JPH0336520B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a method for enzymatically quantifying total bilirubin in human serum and a reagent composition for the assay. More specifically, the present invention relates to a method for quantifying total bilirubin in human serum using bilirubin oxidase and a reaction promoter in combination, and a reagent composition for the assay. Measurement of bilirubin present in serum has a high degree of clinical significance, and is used, for example, to differentiate jaundice. [Prior art] Conventionally, the method for quantifying bilirubin has been to colorimetrically quantify the red color of azobilirubin produced by the reaction between bilirubin and a diazo reagent (Izumi Kanai: Summary of Clinical Testing Methods, 28th edition, Kanehara Publishing, page X). -24, Showa
Chemical methods such as (1953) are mainly used, but this method lacks accuracy because the diazo reagent reacts with serum components other than bilirubin. Furthermore, JP-A-54-151193 discloses a method for measuring bilirubin using an enzyme composition that has the properties of bilirubin produced by Agaricus spisporus. It is only used as a standard, and there is no description of whether it acts on direct or indirect bilirubin present in human serum. [Problems to be Solved by the Invention] In view of these current circumstances, the present inventors continued intensive research and established a method for quantifying total bilirubin in human serum using an enzymatic method. Specifically, the present invention relates to a method for quantifying total bilirubin in human serum using bilirubin oxidase and a reaction promoter in combination, as well as a reagent composition thereof. [Means and effects for solving the problems] The quantitative method and composition for quantitative determination of the present invention will be explained in detail below. The presence of bilirubin in biological fluids is roughly divided into direct bilirubin bound to glucuronic acid and the like, and indirect bilirubin bound to albumin (these are collectively referred to herein as total bilirubin). Total bilirubin can be quantified by measuring the reduction in direct and indirect bilirubin caused by the combined use of bilirubin oxidase produced by bacteria of the genus Myrocesium or Coprinus and a reaction promoter. The enzymatic chemical properties of bilirubin oxidase NS-1 produced by Myrocesium spp.
As is clear from the description in No. 12032, it does not act on indirect bilirubin bound to albumin, but in order to quantify total bilirubin, it is necessary to measure all types of bilirubin, including direct and indirect bilirubin. Therefore, the present inventors investigated a method for quantifying total bilirubin using bilirubin oxidase NS-1, and found that surfactants, aromatic carboxylic acids, sulfur drugs, and proteases
It has been found that by coexisting one or more species in the reaction system, total bilirubin can be quantified by acting on indirect bilirubin. It is not clear what effect these additives have, but in any case, bilirubin oxidase NS
-1 and indirect bilirubin. Therefore, these additives are referred to herein as reaction promoters. Specific examples of these reaction accelerators include surfactants such as sucrose fatty acid esters, bile acids, bile salts, dodecyl sulfates, p-toluenesulfonic acid, cetylpyridiinium chloride, and nonylphenol ethoxylates. , polyoxyethylene-polyoxypropylene condensate, etc.Aromatic carboxylic acids such as salicylic acid and sulfolithylic acid; sulfur drugs such as sulfanilamide and sodium acetosulfamine; proteases such as Pronase P (manufactured by Kaken Chemical Co., Ltd.); ) etc. Table 1 shows the effects of these reaction accelerators. That is, a reaction solution having the following composition was reacted at 37°C, and the decrease in absorption at 440 nm was measured for 3 minutes after the start of the reaction, and the values corrected per minute are shown in the same table. The indirect bilirubin used as a substrate was Bilirubin Control manufactured by Dade. 0.1M Tris-HCl buffer (PH8.4) 3ml Bilirubin control (20mg/dl) 20μ Bilirubin oxidase NS-1 (15u/ml)
20μ Reaction promoter (10% aqueous solution) (Pronase P is 0.1% aqueous solution) 50μ

[Table] As can be seen from Table 1, particularly preferred reaction accelerators are sodium cholate, sodium dodecyl sulfate, and salicylic acid. Suitable concentrations of these reaction promoters in the reaction solution are as shown in Table 2, using sodium dodecyl sulfate and sodium cholate as examples. The method was carried out in accordance with the method shown in Table 1 above.

Table 2 shows that in the case of sodium dodecyl sulfate, a concentration of 0.05 to 0.1% in the reaction solution is suitable, and in the case of sodium cholate, a concentration of 0.5 to 0.7% is suitable. Furthermore, in order to investigate whether the addition of a reaction accelerator is effective for bilirubin oxidase of different origin as a means for practically advantageously carrying out the quantitative determination of total bilirubin using bilirubin oxidase in the method of the present invention, the above-mentioned Myrocesium spp. In addition to the method of using a reaction promoter in combination with bilirubin oxidase produced by bacteria, a reaction promoter was used in combination with bilirubin oxidase produced by Coprinus spp. As a result, it was found that although the method for quantifying total bilirubin using bilirubin oxidase produced by Coprinus spp. reacts without a reaction accelerator, it is desirable to add it to speed up the reaction. That is, the effect of the combined use of reaction promoters was observed with bilirubin oxidase of any origin. Next, the reagent composition for quantifying bilirubin used in the method of the present invention will be explained. The amount of enzyme contained in the reagent composition is 0.01mu/ml to 20u/ml for bilirubin oxidase activity.
ml can be used. The most preferable is 0.01
~10u/ml (in reaction solution). For example, the reaction accelerator is 0.02 to 0.2%, preferably 0.05 to 0.1%, in the reaction solution in the case of sodium dodecyl sulfate, and 0.1 to 1.0%, preferably 0.5 to 0.7% in the case of sodium cholate. Other buffers, enzyme stabilizers, etc. may be added as necessary. The enzyme activity measurement method, test examples, and examples will be described below. Bilirubin oxidase activity measurement method 0.2M containing 1mM of ethylenediaminetetraacetic acid
Dissolve 5 mg of reagent bilirubin (manufactured by Wako Pure Chemical Industries, Ltd.) in 250 ml of Tris-HCl buffer (PH8.4), react this 2 ml with 0.2 ml of enzyme solution at 37°C, and measure the decrease in absorbance at 440 nm. One unit is the amount of enzyme that oxidizes 1 micromole of bilirubin per minute. Test Example 1 Preparation of bilirubin oxidase NS-1 Myrothecium bercariae
verrucaria) MT-1 (FERM-BP No. 653) strain was cultured in a glucose/potato medium to obtain a culture solution containing bilirubin oxidase NS-1.
The culture solution was sequentially subjected to ammonium sulfate analysis, dialysis, activated carbon treatment,
QAE-Sephadex A-50 column chromatography, ultrafiltration and Sephadex G-100
Column chromatography was performed to obtain purified bilirubin oxidase NS-1. This specimen was unique in polyacrylamide gel disk electrophoresis. Test Example 2 Preparation of bilirubin oxidase produced by Coprinus genus Coprinus cinereus
IFO8371 strain was cultured in potato glucose medium,
The culture solution was sequentially subjected to ammonium sulfate analysis, dialysis, DEAE-cellulose column chromatography, DEAE-Sepharose column chromatography, ultrafiltration,
Purified bilirubin oxidase was obtained by performing Sephadex G-100 column chromatography. The optimum temperature for this enzyme is around 30℃, and the isoelectric point is 3.8.
It is. And other enzyme chemical properties are bilirubin oxidase NS- produced by Myrocesium spp.
Similar to 1. Test Example 3 Effect of sodium cholate on total bilirubin measurement using bilirubin oxidase produced by Coprinus spp. Reaction solutions with the following compositions (1) and (2) were reacted at 37°C.
The decrease in absorption at 440 nm was measured in minutes after the reaction. The results are shown in FIG. Reaction solution (1) 0.1M phosphate buffer (PH7.0) 3.0ml Coprinus bilirubin oxidase
0.15 units Sodium cholate 0.6% Serum 100μm Reaction solution (2) Use reaction solution (1) with sodium cholate removed. As is clear from FIG. 2, a reaction promoting effect is observed in the presence of sodium cholate. Test Example 4 Substrate specificity of various enzymes The substrate specificity of bilirubin oxidase of the present invention was measured. The enzymes used were bilirubin oxidase NS-1 (abbreviated as A, hereinafter the same) prepared in Test Examples 1 and 2, and bilirubin oxidase B produced by a bacterium of the genus Coprinus. Various substrates (unconjugated bilirubin was used) shown in Table 3 at a concentration of 7mM were reacted with a certain amount of the above enzyme at 37°C, and the enzyme activity was measured. The results are shown in Table 3. The table lists the 100 enzymes with the highest activity for each substrate.
It is expressed as relative activity.

〔Effect of the invention〕

According to the present invention, a method for measuring total bilirubin by an enzymatic method using bilirubin oxidase has been completed for the first time, and this has facilitated the diagnosis of jaundice and the like in daily clinical examinations.

[Brief explanation of drawings]

FIG. 1 shows changes in the absorption pattern when the enzyme used in the present invention acts on bilirubin. FIG. 2 shows the reaction promoting effect of sodium cholate in measuring total bilirubin using bilirubin oxidase produced by a bacterium of the genus Coprinus, and FIG. 3 shows a calibration curve for bilirubin. FIG. 4 is a diagram showing the amount of bilirubin oxidase NS-1 in the reagent composition of the present invention and the reaction rate. FIG. 5 and FIG. 6 are diagrams showing the relationship between the results of quantifying total bilirubin in serum by the method of the present invention and the diazo method, respectively.

Claims (1)

[Scope of Claims] 1. Acting on human serum in combination with bilirubin oxidase and one or more reaction promoters selected from the group consisting of surfactants, aromatic carboxylic acids, sulfur drugs, and proteases, A method for quantifying total bilirubin in human serum, which is characterized by optically measuring changes in bilirubin in human serum. 2. The method for quantifying total bilirubin in human serum according to claim 1, wherein the bilirubin oxidase is an enzyme produced by a strain belonging to the genus Myrocesium or the genus Coprinus. 3. A reagent composition for quantifying total bilirubin in human serum, comprising bilirubin oxidase and one or more reaction promoters selected from the group consisting of surfactants, aromatic carboxylic acids, sulfur drugs, and proteases. 4. The quantitative reagent composition according to claim 3, wherein the bilirubin oxidase is an enzyme produced by a strain belonging to the genus Myrocesium or the genus Coprinus.