JP3082374B2 - Stabilizer of 1,2-dioxetane derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a compound represented by the general formula:

Embedded image (Wherein, ^{R 1} represents a hydrogen atom or a halogen atom, ^{R 2} is a lower alkyl group, Ar is phenylene or naphthalene - diyl group, ^{R 3} is group or represented by -OPO ₃ ^2- · 2M ⁺
Is a galactosyl group, wherein M is a sodium atom, a potassium atom, or a group represented by NH ₄ . The present invention relates to a stabilizer for a 1,2-dioxetane derivative represented by the formula:

[0002]

2. Description of the Related Art Enzyme immunoassay (hereinafter referred to as EIA)
Is a labeled immunoassay in which the amount of color development, the amount of fluorescence or the amount of luminescence generated by the reaction between a labeled enzyme and a substrate is measured to determine the enzymatic activity, and the amount of the measurement object is measured based on the result. .

Among EIAs, a chemiluminescent enzyme immunoassay (hereinafter referred to as CLEIA) for measuring the amount of luminescence using a luminescent substrate has recently attracted particular attention due to the short time required for the measurement and high sensitivity. . In CLEIA,
A method using peroxidase (POD) as an enzyme and luminol and hydrogen peroxide as substrates for this enzyme is known (see “Enzyme Immunoassay”, Medical Shoin 1987). However, these methods have problems such as a short emission time and a high background. Therefore, as a result of a study for achieving a high sensitivity by a simple operation with a long light emission time, a 1,2-dioxetane derivative represented by the above general formula (I) was found as a substrate (Japanese Patent Application Laid-Open No. 3-72489, etc.). reference).

[0004]

The 1,2-dioxetane derivative represented by the general formula (I) is an excellent luminescent substrate for highly sensitive measurement in CLEIA, but causes degradation in a solution. Therefore, there is a disadvantage that the measurement sensitivity of CLEIA is reduced.

[0005]

Accordingly, the present inventors have found that it can be stabilized by adding a sugar to the 1,2-dioxetane derivative represented by the above general formula (I), and completed the present invention. The present invention is a stabilizer for a 1,2-dioxetane derivative represented by the above general formula (I), comprising a sugar.

[0006] Sugars that can be used in the present invention include:
For example, lactose, mannose and trehalose can be mentioned. The saccharide is present in an amount of from 0.01% to 1,2-dioxetane derivative represented by the general formula (I).
An amount of 5.0% can be used.

As shown in the structural formula, the 1,2-dioxetane derivative represented by the general formula (I) has an adamantyl group in which ^one carbon atom of 1,2-dioxetane is substituted by R ¹ with a spiro bond. and a further compound oR ² and Ar-R ³ is bound to another carbon atom. R ¹ described above may be a hydrogen atom or a halogen atom such as a chloro atom, a bromo atom or an iodine atom. The -OR ² becomes group, 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. Further Ar-R ³ are those wherein Ar is a benzene ring or a naphthalene ring, the ring on the R ³ becomes substituents are those having, for example, phosphoric acid ester group or a galactosyl group. 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.

[0008] More specific examples of compounds of general formula (I) 3- [4-methoxy-spiro (1,2-dioxetane-3,2'-tricyclo [3,3,1,1 ^3,7] Decane) -4-yl] phenylphosphate disodium salt (A
MPPD), 7- [4-methoxyspiro (1,2-dioxetane-3,2'-tricyclo [3,3,1,
1 ^3,7 ] decane) -4-yl] naphthyl-2-yl phosphoric acid disodium salt (AMNPD), 3- [4-methoxyspiro (1,2-dioxetane-3,2′-tricyclo [3,3, 1,1] ^decane) -4-yl] phenyl beta-D-galactopyranose (AMPGD) 3- [4- methoxy-spiro [1,2-dioxetane -
3,2 '-(5'-chloro) tricyclo [3,3,1,
^1,3,7 ] decane] -4-yl] phenyl phosphate disodium salt (Cl-AMPPD), 3- [4-methoxyspiro [1,2-dioxetane-3,2 '-(5'-chloro) tricyclo [3,3,1,1 ^3,7 ] decane] -4-
Yl] phenyl β-D-galactopyranose (Cl-
AMPGD), 3- [4- methoxy-spiro (1,2-dioxetane-3,2 '- (5'-methoxy) tricyclo [3,3,1,1 ^3,7] decane) -4-yl] phenyl phosphate Acid disodium salt and the like.

The 1,2-dioxetane derivative solution represented by the general formula (I) to which the saccharide of the present invention is added as a stabilizer is used as a substrate solution in CLEIA, and the antigen or antibody to be measured in the sample is used. Can be used for measurement. When the antigen in the sample is measured by CLEIA, for example, the antigen-bound solid phase, the enzyme-labeled antibody, and the sample are mixed and reacted for 1 to 30 minutes. The reaction temperature in the practice is between 4 ° C and 40 ° C, preferably 25 ° C.
３８38 ° C. Next, after washing the unreacted enzyme-labeled antibody, the amount of the enzyme of the enzyme-labeled antibody bound to the solid phase is reduced by adding a substrate solution containing the 1,2-dioxetane derivative represented by the general formula (I) and the stabilizer. By measuring the amount of luminescence, the antigen in the sample can be quantified.
The measurement is carried out by reacting at room temperature to 40 ° C. for 0.1 to 20 minutes and measuring the amount of luminescence by a detector.

The solution containing the 1,2-dioxetane derivative represented by the above general formula (I) is prepared by adding the 1,2-dioxetane derivative represented by the above general formula (I) to an aqueous solution in an amount of 0.1%.
It can be obtained by dissolving to a concentration of 1 mg / ml to 0.5 mg / ml, and adding the stabilizer to 0.01% to 5.0% to prepare. As the aqueous solution, various buffer solutions can be used, and examples thereof include a diethanolamine buffer solution.

In addition, 1, represented by the general formula (I),
After dissolving the 2-dioxetane derivative and the stabilizer in an aqueous solution, it can be lyophilized and stored. When used as a substrate solution, it can be carried out by adding a reconstitution solution containing the above-mentioned buffer solution.

The enzymes used for CLEIA include alkaline phosphatase, β-galactosidase and the like, and can be used according to the 1,2-dioxetane derivative represented by the above general formula (I). Enzyme labeling of the antibody can be easily performed by the active ester method (see “Enzyme immunoassay”, Medical Shoin 1987).

In the aqueous solution containing the 1,2-dioxetane derivative represented by the general formula (I) and the stabilizer, an enhancer may be added to further enhance the emission intensity and extend the emission time. This is advantageous for sensitivity measurement. Examples of the enhancer include poly (vinylbenzyltributylammonium chloride) (TBQ), poly [vinyl (benzyldimethylammonium chloride)] (BDMQ), poly (vinylbenzyltrimethylammonium chloride) (TMQ), and poly (vinylbenzyltriethylammonium). Chloride) (TEQ)
And fluorescein. These can be used alone or as a mixture.

[0014]

The present invention will be described in more detail with reference to the following examples. Example 1 Stability Effect in Solution 0.4 with 0.5% Lactose or Mannose
A mg / ml MCl-AMPPD solution was prepared with a 0.1 M jetanolamine buffer (pH 10). Also, 0.
08% enhancer (TBQ) solution is also Cl-AMPPD
It was prepared under the same conditions as the solution. The two solutions were then provided for stability testing. Cl containing no sugar as a comparative example
-A mixed solution of AMPPD and enhancer was used. 37
Stored at 7 ° C. for 7 days or 14 days, respectively. A mixture of the stored Cl-AMPPD solution and the enhancer solution was diluted 20-fold with a 0.1 M diethanolamine buffer solution, and 300 µl of this Cl-AMPPD solution was 1.3 x
It was completely hydrolyzed with 10 ^-11 mol of alkaline phosphatase, and the luminescence was measured with a luminometer. Table 1 shows the results when the light emission amount before storage was 100%.

[0015]

[Table 1]

Example 2 Stability Effect in Lyophilization 0.2 mg / ml MCl-A containing 0.5% lactose
An MPPD solution was prepared with purified water and freeze-dried. A 0.04% enhancer (TBQ) solution was prepared with a 0.1 M diethanolamine buffer (pH 10) as a reconstitution solution. The two reagents were then provided for stability testing. Cl-AMPP without lactose as a comparative example
A mixed solution of D and an enhancer was used. After being stored at 37 ° C. for 7, 14, 21 and 28 days, respectively, the Cl-AMPPD lyophilized reagent was reconstituted with a reconstituted reconstituted solution, and the solution was reconstituted with 0.1 M jetanolamine buffer for 20 days. Dilute two times. This Cl-AMP
300 μl of the PD solution was completely hydrolyzed with 1.3 × 10 ⁻¹¹ mol of alkaline phosphatase, and the luminescence was measured using a luminometer. FIG. 1 shows the result when the light emission amount before storage was 100%.

Example 3 Stability Effect in Freeze-Drying A Cl-AMPPD solution containing an enhancer (TBQ) was prepared in purified water containing 0.5% lactose and freeze-dried. Further, a 0.1 M diethanolamine buffer solution (pH 10) was prepared as a restoring solution. The two reagents were then provided for stability testing. As a comparative example, a mixed solution of Cl-AMPPD containing no lactose and an enhancer was used. After storage at 37 ° C. for 7, 14, 21 and 28 days, respectively, the Cl-AMPPD
And the freeze-dried reagent of the enhancer are reconstituted with the same reconstituted reconstitution solution, and this solution is diluted 20-fold with 0.1 M diethanolamine buffer. This Cl-AMPPD solution 30
0 μl was completely hydrolyzed with 1.3 × 10 ⁻¹¹ mol of alkaline phosphatase, and the luminescence was measured using a luminometer. FIG. 2 shows the result when the light emission amount before storage was set to 100%.

[0018]

According to the present invention, when a saccharide such as lactose, mannose or trehalose is added to a solution containing a 1,2-dioxetane derivative represented by the above general formula (I),
Extremely stable. Therefore, when this solution is used for CLEIA, highly accurate measurement can be performed.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the measurement of the lapse of time and the amount of luminescence when stored after freeze-drying using lactose as a stabilizer.

FIG. 2 is a diagram showing the measurement of the lapse of time and the amount of luminescence when a lactose is used as a stabilizer, an enhancer is added, freeze-dried and stored.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI // C09K 11/06 635 C09K 11/06 635 690 690 C12Q 1/42 C12Q 1/42 G01N 21/76 G01N 21/76 (58 ) Field surveyed (Int. Cl. ⁷ , DB name) C07D 321/00 C07H 3/00-3/10 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A general formula using a sugar. Wherein R ¹ is a hydrogen atom or a halogen atom, R ² is a lower alkyl group, Ar is a phenylene group or a naphthalene-
Diyl group, R ³ is a group or a galactosyl group represented by -OPO ₃ ^2- · 2M ^+, wherein M is sodium atom,
It is a potassium atom or NH ₄ . ) 2. The stabilizer according to claim 1, wherein the sugar is lactose, mannose or trehalose.