JPS647759B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a stable peroxidase composition, and particularly to the production and storage of labeled enzymes and peroxidases used in enzyme immunoassays. (Prior Art) In order to stabilize free peroxidase or peroxidase bound to other components (e.g., immunologically active substances) for long periods of time, stabilization is carried out using methods such as freeze-drying. , 8-anilino as described in Japanese Patent Publication No. 58-5668
Stabilization methods have been described in which an effective amount of 1-naphthalenesulfonic acid is added or, as described in JP-A-58-23786, an effective amount of 4-amino-antipyrine is added. (Problems to be Solved by the Invention) However, if substances such as 8-anilino-1-naphthalenesulfonic acid and 4-aminoantipyrine are not used, free peroxidase or other components (e.g., immunoactive substances) may be bound to it. It is difficult to stabilize peroxidase for a long period of time, and it is extremely unstable, especially in a low concentration aqueous solution state, and maintaining the enzyme activity necessary for measurement is of important significance in enzyme immunoassay. The present inventors have arrived at the present invention as a result of intensive research to develop a method for stabilizing peroxidase that does not rely on the above-mentioned 8-anilino-1-naphthalenesulfonic acid or 4-aminoantipyrine. (Means for Solving the Problems) The present invention is a stable peroxidase composition characterized in that it contains peroxidase, a medium containing serum or serum proteins, and a halogenated alkylphenol, and does not contain hydrogen peroxide. The molecular formula of the halogenated alkylphenol used in the present invention has the structure shown below. (In the formula, X is a halogen atom, R has 1 to 1 carbon atoms,
10 alkyl groups, m=1-4, n=1-4. ) Examples of halogenated alkylphenols include 4-chloro-m-cresol, 4-chloro-o
-cresol, 4-bromo-m-cresol,
Examples include 2-ethyl-4-chlorophenol. The amount of the halogenated alkylphenol added is about 0.01 to 5 weight to volume (W/
V)%, preferably 0.02 to 3 weight to volume (W/
V)%. Furthermore, even if an amount is added that exceeds the solubility, a precipitate will be generated in the aqueous medium, so it cannot be used. Examples of serum include rabbit serum, human serum, horse serum, fetal bovine serum, and bovine serum. Serum proteins include bovine serum albumin, rabbit serum albumin, and many others. The amount of serum or serum protein added is 5 to 50% by volume based on the total composition as serum, and 0.1 to 10% by weight (W/V) based on the total composition for dry fine powder of serum protein. It is preferable. Examples of the medium containing serum or serum proteins include water or a buffer solution. Although the stabilizing effect of halogenated alkylphenol on peroxidase in the composition of the present invention is unknown, it is thought that serum proteins and halogenated alkylphenol act on peroxidase to maintain its special structure. Peroxidase may be in a free state or bound to an immunoactive substance. Alternatively, it may be a mixture of a free state and a bound state. Immunologically active substances include haptens, multivalent antigens,
Examples include antibodies. Haptens include thyroxine, histamine, digoxin, adrenaline,
Prostaglandins, steroid hormones such as progesterone, estradiol, testosterone, estriol, etc., and antibiotics such as penicillin are used. As multivalent antigens, hormones such as growth hormone, proteins such as albumin, globulin, α-fetoprotein, etc., and various bacteria and viruses such as streptococcus, hepatitis virus, rubella virus, etc. are used. As an antibody, by a conventionally known method,
Antibodies (eg, anti-insulin antibodies, anti-α-fetoprotein antibodies, etc.) obtained by immunizing mammals such as goats and rabbits with the above hapten or antigen are used. Antibodies produced by the hybridoma method can also be used in the same manner. In addition, the bonding form of the immunoactive substance and the peroxidase conjugate includes covalent bonding, and various known methods such as a method of covalently bonding the amino group of the immunoactive substance and the amino group of peroxidase using glutaraldehyde as a crosslinking agent can be mentioned. (Avrameas, S.; Immunochemistry, 6 :43-
52, [1969]) and a method in which sugar chains contained in peroxidase are oxidatively cleaved with periodic acid, an aldehyde group is introduced, and then a Schiff base is created and bonded to the amino group of an immunoactive substance (Nakane, PK, & Kawaoi,
A.; J. Histochem. Cytochem, 22 : 1084-1091,
[1974] etc. can be used to create covalent bonds. The enzymatic activity of free peroxidase or peroxidase to which an immunoactive substance is covalently bound can be determined using hydrogen peroxide and a suitable coloring agent, such as o-aminophenol, 4-aminoantipyrine and phenol, or o-phenylenediamine. It is possible to quantify the enzyme activity by color methods. Further, the composition of the present invention may contain an optional component having a buffering effect, or may contain sodium chloride at a physiological saline concentration. Phosphate buffer, Tris buffer, etc. are used as the buffer, and an aqueous solution having a pH of 6 to 8 is preferable. When preparing an aqueous composition, the order of addition of each substance is not particularly limited. It is also possible to add and use antibiotics such as penicillin G, ampicillin, chloramphenicol, tetracycline, and amphotericin B. Advantageous test reagents using the stabilized peroxidase compositions according to the invention include immunodiagnostic reagents, especially enzyme immunoassays. Furthermore, it can be used as a diagnostic agent other than enzyme immunoassay, for example, as a reagent for glucose oxidase method for quantifying glucose. (Examples) The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples. Example 1 Horseradish peroxidase (manufactured by Toyobo Co., Ltd., grade 1-C) was mixed with 10% by volume of domestic rabbit serum and 4
- Chlorometacresol 0.02 weight to volume (W/
V) 0.01M phosphate buffered saline containing %
(abbreviated as 0.01M PBS) dissolved in PH6.0. As a control, a sample containing no 4-chlorometacresol and a sample containing no rabbit serum were prepared and subjected to the following enzyme stability test. Further, the concentration of horseradish peroxidase was 1.0 μg/each. Each solution was passed through a 0.22 μm sterile filter, placed in 5 ml sterile bottles, and stored at 4°C and 40°C. 0, 7 days,
Enzyme stability was examined for 14 days. Stability was expressed as a percentage of the enzyme activity when stored at 40°C relative to the activity when stored at 4°C. Enzyme activity measurements were performed using 0.5% citrate phosphate 0.1M buffer at pH 5.0 containing 0.02% by weight to volume (W/V) hydrogen peroxide and 3mg/ml o-phenylenediamine dihydrochloride (hereinafter referred to as OPD). ml of the above solution, react for 30 minutes at room temperature, and add 1N sulfuric acid.
After stopping the reaction by adding 2.0 ml, the absorbance was determined by measuring the absorbance at 492 nm. The results are shown in Table 1.

[Table] Example 2 The stabilizing effect of horseradish peroxidase was examined in the same manner as in Example 1, except that 4-chlorometacresol in Example 1 was replaced with a halogenated alkylphenol shown in Table 2. The results are shown in Table 2.

【table】

[Table] Example 3 Periodate oxidation method [Nakane, PK, &
Kawaoi.A.; J.Histochem.Cytochem. 22 , 1084−
1091, (1974)] for rabbit immunoglobulin G.
(hereinafter abbreviated as rabbit IgG) and horseradish peroxidase is gel-filtered using Cephadex G-200 to remove free peroxidase, and the bound peroxidase coexists with free peroxidase and bound peroxidase without gel-filtering. For each of 4-chloro-m
- Contains cresol, 0.025% weight-to-volume (W/V) and rabbit serum 1%, bovine serum albumin 1% weight-to-volume (W/V) chloramphenicol, 0.1% weight-to-volume (W/V). The stability of peroxidase in solution was investigated using the procedure described in Example 1 when it was added to 0.02M phosphate buffered saline and when only 4-chloro-m-cresol was removed as a control example. The results are shown in Table 2.

[Table] (Effects of the Invention) According to the present invention, peroxidase can be stabilized at low concentrations for a long period of time.

Claims (1)

[Scope of Claims] 1. A stable peroxidase composition, characterized in that it contains peroxidase, a medium containing serum or serum proteins, and a halogenated alkylphenol, but does not contain hydrogen peroxide. 2. The stable peroxidase composition according to claim 1, wherein the peroxidase is peroxidase in a free state and/or peroxidase in a state in which an immunoactive substance is covalently bound.