JPH08154699A - Cypridinoid luciferin solution and method for stabilizing cypridinoid luciferin - Google Patents

Abstract

PURPOSE: To obtain the subject solution, containing a cypridinoid luciferin and an acidic solute, capable of enhancing the stability of an aqueous solution of the cypridinoid luciferin, improved in luminous efficiency and stability of measured values and useful for a high-sensitivity sensing system, etc., for an enzyme immunoassay. CONSTITUTION: A cypridinoid luciferin and an acidic salute such as formic, propionic, malic, tartaric, ethylenediaminetetraacetic or citric acid are dissolved in a 10mM phosphoric acid buffer physiological saline solution, etc., at pH<=6.0 to improve the stability of the cypridinoid luciferin in an aqueous solution, further the luminous efficiency and the stability of measured values due to the ability of addition to the reactional system with a good accuracy. Thereby, the objective stabilized cypridinoid luciferin solution useful for a high-sensitivity sensing system, etc., in an enzyme immunoassay for labeling an antibody against a substance which is contained in a humor such as human blood serum to be measured with an enzyme, capturing the substance, carrying out the immunological reaction with the enzyme-labeled antibody, measuring the enzymic activity thereof and measuring the presence or amount of the substance is obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for stabilizing luciferin of Cypridina luminescent system in an aqueous solution.

[0002]

2. Description of the Related Art In recent years, chemiluminescence and bioluminescence have been attempted as a highly sensitive detection system for enzyme immunoassay, and bioluminescence is particularly drawing attention because of its high quantum yield and rapid luminescence reaction. For bioluminescence, luminescent systems such as firefly, medusa, and firefly have been tried. The firefly luminescent system requires ATP (adenosine triphosphate) when luciferin emits light by luciferase, which is a luminescent enzyme, so the luciferin aqueous solution is quite stable in the absence of ATP, but the three substances Since it is a complex reaction of, the control of the reaction is difficult and the measurement system becomes complicated.

[0003] In the luminescent system of Owan jellyfish, the luminescent enzyme aequorin forms a complex with a luminescent substance and emits light by calcium ions. Therefore, it is said that it is very stable in the absence of calcium. Is difficult to completely remove, and there is a problem in the stability of the light emitting system.

On the other hand, the Cypridina firefly luminescent system is a simple luminescent reaction of Cypridina luciferase and Cypridina luciferin, which is very advantageous as a highly sensitive detection system for enzyme immunoassay, but Cypridina luciferin is in an aqueous solution. Since it is unstable, it is frozen and stored as an alcohol solution such as butanol.

[0005]

When a large amount of alcohol such as butanol is mixed in the luminescence reaction in the Cypridina luminescent system, the luciferase activity may be lost and the luminescence may be inhibited. Therefore, the amount of alcohol added becomes very small, and the measurement accuracy deteriorates. Furthermore, there is concern that the luciferin concentration may change due to the evaporation of alcohol. An object of the present invention is to provide Cypridina luciferin which is excellent in measurement accuracy without inactivating luciferase.

[0006]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors examined conditions for stabilizing Cypridina luciferin in an aqueous solution. The above object is achieved by the present invention described below. That is, the present invention relates to a Cypridina luciferin solution characterized by containing Cypridina luciferin and an acidic solute.

As a result of examining the optimum conditions for the pH and solute of the Cypridina luciferin solution, in order to stabilize Cypridina luciferin, a stabilizing effect was exhibited when the pH of the Cypridina luciferin solution was 6.0 or less, and pH 4.0-4.0.
It was found that the stabilization effect was further increased with 5.0.

The Cypridina luciferin solution of the present invention preferably uses water as a solvent, but the solvent can be appropriately selected as necessary.

The Cypridina luciferin solution of the present invention is characterized by containing an acidic solute, but the acidic solute is not particularly limited, and preferably the Cypridina luciferin solution can be adjusted to pH 6.0 or less. It is a thing. More preferably, formic acid, propionic acid, malic acid, tartaric acid or a salt thereof, or a compound having a chelating effect such as citric acid or ethylenediaminetetraacetic acid (EDTA) or a salt thereof is used. Among these, citric acid, ethylenediaminetetraacetic acid (EDTA), etc.
The stabilizing effect can be enhanced by using a compound having a toxic effect or a salt thereof. Further, these solutes may be used together. The addition amount of these is not particularly limited and can be appropriately selected so that the pH of the luciferin aqueous solution falls within a predetermined range.

The salt used in the present invention is not particularly limited, but examples thereof include sodium, potassium, ammonium, primary amine, secondary amine, tertiary amine, and quaternary ammonium.

Furthermore, by removing dissolved oxygen from the aqueous solution by degassing under reduced pressure, the stabilizing effect is further enhanced. The method for removing dissolved oxygen is not particularly limited. Examples of the method include degassing under reduced pressure, boiling, or blowing nitrogen gas.

According to the present invention, luciferin is stabilized to some extent by adding formic acid, propionic acid, malic acid, tartaric acid or a chelate compound or its salt to an aqueous solution of Cypridina luciferin.

In addition to the acidic solute, sodium chloride or the like may be added for the purpose of adjusting ionic strength or salt concentration.

[0014]

The present invention will be described in detail with reference to the following examples.

Example 1 Cypridina luciferin was added at 3.3 μg / m in the following solvent.
It was dissolved in a concentration of 1 and stored at room temperature. 50 μl of a stock solution was added to a solution of Cypridina luciferase in 100 mM sodium phosphate, 50 mM NaCl (pH 7.2) (3 ng /
ml) to 200 μl, and after 10 seconds with a luminometer (BLR-201 manufactured by Aloka Co., Ltd.), the percentage of the ratio between the value measured for 10 seconds and the value similarly measured with a butanol solution of Cypridina luciferin was activated. The residual rate was taken, whereby the stability was compared at pH.

Solvent 1: 20 mM sodium acetate, 130 mM sodium chloride pH = 3.0 Solvent 2: 〃 〃 pH = 4.5 Solvent 3:10 mM sodium phosphate, 140 mM sodium chloride pH = 6.0

[Table 1]

Example 2 Cypridina luciferin was added to the following solvent at 3.3 μg / ml.
It was dissolved in the solution of the above concentration and stored at 4 ° C. for 3 days.

The activity residual ratio was determined in the same manner as in Example 1, and the stability was compared at pH.

Solvent 1:10 mM sodium citrate, 140 mM sodium chloride pH = 4.0 Solvent 2: 〃 〃 pH = 4.5 Solvent 3: 〃 〃 pH = 5.0 Solvent 4: 〃 〃 pH = 7.0 Solvent 5: 〃 〃 pH = 8.0

[Table 2]

Example 3 Cypridina luciferin was added at 3.3 μg / m in the following solvent.
It was dissolved in a concentration of 1 and stored at 4 ° C. With respect to the storage solution, the residual activity ratio was determined in the same manner as in Example 1, and the stability was compared using solutes.

Solvent 1:10 mM sodium citrate, 140 mM sodium chloride pH = 4.5 Solvent 2:20 mM sodium formate, 130 mM sodium chloride pH = 4.5 H = 4.5 Solvent 3:20 mM sodium propionate, 130 mM
Sodium chloride pH = 4.5 Solvent 4:20 mM sodium malate, 130 mM sodium chloride pH = 4.5 Solvent 5:20 mM sodium tartrate, 130 mM sodium chloride pH = 4.5 Solvent 6:10 mM sodium EDTA, 140 mM sodium chloride pH = 4.5

[Table 3]

As shown in the above table, formic acid, propionic acid,
Although salts of malic acid and tartaric acid have a stabilizing effect, citric acid and a salt of a compound having a chelating effect of EDTA showed long-term stability.

Example 4 Cypridina luciferin was added to the following solvent at 3.3 μg / ml.
The solution was dissolved in the above solution, boiled 3 times under reduced pressure at room temperature, returned to normal pressure in a nitrogen stream and stored at 4 ° C.

With respect to the preservation solution, the residual activity ratio was determined in the same manner as in Example 1 and compared with the case where no deoxidation was performed.

Solvent: 10 mM sodium citrate, 140 mM sodium chloride pH = 4.5

[Table 4] Although deoxidation was insufficient with this treatment, a stabilizing effect was clearly recognized as compared with the case of no treatment.

[0026]

EFFECT OF THE INVENTION Enzyme immunoassay, that is, an antibody against a substance to be measured contained in a body fluid such as human serum is labeled with an enzyme, the substance to be measured is captured, and then an immunoreaction is performed with the enzyme-labeled antibody, The use of Cypridina luminescence as a highly sensitive detection system when measuring the presence or amount of the substance by measuring its enzyme activity is achieved by simply adding a Cypridina luciferin solution to the labeled antibody Cypridina luciferase, that is, lucifera. -Since it is a simple reaction between luciferin and luciferin and the luminescence reaction is rapid, it is very advantageous and expected. Improving the stability of Cypridina luciferin in aqueous solution, which is a problem of this luminescence system, does not inhibit luminescence,
Since it can be added to the reaction system with high accuracy, the superiority of the entire detection system such as improvement of luminous efficiency and stability of measured values will be improved.

Claims (11)

[Claims] 1. A Cypridina luciferin solution containing Cypridina luciferin and an acidic solute. 2. The Cypridina luciferin solution according to claim 1, wherein the acidic solute is formic acid, propionic acid, malic acid, tartaric acid, a chelate compound or a salt thereof. 3. The Cypridina luciferin solution according to claim 2, wherein the chelate compound is ethylenediaminetetraacetic acid, citric acid or a salt thereof. 4. The Cypridina luciferin solution according to claim 1, which has a pH of 6.0 or less. 5. The Cypridina luciferin solution according to claim 4, which has a pH of 4.0 to 5.0. 6. The pH of the Cypridina luciferin solution is set to 6.
A method for stabilizing Cypridina luciferin, characterized in that it is 0 or less. 7. The pH of the Cypridina luciferin solution is 4.
The method for stabilizing Cypridina luciferin according to claim 6, which is 0 to 5.0. 8. The Cypridina luciferin solution contains formic acid, propionic acid, malic acid, tartaric acid or a chelate compound or a salt thereof as a solute.
Alternatively, the method for stabilizing Cypridina luciferin according to item 7. 9. The method for stabilizing Cypridina luciferin according to claim 8, wherein the chelate compound is ethylenediaminetetraacetic acid, citric acid or a salt thereof. 10. The method for stabilizing Cypridina luciferin according to claim 6, wherein water is used as a solvent for the Cypridina luciferin solution. 11. The method for stabilizing Cypridina luciferin according to claim 6, wherein dissolved oxygen is removed from the Cypridina luciferin solution.