JPS6131096A - Method for determining bilirubin - Google Patents

Abstract

PURPOSE:To determine accurately and rapidly bilirubin, by using 3-methyl-2-benzothiazoline-hydrazone and a novel bilirubin oxidase M-1 is determining the bilirubin in a test solution. CONSTITUTION:3-Methyl-2-benzothiazoline-hydrazone and a novel bilirubin oxidase M-1 are reacted with a test solution, blood serum or urine, etc. containing bilirubin. The reaction solution is then acidified to measure optically the formed blue dyestuff and determine the bilirubin. The bilirubin oxidase M-1 oxidizes the bilirubin in the presence of oxygen to form biliberdin. The optimum pH is about 5.5, and the optimum temperature is about 50 deg.C. The molecular weight is about 83,000 measured by the gel filtration method.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for quantifying bilirubin in a test fluid, such as a body fluid, using the novel bilirubin oxidase M-1.

[Conventional technology]

Conventionally, the most popular method for quantifying bilirubin in body fluids is the diazo method, a chemical measurement method using diazonium salts such as diazosulfanilic acid (Izumi Kanai,
Co-authored by Masamitsu Kanai, Summary of Clinical Testing Law, 28th edition, No. M-24
Page, published by Metal Publishing in 1978). However, such 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.
It has been pointed out that there are many problems in terms of maintenance, management, and accurate measurement.

Furthermore, recently, a method for quantifying bilirubin in body fluids using bilirubin Φ oxidase is being developed. Agaricus bisporus (A
There is a description that a bilirubin-specific fungal enzyme composition prepared from B. garicua bisporus has the effect of specifically reacting with bilirubin to cause a color change. In the reaction between the enzyme composition and bilirubin, hydrogen peroxide may or may not be produced depending on the method for preparing the enzyme composition. Therefore, a method has been proposed for quantifying bilirubin by applying the enzyme composition to a bilirubin-containing specimen and optically measuring the amount of hydrogen peroxide produced or changes in bilirubin. It is very unclear whether the reaction on bilirubin is catalyzed by a single enzyme or by a system of multiple enzymes. 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. Also, JP-A-59-1
No. 7999 states that when bilirubin oxidase produced by the genus Myrocesium (see JP-A-58-141783 for this bilirubin oxidase) is used to quantify bilirubin in body fluids, bilirubin in serum cannot be determined by using this enzyme alone. Since it does not act on albumin-bound bilirubin in its existing form, a method has been reported for quantifying bilirubin by optically measuring changes in bilirubin with the addition of a surfactant, preferably sodium cholate, as a reaction accelerator. Station that has been. The present inventors previously developed a novel lupine oxidase M-1 produced by the basidiomycete, Trachyderma tsunodae
We have proposed a method for quantifying bilirubin in which a reagent composition for bilirubin containing bilirubin (see No. 8-218895) is applied to a bilirubin-containing test solution and the resulting change in bilirubin is optically measured (Japanese Patent Application No. 10560/1982).
No. 4). This method is superior to conventional methods in that it is possible to quantify bilirubin even without the addition of a reaction accelerator, and in the case where a reaction accelerator is used, sodium deoxycholate, which is about 13 times more effective than sodium cholate, is used.

Although the enzymatic method described above is superior to chemical methods in that accurate quantification is possible due to the specificity of the reaction, development of a more rapid measurement method is desired due to its character as a clinical diagnostic method.

[Problem that the invention seeks to solve]

The purpose of the present invention is to develop a new method for quantifying bilirubin in a test solution, and to provide a method for quickly, accurately, and easily quantifying bilirubin using the novel bilirubin oxidase M-1. .

[Means for solving problems]

To summarize the present invention, the present invention relates to a method for quantifying bilirubin, and the present invention relates to a method for quantifying bilirubin.
(abbreviated as TH) and novel bilirubin oxidase M-1, and then acidified and optically measuring the blue pigment produced. As a result of extensive research into methods for quantifying bilirubin, the present inventors found that novel bilirubin oxidase M-1 catalyzes the condensation reaction between MBTH and bilirubin, and the resulting condensed substance is colorimetrically quantified under acidic conditions. We have discovered that bilirubin can be quantified and that in the presence of the novel bilirubin oxidase M-1, the condensation reaction between MBTI (and bilirubin) proceeds more rapidly than the oxidation of bilirubin.

First, each property of the novel bilirubin oxidase M-1 used in the present invention will be shown.

Enzyme-chemical and physicochemical properties of novel bilirubin oxidase M-1 (1) Action: This enzyme liquefies bilirubin (albumin-bound type, glucuronic acid-bound type, free type) in the presence of oxygen to produce biliverdin. Then, after passing through a pale green substance and a pale red substance,
Although it has the effect of producing a colorless substance, it does not produce hydrogen peroxide.

(2) Substrate specificity: Acts on bilirubin. It also acts on biliverdin,
This is about 12% of the oxidation rate of bilirubin. It also acts on hydroquinone, pyrocatechol, para-phenylenediamine, pyrogallol, 4-aminoantipyrine and monophenol. It has no effect on hemin, vitamin l1tt and hemoglobin.

(3) Optimum pH and pH stability: The optimal pH of this enzyme is around 5.5, and when treated at 37°C for 60 minutes at each pH, the pH was 5.
It is stable between ~pH9.

(4) Optimum temperature and thermostability: The optimal temperature of this enzyme is around 50°C, and when treated for 10 minutes at each temperature at pH 7.0,
It is stable up to C.

(5) Molecule lit: The molecular weight of this enzyme is about 83,000 when measured by gel filtration using Sephacryl S-200 (manufactured by Pharmacia).

(6) Uniformity: Disk electrophoresis was performed using a 7.5% polyacrylamide gel (pH 9,4), and protein staining and activity staining were performed. A single protein staining band was observed. Furthermore, since this enzyme has the effect of permanently producing 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: Pharmalite (pH 3 to pH 10, manufactured by Pharmacia)
The isoelectric point of this enzyme is 3.92±0.05 as determined by focal electrophoresis using .

(8) Effects of metal ions, inhibitors, etc.: This enzyme contains L-ascorbic acid, azide + ) IJium, dithiothreitol, potassium cyanide, ■, -cysteine, EDT
It is inhibited by A1Fθ'+ etc. (Table 1).

Table 1 Additive inhibition rate (
(6) L-Asfulubi 71! ! (1mM)
100 Sodium azide C
rt > 60 (5mM) 10
0 dithiothreitol (1mM)
86 Potassium cyanide (p)
84L-cystine (tt
) 74°DTA
Cry) 591・. tithe
(=) 57 reduced glutathione (〃) 380-phenanthroline C1/) 342-mercaptoethanol (〃) 31α, C1-dipyridyl (
#) 30 iodoacetic acid
(tt) 10(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 a blue protein.

(10) Disk electrophoresis was performed using a 5% polyacrylamide gel (pH 9.4) with a low sugar content, and PAS staining t' [Analytical Biochemistry (Anal.

Biochem, ) Vol. 30, p. 148 (1969)
], 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) Preparation lt: 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 from the decrease in absorption of bilirubin at 460 nm. 10. Omega high bilirubin control serum (manufactured by Highland, USA) 01 ml, phosphate buffer (pH 7.0) 300 phylomoles and appropriately diluted enzyme solution 0.1 mJ, reaction volume 30 m/37°C.
The reaction was allowed to proceed for 10 minutes and the absorption decrease <460 nm based on bilirubin was measured. Shinbiki bilirubin-oxidase M-
One unit of 1 is expressed as the amount of enzyme that oxidizes 1 micromole of bilirubin per minute in the above reaction system.

The above novel bilirubin oxidase M used in the present invention
-1 with conventional bilirubin oxidase,
As shown in Table 2.

The method for producing the novel bilirubin oxidase M-1 is described in Japanese Patent Application No. 58-218895.

Method for Assaying Bilirubin The present inventors have conducted intensive studies on methods for quantifying bilirubin in body fluids, and have discovered that the novel bilirubin oxidase M-1 further catalyzes the oxidative condensation of MBTH and bilirubin. According to the present invention, bilirubin in bilirubin-containing test fluids, such as body fluids such as serum and urine, can be quantified by utilizing the above properties.

For example, apply METH to 10 to 10 apl- of a sample such as serum.
o, 1-3 micromolar, pH 5,0-7.0 buffer and novel bilirubin oxidase M-1, o, o
os~0.1 unit is added and reacted at 20-40°C, preferably 37°C, for 1-20 minutes, preferably 3-6 minutes, producing a red dye with maximum absorption around 520 nm and 550 nm. .

Under acidic conditions, such as hydrochloric acid or sulfuric acid, this red pigment converts to a blue pigment with maximum absorption at ilQnm and a large molecular extinction coefficient. Bilirubin can be quantified.

For example, Omega High Bilirubin Control Serum (20■
/dt) (manufactured by Highland, USA) 100pt, MB
TH1 micromolar, 0.3M citrate buffer (pH 5
,5) Adding 1.9 WLl and 10,05 units of the novel bilirubin-oxidase M, with a total liquid volume of 2.0-3
520 nm and 550 nm after 15 minutes reaction at 7°C.
Generates a red dye with maximum absorption near nm. When IN Hotl, 0- is added to this substance and brought under acidic conditions, a blue dye having maximum absorption at 610 mm and a large molecular extinction coefficient is produced (see Figure 1). That is, the first
The figure is a graph showing the relationship between wavelength (n) (horizontal axis) and rM luminous intensity (vertical axis). In FIG. 1, the absorption spectrum of the red dye is shown as a dotted line, and the absorption spectrum of the blue dye is shown as a solid line.

〔Example〕

The method for quantifying bilirubin according to the present invention will be illustrated below with examples, but the present invention is not limited to the following examples.

Example 1 5η/dt, 10η7'az, xs■/at, 20ya
Prepare each bilirubin solution of p/dt [Albumin Binding Gourd - Manufactured by Kagaku Yakuhin Co., Ltd.], and add 0.1 liter of each solution.
0.3M citrate buffer (pH 5,5) 1.0wt1
．． l OmM MBTHQ, I ml and 10.05 units of novel bilirubin oxidase M- were added, each bringing the total volume to 2. After reacting at 37° C. for 15 minutes, add IN Hot 1 and Ottrl and measure the absorbance at 610 nm (OD sample). Separately, as a control, the absorbance at 610 nm 11i (
OD blank) was measured, and △ODa+o (OD sample - 〇D blank) was determined. The results are shown in Figure 2. In other words, in Figure 2, the calibration curve is
) (horizontal axis) and absorbance difference (80Ds+o) (vertical axis).

As is clear from FIG. 2, 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 absorbance at 610 nm.

Example 2 Omega high bilirubin control serum (manufactured by Highland, Inc., USA) was used in place of the bilirubin (albumin-binding type, manufactured by Daiichi Chemical Co., Ltd.) used in Example 1, and the bilirubin dilution series was similarly adjusted. Then, the reaction was carried out in the same manner as in Example 1, ΔQDa+· was determined, and a calibration curve was prepared. The results are shown in Figure 3. In other words, Figure 3 shows the calibration curve between the bilirubin concentration (P/d/S) (horizontal axis) and the absorbance difference (ΔODs+o
) (vertical axis).

〔Effect of the invention〕

As explained above, the present invention provides a novel method for quantifying bilirubin using novel bilirubin oxidase M-1 and MBTH. The method of the present invention has the remarkable effect that bilirubin in a test fluid can be determined quickly, accurately, and easily.

[Brief explanation of drawings]

1st v! J is a graph showing the absorption spectrum (radio) of the reaction product (red dye) of bilirubin and MBTH by the novel bilirubin oxidase M-1 and the absorption spectrum (solid line) of the blue dye produced under hydrochloric acid acidity. FIG. 12 is a graph showing the calibration curve in Example 1 of the quantitative method of the present invention in terms of the relationship between bilirubin concentration and absorbance. FIG. 3 is a graph showing the calibration curve in Example 2 as a relationship between bilirubin concentration and absorbance.

Claims (1)

[Claims] 1. 3-Methyl-2-benzothiazolinone-hydrazone and novel bilirubin oxidase M-1 are added to the bilirubin-containing test solution to carry out the reaction, then the reaction solution is made acidic, and bilirubin is optically quantified. A method for quantifying bilirubin, characterized by: