JP2640941B2 - Method for stopping enzyme activity of galactosidase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Applications] There are two types of galactosidase, α- or β-galactosidase, which are widely distributed in animals, plants and microorganisms, and lactose, alkyl and aryl-β-D
-It is used when performing a hydrolysis reaction of galactoside or the like. Furthermore, in recent years, with the development of enzyme immunoreactions, it has been increasingly used as a labeling enzyme for antibodies or antigens together with peroxidase, alkaline phosphatase and the like.

(Prior art)

In the enzyme immunoreaction, as a substrate for galactosidase, o-nitrophenyl-β-D-galactopyranoside as a chromogenic substrate and 4-methylumbelliferyl-β-D-galactoside as a fluorescent substrate are mainly used. When these substrates are used, a sodium carbonate solution or a high pH buffer is used as a method for inactivating the enzyme by shifting the pH of the reaction solution to the alkaline side as a solution for stopping the enzyme activity. Further, the enzyme is inactivated by shifting the pH of the reaction solution to the acidic side.

[Problems to be solved by the invention]

However, when these acid or alkali solutions are used as reaction stop solution in kits, etc., if they do not adhere to the skin or clothing due to inaccuracies in operation, they may cause skin irritation or clothing damage. Extreme caution is required.

Furthermore, depending on the substrate used, the reactivity with galactosidase is very high, such as chlorophenol red-β-D-galactopyranoside, but the pH is stable only around neutral, and the pH is shifted to the alkaline or acidic side. In the case of transfer, there is a case where the conventional stop solution cannot be used because self-decomposition, color change and the like are remarkable. Further, in order to express the enzyme activity again, it is necessary to stop the enzyme activity around a neutral pH without denaturing and inactivating the enzyme due to a change in pH.

[Means for solving the problem and its operation]

Therefore, the present inventors have conducted intensive studies as a simple method for preparing a galactosidase activity stopping solution, and found that a substrate or an enzyme reaction product of galactosidase such as lactose or galactose was mixed with ethylenediaminetetraacetic acid, nitrilotriacetic acid,
It has been found that the activity of galactosidase can be stopped for a long time at a neutral pH by using a solution in which a general metal chelating agent such as uramil diacetate is mixed, and the present invention has been completed. After the reaction is stopped, the concentration of galactose and lactose is more than 10 times that of the substrate used, and the concentration of the chelating agent is 5 mM.
With the above combination, the activity expression can be stopped for a long time. However, it is impossible to efficiently stop the expression of activity at a similar low concentration over a long period of time using only one of them. The use of the stop solution of the present invention makes it possible to use substrates which were significantly affected by fluctuations in pH and could not be used in enzyme immunoassays, etc., and which have high measurement sensitivity and reduced reaction time. The development of the law became possible.

 Hereinafter, the present invention will be described with reference to Examples.

Example 1 Nerve-specific elanase (NSE) was measured by a sandwich-type enzyme immunoassay using polystyrene beads as an antibody-insoluble carrier and β-galactosidase from Escherichia coli (Boehringer Mannheim, West Germany) as an antibody labeling enzyme. As a substrate of β-galactosidase, 0.5 ml of 0.1% chlorophenol red-β-D-galactopyranoside (Boehringer Mannheim, West Germany) was used. As an enzyme reaction stop solution, a solution in which galactose was added to a concentration of 0.25% to a 50 mM sodium phosphate buffer, a solution in which ethylenediaminetetraacetic acid was added to a concentration of 6.25 mM, and a mixture of galactose and ethylenediaminetetraacetic acid in the same concentration FIG. 1 shows the change in the absorbance of the enzyme reaction solution when 2 ml of each mixture was used.

When an enzyme reaction stop solution to which only galactose or ethylenediaminetetraacetic acid was added was used, galactosidase activity could not be completely stopped, and the absorbance of the enzyme reaction solution gradually increased. However, when a stop solution to which both were mixed and added was used, the activity could be completely stopped for a long time, and no increase in absorbance was observed.

EXAMPLE 2 Polystyrene beads antibody immobilizing carrier, beta-galactosidase (Boehringer Mannheim, West Germany) from Escherichia coli was measured S-100a ₀ protein in a sandwich-type enzyme immunoassay using as the antibody labeling enzyme. β
As a substrate for galactosidase, 0.5 ml of 0.1% chlorophenol red-β-D-galactopyranoside (Boehringer Mannheim, West Germany) was used as in Example 1. Lactose was added to a 50 mM sodium phosphate buffer to a concentration of 0.5%, ethylenediaminetetraacetic acid was added to a concentration of 6.25 mM as an enzyme reaction stop solution, and lactose and ethylenediaminetetraacetic acid were added to the same concentration. FIG. 2 shows the change in the absorbance of the enzyme reaction solution when 2 ml of each mixture was used.

When an enzyme reaction stop solution to which only lactose or ethylenediaminetetraacetic acid was added was used, galactosidase activity could not be completely stopped, and the absorbance of the enzyme reaction solution gradually increased. However, when a stop solution to which both were mixed and added was used, the activity could be completely stopped for a long time, and no increase in absorbance was observed.

Example 3 o-nitrophenyl-β- as a substrate was added to 1 ml of a 50 mM sodium phosphate buffer containing 10 mu of β-galactosidase (Boehringer Mannheim, West Germany) at 50 mM, pH 7.0.
0.25 ml of a D-galactopyranoside solution (4.5 mg / ml) was added, and the change in absorbance at 420 nm was observed. 1.25 ml of the stop solution was added simultaneously with the addition of the substrate. As a stop solution, 1% galactose, 10 mM nitrilotriacetic acid, and 50 mM pH 7.0 sodium phosphate buffer containing the same concentration of both were compared with the case where only a buffer containing nothing was added. The change in absorbance of each reaction solution is shown in FIG. By using a mixture of galactose and nitrilotriacetic acid, the activity of β-galactosidase was completely stopped, and no increase in the absorbance of the enzyme reaction solution was observed.

Example 4 By the same operation as in Example 1, the reaction was stopped with an enzyme reaction stopping solution containing 0.25% of galactose and 6.25 mM of ethylenediaminetetraacetic acid, and polystyrene beads bound to β-galactosidase as an antibody labeling enzyme were added to 50 mM, pH7. .0
After washing with sodium phosphate buffer, transfer to a test tube containing 0.5 ml of a 0.1% chlorophenol red-β-D-galactopyranoside solution as a substrate solution. The same enzyme activity as the bound β-galactosidase was observed. The above results indicate that β-galactosidase is not inactivated by the present stop solution.

〔The invention's effect〕

The safety of the enzyme reaction stop solution has been improved, and the use of a highly sensitive substrate of galactosidase, which was difficult to apply due to the extremely poor safety of enzyme immunoassays except at neutral pH, has become possible. The rise and shortening of the reaction time were obtained.

[Brief description of the drawings]

FIG. 1 shows that chlorophenol red-β-D-
In the reaction using galactopyranoside, 0.25% galactose, 6.25 mM ethylenediaminetetraacetic acid and a mixed solution of both are used as a stop solution for the enzyme reaction. B shows a case where a 0.25% galactose solution was used as a stop solution, B shows a case where a 6.25 mM ethylenediaminetetraacetic acid solution was used as an enzyme reaction stop solution, and C shows a case where both were used together. Also, in the figure,-●-is a measured value of the sample of NSE5ng / ml,-○-is a measured value of the sample of NSE15ng / ml, Is the sample measurement of NSE 45 ng / ml. Figure 2 is 0.5 percent lactose as an enzyme reaction stop solution in the reaction using the chlorophenol column de-beta-D-galactopyranoside as a substrate of an enzymatic reaction in an enzyme immune response S-100a _0, 6.25mM ethylenediaminetetraacetic acid 5 shows a comparison of changes in absorbance with time when a mixture of the two was used, A: when a 0.5% lactose solution was used as a stop solution for enzyme reaction, B: 6.25 mM ethylenediamine tetrachloride as a stop solution for enzyme reaction. When an acetic acid solution is used, C indicates the case where both are used together. In the figure,
Is the measured value of the sample of S-100a ₀ 500 pg / ml,
- is a measurement of the sample S-100a ₀ 3ng / ml, Is a sample measurements of S-100a ₀ 9ng / ml. Figure 3 shows a 1% galactose solution, a 10 mM nitrilotriacetic acid solution, and a 10 mM nitrilotriacetic acid solution as an enzyme reaction stop solution in 50 mM, pH 7.0 sodium phosphate buffer containing 10 mu β-galactosidase (Boehringer Mannheim, West Germany). The change in the absorbance of the enzyme reaction solution when a solution containing the same concentration was used was compared with the case where a 50 mM, pH 7.0 sodium phosphate buffer solution containing nothing was used as a stop solution. In the figure,-○-,-●-,-△-, and-□-contain 50 mM, pH 7.0 sodium phosphate buffer, the same buffer containing 1% galactose, and 10 mM nitrilotriacetic acid as stop solutions, respectively. The case where the same buffer and the same buffer containing both at the same concentration is used is shown.

Claims (4)

(57) [Claims] (1) The use of a reaction terminating solution containing either an enzyme substrate or an enzyme reaction product and a chelating agent allows the pH of the reaction solution to be adjusted to α-galactosidase or β-galactosidase without shifting to the alkaline side or the acidic side. A method for stopping the enzyme activity of galactosidase, wherein the enzyme activity of galactosidase is stopped for a long time without inactivating it. 2. The method according to claim 1, wherein the enzyme substrate or the enzyme reaction product is lactose and glassose, respectively. 3. The enzyme activity of galactosidase according to claim 1, wherein the chelating agent is a metal chelating agent such as ethylenediaminetetraacetic acid, nitrile triacetic acid, uramil diacetate, etc., which coordinates with a metal ion to form a chelating compound. Stop law. 4. The method according to claim 1, wherein α-galactosidase or β-galactosidase is a labeling enzyme for an antigen or an antibody.