JPH07213284A - Method for stabilizing antigencity of myeloperoxidase - Google Patents

Abstract

PURPOSE:To stabilize the antigenicity of a myeloperoxidase useful for diagnosing, etc., renopathy and enable the preservation for a long period (e.g. >=6 months) by adding a specific amount of dextran as an antigenicity stabilizer thereto. CONSTITUTION:This method for stabilizing the antigenicity of myeloperoxidase is to add dextran as an antigenicity stabilizer in an amount of 0.05-4wt./vol.% thereto. The dextran preferably contains >=65% alpha-1,6-bonds and has preferably about 30000-90000 molecular weight. Thereby, the myeloperoxidase is stabilized against even to heat.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for stabilizing the antigenicity of myeloperoxidase.

[0002]

BACKGROUND OF THE INVENTION Myeloperoxidase is an enzyme having heme similar to heme a, which is mainly present in neutrophils of leukocytes and plays a role of detoxifying bacteria and the like through halide ions. In 1982, fluorescent antibody indirect method (IIF
Method, Davies et al. Reported that anti-neutrophil cytoplasmic antibody (ANC
A) is reported, and Cytoplasmic-AN is reported from the staining pattern.
CA (C-ANCA) and perinuclear-ANCA (P-ANC)
It was divided into two subsets of A). This P-ANCA
Myeloperoxidase is one of the corresponding antigens of Escherichia coli, and has come to be used as an antigen in immunological action.

In recent years, it has been noted that it is useful to measure an anti-myeloperoxidase antibody such as P-ANCA in a blood test for the diagnosis of renal disease, and the enzyme used myeloperoxidase as an antigen in an antigen-antibody reaction. Immunoassay (EIA) reagents are commercially available. This is a method in which myeloperoxidase is attached to the surface of a synthetic resin plate or the like by adsorption, the sample is put in a dried state, and a coloring agent or the like is added to measure the absorbance.

[0004]

However, myeloperoxidase is stable under normal conditions (freeze-dried state), but is antigenic (immunological action) in aqueous solution or in a fixed state.
Is unstable and loses its antigenicity immediately, so it was impossible to store it for a long time (for example, 3 months or more). Therefore, it is difficult to keep the reagent performance after reagent preparation constant and difficult to store because the enzyme immunoassay reagent that uses myeloperoxidase as an antigen in immunological action changes its antigenicity over time. There was a flaw.

The present invention provides a method for stabilizing the antigenicity of myeloperoxidase, which allows a reagent using myeloperoxidase as an antigen in immunological action to be stored for a long period (for example, 6 months or more) without impairing the reagent performance. The purpose is to provide.

[0006]

The present invention provides a method for stabilizing the antigenicity of myeloperoxidase by the addition of an antigenicity stabilizer, wherein dextran is added in an amount of 0.05 to 4 W /
The gist is a method for stabilizing the antigenicity of myeloperoxidase, which is characterized in that it is added in an amount of V%.

The present invention uses dextran as an antigenic stabilizer. With dextran, D-glucose is α
It is a viscous bacterial polysaccharide composed mainly of -1,6 bonds.

Dextran used in the present invention is α-
Bonds other than 1,6 bonds, such as α-1,3 bonds and α-
Although 1,2 bonds and the like may be contained, it is preferable that α-1,6 bonds account for 65% or more. The degree of polymerization varies, but the molecular weight is low, for example, about 30,000.
It is preferably about 0 to 90,000.

In the present invention, dextran is 0.05
It is effective when added in an amount of W / V% or more. There is no problem even if the amount of dextran is 4 W / V% or more, but a high-concentration dextran solution has a high viscosity and is difficult to adopt.

To use the myeloperoxidase of the present invention as an antigen, for example, myeloperoxidase may be attached to a plate-like insoluble carrier and, if necessary, blocked using albumin made of bovine blood, gelatin, casein or the like. Good.

As the insoluble carrier, glass, agarose, cellulose, polyacrylamide, polystyrene, homopolypeptide, latex, polycarbonate, polypropylene, aminoalkyl silica glass, silicone rubber or the like is used, and the surface thereof is covalently bonded, Myeloperoxidase is attached by an ionic bond method, physical adsorption, etc., and then dried to form a solid phase.

[0012]

In the present invention, by adding dextran to myeloperoxidase, the quality of myeloperoxidase does not change even after long-term storage, and the function of myeloperoxidase as an antigen is not lost. .

[0013]

[Example] A myeloperoxidase (manufactured by Kamiya Biomedical) having a protein concentration of 5 μg / ml was adjusted with a phosphate buffer (pH 6.0), and 200 μl / well was added to each microtiter plate (Nunc, 96-wells). Dispense and leave at 4 ° C for 18 hours, then 0.05% Tween-
It was washed with a phosphate buffer solution (pH 7.2) containing 20. Then dextran (molecular weight of about 30,000 to 90,000)
0, manufactured by Nacalai Tesque, Inc., in phosphate buffer (pH 7.
After adjusting the concentration as shown in Table 1 in 0), 200 μl / well was added to each plate. After reacting at 25 ° C. for 60 minutes, the solution was removed and air-dried in a desiccator.

These plates were stored at 40 ° C., and immediately after drying (0 days), one week and two weeks later, the serum of patients with microscopic polyarteritis nodosa (containing anti-myeloperoxidase antibody) was used as a sample on each plate. Phosphate buffer (pH
Diluted 50 times with 7.3) and dispensed 200 μl / well.

After reacting at 25 ° C. for 60 minutes, 0.05% Tw
Alkaline phosphatase-labeled anti-human IgG antibody (manufactured by Sigma), which was washed with a phosphate buffer (pH 7.2) containing een-20 and adjusted with a phosphate buffer (pH 7.3).
Was dispensed at 200 μl / well.

After reacting at 25 ° C. for 60 minutes, 0.05% Tw
After washing with a phosphate buffer (pH 7.2) containing een-20, diethanolamine-HCL buffer (pH 9.
The disodium p-nitrophenylphosphate (manufactured by Sigma) prepared in 8) was added as a color former in an amount of 200 μl / well, and the difference in absorbance between 0 minutes and 60 minutes was measured. The absorbance is measured at 405 nm and the microplate reader Ty is used.
pe-MTP-120 (made by Corona) was used.

The value immediately after drying was taken as 100%, and the relative value of each was calculated in%, and the stability was examined by the change with time when kept at 40 ° C. The average value of 3-wells was calculated from the results, and the measurement results are shown in Table 1.

As a comparative example, a myeloperoxidase plate was prepared in the same manner as in the example, and the stability was examined by performing the same measurement without adding dextran. The results are also shown in Table 1.

From the results shown in Table 1, 7 days after the drying and 1
On day 4, the residual antigenicity is higher due to the addition of dextran, as compared with the case where no addition is made. From this, it can be said that effective addition of dextran is effective for the stability of myeloperoxidase.

[0020]

[Table 1]

[0021]

INDUSTRIAL APPLICABILITY According to the present invention, myeloperoxidase is remarkably stable in antigenicity, and since its action also has a high temperature, myeloperoxidase is also stabilized against heat. Therefore, even a reagent using myeloperoxidase as an antigen in an immunological action can be effectively used without wasting the reagent because the reagent can be stored for a long period of time without impairing the reagent performance.

Claims (1)

[Claims] 1. A method for stabilizing the antigenicity of myeloperoxidase by adding an antigenic stabilizer, wherein dextran is added in an amount of 0.05 to 4 W / V%. How to stabilize.