JPH0357751B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to stable enzyme compositions. More specifically, the present invention relates to the stabilization of peroxidase enzymes, either alone or in combination with peroxidase and an immunologically active substance. Peroxidase has come to be used for a variety of purposes in recent years, particularly as a diagnostic reagent in clinical laboratory departments, and with the advancement of enzyme immunoassay, the importance of peroxidase as a labeling enzyme for this assay has increased. is increasing. However, peroxidase is extremely unstable in a low concentration aqueous solution state, even when bound to other components (e.g., immunoactive substances) or in its free state, and it is difficult to maintain the enzymatic activity necessary for measurement. Very difficult. Therefore, in order to maintain peroxidase activity for a long period of time, it is generally recommended to add 8-anilino-1-naphthalenesulfonic acid as shown in Japanese Patent Publication No. 58-5668, or to use methods such as freeze-drying. However, it has been revealed that even in the case of freeze-drying, the enzymatic activity of peroxidase is considerably reduced during drying. For these reasons, peroxidase has to be used in a very limited manner. As a result of our research to develop a method for stabilizing peroxidase that does not have the above-mentioned drawbacks, the present inventors discovered that peroxidase should be combined with serum proteins and polyalkylene glycol, and have already proposed it (Japanese Patent Application No. 136126/1982). . However, although peroxidase is stable for a long time in the above composition,
A precipitate forms during long-term storage. The present inventors have arrived at the present invention as a result of various studies on stabilizers that do not form precipitates during long-term storage. That is, the present invention is a stable enzyme composition containing peroxidase, serum proteins whose lipid components have been extracted and removed using a halogenated hydrocarbon, and polyalkylene glycol. The stabilizers of the invention in particular significantly stabilize peroxidase in solution, with almost no precipitate formation observed during long-term storage. Examples of the polyalkylene glycol used in the present invention include polyethylene glycol, polypropylene glycol, ethylene oxide/propylene oxide copolymer, polytetramethylene glycol, and the like. The molecular weight of the polyalkylene glycol used in the present invention is about 1000 to
20,000, preferably about 4,000 to 6,000.
The amount added is about 1 to 10% by weight to volume (W/V), preferably 3 to 6% by weight to volume (W/V), based on the aqueous composition. 10 Weight to capacity (W/
Addition exceeding V)% causes precipitation of serum proteins and is not preferred. In addition, various types of serum proteins include bovine albumin, human albumin, bovine serum, human serum, horse serum, and rabbit serum. The halogenated hydrocarbon used in the present invention is a hydrocarbon having 2 to 5 carbon atoms and containing hydrogen atoms.
Those in which 50% or more are substituted with halogen atoms are preferred. Halogen atoms include chlorine, bromine,
Examples include iodine. Preferred halogenated hydrocarbons include 1,1,2-trichloro-1,2,2
-Trifluoroethane, perchrene, trichrene, etc. are exemplified. When the above serum protein is in powder form, it is made into an aqueous solution of 1 to 10% by weight/volume, and 1 volume of serum protein is mixed with a halogenated hydrocarbon, such as 1,1,2-trichlor 1,2,2-trifluoroethane 1,5 Mix 5 volumes and stir well to extract lipid components in serum proteins. After centrifugation at 3000 rpm for 10 minutes, the supernatant portion of serum proteins is preferably used in an amount of 1 to 50% by volume based on the aqueous composition. Polyethylene glycol has been used as a precipitant for immune complexes in immunoassays using radioisotopes, but there have been no examples of its use for stabilizing enzymes, particularly peroxidase. The stabilizing effect of peroxidase in the composition of the present invention is thought to be due to the action of serum proteins and polyalkylene glycol treated with halogenated hydrocarbons on peroxidase, thereby maintaining its special structure. Peroxidase may be in a free state or in a bound state bound to an immunologically active substance.
Further, 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 the antibody, an antibody (e.g., anti-insulin antibody, anti-α-fetoprotein, etc.) obtained by immunizing a mammal such as a goat or rabbit with the above-mentioned hapten or antigen by a conventionally known method is used. It will be done. 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.; Imminochemistry, 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 immunologically active substance (Nakane, PK, &
Kawaoi, A.; J.Histochem.cytochem, 22 :
1084-1091, [1974]), etc., can be used to create a covalent bond. The enzymatic activity of free or covalently bound peroxidase is determined by combining hydrogen peroxide with a suitable coloring agent, e.g. o-aminophenol,
It is possible to quantify the enzyme activity by a colorimetric method using 4-aminoantipyrine, phenol, o-phenylenediamine, etc. 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. As a buffer,
Phosphate buffer, Tris buffer, etc. are used, 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. Advantageous test reagents using the stabilized peroxidase compositions according to the invention include immunodiagnostic reagents, especially enzyme immunoassays. Further, it can be used as a diagnostic agent other than enzyme immunoassay, for example, as a reagent for glucose oxidase method for quantifying glucose. The present invention will be explained below with reference to Examples.
The present invention is not limited to these examples. Example 1 1.5 volumes of 1,1,2-trichloro-1,2,2-trifluoroethane was mixed with 1 volume of rabbit serum, stirred, and centrifuged at 3000 rpm. Rabbit serum 10% by volume, horseradish peroxidase (manufactured by Toyobo Co., Ltd., grade 1-C) and polyethylene glycol #4000 (manufactured by Hanui Chemical Co., Ltd.)
It was dissolved in 0.01M phosphate buffer (hereinafter abbreviated as 0.01MPB) containing 5% weight to volume (W/V). Control Example 1 contains the above rabbit serum but does not contain polyethylene glycol #4000, and Control Example 2 contains rabbit serum containing polyethylene glycol #4000 but without any treatment. It was subjected to a sex test. Further, the concentration of horseradish peroxidase was 1.0 μg/in each case. Each solution was passed through a 0.22 μm sterile filter;
It was placed in 5 ml sterile bottles and stored at 4°C and 40°C. Enzyme stability was examined on days 0, 7, 14, 21, and 28. Stability was expressed as a percentage of the enzyme activity when stored at 40°C relative to the activity when stored at 4°C. In addition, the stability of the composition liquid is 2 months, 4 months, 6 months, and 9 months at 4℃.
The samples were stored for 12 months and the formation of precipitates was visually determined. In addition, the enzyme activity is hydrogen peroxide 0.02% and o-phenylenediamine dihydrochloride (hereinafter abbreviated as OPD).
Add 50μ of each of the above solutions to 0.5ml of 0.1M citrate phosphate buffer at pH 5.0 containing 3mg/ml of
After leaving at room temperature for 30 minutes, add 2.0 ml of N sulfuric acid and adjust to 492 nm.
The absorbance was measured at The results are shown in Tables 1 and 2.

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[Table] Judgment -: No precipitate formed.
±: Slight formation of precipitate is observed.
+: Yes.
Example 2 Polyethylene glycol #1000 (manufactured by Hanui Chemical Co., Ltd.)
The stability was investigated using the method described in Example 1. The concentration of polyethylene glycol #1000 is 8
It was expressed as weight to volume (W/V)%. The result is the third
Table 4 shows the results. In addition, Control Examples 1 and 2 are Example 1
The mixture was prepared in the same manner as in Control Examples 1 and 2.

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[Table] Judgment -: No precipitate formed.
±: Slight formation of precipitate is observed.
+: Yes.
Example 3 Periodate oxidation method (NaKane, PK &
Kawaoi, A.: J. Histochem. Cytochem. 22 , 1840.
-1091, [1974]) for rabbit immunoglobulin G
Horseradish peroxidase bound to rabbit IgG (hereinafter referred to as rabbit IgG) was passed through a gel using Sephadex G-200 to remove free peroxidase, and the bound peroxidase was not passed through a gel, and the free peroxidase and bound peroxidase coexisted. Each case was investigated for stability as well as precipitate formation. That is, 0.05M phosphate buffered saline (hereinafter referred to as 0.05MPBS) containing 4% by weight (W/V) of polyethylene glycol #6000 (manufactured by Hanui Chemical Co., Ltd.) and 10% by volume of domestic rabbit serum treated in the same manner as in Example 1. (abbreviated as ) and control example 1
Using the procedure described in Example 1, a control example 2 in which only polyethylene glycol #6000 was removed from the above composition, and a control example 2 in which untreated rabbit serum was used in place of the rabbit serum in the above composition. The stability of peroxidase in solution and the presence or absence of precipitate formation were investigated. The results are shown in Tables 5 and 6.

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[Table] Example 4 Domestic rabbit serum and 10% weight to volume (W/V) bovine serum albumin (Fraction V) were mixed at 1,
Using 1,2-trichloro-1,2,2-trifluoroethane, 1,1,
2-Trichloro-1,2,2-trifluoroethane
The mixture was mixed and stirred in a volume of 1.5, centrifuged at 3000 rpm, and the supernatant serum protein solution was collected. Using the serum protein solution thus collected, the following peroxidase solution was prepared. Horseradish peroxidase and polypropylene glycol bound to the rabbit IgG used in Example 3, diol type average molecular weight 2000 (manufactured by Wako Pure Chemical Industries, Ltd.) 5% by weight to volume (W/V), and 1,1 , 2% by volume of rabbit serum treated with trichloro-1,2,2-trifluoroethane and 1% by weight to volume (W/V) of bovine serum albumin.
Dissolved in 0.05MPBS. Control example 1 contains the above rabbit serum but does not contain polypropylene glycol; control example 2 shows the stability of peroxidase when untreated rabbit serum and bovine serum albumin are used instead of the treated serum protein solution. Tables 7 and 8 show the results of examining the presence or absence of precipitate formation at 4°C using the method of Example 1.

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[Table] Example 5 Mix 1 part domestic rabbit serum with 2 volumes of tetrachlorethylene or 2 volumes of trichlorethylene.
Stirred and centrifuged at 3000 rpm, the supernatant contained 10% by volume of rabbit serum, horseradish peroxidase (manufactured by Toyobo Co., Ltd., grade 1-C), and polyethylene glycol #4000 (manufactured by Hanui Chemical Co., Ltd.) 5
It was dissolved in 0.01M phosphate buffered saline (hereinafter abbreviated as 0.01MPBS) containing % weight to volume (W/V). Control example 1 does not contain polyethylene glycol #4000, control example 2 contains untreated rabbit serum, and control example 3 does not contain polyethylene glycol #4000 treated rabbit serum. Then, it was subjected to the same enzyme stabilization test as in Example 1. The results are shown in Tables 9 and 10.

【table】

[Table] Judgment: - No formation of precipitate
± slight formation of precipitate
+ Formation of precipitate is observed.

Claims (1)

[Scope of Claims] 1. A stable enzyme composition containing peroxidase, serum proteins extracted with a halogenated hydrocarbon, and polyalkylene glycol. 2. The stable enzyme composition according to claim 1, wherein the peroxidase is a free peroxidase and/or a bound peroxidase. 3. The stable enzyme composition according to claim 2, wherein the bound peroxidase is a conjugate of peroxidase and an immunoactive substance.