JPH06121683A - Stabilized peroxidase composition - Google Patents

Abstract

PURPOSE:To provide the subject composition highly excellent in peroxidase stability. CONSTITUTION:The objective composition comprising (A) sodium chloride, (B) bovine serum albumin, (C) a phosphate buffer solution, (D) horseradish peroxidase and (E) 2-(4-hydroxyphenyl)ethylamine hydrochloride. This composition has a horseradish peroxidase residual activity of 84% even after a lapse of 48 days at 37 deg.C.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a stabilized peroxidase composition which can be used in immunoassays.

[0002]

Description of the Prior Art In recent years, enzyme immunoassays and immunohistochemical staining methods have been used to detect various proteins in body fluids and living tissues.
As a method for detecting hormones and the like with high sensitivity, it has become important in fields such as clinical examinations and medical research. As an enzyme widely used in these methods, there is a peroxidase capable of oxidizing a chromogen in the presence of hydrogen acceptor hydrogen peroxide to produce a dye. It is preferable that the activity of peroxidase is not likely to decrease even if it is stored for a long period of time in practical use. It is known that the activity is decreased by long-term storage in.

Therefore, phenol (JP-A-60-192260), serum protein and halogenated alkylphenol (JP-B-64-7759) are used as stabilizers for stable storage of peroxidase for a long period of time.
Gazette), para-hydroxyphenylcarboxylic acid (Japanese Patent Publication No. 63-24677), alkoxyphenol (Japanese Patent Laid-Open No. 64-63380), a group or amide having a Hammett sigma value of -0.20 or less or +0.24 or more. Phenols having a group (JP-A-2-42982) are disclosed. However, even compositions containing these stabilizers are not sufficiently stable due to long-term storage of peroxidase.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a stabilized peroxidase composition having excellent peroxidase stability.

[0005]

As a result of studies to solve the above-mentioned problems, the present inventors have found that a phenol derivative significantly enhances the stability of peroxidase, and completed the present invention. . That is, the present invention provides the following formula (I):

[0006]

[Chemical 2]

(In the formula, X represents a hydroxyl group or an amino group,
n represents 1 or 2. The present invention relates to a stabilized peroxidase composition containing a phenol derivative represented by (4) and peroxidase.

The present invention will be described in detail below. The stabilized peroxidase composition of the present invention has remarkable peroxidase stability and contains at least one peroxidase and at least one phenol derivative (I). As the phenol derivative (I), −
The substitution position of (CH ₂ ) _n —X is not particularly limited, but preferably p-hydroxybenzyl alcohol, p-hydroxybenzylamine or its hydrochloride, 2- (4-hydroxyphenyl) ethyl alcohol, 2- (4-). Examples thereof include hydroxyphenyl) ethylamine and its hydrochloride; and more preferred are p-hydroxybenzyl alcohol, 2- (4-hydroxyphenyl) ethyl alcohol, 2- (4-hydroxyphenyl) ethylamine and its hydrochloride. Good.

Peroxidase is an enzyme that catalyzes the oxidation reaction of various substrates by using hydrogen peroxide as a hydrogen acceptor. For example, there are those obtained from horseradish, milk, yeast, leukocytes, erythrocytes, etc .; Those derived from wasabi are preferred.

As the phenol derivative (I) and peroxidase, commercially available products can be used, but those which are difficult to obtain can be obtained by a conventional method. Peroxidase can be used alone or in combination with a carrier. The carrier may be an antibody, an antigen (eg, protein, sugar,
Lipids, nucleic acids, haptens, etc.). Examples of the antibody include monoclonal antibody, polyclonal antibody, Fab of monoclonal antibody, Fab of polyclonal antibody, Fab ′ of monoclonal antibody, Fab ′ of polyclonal antibody, and the like;
Fab 'of a monoclonal antibody is preferred, and more preferably, anti-morphine monoclonal antibody (JP-A-63-23).
Fab 'of MO5) described in Japanese Patent No. 4826 is preferable.

The bond between the peroxidase and the carrier is preferably a covalent bond. For example, a method of combining an amino group with an amino group using glutaraldehyde as a cross-linking agent, an aldehyde produced by cleaving the sugar chain of peroxidase with periodic acid. Examples of the method include a method of binding a group to an amino group, a method of binding a thiol group to an amino group with sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate as a crosslinking agent. Preferably, a method of binding a thiol group and an amino group using sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate as a crosslinking agent.

The composition of the present invention includes, in addition to those described above, a buffer solution, physiological saline, serum, biopolymer protein,
Sugars, surfactants, preservatives, etc. can be added.

The compositions of the phenol derivative (I) and peroxidase in the composition of the present invention are as follows. In the composition solution, the phenol derivative (I) is 0.0
1-5% (w / v), preferably 0.1-1% (w /
v); peroxidase from 0.01 μg / ml
It is 10 mg / ml. The composition powder is the composition solution obtained by removing the solvent by freeze-drying or the like.

Examples of the buffer solution include Tris buffer solution,
Examples thereof include a phosphate buffer solution, an acetate buffer solution, a carbonate buffer solution; the composition solution has a pH of 4 to 9, and preferably a pH of 6 to 8. Examples of the biopolymer proteins include ovalbumin, rabbit, horse, bovine, human-derived serum proteins, serum albumin and casein, but serum albumin is preferable, and bovine serum albumin is more preferable; The proportion of this composition is 0.1-10% (w / w), preferably 0.1-5%
(W / w), more preferably 0.5 to 2% (w / w)
Is good.

Examples of the serum include those derived from rabbits, horses, cows and humans; the proportion of this composition is preferably 1 to 50% (w / w). Examples of saccharides include sucrose and lactose; the proportion of this composition is 0.1 to 10% (w
/ W), preferably 1 to 5% (w / w). Examples of the surfactant include Tween 20 and the like; the ratio of this composition is 0.01 to 0.1 (w /
w) is good. Examples of preservatives include caisson CG
(Made by Rohm and Haas Co.), thimerosal, sodium azide, etc .; the ratio of this composition is 0.001-1 (w / w), preferably 0.01-.
0.1 (w / w) is preferable.

The thus-obtained stabilized peroxidase composition can be used by combining the above-mentioned compositions individually or in an appropriate combination according to the purpose. . INDUSTRIAL APPLICABILITY The stabilized peroxidase composition of the present invention can stably retain the peroxidase activity even after long-term storage at −60 to 40 ° C. regardless of whether or not the peroxidase is bound to the carrier. That is, even if the stabilized peroxidase composition of the present invention is stored at 4 to 37 ° C. for 50 days,
The carrier having a residual activity of not less than%; the carrier to which peroxidase binds is preferably an anti-morphine monoclonal antibody (MO5 described in JP-A-63-234826).

[0017]

EXAMPLES The present invention will be specifically described below with reference to examples. It should be noted that these examples do not limit the scope of the present invention. Example 1 A horseradish peroxidase (Boehringer Co.) was added to 0.01 M phosphate buffer (pH 7.3) containing 0.14 M sodium chloride, 1% bovine serum albumin and 0.1% Caisson CG (Rohm and Haas). Made) is 100n
It was added so that the concentration would be g / ml, and 2- (4-hydroxyphenyl) ethylamine hydrochloride was dissolved so as to be 0.1%. This peroxidase composition was placed at 37 ° C.
25 μl at the time described in Table 1 (0 to 49 days)
Each of them is taken out and added to 1 ml of a substrate solution [0.05M citrate buffer solution (pH 5.0) containing 0.014% hydrogen peroxide and 0.01% o-phenylenediamine], and the mixture is allowed to stand at room temperature for 3.5 minutes. It was made to react. After that, 0.5m of 2N sulfuric acid
The reaction was stopped by adding 1 and the absorbance at 492 nm was measured. The results are shown in Table 1 as a relative value with the residual peroxidase activity when the absorbance immediately after the preparation was shown as 100%.

[0018]

[Table 1]

Example 2 Horseradish peroxidase was added to 0.01M phosphate buffer (pH 7.3) containing 0.14M sodium chloride, 1% bovine serum albumin and 0.1% caisson CG (Rohm and Haas). Fa of anti-morphine antibody labeled with
b ′ was added at 20 μg / ml, and the phenol derivative (I) shown in Table 2 was dissolved so as to be 0.1%. The peroxidase composition was kept at 37 ° C., and 50 μl each was withdrawn at the time (0 to 22 days) described in Table 2 to prepare a morphine standard solution (0.046 ng / ml).
The absorbance of was measured. The results are shown in Table 2 as relative values with the residual peroxidase activity when the absorbance immediately after the preparation was shown as 100%.

[0020]

[Table 2]

The enzyme-labeled Fab 'was prepared by a conventional method using an anti-morphine antibody (MO5, which is a monoclonal antibody described in JP-A-63-234826) [eg, enzyme immunoassay, 3rd edition, 83]. To the method described on page 92 (Medical Shoin)], and after obtaining Fab ′ of this antibody, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (manufactured by Pierce) Was combined with horseradish peroxidase (produced by Boehringer) as a cross-linking agent.

Further, the measurement of the concentration of morphine is conducted in Japanese Patent Application No. 3-
According to the enzyme immunoassay method described in 333843, the measurement was performed as follows. After adding 50 μl of morphine standard solution to a microtiter plate (manufactured by Nunc) on which an anti-idiotype antibody was immobilized, 50 μl of the above-mentioned peroxidase composition was added and stirred, and the mixture was allowed to stand at room temperature for 1 hour.
Next, PBS containing 0.01% Tween 20 [0.
14M sodium chloride, 0.01M phosphate buffer (pH
7.3)], washed with 20 mg of o-phenylenediamine and 10 μl of 35% H ₂ O ₂ in 25 ml of a citrate buffer solution (pH 5.0), and 100 μl of the solution was poured into the well and shielded from light at room temperature. After standing for 15 minutes, 50 μl of 2H ₂ SO ₄ was added to stop the reaction. It was carried out by measuring the absorbance of the morphine standard solution (0.046 ng / ml) of this reaction solution at 492 nm using a microplate photometer.

Example 3 The phenol compound (I) in Example 1 was added to 2- (4-
The same procedure as in Example 1 was carried out except that hydroxyphenyl) ethylamine hydrochloride was dissolved so as to have a concentration (0 to 1.0%) as shown in Table 3. The results are shown in Table 3.

[0024]

[Table 3]

[0025]

INDUSTRIAL APPLICABILITY The novel stabilized peroxidase composition of the present invention has very excellent peroxidase stability.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kokatsu Ito 10 1978, Kogushi, Ube City, Ube, Yamaguchi Prefecture Ube Industries Co., Ltd.

Claims (1)

[Claims] 1. The following formula: (In the formula, X represents a hydroxyl group or an amino group, and n is 1 or 2
Represents ] The stabilized peroxidase composition containing the phenol derivative shown by these, and peroxidase.