JP2001181291A - Stabilizer for 1,2-dioxetane derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a stabilizer capable of preventing the decomposition of 1,2-dioxetane derivative in an aqueous solution, having high excipient effect of additives and enabling easy preparation of an agent. SOLUTION: The stabilizer for 1,2-dioxetane derivative is composed of a polysaccharide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a compound represented by the following general formula 1 wherein R1 is a hydrogen atom or a halogen atom, R2 is a lower alkyl group, Ar is a phenyl-triyl group or a naphthalene-triyl group, and R3 is a hydrogen atom. An atom or a halogen atom, R4 is a group represented by -OPO32-2M + or a galactosyl group, wherein M is a sodium atom,
The present invention relates to a stabilizer for a 1,2-dioxetane derivative represented by a potassium atom or NH4), and more particularly, to the stabilizer comprising a polysaccharide.

[0002]

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[0003]

2. Description of the Related Art Enzyme immunoassay (hereinafter abbreviated as EIA)
Is a method for measuring the amount of change in color development, fluorescence, luminescence, and the like caused by a product generated by a reaction between an enzyme labeled on an antibody or an antigen and its substrate, and quantifying the measurement target. EI
In A, a method for measuring the amount of luminescence associated with the decomposition of a luminescent substrate, called a chemiluminescent enzyme immunoassay (hereinafter abbreviated as CLEIA), has a lower background in principle than the colorimetric method and the fluorescent method. It is considered that high-sensitivity measurement is possible.

However, CLEIA (enzyme immunoassay, Medical Shoin, 1987) using luminol and hydrogen peroxide as substrates and peroxidase (POD) as a labeling enzyme.
In other words, there is a problem that the emission time is short, the relationship between the amount of enzyme and the amount of emission is not linearly proportional, and the background emission is high. In recent years, research on a labeling enzyme and a luminescent substrate has been advanced, and an excellent CLEIA excellent in quantification and capable of maintaining luminescence for a long time in a low background has been proposed. As such an excellent CLIEA, for example, alkaline phosphatase (hereinafter abbreviated as ALP) or β-galactosidase is used as an enzyme label, and 1,2 represented by the above general formula 1 is used as a substrate.
-CLEIA using a dioxetane derivative (see Patent No. 1971238).

[0005]

The 1,2-dioxetane derivative represented by the general formula 1 is an excellent luminescent substrate for ALP and the like, but is easily decomposed in a solution, so that the measurement sensitivity in CLEIA is low. descend. In order to prevent this, a method has been proposed in which a sugar is added to a solution of a 1,2-dioxetane derivative to stabilize the solution (see JP-A-5-140146). Formulation was difficult.

Accordingly, the present invention provides a stabilizer which can prevent the decomposition of a 1,2-dioxetane derivative represented by the above general formula 1 in an aqueous solution, and has a high shaping effect of an additive and can be easily formulated. The purpose is to provide.

[0007]

Means for Solving the Problems The invention of claim 1 made to achieve the above object relates to a stabilizer for a 1,2-dioxetane derivative represented by the above general formula 1, It is characterized by becoming. The invention of claim 2 of the present application is directed to the invention of claim 1, wherein the polysaccharide is at least one selected from dextran, pullulan, and ficoll. Hereinafter, the present invention will be described in detail.

The stabilizer of the present invention exerts an effect of suppressing the decomposition of the 1,2-dioxetane derivative represented by the general formula 1 in an aqueous solution. In the 1,2-dioxetane derivative represented by the general formula 1, an adamantyl group substituted with R1 is spiro-bonded to one carbon atom of 1,2-dioxetane, and OR2 and Ar are further bonded to another carbon atom. -R3 is a compound bound thereto. R1
May be a hydrogen atom, or may be a halogen atom such as a chloro, bromo or iodine atom. The group represented by -OR2 is a lower alkoxy group having 1 to 4 carbon atoms, and examples thereof include a methoxy group, an ethoxy group, a propoxy group, and a butoxy group. Ar-
R3 is one in which Ar has a benzene ring or a naphthalene ring, and R3 has a substituent such as a phosphate ester group or a galactosyl group on the ring. When it is a phosphate group, it naturally accompanies a divalent cation, and the cation source may be any of a sodium cation, a potassium cation, and an ammonium group.

As specific examples of the 1,2-dioxetane derivative represented by the general formula 1, 3- [4-methoxyspiro (1,2-dioxetane-3,2'-tricyclo [3,3]
3,1,13,7] decane) -4-yl] phenyl phosphate disodium salt, 3- [4-methoxyspiro (1,2
-Dioxetane-3,2 '-(5'-chloro) tricyclo [3,3,1,13,7] decane) -4-yl] phenyl phosphate disodium salt, 2-chloro-5- [4-methoxy Spiro (1,2-dioxetane-3,2 '-(5'
-Chloro) tricyclo [3,3,1,13,7] decane)
-4-yl] -1-phenylphosphate disodium salt, 2
-Chloro-5- [4-methoxyspiro (1,2-dioxetane-3,2'-tricyclo [3,3,1,13,7]
Decane) -4-yl] -1-phenylphosphate disodium salt, and the like.

The stabilizer of the present invention capable of stabilizing the 1,2-dioxetane derivative is, for example, dextran,
It is a polysaccharide such as pullulan or ficoll. These polysaccharides have a high excipient effect and can be formulated into a well-formed formulation. The polysaccharides can be used alone or as a mixture of two or more kinds. The amount of the polysaccharides used is 5.0% to 10.0% by weight based on the aqueous solution of the 1,2-dioxetane derivative. . Dextran has various molecular weights ranging from thousands to hundreds of thousands, and any of them can be used as the stabilizer of the present invention.

The stabilizer of the present invention has an effect of suppressing the decomposition of the 1,2-dioxetane derivative only by adding it to the 1,2-dioxetane derivative solution. The solution of the 1,2-dioxetane derivative to which the polysaccharide has been added is directly used as CLEI.
The substrate liquid for A can be used for measurement of an antigen or antibody as a measurement target in a sample. For example, CLEIA
When the antigen in the sample is measured by the method, the solid phase to which the antibody is bound, the antibody labeled with ALP and the sample are mixed with the sample, and the mixture is mixed at 4 to 40 ° C., preferably 25 to 38 ° C. for 5 to 30 minutes. After the reaction and removal of the unbound ALP-labeled antibody, the enzyme amount of the ALP-labeled antibody bound to the solid phase was determined by the 1,2-
The luminescence may be measured by adding a dioxetane derivative solution. The measurement of luminescence may be carried out in the course of an enzymatic reaction from room temperature to 37 ° C. for 0.1 to 20 minutes or after the enzymatic reaction using a commercially available detection device. The 1,2-dioxetane derivatives are enzyme substrates for ALP and β-galactosidase, and these 1,2-dioxetane derivatives can be used as substrates in CLEIA using such enzymes as labels.

The 1,2-dioxetane derivative solution for CLEIA is prepared by dissolving the 1,2-dioxetane derivative in an aqueous solution so as to have a concentration of 0.1 mg / ml to 0.5 mg / ml. 5.0 parts by weight of the agent
% To 10.0%. 1,2-
As the aqueous solution for dissolving the dioxetane derivative, various buffer solutions can be used, and examples thereof include a diethanolamine buffer solution.

The 1,2-dioxetane derivative solution containing the stabilizer of the present invention can be lyophilized and stored. Therefore, when implementing CLEIA, etc., CLEI
Immediately before A, a 1,2-dioxetane derivative solution containing a stabilizer may be prepared and used, or a freeze-dried 1,2-dioxetane derivative containing a stabilizer prepared and freeze-dried in advance may be used as a CLEIA. It may be restored just before use. As a restoring agent, for example, diethanolamine buffer,
Various buffer solutions for dissolving the 1,2-dioxetane derivative can be used without limitation.

The stabilizer of the present invention does not interact with various additives generally added to a 1,2-dioxetane derivative solution in CLEIA, and thus can be coexisted therewith. Examples of the additive include an enhancer that is added to enhance emission intensity and extend emission time. Enhancers include, for example, poly (vinylbenzyltributylammonium chloride), poly [vinyl (benzyldimethylammonium chloride)], poly (vinylbenzyltrimethylammonium chloride), poly (vinylbenzyltriethylammonium chloride), and quaternary ammonium salt polymers (eg, Sapphire I, manufactured by Tropix
I, Emerald II, etc.) and fluorescein.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Stability effect in solution 0.04% of enhancer (quaternary ammonium salt polymer; Emerald II, manufactured by Tropix) and 0.2 mg of 1,2-dioxetane derivative (2-chloro-5- [4-methoxyspiro (1,2-dioxetane-
3,2 '-(5'-chloro) tricyclo [3,3,1,
[13,7] decane) -4-yl] -1-phenylphosphate disodium salt; CDP-Star, manufactured by Tropix
Was dissolved in a 0.1 M diethanolamine buffer solution (pH 9.5), and the sugar 9% shown in Table 1 or the stabilizer 9 of the present invention was used.
% Of the 1,2-dioxetane derivative was investigated. For comparison, the same solution was used except that sugar or the stabilizer of the present invention was not added.

After storage at 40 ° C. for 4 days, 50 μl of each solution was hydrolyzed with 1.0 × 10 ⁻¹⁶ mol of ALP, and the luminescence was measured using a luminometer. Table 1 shows the results when the light emission amount before storage was 100%. As is clear from Table 1, among the stabilizers of the present invention, the solution to which dextran was added had improved stability as compared with the control.

[0018]

[Table 1]

Example 2 Stability effect in freeze-drying 0.04% enhancer (Eme, Tropix)
rald II) and 0.2 mg of a 1,2-dioxetane derivative (CDP-Star, manufactured by Tropix) were dissolved in a 0.1 M diethanolamine buffer solution (pH 9.5), and 9% of the saccharide shown in Table 2 or the present invention was used. 9% stabilizer
After addition, the mixture was freeze-dried. For comparison, the same solution was also freeze-dried except that no sugar or the stabilizer of the present invention was added.

After storing at 40 ° C. for 4 days, each freeze-dried product was stored at 40 ° C. for 4 days at 0.1M.
Diethanolamine buffer (pH 9.5) was added for reconstitution. 50 μl of each reconstituted solution was added to 1.0 × 10
The mixture was hydrolyzed with ^-16 mol of ALP, and the luminescence was measured using a luminometer. Table 2 shows the results when the light emission amount before storage was 100%. As is clear from Table 2, the stabilizers of the present invention, dextran, pullulan and ficoll 40, were used.
The freeze-dried product to which 0 or the like was added had remarkably improved stability as compared with the control.

[0021]

[Table 2]

[0022]

According to the present invention, the decomposition of the 1,2-dioxetane derivative is suppressed only by adding it to the solution containing the 1,2-dioxetane derivative represented by the general formula 1, and the compound is stabilized for a long time. A stabilizing agent is provided. Therefore, if the 1,2-dioxetane derivative solution containing the stabilizer of the present invention is used for CLEIA, it is possible to carry out highly accurate CLEIA which is not affected by the decomposition of the 1,2-dioxetane derivative during storage. It is.

Claims (2)

[Claims] 1. A stabilizer for a 1,2-dioxetane derivative comprising a polysaccharide. 2. The stabilizer according to claim 1, wherein the polysaccharide is at least one selected from dextran, pullulan and ficoll.