JPH0675072B2 - Indirect bilirubin assay and kit - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for quantifying indirect bilirubin and a kit used therefor.

Bilirubin is a metabolite of red blood cells and is an unnecessary component in the living body. This is excreted from the liver and becomes urobilin or stercobilin and is excreted outside the body. By knowing the amount of bilirubin in the blood, it is possible to know the degree of impairment of liver function. Although bilirubin is bound to protein in serum, there are two types of bilirubin, which are loosely bound and indirect bilirubin that is tightly bound. By knowing the amount of these, it is possible to know the type of liver disease. You can

BACKGROUND ART A method for quantifying the total amount of indirect bilirubin and direct bilirubin by quantifying an azo dye produced by reacting bilirubin with a diazo reagent in the presence of a reaction accelerator such as a surfactant is known. .

It is known to directly quantify only bilirubin by carrying out the above reaction in the absence of a reaction accelerator.
The amount of indirect bilirubin can be known by determining the difference between the two.

Also, a method for measuring the decrease in yellow color tone of bilirubin using bilirubin oxidase is known (JP-A-56-2).
7656).

Furthermore, the pH is lower than the optimum pH of bilirubin oxidase.
A method for quantifying direct bilirubin, which utilizes the fact that only bilirubin is directly decomposed and indirect bilirubin is not decomposed under the action of bilirubin oxidase under the conditions, is known (JP-A-59-125899). Indirect bilirubin can be quantified by subtracting this from the total amount of bilirubin.

Problems to be Solved by the Invention The method using a diazo reagent is not suitable for direct bilirubin determination with a small amount of sensitivity. In addition, the serum sample contains components such as lipids that cloud the reaction liquid. When a surfactant suitable for dissolving this is used, indirect bilirubin is also diazotized, and direct bilirubin alone cannot be quantified.

On the other hand, the method using bilirubin oxidase requires measurement before and after the enzymatic reaction and requires a large amount of sample. Also, the sensitivity for measuring the change in absorbance due to bilirubin itself is lower than the method using a diazo reagent.

Means for Solving the Problems The present invention converts direct bilirubin in a sample containing direct and indirect bilirubin to biliverdin by the action of bilirubin oxidase in the presence of a pH 1-8 buffer and then remains. The present invention relates to a method for quantifying indirect bilirubin, which comprises reacting indirect bilirubin with a diazo reagent in the presence of a reaction accelerator to produce an azo dye and quantifying the azo dye.

In carrying out the present invention, bilirubin oxidase is added to a sample containing bilirubin and the reaction is carried out in an appropriate buffer (hereinafter referred to as the first reaction), and then a diazonium salt is used as a diazo reagent to form indirect bilirubin. An equimolar amount or more, usually 10 to 1000 times mol and a reaction accelerator are added and reacted (hereinafter referred to as the second reaction).

The first reaction is carried out at 15 to 45 ° C, preferably 30 to 40 ° C, and 2
~ 30 minutes, usually about 5 minutes.

The second reaction is carried out at 5 to 50 ° C, preferably 20 to 40 ° C, and 2
~ 30 minutes, usually about 5 minutes.

After the reaction, the absorption of the colored reaction solution is measured at the maximum absorption wavelength (λmax) of the produced dye using the reagent blank as a control, and indirect bilirubin in the sample can be quantified using the calibration curve previously obtained from the test for a known amount. .

As the bilirubin oxidase used in the first reaction, enzymes derived from microorganisms such as genus Milocesium and genus Trachyderma can be easily obtained, and these are used at 0.1 to 10 U / ml.

The buffer used in the first reaction and the second reaction has a pH of 1 to 1
1, such as hydrochloric acid-potassium chloride, citric acid, acetic acid, phosphoric acid, lactic acid, tartaric acid, tris-hydrochloric acid, succinic acid,
Oxalic acid, Good's, etc. are used at 0.005 to 2 M / L.

As the diazo reagent used in the second reaction, any diazo reagent can be used as long as it reacts with indirect bilirubin to quantitatively form a stable dye, and examples thereof include P-sulfobenzenediazonium salt and 2,4-dichlorophenyldiazonium. salt,
2,5-dichlorophenyldiazonium salt, general formula (I) A diazonium salt such as a compound represented by the formula (wherein X represents halogen such as F, Cl and Br) [hereinafter referred to as compound (I)] is used.

As these diazonium salts, stabilized salts such as 1,5-naphthalenedisulfonate are preferably used.

As the reaction accelerator, for example, caffeine, dyphylline,
Benzoate, dimethyl sulfoxide, acetate, urea, acetamide, salicylate, surfactant such as Triton X-100, etc. are contained in 0.1 to 100 mg / ml.

In carrying out the procedure, it is convenient to prepare a first reagent solution in which bilirubin oxidase is dissolved in a buffer and a second reagent solution in which a diazonium salt and a reaction accelerator are dissolved in a buffer and to carry out the reaction. is there.

According to the present invention, a kit for quantifying indirect bilirubin is provided. This kit comprises a first reagent consisting of a combination of bilirubin oxidase and a buffer and a diazonium salt, and a second reagent consisting of a combination of a reaction accelerator and a buffer. Second
Some of the reagent diazonium salts are unstable, in which case the diazonium salt may be replaced by a compound for preparing the diazonium salt.

Examples of compounds for preparing a diazonium salt include 2,4-dichloroaniline, 2,5-dichloroaniline, and a compound of formula (II) The compound represented by (X has the same meaning as above) [hereinafter referred to as compound (II)] is combined with sodium nitrite for diazotization and an acid such as hydrochloric acid or sulfuric acid.

The respective compounds in these kits are combined so as to be in the concentration range used in the above reaction.

According to the method of the present invention, since a large amount of reaction accelerator can be used in the second reaction, it is possible to add a sufficient amount of surfactant to dissolve the turbid components in the sample, which can be used for the reagent blank test. There is no need, it is possible to measure a small amount of sample and it is simple.

Examples showing aspects of the present invention are shown below.

Example 1. As the first reagent, 100 units of 0.1 M citrate buffer (pH 4.0) in which 100 units of bilirubin oxidase (derived from a microorganism of the genus Trachyderma, manufactured by Takara Shuzo Co., Ltd.) was dissolved is prepared. As a second reagent, 0.05M phthalate buffer (pH3.0)
A solution is prepared by dissolving 1 g of Triton X-100, 250 mg of acetoamide, and 50 mg of compound (I) (where X = Cl is shown) as a diazonium salt in 100 ml.

A bilirubin standard solution (omega bilirubin control) is added to the first reagent, and after heating at 37 ° C for 5 minutes, the second reagent is added. After heating at 37 ° C. for 5 minutes, the absorbance is measured at λmax 540 nm using a reagent blind test as a control. Table 1 shows the relationship between the concentration of indirect bilirubin and the absorbance.

It is clear from Table 1 that the relationship between the concentration of indirect bilirubin and the absorbance is linearly proportional.

Example 2 As a first reagent, 100 units of 0.1 M citrate buffer (pH 4.0) containing 100 units of bilirubin oxidase (derived from a microorganism of the genus Trachyderma, manufactured by Takara Shuzo Co., Ltd.) is prepared.
As a second reagent, 1 g of Triton X-100, 250 mg of acetoamide and 100 mg of 0.05M phthalate buffer (pH 3.0) as P-sulfobenzenediazonium, 1,5-naphthalenedisulfonate sodium salt, 2, 4-
A solution is prepared by dissolving 50 mg of dichlorophenyldiazonium, sodium 1,5-naphthalenedisulfonate, 2,5-dichlorophenyldiazonium, sodium 1,5-naphthalenedisulfonate, and compound I (X = Cl) in an amount of 50 mg each.

The first reagent is added to each of the 5 types of serum samples, and after heating at 37 ° C. for 5 minutes, each second reagent is added. After heating at 37 ° C for 5 minutes, the absorbance was measured at λmax 540nm using a reagent blind test as a control, and the indirect bilirubin concentration in the serum sample was obtained from the calibration curve prepared in advance using the bilirubin standard solution. . The measured values are shown in Table 2 together with the values obtained by the bilirubin B test "Wako" [manufactured by Wako Pure Chemical Industries, Ltd.] which is a measuring reagent by the known diazo method. However, the indirect bilirubin value of the bilirubin B test "Wako" is a value calculated by subtracting the direct bilirubin value from the total bilirubin value.

Regardless of the type of diazonium salt, it shows a good correlation with the "Wako" kit using the alkaline azobilirubin blue method.

EFFECTS OF THE INVENTION According to the method of the present invention, indirect bilirubin can be quantified more accurately.

Claims (2)

[Claims] 1. Direct bilirubin in a sample containing direct and indirect bilirubin is converted into biliverdin by the action of bilirubin oxidase in the presence of a buffer having a pH of 1 to 8, and the remaining indirect bilirubin and diazo reagent are then converted. Reacting in the presence of a reaction accelerator to form an azo dye,
A method for quantifying indirect bilirubin characterized by quantifying this. 2. A, bilirubin oxidase and pH 1-8
A kit for quantitative determination of indirect bilirubin, which comprises a first reagent consisting of a combination of the above buffers and a second reagent consisting of B, a diazo reagent, a reaction accelerator and a buffer.