JPH0379998B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a reagent and method for measuring total and direct bilirubin using an enzyme and potassium ferricyanide. BACKGROUND OF THE INVENTION The measurement of bilirubin in serum has been clinically performed for a very long time as an index for diagnosing diseases of the hepatobiliary system, etc., and has become one of the most universal and essential measurement items in clinical tests. Bilirubin is a typical in-vivo pigment, and is primarily produced from hemoglobin produced by the decay of waste red blood cells. This free bilirubin becomes protein-bound or becomes conjugated (direct) bilirubin by a bilirubin conjugating enzyme in liver microsomes and is excreted from hepatocytes into bile.
Bilirubin excreted into the intestines is partially absorbed and circulated into the enterohepatic system, but the majority is excreted from the body as urobilin bodies. An increase in serum bilirubin is caused by disturbances or changes in the flow from production to excretion, and when these become significant, jaundice occurs. There are two types of bilirubin: the above-mentioned conjugated (direct) type and unconjugated (indirect) type. Direct bilirubin is indirect bilirubin with one or two glucuronic acids bound to the propionic group in the side chain. The combination of bilirubin and indirect bilirubin is called total bilirubin. The method for measuring serum bilirubin is as follows:
Generally, Evelyn-Malloy
A colorimetric method using a diazo reagent was adopted by et al. In this method, the diazo reaction proceeds quickly for the diglucuronide of direct bilirubin (therefore also referred to as 1-minute bilirubin), whereas the reaction proceeds slowly for monoglucuronide (therefore also referred to as delayed direct reaction bilirubin). Therefore, it has the disadvantage that bilirubin cannot be directly measured accurately and precisely unless the diazo reaction time is accurately specified. Recently, methods for measuring bilirubin using enzymes have been developed, such as the method using hydrogen peroxide and peroxidase by Jacobson et al. [Jacobson, G. &
Wennberg, RP; Clin.Chem., 20 , 783 (1974)
], method for measuring the reduction of yellow tone of bilirubin using mushroom-derived bilirubin oxidase by Tai-Win Wu
Furthermore, Murao et al. have reported that bilirubin oxidase is obtained by microorganisms of the genus Myrothecium [Murao et al.
S. & Tanaka, N.;Agrical.Biol.Chem., 45,
2383 (1981) and Japanese Patent Application Publication No. 159487/1987]. Such enzymatic methods have high accuracy in bilirubin measurement,
Although it is excellent in terms of analytical accuracy, etc., it usually requires more than one unit of enzyme per test, so the reagent cost is high, and it is not satisfactory in this respect. In view of the above-mentioned problems, the present inventors have conducted intensive studies on a method for quantifying bilirubin accurately and quickly at low cost, and have found that by using potassium ferricyanide in combination with bilirubin oxidase, the dose of oxidase can be significantly reduced. They discovered that it is possible and completed the present invention. That is, the present invention provides bilirubin oxidase,
In particular, the present invention provides a method for measuring total bilirubin and direct bilirubin by a combined method using bilirubin oxidase derived from a microorganism of the genus Myrocesium and potassium ferricyanide as an oxidizing agent, and a reagent for the measurement. To measure bilirubin according to the present invention, a buffer solution is added to a bilirubin-containing specimen, and after prewarming, a bilirubin oxidase solution containing potassium ferricyanide is added to oxidize yellow bilirubin to green biliverdin. This is done by measuring using a colorimetric method. According to the research conducted by the present inventors, it has been found that not only total bilirubin but also direct bilirubin can be measured by adjusting the pH of the reaction solution. In other words, PH6~
Total bilirubin can be measured at 9 and direct bilirubin at PH 3.5-4.5. The buffer used here is, for example, 0.1M phosphate buffer or
Examples include 0.1M Tris-HCl buffer solution containing sodium cholate, SDS (sodium dodecyl sulfate), LBS (laurylbenzene sulfate), etc. as a directing agent. For direct bilirubin measurement, 0.1M citrate buffer, 0.1M tartrate buffer, etc. are used. Prewarming the sample after adding buffer to 25-45℃
for 1 to 10 minutes, preferably 3 minutes at 37°C, then add enzyme reagents containing bilirubin oxidase and potassium ferricyanide and incubate for 5 to 20 minutes at 25 to 45°C.
The oxidation reaction is carried out for 10 min, preferably at 37 °C. According to the present invention, the dose of bilirubin oxidase can be reduced to a greater extent than in the known method by the combination of potassium ferricyanide, from 0.005 to 1 unit/test, preferably from 0.01 to
The range is 0.5 units/test. In addition, the dose of potassium ferricyanide is 0.005-1mM/test,
Preferably it is in the range of 0.01-0.2mM/test.
If the dose of bilirubin oxidase is less than 0.005 unit/test, it will take a long time to complete the reaction and the dose of potassium ferricyanide will increase, while if it exceeds 1 unit/test, the effect will be saturated and it is not economical. Also, the dose of potassium ferricyanide
If it is less than 0.005mM/test, the reaction rate becomes slow and impractical, while if it exceeds 1mM/test, the color tone becomes dark and the optical density of the blank increases, which is not preferable. Colorimetric determination is carried out by a conventional method, for example, the photometric density is measured at a wavelength of 460 nm using a commercially available spectrophotometer (for example, Shimadzu Model 220 double beam spectrophotometer) using a reagent blank as a reference. In addition,
At this time, the optical density was measured in the same way using a certain amount (known concentration) of bilirubin-containing serum (hereinafter referred to as standard bilirubin) as a control, and as a blind test, bilirubin oxidase and potassium ferricyanide were added to these sample serum and standard bilirubin. The optical density was measured in the same way for the sample to which a buffer solution containing no
dl). Bilirubin concentration (mg/dl) = A _B
−A _T /A _SB −A _ST ×x……() Where, A _B : Optical density of sample serum (blind test) A _T : Optical density of sample serum (enzymatically reacted) A _SB : Standard Optical density of bilirubin (blind test) A _ST : Optical density of standard bilirubin (enzyme-reacted) x: Bilirubin concentration in standard bilirubin (mg/
dl) To explain the above measurement procedure more specifically, a buffer solution [for example, 0.1M phosphate buffer containing 0.25% sodium cholate (PH7.0)] is added to the sample serum, and this is incubated (for example, at 37°C for 30 minutes). minutes) and
An enzyme reagent containing a predetermined amount of potassium ferricyanide and bilirubin oxidase (for example, potassium ferricyanide 0.1 mM/test, bilirubin oxidase 0.025 unit/test) is added to this solution, and oxidation is carried out at, for example, 37° C. for 10 minutes. The optical density (A _T ) is measured at a wavelength of 460 nm using this as a reference against a reagent blank. On the other hand, the optical density (A _B ) was obtained by performing the same operation except without adding the above enzyme reagent.
Measure. Furthermore, standard bilirubin was used in place of the above sample serum, and the same procedure as above was performed to determine the optical density.
Measure A _ST and A _SB . The reagent for measuring bilirubin of the present invention usually includes () a buffer solution that does not contain bilirubin oxidase and potassium ferricyanide, () an enzyme reagent containing bilirubin oxidase and potassium ferricyanide, and () a certain amount of bilirubin-containing serum (standard bilirubin). It is configured as a kit consisting of. As described above, according to the present invention, due to the combined effect of enzyme and potassium ferricyanide, the amount of expensive enzyme used can be significantly reduced compared to conventional methods, and the reaction can be completed quickly, making it easier for automatic analyzers to use. It became possible to apply it. Next, the present invention will be explained in more detail with reference to Examples. Example 1 (Measurement of total bilirubin) Measurement method: Add 3ml of 0.1M phosphate buffer (PH7.0) containing 0.25% sodium cholate to 100μ of sample serum.
After prewarming at 37°C for 2-3 minutes, add 70μ of enzyme reagent solution containing 0.01mM/test or 0.1mM/test potassium ferricyanide and 0.025 units of bilirubin oxidase/test in the same buffer as above.
and react at 37℃ for 10 minutes. Next, this reaction solution was compared with a reagent blank (a mixture of the buffer solution and enzyme reagent was treated in the same way as the sample) and the wavelength
Measure optical density at 460nm. At the same time, the optical density of a sample blank (a mixture of the sample and buffer solution treated in the same manner) is measured in the same manner using the buffer solution as a control. Experiment 1 (Reproducibility) Patient serum (sample 1, 2) and standard serum (DADE bilirubin control, 20mg/
dl), the reagent described in the measurement method above (the potassium ferricyanide concentration in the enzyme reagent is 0.01mM/
Optical density was measured by test and 0.1mM/test) and procedure. Table 1 shows the results of 10 repetitions.

[Table] As is clear from the table, all of the results showed good reproducibility, confirming that the method of the present invention has high accuracy. Experiment 2 (calibration curve) The same standard serum (20 mg/dl) as above was serially diluted (1/4, 2/4, 3/4, 4/4), and the reagents (in the enzyme reagent) described in the measurement method above were diluted. The potassium ferricyanide concentration was 0.01mM/test, 0.1mM/test) and the optical density was measured by operation. The results are shown in Figure 1 (potassium ferricyanide 0.01mM/test) and Figure 2 (potassium ferricyanide 0.1mM/test).
As is clear from the figure, a straight line passing approximately through the origin was obtained. Experiment 3 (Correlation) Reagent described in the measurement method above (potassium ferricyanide concentration in the enzyme reagent is 0.01mM/test,
0.1mM/test) and the patient serum
Optical densities were determined for 21 specimens by performing the same procedure as in Experiment 1. On the other hand, for the same serum specimen, the optical density was obtained by carrying out the same operation using Bilirubin Kit-N (manufactured by Nippon Shoji Co., Ltd.), which has been commonly used in the past, instead of the reagent of the present invention. Bilirubin control (20 mg/dl) manufactured by DADE was used as the standard serum for this method. Based on these, each measured value was obtained and a correlation diagram between the method of the present invention and the conventional method was created. This is shown in Figure 3 (Potassium ferricyanide
0.01mM/test) and Figure 4 (0.1mM/test)
test). Example 2 (Direct measurement of bilirubin) Example 1 using 0.1M citrate-phosphate buffer (PH4.0) instead of 0.1M phosphate buffer (PH7.0) containing 0.25% sodium cholate The bilirubin concentration was directly measured for three serum samples according to the measurement method described above. The concentration of potassium ferricyanide is 0.1m
M/test was used, and serum containing 7.5 mg/dl of direct bilirubin was used as the standard bilirubin. On the other hand, the direct bilirubin concentration of the same serum specimen was determined using Bilirubin Kit-N. The results are shown in Table 2.

【table】 [Brief explanation of drawings]

Figures 1 and 2 are graphs showing the linearity between the optical density and the serial dilution of standard serum, and Figures 3 and 4 are graphs showing the correlation between the method of the present invention and the conventional method. This is a graph showing.

Claims (1)

[Scope of Claims] 1. A reagent for measuring bilirubin, characterized in that potassium ferricyanide is blended with bilirubin oxidase. 2. The reagent according to item 1 above, wherein the bilirubin oxidase is derived from a microorganism of the genus Myrocesium. 3 Bilirubin oxidase 0.005 to 1 unit/
Test, potassium ferricyanide 0.005-1m
The reagent of item 1 or item 2 above, formulated as M/test. 4. A method for measuring bilirubin using a colorimetric method, which comprises oxidizing bilirubin using bilirubin oxidase and potassium ferricyanide in the specimen.