JPS61239890A - Stabilized peroxidase solution - Google Patents

Abstract

PURPOSE:A stabilized peroxidase solution providing a reagent which is preservable for a long period and has fixed qualities, obtained by adding serum protein and one or more of specific carboxlic acids to a solution of (peroxidase) bonded substance. CONSTITUTION:1l aqueous solution of (peroxidase) bonded substance extracted from a horseradish is blended with serum protein of serum albumin and 10mg-1g parahydroxyphenylcarboxylic acid [e.g., (parahydroxyphenyl)acetic acid and/or propionic acid] shown by the formula (R is CH2, CH2-CH2, CH2-CH2- CH2, CH3-CH-CH2, CH=CH, CH=CH-CH2, or CH3-C=CH).

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is applicable to enzyme immunoassay (hereinafter referred to as EIAJ), which is one of the immunochemical measurement methods, immunohistochemical staining method, etc. The present invention relates to a long-term storage solution of peroxidase or its conjugate to be used.

[Prior Art] In recent years, immunochemical assay methods for measuring various proteins, hormones, etc. that act on living organisms with high sensitivity, and immunohistochemical staining methods for visualizing arbitrary tissues of living organisms, have become popular in clinical examinations. It is gaining importance in various fields. EIA is widely used as one of the immunochemical measurement methods. This measurement method is a method in which an antigen or antibody is labeled with an enzyme, this is reacted with a substrate, and the target protein, hormone, etc. is quantified based on the amount of the compound synthesized by the enzyme.

On the other hand, in immunohistochemical staining, antibodies that specifically bind to proteins present in a particular tissue are labeled with an enzyme using another antibody, and the color is developed by reaction with a substrate. This is a method to prove the localization of components.

A typical enzyme used in these methods is peroxidase extracted from horseradish or the like, which oxidizes a substrate with hydrogen peroxide. This enzyme has a relatively small molecular weight;
Because the reaction is fast, highly sensitive measurements and staining are possible in a short period of time.

[Problems to be solved by the invention] However, peroxidase binds to other components and
Regardless of whether it is in diluted form or not, it has extremely poor storage stability in a diluted solution state, making it difficult to distribute as a reagent product, and its usage is low compared to its usefulness.

As a countermeasure to this problem, efforts have been made to stabilize animal serum by adding it to a peroxidase solution, but this results in inactivation due to hemin interaction with the hemin-binding protein present in serum, making it unsuitable for long-term storage. It was something.

Therefore, the present invention was completed with the aim of providing a peroxidase solution with extremely high storage stability.

[Means for Solving the Problems] The present invention employs the following configuration as a means for solving the above problems.

That is, the gist of the present invention is a stabilized peroxidase solution, characterized in that the solution of peroxidase or peroxidase conjugate contains: serum protein and one or more parahydroxyphenyl carboxylic acids. do.

Here, peroxidase refers to hydrogen peroxide in water.

Enzymes that catalyze the oxidation of various substances are used as preservatives, and include those extracted from horseradish, milk, yeast, white blood cells, red blood cells, etc. In particular, horseradish peroxidase exhibits the stabilizing effect of the present invention. Show noticeably.

Furthermore, the enzyme can be used not only as a single enzyme as extracted, but also as a peroxidase conjugate that has been previously bound to an antibody or the like. For example, it is a conjugate of rabbit or goat peroxidase and peroxidase resulting from an antigen-antibody reaction.

Serum proteins refer to proteins contained in serum such as serum albumin and serum globulin of various animals.

Parahydroxyphenylcarboxylic acid has the formula, for example, where R is a divalent hydrocarbon group, such as CH2, C
H2-CH2, CH2-CH2-CH2, CHa-CH
-CH2, CH-CH, CH-CH-CH2, CHs-
Examples include C-CH.

Among the compounds represented by the above formula, barahydroxyphenylacetic acid, 3-parahydroxyphenylcarboxylic acid and 2-barahydroxyphenylcarboxylic acid,
This is a more preferred compound in terms of peroxidase stability.

The above-mentioned components are usually dispersed in a medium, and water is usually used as the medium, and buffering salts and the like are dissolved as necessary.

The concentration of parahydroxyphenylcarboxylic acid is highly stable at a concentration of 101 (] to 1 g per 1 solution, and within this range, 500
+ac+ to 10 is particularly stable.

[Function] Although the function of the present invention has not yet been clarified, it is likely to be as follows.

As mentioned above, peroxidase interacts with hemin with serum proteins, causing inactivation.

However, due to the presence of parahydroxyphenylcarboxylic acid, it is thought that this acts on the site of hemin interaction in serum proteins.

As a result, it is thought that the hemin interaction between peroxidase and serum proteins is disrupted, and all of the stabilizing effects of serum proteins are brought out.

[Effects of the Invention] As described above, in the present invention, parahydroxyphenylcarboxylic acid is added in addition to peroxidase or peroxidase conjugate and serum protein.

Therefore, peroxidase or peroxidase conjugate is extremely stable and can be stored for a long period of time, making it possible to provide reagents of stable and constant quality for various measurements in the fields of medicine, biology, etc., and contributing to accurate diagnosis.

[Example] A peroxidase label corresponding to a concentration of peroxidase (extracted from horseradish) of 0.2 mo/ml was prepared using a peroxidase label 1 10a, 10a, 1 (D), 2 serum albumin (cohn fraction) and 0.25g
/i "Thimerosal" (Th1lerO3a1) (Maruishi Pharmaceutical: preservative) and 0.01 moA/Ω sodium phosphate buffer (pH -8.0) containing 0.1510 mA/m sodium chloride. 200x, 400x, 8
The peroxidase-labeled anti-porcine insulin solution was diluted 00 times and 1600 times to 41 mg. 0.01 each to this peroxidase-labeled anti-porcine insulin solution.
After adding 0/41°0.1o/n, 1 g/n of rose hydroxyphenyl 10 pionic acid (hereinafter expressed as HPPA) or rose hydroxyphenyl acetic acid m<a (hereinafter expressed as HPAA), 0.5 mg~01/n I)H was adjusted to 7.0 mg to 9 mg with di-sodium phosphate solution. These peroxidase-labeled anti-porcine insulin solutions were
Stored at 8°C or 37°C. Zarani HPPA or HPA
To examine the stabilizing effect of A, samples without both reagents were similarly stored at 4-8°C or 37°C, and further stored frozen (-20°C) as a control for peroxidase activity.

After a specific period of time had elapsed, the activity of the peroxidase-labeled anti-porcine insulin solution was measured using the following two methods.

(1) Peroxidase activity stabilization recognition test The stored peroxidase-labeled anti-porcine insulin solution was
,5 citric acid-phosphorus Wi disodium 0.1lo1/
7nmoi/i of H2021 and 160IIIl in A
A solution to which 01/d of glucose was added (hereinafter referred to as I for substrate dissolution)
0.6 mm 0 A,
/fL of orthophenylenediamine solution 0. mg~1l
After incubating for 5 minutes at room temperature, 2N
The reaction was stopped by adding 100 μl of −Hz Soa, and its absorbance (A) was measured. The results are shown in Tables 1 and 2. Table 1 shows those stored at 4°C, and Table 2 shows those stored at 37°C. Here the numbers are HPPA10/
The specific activity is calculated with respect to the activity of the added product. In addition, Fig. 1 shows graphs of changes in specific activity over time for samples stored at 4°C at a dilution rate of 1600, and Fig. 2 for samples stored at 37°C at the same dilution rate.

In this way, those without adding HPPA or HPAA are 1
In contrast, the activity of this example is maintained for a long period of time, whereas the activity is almost lost within a week.

(2) Activity stabilization confirmation test by analytical test Pig insulin 100μL/l i of porcine insulin solution was added to a microplate (Dynatic Imron ■) sensitized with guinea pig IoG at a concentration of 35μQ/Shortate.
After reacting at room temperature for 1 hour and washing, 100 μl each of the stored peroxidase-labeled porcine insulin solution was added and reacted for 1 hour at room temperature. For substrate dissolution after washing
After adding 100μ of a solution of 3m+ofL/u of orthophenylenediamine dissolved in a buffer solution and reacting for 30 minutes at room temperature, the reaction was stopped by adding 100μ of a 2N-Hz SOa solution, and its absorbance (Ahu2 ) was measured.

In order to measure the stabilizing effect of HPPA or HPAA, the results were compared with those stored frozen (-20°C).
It is shown in Table and Table 4. Table 3 shows those stored at 4°C, and Table 4 shows those stored at 37°C.

In addition, graphs of specific activities compared to those stored frozen are shown in FIGS. 3 to 10. Figures 3 to 6 represent samples stored at 4°C, with Figure 3 diluted 200 times and Figure 4 diluted 400 times.
Figure 5 shows the 800-fold dilution, and Figure 6 shows the 1600-fold dilution. In addition, Figures 7 to 10 show samples stored at 37°C, with Figure 7 showing 200-fold dilution and Figure 8 showing 400-fold dilution.
FIG. 9 shows the 800-fold dilution, and FIG. 10 shows the 1600-fold dilution.

In this way, those without adding HPPA or HPAA are 1
On day 0, it hardly shows any activity, whereas
The samples of this example can be used for analytical tests even after that, and especially those stored at 4°C can be used for precise analysis even after 60 days.

A similar test was conducted using peroxidase itself, and the results showed that the peroxidase had extremely high stability, similar to the case with Habo.

[Brief explanation of drawings]

Figure 1 shows 160 of the storage stability confirmation tests at 4°C.
Graph showing oral changes in specific activity for 0x dilution; Figure 2 is a similar graph at 37°C; Figures 3 to 6 are 4
3 is a graph showing a 200-fold dilution, FIG. 4 is a 400-fold dilution, and FIG. 5 is an 800-fold dilution. , FIG. 6 shows a graph of a 1600-fold dilution, and FIGS. 7 to 10 are graphs showing changes over time in an activity stabilization confirmation test by an analytical test at 37°C,
FIG. 7 shows a graph for 200 times dilution, FIG. 8 shows a graph for 400 times dilution, FIG. 9 shows a graph for 800 times dilution, and FIG. 10 shows a graph for 1600 times dilution.

Claims (1)

[Scope of Claims] 1. A stabilized peroxidase solution comprising: a serum protein; and one or more parahydroxyphenyl carboxylic acids in a solution of peroxidase or peroxidase conjugate. 2. The stabilized peroxidase solution according to claim 1, wherein peroxidase as a reaction raw material for peroxidase or peroxidase conjugate is extracted from horseradish. 3. The stabilized peroxidase solution according to claim 1 or 2, wherein the peroxidase conjugate is a conjugate of peroxidase and an antibody using the peroxidase as an antigen. 4. The stabilized peroxidase solution according to any one of claims 1 to 3, wherein 1 liter of solution contains 10 mg to 1 g of parahydroxyphenylcarboxylic acid. 5. The stabilized peroxidase solution according to any one of claims 1 to 4, wherein the parahydroxyphenylcarboxylic acid is parahydroxyphenylacetic acid and/or parahydroxyphenylpropionic acid.