JPH0214035B2 - - 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, stabilization is generally carried out using methods such as freeze-drying, but it is also clear that even in this case, the enzymatic activity of peroxidase is considerably reduced during freeze-drying. is being used. For these reasons, peroxidase has to be used in a very limited manner. The present inventors have arrived at the present invention as a result of intensive research aimed at developing a method for stabilizing peroxidase that does not have the above-mentioned drawbacks. That is, the present invention is a stable enzyme composition comprising peroxidase, serum protein, and polyalkylene glycol. The stabilizers of the invention particularly significantly stabilize peroxidase in solution. 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 of more than V)% is not preferred as it may cause precipitation of serum proteins. Further, various serum proteins include bovine albumin, human albumin, bovine serum, human serum, horse serum, etc., but rabbit serum is preferable.
The amount of serum protein added to the aqueous composition is preferably 5 to 30% by weight (W/V) in the case of a dry fine powder, and 5 to 50% by volume in the case of serum. Polyethylene glycol is used as a precipitant for immune complexes in immunoassays using radioisotopes, but there are no examples of its use for stabilizing enzymes, especially peroxidase. . The stabilizing effect of peroxidase in the composition of the present invention is thought to be due to serum proteins and polyalkylene glycol acting on peroxidase to maintain 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, 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.; 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 a covalent bond. The enzymatic activity of free or covalently bound peroxidase is determined by the addition of hydrogen peroxide and a suitable coloring agent, such as 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, 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 polyethylene glycol #4000 (manufactured by Hanui Chemical Co., Ltd.) 5 weight to volume (W/V)
% and rabbit serum at 10% by volume (hereinafter abbreviated as 0.01 MPB). Control Example 1, which did not contain polyethylene glycol #4000, and Control Example 2, which did not contain rabbit serum, were prepared and subjected to the following enzyme stability test. In addition, the concentration of horseradish peroxidase was 1.0μ, respectively.
It was g/. Each solution was passed through a 0.22μm sterilizer and 5ml
The samples were placed in sterilized 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. The enzyme activity was determined using 0.02% hydrogen peroxide and 3% o-phenylenediamine dihydrochloride (hereinafter abbreviated as OPD).
PH5.0 Citrate Phosphate containing mg/ml
Add 50μ of each of the above solutions to 0.5ml of 0.1M buffer,
After leaving it at room temperature for 30 minutes, add 2.0ml of 1N sulfuric acid and dilute to 492nm.
The absorbance was measured at The results are shown in Table 1.

[Table] 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 second result is
Shown in the table.

[Table] Example 3 The stability of polyethylene glycol #20000 (manufactured by Hanui Chemical Co., Ltd.) was investigated by the method described in Example 1. The concentration of polyethylene glycol #20000 is
2% by weight to volume, and the results are shown in Table 3.

[Table] Example 4 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. For each case, 0.05 M containing polyethylene glycol #6000 (manufactured by Hanui Chemical Co., Ltd.) 4% by weight to volume (W/V) and 10% by volume of rabbit serum.
In Example 1, when added to phosphate buffered saline (hereinafter abbreviated as 0.05MPBS), as Control Example 1, only polyethylene glycol #6000 was removed, and as Control Example 2, only rabbit serum was removed. The stability of peroxidase in solution was investigated using the procedure described. The results are shown in Table 4.

[Table] Example 5 Rabbit IgG-conjugated horseradish peroxidase described in Example 4 was mixed with polyethylene glycol #1000 described in Example 2, 0.05% containing 10% weight-to-volume (W/V) and 10% by volume rabbit serum. Dissolved in MPBS. Control example 1 does not contain polyethylene glycol #1000, control example 2 does not contain rabbit serum, and control example 3 uses polyethylene glycol #200 instead of polyethylene glycol #1000.
(manufactured by Hanui Chemical Co., Ltd.) containing 10% by weight to volume (W/V), the stability of peroxidase in the solution was investigated using the operating method described in Example 1. The measurement results are shown in Table 5.

[Table] Example 6 Horseradish peroxidase and human immunoglobulin G were combined by the method described in Example 4, and the bound peroxidase was separated by gel filtration using Sephadex G-200. This conjugated peroxidase was combined with polyethylene glycol #4000, containing 5% weight-to-volume (W/V) and bovine serum albumin 10% weight-to-volume (W/V).
Dissolved in 0.05MPBS. Control example 1 does not contain polyethylene glycol #4000, control example 2
The stability of peroxidase in a solution containing no bovine serum albumin was examined using the procedure of Example 1. The results are shown in Table 6.

[Table] Example 7 The peroxidase bound to human immunoglobulin G described in Example 6 was mixed with polypropylene glycol, diol type average molecular weight 2000 (manufactured by Wako Pure Chemical Industries, Ltd.) 5% by weight to volume (W/V), and It was dissolved in 0.02M PBS containing 10% by volume of rabbit serum.
The stability of peroxidase in the solution was examined using the procedure described in Example 1 for Control Example 1, which did not contain polypropylene glycol, and Control Example 2, which did not contain rabbit serum. The results are shown in Table 7.

【table】

Claims (1)

Claims: 1. A stable enzyme composition comprising peroxidase, serum protein 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.