JPS58198300A - Analytical process - Google Patents

Abstract

PURPOSE:When components in a liquid sample are determined utilizing the catalytic actions of enzymes, enzymes immobilized on spherical cellulose supports are used to enable their repeated use with prolonged enzyme life. CONSTITUTION:When components in a liquid sample are determined utilizing the catalytic actions of enzymes, e.g., in the determination of bile acid using 3alpha-hydroxysteroid dehydrogenase or of cholesterol using cholesterol esterase, enzymes immobilized on spherical cellulose supports with particle sizes of 1- 10mm. are used.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an analytical method, particularly an analytical method that utilizes the catalytic action of an enzyme.

Conventionally, K for analyzing organic substances, particularly biological substances such as bile acids, has been used to analyze the reaction between bile acids and nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD), as seen in Tokusho@56-39199. The enzyme 3a-hydroxysteroid dehydrogenase (hereinafter 3a-H5)
(abbreviated as D), the above NAD is converted to N by the catalytic action of
ADHK is reduced, and the NADH is oxidized to NADK by the action of diaphorase in the presence of resazurin, and at the same time, redezurin is reduced to produce resorufin;
Bile acids in a sample have been quantified by measuring the fluorescence of resorufin.

Similarly, it is known that the enzyme cholesterol esterase is used to measure cholesterol, and the enzyme glutathione peroxidase is used to measure lipid peroxide.

However, in the conventional method described above, the enzyme is added to the sample in the form of a solution, which is difficult to recover, and in most cases the enzyme is expensive and disposable after only one use. It had been.

In view of the above-mentioned current situation, the present invention provides an analytical method that can be effectively used over and over again without making expensive enzymes disposable.

This was done with the purpose of
A method that utilizes the catalytic action of enzymes when quantifying ls, min in a sample solution, characterized in that an enzyme immobilized on a spherical cellulose support is added to the sample solution in order to carry out the catalytic action. It depends on the analysis method.

The cellulose support used in the present invention is spherical, and a particle size of about 1 to 10 square centimeters is preferably used from the viewpoint of handling, such as when the support is held between bottles.

Furthermore, since the analysis method of the present invention is applied to analysis using the catalytic action of an enzyme, the enzyme used for analysis of the component to be analyzed is selected depending on the type of component to be analyzed in the sample liquid. For example, 3α-H3D and diaphorase can be used in the analysis of bile acids, cholesterol esterase can be used in the analysis of cholesterol, and glutathione peroxidase can be used in the analysis of lipid peroxides.

In order to immobilize an enzyme on a cellulose support, any method of chemically bonding the enzyme to the surface of the support can be employed, such as a method using I.logenide cyanide or a method using epoxide. Depending on the nature of the enzyme to be immobilized, immobilization may be performed after introducing a suitable spacer such as hexamethylene diamine.

He then immobilized the enzyme on a cellulose support and stored it at a temperature of around 4°C in a PHK-adjusted buffer that could keep the enzyme stable. It is more preferable in terms of preservability to contain sodium 411ate in a proportion of QO 5% or less and a preservative such as sodium azide in a proportion of a1% or less.

When using the enzyme immobilized on the cellulose support mentioned above, it is removed from the enzyme storage container using a bottle set, preferably washed with the buffer used for analysis, and then used for analysis. The use of the identified enzyme is carried out by adding an appropriate number of cellulose supports on which the enzyme is immobilized using a bottle set instead of the solution enzyme at the stage of adding the solution enzyme in the conventional analysis method. . When added to a sample, the desired reaction proceeds due to the catalytic action of the # element immobilized on the cellulose support, similar to when a solution enzyme is added to the sample in the conventional method. For example, in the analysis of bile acids, when a cellulose support on which 3α-H3D and diaphorase are each immobilized is added to the sample solution, the action of 3α-H3D causes the bile acids in the sample solution to Reacts with NAD added in advance to form 3-keto bile acid and NADH
is generated, and further, by the action of diaphorase, the above N
At the same time as ADH is oxidized to NAD, resazurin added in advance to the sample solution is reduced to produce resorufin in an equimolar amount to the bile acid in the sample solution, and the fluorescence of the resorufin thus produced is measured. Quantitative analysis of bile acids is performed using this method.

Enzymes immobilized on cellulose supports are not consumed by the catalytic action described above, so if they are removed from the reaction system after use, washed, and stored, they can be used over and over again for the same purpose. It is possible.

The analysis □ method of the present invention is as described above, and in particular, in the analysis method that utilizes the catalytic action of an enzyme, an enzyme immobilized on a spherical cellulose support is added to the sample solution, so it is different from the conventional sample. Compared to analytical methods that add enzymes in solution form to liquids, this method allows for repeated use of expensive enzymes without having to be thrown away, and is therefore more economical.

Furthermore, since the support is cellulose, there is little non-specific adsorption of substances in the sample, and the immobilized enzyme is stable over a wide range of pH 9, so the life of the immobilized enzyme is long.

The following is a description based on examples.

Example 1 Using cellulose particles (particle size: about 3 sm) as a support, 30 liters of 2M #2 thorium oxide aqueous solution was added to the cellulose particles 3F and stirred, and cyanide bromide 3F was dissolved in advance in this. 3 ml of acetonetrile was added and reacted for 90 seconds with vigorous stirring. The cellulose particles activated in this way are
After washing with H9,s), 20 liters of QIM Tanshun buffer (PH9,5) in which 3α-H3D50 was dissolved was added, and the mixture was stirred at room temperature for 1 hour to react. Next, by the above treatment, in order to lock the active sites still present in the cellulose particles in which 3a-H5D was identified, α05%2-
The reaction was carried out at 4° C. for 2 hours in α1M) Lis-HCl buffer (PH&O) containing mercaptoethanol. The thus obtained cellulose particles on which 3a-HSD was immobilized were mixed with QIM acetate buffer containing Q5M sodium chloride CPH 0), ion-exchanged water and QIM acetate buffer containing Q5M sodium chloride. /l (after repeated washing at pH 9,5>), IJ soot-acid buffer (IM) containing α05% 2-mercaptoethanol, α1 mM ethylenediamine 4-acetic acid disodium and aO2% sodium azide was added.
H7,6) K immersion and storage at 4°C.

Ceramic cellulose particles (3w particle size) SPK
Diaphorase 1004 is immobilized and stored.

Dissolve NAD60 Misaki in 10-water and add 100 μM to this.
In addition to 4 ml of lenazurin-Nm aqueous solution of
shall be.

Add 20 g of human blood (lls/and QIM) and 20 g of lis(bitroxymethyl)aminomethane-hydrochloric acid buffer (PH 5), mix, heat at 65° C. for 30 minutes, and then cool. Add 10 m of reagent A to this, and add the above 3a-H.
After washing the cellulose particles on which 5D was immobilized and the cellulose particles on which diaphorase was immobilized with α06M tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (PH&, 5), eight cellulose particles of each were added. Hold at 30°C for 40 minutes. The fluorescence of resorufin produced by the above procedure is measured for the sample thus obtained using an excitation wavelength of 560 nm and a fluorescence wave of 1c 580 nm, and the concentration of bile acids in the sample is calculated.

After one measurement is completed, the cellulose particles are taken out and QO6M tris(hydroxymethyl)aminomethane-
The above measurement is repeated using the sample that has been thoroughly washed with hydrochloric acid buffer (PH&5). The results obtained in this way are the first
It is a table.

Table 1 As can be seen from the results shown in Table 1, the cellulose particles on which 3a-H5D is immobilized and the cellulose particles on which diaphorase is immobilized do not deteriorate in performance even after repeated use, and the bile This allows the acid to be placed in place with high precision.

Claims (1)

[Claims] L In a method that utilizes the catalytic action of an enzyme to quantify the divisions in a sample liquid, an identified enzyme is added to a spherical cellulose support into the sample liquid in order to carry out the catalytic action. An analysis method characterized by 2. Spherical cellulose is straight! ! 1. The analytical method according to item 1, which is for cod fish of 1 to 10 pieces.