JPS59156300A - Measurement of humor component of organism - Google Patents

Abstract

PURPOSE:To measure a humor component of organism, by using a specimen of humor component of organism pretreated with immobilized with laccase as a solution to be examined, so that laccase is prevented from having bad influence such as interference of bilirubin, etc. on a measurement system of the humor component of organism. CONSTITUTION:For example, enzyme laccase is bonded to a carrier such as cellulose, etc. by covalent bond, etc. to give immobilized laccase, which is packed into column, etc. A humor specimen of organism such as serum, urine, etc. is added to it, reacted and eluted. The prepared specimen is used as a solution to be examined, to determine components such as glucose, cholesterol, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is a method for measuring biological body fluid components, which is characterized in that when measuring biological body fluid components by a known method, a sample obtained by reacting an immobilized laccase with a biological body fluid sample is used for detection. Concerning methods for measuring components. More specifically, the biological body fluid component is characterized in that bilirubin contained in the biological body fluid sample is reduced or eliminated by a reaction between the immobilized laccase and the biological body fluid sample, and interference of bilirubin in the biological body fluid component measurement reaction is avoided or reduced. This is a measurement method. In the method of the present invention, biological fluids include serum, urine, etc., and components to be measured include glucose, cholesterol, uric acid, neutral fats, free fatty acids, -phospholipids, etc.

In recent years, the progress of enzymatic analysis methods in clinical chemistry analysis has been remarkable, and glucose oxidase, cholesterol oxidase, uricase, azircoenzyme A oxidase, choline oxidase, glycerol-3-phosphate, and acid oxidase have been used to measure the various biological fluid components mentioned above. Oxidizing enzymes, such as sarcosine oxidase, are widely used. In these measurement methods using oxidizing enzymes, the content of biological fluid components is determined by colorimetrically quantifying hydrogen peroxide produced by the reaction between oxidizing enzymes and biological fluid components using a peroxidase-chromogenic hydrogen donor system. Desired.

- To give an example, β-D-glucose is converted into rhoglucono-δ-lactone and hydrogen peroxide by the action of glucose oxidase, and hydrogen peroxide converts phenol and 4-aminoantipyrine into hydrogen donors by the action of peroxidase. This is oxidatively condensed to produce a red quinone dye. By measuring this dye with a photometer, the concentration of glucose can be determined.

In the method using the peroxidase-chromogenic hydrogen donor system, various reducing substances present in the sample, administered drugs,
It is known that biological pigments cause interference, but the following is said about the interference mechanism caused by bilirubin (Clinical Chemistry Vol. 8, 6).
3-72, 1979).

(1) Direct effect due to bilirubin's specific absorption (near 460 nm) (2) Bilirubin acts as a hydrogen donor for peroxidase (3) Decomposes by directly acting on the generated color pigment ( 2) is said to have the greatest impact.

The concentration of bilirubin in serum is 1■/d1 in normal people.
However, pathologically, it can reach 20 µ/d1, and its influence on clinical tests is a serious problem.

Known techniques related to these problems include, for example, a method of adding ferrocyanide, ascorbic acid, an EDTA-iron complex, or a bilirubin-specific fungal enzyme composition to the reaction system (Japanese Patent Publication No. 55-25840). Public notice,
JP-A-55-138656, JP-A-55-297
No. 18, JP-A-57-71398, JP-A-Sho 5
4-151193 and Clinical Chemistry Vol. 26, pages 227-231, 1981). However, all of these conventional methods have drawbacks and cannot be said to be fully satisfactory methods.

In the measurement of known biological body fluid components using oxidizing enzymes as described above, the present inventors discovered that by allowing laccase to act on a biological body fluid sample, bilirubin contained in the sample is oxidized and converted to biliverdin, resulting in an almost colorless substance. I learned that interference by bilirubin can be avoided because it changes into a product and does not generate hydrogen peroxide. Laccase (EC1,
10, 3, and 23 are also called polyphenol oxidases, and are enzymes that catalyze the reaction of oxidizing polyhydric phenols to quinones in the presence of molecular oxygen, but it is not known that they are reactive with bilirubin. Based on the above findings, it was possible to reduce or remove bilirubin from biological body fluids using laccase. However, laccase has the ability to react with hydrogen donors such as phenol in the reaction reagent using the oxidizing enzyme and develop a color. It turns out that it cannot be used as is. In order to improve this inconvenience, the present inventors conducted intensive research and found that by using the enzyme laccase in an immobilized manner, it is possible to prevent the adverse effects of laccase on the biological body fluid component measurement system, and in turn, it is possible to use the enzyme repeatedly. They discovered this and completed the present invention.

Laccase is known to exist in sumac sap, fungi, etc., and laccase from any source can be used in the method of the present invention. For example, Agaricus bisporus (Ag
aricus bisporus) (Journal of General Microbiology (J, G
en, Microbiol, Volume 117, Page 327, 1980], Neurospora cranna (Neu
rosporacrassa) (Journal of Bacteriology (J, Bacteriol,
) Volume 120, page 458, 1974], Polyporus versicolor (Polyporus versicolor)
color) (actor chemistry power scandinavian power)
(8cta Chemica 5candinavic
a,) Volume 21, page 2367, 1967], Sumac [Hiochymicae Biology Acta (Bioc.
him, Biophys.

(Acta) Vol. 30, p. 44, 1958] is used.

The method for immobilizing laccase in the method of the present invention is not particularly limited, and any known method can be used. For example, polysaccharides and derivatives such as cellulose, dextran, and agarose,
Alternatively, enzymes may be bonded to a carrier such as polyacrylamide gel, porous glass, or polystyrene by a covalent bonding method or an ionic bonding method, or enzymes may be bonded together using glutaraldehyde,
Isocyaner) iJ derivative bisdiazobenzidine, N,
N'-polymethylene biiodoacetamide, or N
, a method of crosslinking using a multifunctional reagent such as N'-ethylene bismaleimide, a method of enclosing an enzyme in the lattice of a polymer gel such as polyacrylamide gel, polyvinyl alcohol gel, silicone resin, starch, etc. Examples include a method in which enzymes are microencapsulated with a film of a semipermeable polymer such as alpha, polyurea, polystyrene, or collodion. Among these methods, particularly suitable for the method of the present invention is a method in which the enzyme is bonded to agarose, porous glass, or polystyrene as a carrier.

To measure biological body fluid components using the immobilized enzyme obtained as described above, for example, the immobilized enzyme is packed in a column, a sample such as serum is added thereto, reacted, and then eluted with a buffer solution. If the enzyme is immobilized in a polystyrene test tube, the sample can be added to the test tube and reacted, and then the solution can be used as is. Using the sample obtained in this way as a detector,
Glucose, cholesterol, etc. are measured using the iQl enzyme described above, and the concentrations of the components in the sample are determined by comparing with a calibration curve prepared in advance.

When laccase was immobilized and used as described above, it was possible to prevent interference of bilirubin in the measurement of biological fluid components, and it could be stored stably for at least 3 months and could be used repeatedly. .

The present invention will be explained in detail below with reference to Test Examples and Examples, and the laccase unit in the explanation will be defined as follows. That is, 0.000 mg containing 1 mM ethylenediaminetetraacetic acid.
2M) IJS hydrochloric acid buffer ($) l (8,4) 25
0ml! Dissolve 5 mg of bilirubin (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) in the mixture, and react with the enzyme at 37°C for +440 nn.
When the decrease in absorbance at + was measured, the amount of enzyme that oxidized 1 micromole of bilirubin per minute was defined as 1 unit.

Test Example 1 Preparation of immobilized rough case (1) Cyanide odor activated Sepharose 4B (CNBr-activa
After suspending 1 g of ted 5epharose 4B (manufactured by Pharmacia) in 200 yd of 1 mM hydrochloric acid,
Wash thoroughly with 0.14 carbonate buffer (p) 18.3 containing M NaCl (hereinafter referred to as "buffer A"), and add buffer A to this.
Laccase (2 u/mE) dissolved in 1- was added to the mixture, and the mixture was left at 4° C. overnight with gentle stirring. Then buffer A, 0.2M ethanolamine (pH 8,3)
, Buffer A, 0.2M acetate buffer containing 0.5M NaCl (
pH 5,0), 0.05M l-Lis-HCl buffer (p
H7;
m x length 10 mm) to obtain an immobilized enzyme column.

The yield of enzyme activity by the above operation was about 12%.

Test Example 2 Preparation of immobilized laccase (2) Epoxy-activated Sepharose 4B (Epo
xy-activated 5epharose 4B
(manufactured by Pharmacia) in the same manner as in Test Example 2 to obtain an immobilized enzyme column. The yield of enzyme activity by the above operation was about 9%.

Test Example 3 Preparation of immobilized laccase (3) Porous glass beads having amino groups as functional groups (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.)
1% glutaraldehyde (pH 7.4) to Ig 10
- and reacted for 30 minutes at room temperature, washed thoroughly with buffer B, and added laccase (0,'
5 u/me) 1- was added, stirred gently, and left at 4°C for 1-night. Thereafter, in the same manner as in Test Example 1, wash with buffer solutions in order, and finally wash with buffer B to create immobilized enzyme-nocase, fill it into the same column as above, and fill the immobilized enzyme column with Obtained. The yield of enzyme activity by the above operation was about 7%.

Test Example 4 Preparation of immobilized laccase (4) Add several drops of T-aminopropyltriethoxysilane to a polystyrene test tube (13 mm x 100 mm) to coat the inner wall of the test tube, and after washing with water, add 1% glutaraldehyde (p
)l 7.4) 3 ml was added and reacted at room temperature for 15 minutes. After washing the test tube with water, laccase (0.05 u/-) 1- dissolved in buffer B was added and rotated and shaken at room temperature for 4 hours. Thereafter, washing was carried out in the same manner as in Test Example 1 with buffer solutions, and finally with buffer B to obtain an immobilized enzyme test tube. The yield of enzyme activity by the above procedure is approximately 10%.
Met.

Example 1 Measurement of Glucose A 0.1M) lithium-hydrochloric acid buffer (pH 7.0) containing 0.1% sodium dodecyl sulfate was applied to the immobilized enzyme column prepared in Test Example 1 to equilibrate the column. Next, as samples, 5 M glucose aqueous solutions (200 mg/d1) each containing 5110 and 20.30 mg/d1 of bilirubin were passed through the column, and the above buffer was poured off to obtain eluate 5.
00g was obtained. Using the eluate as detection, this 2004 and 1
7u/ml! Glucose oxidase (manufactured by Ohno Pharmaceutical Co., Ltd.)
, 0.3u/-peroxidase (manufactured by Boehringer)
O,I M-phosphate buffer (pH 7,5
) 3- were mixed and reacted at 37°C for 20 minutes, and then the absorbance of the reaction solution at 505 nm was measured.

On the other hand, for comparison, the same glucose measurement procedure as above was performed using a test solution obtained by diluting each sample 10 times without immobilized enzyme treatment.

The results are shown in Figure 1. In the figure, <-0-) represents the results of this example, and (→-) represents the comparative example. It was found that by the method of the present invention, the influence of bilirubin coexisting in the sample can be almost ignored.

Example 2 Measurement of glucose in serum Using the immobilized enzyme column prepared in Test Example 1,
．． The column was equilibrated by flowing 0.1 M) Lis-HCl buffer (pH 7.0) containing 6% sodium salicylate. Next, after adding 50 pβ of the serum sample, the above buffer was poured and the eluate was 50 hj! I got it. The eluate was used as a test solution, and this 200 Pβ was mixed with 0.00 μM containing 17 u/m glucose oxidase (manufactured by Ohno Pharmaceutical), 0.3 u/m Hime peroxidase (manufactured by Boehringer), 0.4 mM 4-aminoantipyrine, and 15 mM phenol. Mix 1M phosphate buffer (pH 7,5) 3me and heat at 37°C.
After reacting for 520 minutes, the absorbance of the reaction solution at 505 nm was measured. The glucose concentration in this sample was determined by comparing this absorbance with a calibration curve prepared in advance by using a standard glucose aqueous solution as a test solution and operating in the same manner as above, and it was found to be 97.0 mg/d1. As a comparison,
When the same procedure was performed using a serum sample that had not been treated with immobilized enzymes as a test solution, the result was 93.1 ng/d! Met. The total bilirubin concentration in the serum sample used was 12.5 mg/
d1, but due to this immobilized enzyme treatment,
It decreased to 3.0 mg/di (24%). Furthermore, the immobilized enzyme column used could be used repeatedly at least 50 times.

Example 3 Measurement of glucose in serum The immobilized enzyme column prepared in Test Example 2 was operated in the same manner as in Example 2 to determine glucose in serum samples.
It was 96.2 mg/aJ. The total bilirubin concentration in the serum sample used was reduced to 3.3 mg/cU (18%) by this immobilized enzyme treatment. Furthermore, the immobilized enzyme column used could be used repeatedly at least 50 times.

Example 4 Measurement of glucose in serum The immobilized enzyme column prepared in Test Example 3 was operated in the same manner as in Example 2 to determine glucose in serum samples.
It was 96.4 mg/di. The total bilirubin concentration in the serum sample used was reduced to 2.8 mg Zdi (22%) by this immobilized enzyme treatment. Furthermore, the immobilized enzyme column used could be used repeatedly at least 50 times.

Example 5 Measurement of glucose in serum 50 pf of the same serum sample used in Example 2 and 0.1
Add M phosphorus M buffer ('p! (7,0) 0.5 rnP and shake at room temperature for 5 minutes. Using the sample obtained here as a test solution, the same procedure as in Example 2 was carried out, and the glucose concentration The total bilirubin concentration in the serum sample used was reduced to 3.0 mg Zdi (24%) by this immobilized enzyme treatment.

In addition, the immobilized enzyme test tube used was tested at least 50 times.
It was possible to use it once.

Example 6 Measurement of total cholesterol in serum A test solution of 200 pf obtained by treating a serum sample with an immobilized enzyme in the same manner as in Example 2! and 1 u/'ml cholesterol esterase (manufactured by Ohno Pharmaceutical Co., Ltd.), '2u/-cholesterol oxidase (same), 6u/mf! Peroxidase (manufactured by Boehringer), '0.4 mM4-
Aminoantipyrine, 15mM phenol and 0
．． 3ml of O, 1M phosphate buffer (pH 7.5) containing 1% Triton X-100! After mixing and reacting at 37° C. for 20 minutes, the absorbance of the reaction solution at 505 nm was measured.

The concentration of Gores delirium in this sample was determined by comparing this absorbance with a calibration curve prepared in advance in the same manner as above using a standard cholesterol aqueous solution as a test solution, and found to be 183 mg/d1. For comparison, a similar procedure was performed using a serum sample that had not been treated with immobilized enzymes as a test solution, and the result was 172-B/dj. The total bilirubin concentration in the serum sample used was 16.8 mg/d1, but it was reduced to 3.5 mg/d by this immobilized enzyme treatment.
i (21%).

Example 7 Measurement of neutral fat in serum 200 g of a test solution obtained by treating a serum sample with an immobilized enzyme in the same manner as in Example 2, 200 u/- riboprotein lipase (manufactured by Ohno Pharmaceutical Co., Ltd.), 0. 3u/me glycerol kinase (same), 4u/-glycerol-3-phosphate oxidase (same), 2u/mR peroxidase (manufactured by Boehringer 2), 0.4mM 4-aminoantipyrine, 15n+M phenol, 0.8mM
Adenosine triphosphate and 0.1% Triton X-
0.1M containing 100) Liss salt buffer (pH 7,5
) 3- were mixed and reacted for 37°C12O minutes, and then the absorbance of the reaction solution at 505 nm was measured. Using this absorbance and standard triolein solution as a test solution,
The neutral fat concentration in this sample was determined by comparing it with a calibration curve prepared in the same manner as above, and was found to be 80.0 mg/d1 as triolein.

For comparison, when a serum sample that had not been treated with immobilized enzyme was operated in the same manner as a detection, 70. 'Omg/8. The total bilirubin concentration in the serum sample used was 1
It was 5.0 mg/di, but it was reduced to 3.5 mg/di (23%) by this immobilized enzyme treatment.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the influence of bilirubin on glucose measurement, and in the figure (→-) indicates the results of the method of the present invention;
〇-) represent comparative examples, respectively. Patent applicant Amano Pharmaceutical Co., Ltd. No. 1]-1111\0. 10 20 Bilirubin agricultural rate (mg/d) 0 1)

Claims (1)

[Claims] 1. A method for measuring components in a biological body fluid, which comprises allowing a biological body fluid sample to act on immobilized laccase, and then using the sample as a test solution.