilirubin by causing an oxidizing agent to act on bilirubin is divided into at least two reagents, and ascorbic acid is used as a first reagent and ascorbic acid is used as a second reagent. A reagent for measuring direct bilirubin or total bilirubin, wherein the reagent contains ascorbate oxidase.