JP2622653B2 - Thyroid hormone aqueous solution - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a thyroid hormone aqueous solution. More particularly, it relates to a thyroid hormone aqueous solution capable of stably storing the activity of thyroid hormone over time.

[0002]

2. Description of the Related Art Hitherto, thyroid hormone has been stored in a dry state because its activity decreases with time in an aqueous solution.

[0003]

In the immunoassay, thyroid hormone is usually a phosphate buffer (about pH 7.2).
Used in. In recent years, there has been an increasing demand for simplification of immunoassay procedures.However, it is necessary to prepare thyroid hormone, which is conventionally in a dry state, into a solution state at the time of use, and it takes extra time for dissolution during the analysis procedure. There was a problem. That is, a thyroid hormone aqueous solution that can be stably stored over time has been desired instead of a phosphate buffer solution whose activity decreases over time.

[0004] In the case of pharmaceutical thyroid hormone preparations, they are used for oral administration of tablets. However, if the thyroid hormone activity is stable even in a solution state, for example, administration in a solution becomes possible.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems, and as a result, have reached the present invention. In other words, the present invention provides sodium salicylate, p-aminobenzenesulfonic acid, N-acetyl -L-phenylalanyl-3,5-diiodo-L-tyrosine, ammonium 8-anilino-1-naphthalenesulfonate and 3,
A thyroid hormone aqueous solution (1) comprising at least one aromatic hapten (a ') selected from 3'-diethylbenzidine dihydrochloride and having an aqueous solution pH of 6.0 to 10.0. ); And a standard reagent for immunoassay comprising the aqueous solution.

In the present invention, the aromatic hapten (a)
As sodium salicylate, p-aminobenzenesulfonic acid, N-acetyl-L-phenylalanyl-
3,5-diiodo-L-tyrosine, 8-anilino-1-
One or more of ammonium naphthalenesulfonate and 3,3'-diethylbenzidine dihydrochloride are used. Particularly preferred among these are sodium salicylate and especially ammonium 8-anilino-1-naphthalenesulfonate (hereinafter abbreviated as ANS).
The hapten is a compound defined as having no immunogenic effect by itself, but becoming an immunogen by binding to a protein.

The concentration of (a) in the aqueous solution (1) of the present invention is generally 0.06 to 50 mg / ml, preferably 0.2 to 50 mg / ml.
10 mg / ml.

The aqueous solution having a pH adjusted in the present invention contains an organic and / or inorganic substance showing acidic or basic properties in the aqueous solution. On the acidic side,
Examples of the acidic substance include an aqueous solution containing hydrochloric acid, sulfuric acid, acetic acid, citric acid, potassium hydrogen oxalate, potassium hydrogen phthalate, and the like. On the basic side, examples of the basic substance include an aqueous solution containing ammonia, sodium hydroxide, potassium hydroxide, disodium hydrogen phosphate, sodium barbital, sodium hydrogen carbonate, and the like. Preferred of these are:
It is an aqueous solution of a buffer solution. The buffer is an aqueous solution having a buffer action, and examples thereof include an acetate buffer, a phthalate buffer, a phosphate buffer, a barbital buffer, and a carbonate buffer.

The pH of the aqueous solution (1) of the present invention is 6.0 to 6.0.
10.0, and preferably 7.2 to 9.5.
If it is less than 6.0, the storage stability of the thyroid hormone aqueous solution is poor, and if it exceeds 10.0, the storage stability is saturated.

The thyroid hormone used in the present invention is a kind of hapten and includes thyroxine (hereinafter referred to as T4), triiodothyronine (hereinafter referred to as T3) and the like. Usually, the aqueous solution (1) or the aqueous solution (2) is used. ) During T
4 is 3-2000 ng / ml, T3 is 0.1-90 ng
/ Ml. Further, the aqueous solution (1) or the aqueous solution (2) of the present invention usually contains proteins such as albumin and sodium chloride as other components. Proteins such as albumin in aqueous solution
150 mg / ml, sodium chloride is usually 2 to 40 mg
/ Ml.

[0011]

EXAMPLES The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.

Examples 1 to 6, Comparative Examples 1 and 2 Preparation of T4 aqueous solution In a 0.02 M phosphate buffer (pH 7.2), 80 mg / ml of albumin and 8.5 mg of sodium chloride were added.
/ Ml to give a basic aqueous solution 1. After the preparation, 1N NaOH or 1N HCl was added so that the pH of the basic aqueous solution 1 became the value shown in Table 1, and ANS was added so that the concentration shown in Table 1 was obtained.
Was added to a concentration of 20 ng / ml or 120 ng / ml.

Examples 7 to 12, Comparative Examples 3 and 4 Preparation of T3 aqueous solution In a 0.02 M phosphate buffer (pH 7.2), 30 mg / ml of albumin and 8.5 mg of sodium chloride were added.
/ Ml to prepare a basic aqueous solution 2. After the preparation, 1N (7) NaOH or 1N (7) HCl was added so that the pH of the basic aqueous solution 2 became the value shown in Table 2, and ANS was added so that the concentration became the one shown in Table 2; It was added to a concentration of 1.0 ng / ml or 8.0 ng / ml.

Evaluation Regarding the storage stability of the aqueous thyroid hormone solution obtained in the examples, the thyroid hormone activity was evaluated by measuring the residual activity rate over time.

T4 activity measurement method Dry T4 having a known concentration was prepared at the time of use in a 0.02 M phosphate buffer (pH 7.2) and used as a standard solution. Next, 100 μl of a standard solution or T4 aqueous solution was added to 6 mg /
300 μl of a barbital buffer containing 2 ml of albumin and 2 μg / ml of peroxidase-labeled T4 (hereinafter referred to as P1) and one anti-T4 polyclonal antibody-bound glass bead (hereinafter referred to as G1) are sequentially dispensed into a test tube. And allowed to react in a test tube at 37 ° C. for 15 minutes.
A 4-G1-P1 complex was formed. After the reaction, the liquid in the test tube was removed with an aspirator, and the T4-G1-P1 complex was washed three times with 3 ml of saline.

Furthermore, 0.2 mg / ml hydrogen peroxide and 3 mg were added to one T4-G1-P1 complex in a test tube.
300 ml of a citrate-phosphate buffer solution containing ortho-phenylenediamine / ml at 37 ° C. for 15 minutes at 37 ° C. After the reaction, 3 ml of 1.5 N sulfuric acid is added to stop the enzyme reaction, and the solution is absorbed. Using a spectrophotometer 49
Photometry was performed at 2 nm.

Finally, the concentration value and the photometric value of each standard solution are plotted on a graph paper to prepare a calibration curve.
The concentration value for the photometric value of the four aqueous solutions was calculated. This concentration value was defined as T4 activity.

T3 activity measurement method T3 in a dry state having a known concentration was prepared in a 0.02 M phosphate buffer (pH 7.2) at the time of use, and used as a standard solution. Next, 100 μl of the standard solution or T3 aqueous solution was added to 6 mg /
300 μl of a barbital buffer solution containing ml of albumin and 6 μg / ml of peroxidase-labeled T3 (hereinafter referred to as P2), and one glass bead (hereinafter referred to as G2) conjugated with an anti-T3 polyclonal antibody were sequentially dispensed into a test tube. Immunized at 37 ° C. for 15 minutes in a test tube;
A -G2-P2 complex was formed. After the reaction, the liquid in the test tube was removed with an aspirator, and the T3-G2-P2 complex was washed three times with 3 ml of saline.

Furthermore, 0.2 mg / ml of hydrogen peroxide and 3 mg of T3-G2-P2 complex were added to one T3-G2-P2 complex in a test tube.
300 ml of a citrate-phosphate buffer solution containing ortho-phenylenediamine / ml at 37 ° C. for 15 minutes at 37 ° C. After the reaction, 3 ml of 1.5 N sulfuric acid is added to stop the enzyme reaction, and the solution is absorbed. Using a spectrophotometer 49
Photometry was performed at 2 nm.

Finally, the concentration value and the photometric value of each standard solution are plotted on a graph paper to prepare a calibration curve.
The concentration values for the photometric values of the three aqueous solutions were calculated. This concentration value was defined as T3 activity.

Residual activity rate Using the T4 aqueous solutions prepared in Examples 1 to 6 and Comparative Examples 1 and 2, and the T3 aqueous solutions prepared in Examples 7 to 12 and Comparative Examples 3 and 4, the respective activities on the first day were determined. The activity after storage at 7 ° C. for one year was measured, and the activity on the first day was defined as 100%.
The remaining activity after one year is shown in Tables 1 and 2 in percentage.

[0022]

[Table 1]

[0023]

[Table 2]

In Tables 1 and 2, the residual activity ratio after one year is remarkably improved in Examples, and it is understood that the storage stability of the aqueous solution of the present invention is excellent.

[0025]

According to the present invention, thyroid hormone activity can be maintained in an aqueous solution state for a long time. Therefore, it is suitable for applications requiring storage stability such as standard reagents in immunoassays, and can be developed for pharmaceutical applications.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location G01N 33/53 G01N 33/53 E

Claims (5)

(57) [Claims] (1) sodium salicylate, p-aminoben
Zensulfonic acid, N-acetyl-L-phenylalanyl
-3,5-diiodo-L-tyrosine, 8-anilino-1
-Ammonium naphthalenesulfonate and 3,3'-
Selected from diethylbenzidine dihydrochloride
A thyroid hormone aqueous solution (1) containing at least one aromatic hapten (a) and having an aqueous solution pH of 6.0 to 10.0. 2. The aqueous solution according to claim 1, wherein the pH of the aqueous solution is 7.2 to 9.5. 3. The aqueous solution according to claim 1, wherein (1) is an aqueous solution of a buffer solution. 4. The aqueous solution according to claim 1, wherein (a) is ammonium 8-anilino-1-naphthalenesulfonate. 5. An aqueous solution according to claim 1,
Standard reagents in immunoassays.