JPS5876100A - Use of laccase - Google Patents

Abstract

PURPOSE:In the measurement of components in body fluids using coloring method of oxidase-peroxidase-hydrogen donor as a color raw material, to improve analysis precision, by separating previously a hydrogen donor in a specimen to be tested by laccase. CONSTITUTION:In a determination wherein an oxidase specific to the desired components (glucose, cholesterol, etc.) in body fluids (serum or urine) is added to a specimen to be tested to give hydrogen peroxide, which is reacted with peroxidase derived from a horseradish, and the amount of the resultant oxidizing coloring compound is measured to determine the desired components, laccase (preferably derived from coriolus) is previously added to the specimen to be added, main hydrogen donors (bilirubin, ascorbic acid, urea, hemoglobin) in body fluids are decomposed, so that obstructions caused by them are removed. A laccase-containing buffer solution (pH of 4-7) is added to the specimen to be tested and reacted with it is 37 deg.C for 1-20min, the reduction ratio in absorbance at 460nm of bilirubin is measured to determine bilirubin in the specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for using laccase O, which has the activity of degrading bilirubin, ascorbic acid, uric acid, and hemoglobin, which are mainly hydrogen donors, in body fluids.

More specifically, in order to accurately measure the two-purpose acid content in a system in which the components in body fluids such as serum or urine are kept constant, there is a method in which untargeted O components are decomposed using laccase. And laccase O has the property of degrading bilirubin.

As a method for measuring O components in body fluids such as serum or urine, an oxidase (oxidase) that specifically reacts with the target component is used to react with hydrogen peroxide, which is the O reaction product, and horseradish-derived O /(- oxidase acts,
A measurement method using a system that converts a chromogen, which is a hydrogen donor, into an oxidative color-forming compound has become widespread and has been incorporated into daily testing. Example JLtf cholesterol, crucose,
is.

Measurements of phospholipids, neutral fats, and free fatty acids are mainly carried out by the methods described above.

Since this method uses an enzymatic reaction, it has good specificity, is quick and simple, and is a method with excellent Fiability as a daily test. However, there is a reducing substance that is an O component other than the purpose that exists in the specimen sample and body fluid, that is, bilirubin,
Because ascorbic acid, uric acid, hemoglobin, etc. act as hydrogen donors.

The peroxidase-hydrogen donor chromogen O coloring reaction, which is a hydrogen peroxide O detection system, is affected by the shadow 411 and has the drawback that accurate measurement values of the target component O cannot be obtained. In particular, bilirubin and ascorbic acid are the main causes.

It is good that efforts are being made to avoid such negative effects. One person is 1 just like the O-reducing substance in the body casing.
Although it is a method to improve the chromogen that acts as a hydrogen donor, no promising results have been observed yet. The other method is to decompose and remove O-reducing substances from body fluids. For example, in the case of bilirubin D, a method of oxidizing it with a2-benzoic acid [Japanese Journal of Medical Technology (Japan*se Journal of M
@1ioax〒@OMOIOg7 ) &s Volume 0, 3
issue, SOS page, (19151)], in the case of ascorbic acid O, there is a method of treatment under strong alkaline O conditions, a method of pretreatment with Cu + 4- ions or 1. !4?20) and a reaction treatment method using an enzyme called ascorbic acid oxidase, which has already been put into practical use in some places [Clinical Chemistry (Olinfoalohamistry), 24
Vol., 250 pages (1978)] are being considered.

However, the purpose of these methods is to simultaneously degrade bilirubin, ascorbic acid, uric acid, and hemoglobin as reductive interfering substances in a single treatment. , there is no known method to avoid these D effects in the measurement system.

On the other hand, the measurement of bilirubin O, which coexists in body fluids, has a high degree of clinical significance in itself and is used to differentiate, for example, the mechanism of hepatobiliary obstruction.
The measurement method used in routine testing is a chemical method, which uses diazo and nium salts such as diazosulfanilic acid. The so-called diazo method is the mainstream. In general, there are not many reports regarding bilirubin O regarding fermentation methods that allow easy and specific measurement under mild conditions. In part, as mentioned above, bilirubin D acts as a surface hydrogen donor.
There is a method that indirectly utilizes the interference effect in a peroxidase-hydrogen donor chromogen coloring system.

Hire originally meant O, so it is difficult to say that it is an O enzyme method. Also %l1
1975-IF11? As described in No. S, there is a measurement method that uses bilirubin-specific resistant enzyme compositions; There may be no. Therefore, if we discuss the enzymatic reaction stoichiometrically.

It is doubtful whether the enzyme in question is a single enzyme, and it is thought that there is a problem in terms of supplying the enzyme with uniform quality, which is an essential condition for enzymatic methods.

As a result of extensive research, the inventors found that lacquer (
is used to decompose O-reducing substances in body fluids.

A method for avoiding OB shadow contamination in a reducing substance O coloring reaction system and a method for measuring O bilirubin in body fluids using Lacquer-V 1. Heading 1. The present invention has been completed. The present invention will be explained in detail below.

Laccase is generally an enzyme (1
・10.3.2).

The lacquer 41 used in the present invention may be any laccase as long as it has the properties shown below, but is preferably O-laccase derived from the genus Coriolus. To explain the present invention, typical properties of the present enzyme will be described.

(1) Bilirubin, which has an absorption maximum of 460 nm, is oxidized to biliverdin, which has an absorption maximum of 480 nm and a smaller extinction coefficient than bilirubin, and is further oxidized to an unknown substance that has no absorption.

In the case of hydrogen oxide, dissolved oxygen in the reaction solution is consumed, but hydrogen peroxide is not produced. In other words, it eliminates the color tone and reducing power of bilirubin.

C2) In addition to bilirubin, ascorbic acid, uric acid, hexagonal globin, etc. also serve as substrates. This fact has been confirmed by the measurement method based on the reducing power of ascorbic acid, the measurement of O ultraviolet number 1flO at 2 9 S nm under alkaline conditions, and the production of methemoglobin O by absorption spectroscopy of hemoglobin solution. . Furthermore, even when these serve as a base, no hydrogen peroxide O is produced.

(3) Glucose, cholesterol, purine, glycerol, glycerol-tri-phosphate and acyl-Co
It does not show any effect on mu etc.

(4) Enzyme protein has a molecular weight of s a o o o, a sugar content of 4, a copper content of 9 G, and an isoelectric point of 41°.
It is generally thought to belong to a group of enzymes called blue proteins.

(5) Full & reaction optimum p111115~L
Typical reaction inhibitors include sodium azide, ferrous sulfate, potassium ferrocyanide, and potassium monofluoride. In particular, sodium azide completely inhibits the reaction at a final concentration of 1 mM.

(6) A chromogen suitable for the peroxidase-hydrogen donor chromogen color method used for hydrogen peroxide detection.

For example, 4-7 minoantipyrine (hereinafter abbreviated as 4-mu)-phenol coupling system, 4-mu-/ethyl 7-component system, 4-mu iris-ethyl-1-hydroxyethyl theory-toluicine la and US - Meterni-2-benzothiazolinone-kando-2-dimethylaniline, etc. are oxidized and condensed to develop color under conditions where there is no volume of peroxide.

According to the present invention, as is clear from the properties of the laccase, glucose, cholesterol, choline, glycerol, In the method of measuring various biological components of glycerol-triacyl-phosphate-0oA, by treating the specimen with lacquerase in advance, bilirubin, ascorbic acid, uric acid, and hemoglobin III, which coexist in 9 specimens, can be detected.
IO-reducing interference substances
Avoid the negative influence of O on these O coloring systems and measure the target component O.
It can be done very accurately. Furthermore, by adding a reaction inhibitor as shown in (5) after pretreatment with laccase, it is possible to stop the action of laccase O on the chromogen and to accurately reflect only the chromogen in the chromogen. can.

As a processing method, (1) O specimen sample 1 such as serum or urine
11 containing 11 to 1 unit of laccase in 0 to 100 pj
1H4o~7.00 buffer 2) H&5~10
Add 0 acetate buffer 11-1-1 to 1-1 at a temperature of around 37℃.
After reacting for 20 minutes, preferably 5-10 minutes, (1
) Add an aqueous solution containing lacquer 4Illl harmful agent at a concentration of 1 to 50 mmoj/J, preferably 5 mmoj/JO, and after this, various biological component O measurement methods can be carried out. Further, in the above O treatment method, a laccase inhibitor may be added to the O reagent for measuring various body fluid components, and then added to the laccase treated specimen together with the reagent.

Furthermore, according to the present invention, the above-mentioned laccase O properties (1
) can be used to immobilize O-bilirubin in body fluids.

To quantify bilirubin O, the reaction was carried out according to the treatment method O (:) with laccase described above, and then O bilirubin 0
absorbance reduction rate at 460 mm] or measure the bilirubin O content in the specimen, or measure S-methyl-2-benzothiazolinone and dorazine (hereinafter abbreviated as MBTH).

), a bilirubin decomposition product, and the blue pigment produced by the O reaction. For example, add 105 to 15 units of laccase L and 1 to 10 units of MBTH to 10 to 100 μl of an O specimen sample such as serum. Buffer containing pmoJes.

Preferably pH 5~&OID acetate buffer a 15~&O
d for 1 to 20 minutes at a temperature of around 57°C.

Preferably, this is carried out by reacting for 5 to 15 minutes, acidifying with hydrochloric acid, or adding a solution of ferric chloride under the acidity of hydrochloric acid, and then measuring the absorption maximum 6 j Onu O absorbance.

The present invention will be explained in more detail with reference to Examples below.
These are not O, although they limit the invention.

Example 1 Example of avoiding the influence of bilirubin and ascorbic acid O during neutral fat measurement 1) Pretreatment agent in reagent: 50 m containing 500 units/j of laccase
a moj/J acetate buffer (pH 5,5).

Oi) Neutral fat measurement reagent 2 Commercially available O Libidosu 550 kit (Trademark, manufactured by Ono Pharmaceutical Co., Ltd.). The reagent composition is lipoprotein lipase and glycerol kinase.

Daliserophosphate oxidase, peroxidase.

These are adenosine triphos 7ate, 4-aminoantipyrine, ILII-diethyl-m-)luidine, and adenosine triphosate.

[Phase] Laccase inhibitor: Sodium azide 100mw
Aqueous solution containing hoj/j.

2) Procedure Add 20 pi of serum sample to a test tube, add reagent (1) α5-
After incubating at 37°C for 10 minutes, 5-5 times of the reagent O1) and ωO mixture [mixing ratio (ii):(hl-20:1) were added, and the mixture was allowed to react at 37°C for 15 minutes according to a conventional method.

Absorbance was measured at s o nm. It was also measured by a conventional method using the same WO serum sample.

Next, bilirubin (manufactured by Dai-ichi Chemical Co., Ltd.) and asculpic acid (manufactured by Torijin Pharmaceutical Co., Ltd.) were added to the serum sample at 20wII/44ml each.
01 added at a ratio of 5 mf/ajO and 0 added with both were prepared and measured using the method of the present invention and the conventional method as test samples to examine the effect of method O of the present invention.

The results are shown in the table below.

Table 1 Influence Wa of reducing substances in specimen samples according to the method of the present invention and the conventional method
) tloo-, and other cases of O are expressed as relative values to this.

Example 2 Bilirubin and uric acid O treatment with laccase Example 1) Bilirubin (manufactured by Dai-Kyakuyaku) 020 tsp/1j solution 300
pi and 10. Ommoj/jO acetate buffer (pH 45
), the degree of decrease in absorbance at 440 nm K by adding 11 units of laccase to a mixed solution consisting of S-, flz57
It was kept warm at ℃ and observed over time.

The results are shown in FIG. As shown in Figure 1, bilirupine O oxidation by laccase is evident. or.

When lacquer (along with KMBTil) is present in this reaction system, a compound with a maximum absorption at 520 nm is produced (see Figure 2). High molecular extinction coefficient (
A compound with a 610 nm K maximum absorption was produced.

2) 4 test tubes each containing uric acid 01G 11f/
Pour 100 JIji of dJ solution into 5 test tubes and 50 wa moj/j D Linshun buffer solution (pHLD).
Add 250μj, add 102 of the lacquer and add α2 monofilament, add 01 of the lacquer at 3'7℃ for 10
Allowed to react for minutes. After that, put 100 in each of the 4 test tubes.
m moj/j O) Lis-HCl buffer i [(pHto
) sWll was added and uric acid 02 under alkaline conditions.
The change in O absorption at 95 nm was observed. The results are shown in Figure S.Example 3 Bilirubin O effect during neutral fat measurement 0 cases For 40 serum samples, bilirubin (manufactured by Dai-Kyakuyaku) was added at a ratio of 20 x 7 dt to each sample. Neutral fat was measured using added 4O and non-added 4O as samples. Reagents and operating methods were used in accordance with Example IK, and the comparative correlation between the conventional method, the method of the present invention, and O was examined. 4th result
As shown in the figure. In the conventional method, the correlation O regression line is y-IB12x
riy-(L985-&2(n-
40,rsma? ? 7), indicating that the influence of bilirubin O, which was measured at a low value in the conventional method, was improved by the method of the present invention.

Example 4 Quantification of Bilirubin O 1) Quantification of bilirubin 0460nw* by absorption reduction method.

Using bilirubin (manufactured by Dai-ichi Chemical Co., Ltd.), OwIg/4
j, 511F/aj, 10wII/aj, 15
1QF/14゜2011f/(1jO) Create a dilution series, collect 020 pi of each in a test tube, and 10
．． Om moj/j O phosphate buffer (pli&5)
2m was added. ζOO liquid 0440! The absorbance at IIIK was measured, and then 2 units of laccase Oα0 were added, and the O absorbance was determined at Km every minute from the right edge of the addition. The results are shown in IR5 diagram.

2) Quantification by MBTH and O dye generation method.

Same as 1) Ig! Create a bilirubin O dilution series using
Collect in a sosoμAt test tube and add MBTH5m moj
es, laccase 50 units/It) containing 1
00 wa wSoj/j O acetate buffer i [(pH 45
)2□□)- was added and kept warm at s7°OK for 15 minutes. After keeping warm,
asMtJ[@01- was added, and its O absorbance was measured using water as a control. The results are shown in Figure 6.

As is clear from the examples, by using the O-laccase of the present invention, it is possible to decompose and remove the reducing interfering substances contained in body fluids, such as bilirubin, ascorbic acid, uric acid, and hemoglobin, with a simple treatment method. These effects on various component analyzes [can be avoided. It is also understood that bilirubin can be easily immobilized by utilizing its reactivity.

[Brief explanation of the drawing]

FIG. 1 shows the change in bilirubin O absorption spectrum O that disappears due to the laccase O reaction in Example 1. FIG. 2 shows changes in the absorption spectrum of bilirubin in an example of the reaction between O-bilirubin and laccase O in the presence of MBIH, and the O arrow in the figure indicates that the mixture was made acidic with hydrochloric acid. #! Figure 3 shows changes in the absorption spectrum O of uric acid due to laccase. FIG. 4 shows a comparison of actual measured values of triglyceride O in serum between the conventional method and the method of the present invention when bilirubin was added to various serums and when bilirubin was not added. Figure 5 shows a straight line for quantifying bilirubin 0 by the absorption reduction method, and the horizontal axis represents the rate of decrease in absorbance of 0 per minute at 460 nmK. FIG. 6 shows a straight line for quantifying bilirubin O using MBTH and the O dye production method. (3 people) Length (nm) Length (nm) Rod 3 Length (nm) Figure 4-fs5 Figure 6

Claims (1)

[Scope of Claims] 1) A method for degrading bilirubin, ascorbic acid, urea and/or hemoglobin 1, which are the main hydrogen donors in body fluids, using laccase. 2) Used for O analysis of various O components in body fluids. According to the measurement method of the target component oxidase-peroxidase-hydrogen donor chromogen reaction system, it is characterized by decomposing and removing bilirubin, ascorbic acid, uric acid, and hemoglobin coexisting in the 6!1J constant sample. Scope of patent claim O stated in paragraph 1
Method. 3) The method according to claim 8, wherein a pH 40 to z00 buffer containing laccase is added to the measurement sample, and the mixture is reacted at 37°C for 1 to 20 minutes, after which a laccase inhibitor is added. The method described in Paragraph 1 of Patent Application FI5, characterized in that bilirubin in a body fluid is immobilized. 5) Measurement sample contains laccase at pH 40-7.0O
Add a buffer solution, react at 57°C for 1 to 20 minutes, and then turn O
4. The method according to claim 4, which comprises measuring the absorbance reduction rate of bilirubin at 0440 nmfC. 6) Add a pH 40 to 7.00 buffer solution containing laccase and 3-methyl-2-benzothiazolinone-hydrazine to the measurement sample, react at 7°C for 1 to 20 minutes, and then acidify with hydrochloric acid. The absorbance at nm fC is I
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