JP3227688B2 - Method for stabilizing myeloperoxidase antigenicity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

[0002]

2. Description of the Related Art Myeloperoxidase is an enzyme having a heme similar to heme a, which is mainly present in neutrophils of leukocytes and plays a detoxifying effect on bacteria and the like via halide ions. In 1982, the fluorescent antibody indirect method (IIF
Davies et al. Reported that antineutrophil cytoplasmic antibodies (ANC)
A) was reported and Cytoplasmic-AN
CA (C-ANCA) and perinuclear-ANCA (P-AN
CA) were divided into two subsets. This P-ANC
One of the corresponding antigens of A is myeloperoxidase,
It has come to be used as an antigen in immunological effects.

[0003] In recent years, it has been noted that measurement of an anti-myeloperoxidase antibody such as P-ANCA in a blood test is useful for diagnosis of kidney disease, and an enzyme using myeloperoxidase as an antigen in an antigen-antibody reaction has been noted. Reagents by immunoassay (EIA) are commercially available. In this method, myeloperoxidase is adhered to the surface of a synthetic resin plate or the like by adsorption or the like, and a sample is put in a dried state, and a color former or the like is added, and the absorbance is measured.

[0004]

However, myeloperoxidase is normally stable (lyophilized state), but antigenic (immunological action) in aqueous solution or immobilized state.
Is unstable and immediately loses its antigenicity, making it 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 antigenicity of an enzyme immunoassay using myeloperoxidase as an antigen in the immunological action changes over time. There were drawbacks.

The present invention stabilizes the antigenicity of myeloperoxidase, and can store an immunoassay reagent using myeloperoxidase as an antigen in an immunological action for a long period (for example, 6 months or more) without impairing the reagent performance. The aim is to provide a method.

[0006]

SUMMARY OF THE INVENTION The present invention relates to a myeloperu.
0.00005 to 14 W / V% cyclode
The gist of the present invention is to provide a method for stabilizing the antigenicity of myeloperoxidase, which comprises adding a xytrin solution .

[0007] The present invention uses cyclodextrin as an antigenic stabilizer. Cyclodextrin is a cyclic polysaccharide in which six or more glucose molecules are α-1,4-glucosidic bonded. So far, a glucose unit has six α-cyclodextrins, seven β-cyclodextrins, eight γ-cyclodextrins, nine δ-cyclodextrins, and a branch in which glucose or more α-1,4 glucan is attached to the ring. Cyclodextrins are known.

[0008] The cyclodextrin used in the present invention may be any of α-cyclodextrin , β-cyclodextrin, γ-cyclodextrin, δ-cyclodextrin, and branched cyclodextrin, and may be a mixture of these in any ratio.

The present invention is effective when a cyclodextrin solution of 0.00005 W / V% or more is added to myeloperoxidase. However, the amount of β cyclodextrin, γ cyclodextrin, or δ cyclodextrin is preferably 0.05 to 4 W / V%. The cyclodextrin may be used in an amount of 14 W / V% or more, but the solubility of cyclodextrin in water is 14.5 g / 100 ml for α-form and 1.85 g / 100 m for β-form.
The l and γ-forms are 23.2 g / 100 ml and the δ-form is “very soluble” depending on the type.

The immunoassay reagent using myeloperoxidase as an antigen of the present invention may be prepared by adhering myeloperoxidase to a plate-shaped insoluble carrier and , if necessary, performing blocking using bovine serum albumin, gelatin, casein or the like. .

Examples of the insoluble carrier include glass, agarose, dextran, cellulose, polyacrylamide, polystyrene, homopolypeptide, latex, polycarbonate, polypropylene, aminoalkyl silica glass,
A carrier such as silicon rubber is used. Myeloperoxidase is adhered to the surface of the carrier by a covalent bonding method, an ion bonding method, physical adsorption or the like, and then dried and solidified.

[0012]

[Example 1]

Protein concentration of 5 using phosphate buffer (pH 6.0)
A μg / ml myeloperoxidase (manufactured by Kamiya Biomedical) solution was prepared, and 200 μl / well was placed in a microtiter plate (manufactured by Nunc, 96-wells).
After being left at 4 ° C. for 18 hours, 0.05% Tween was added.
The plate was washed with a phosphate buffer (pH 7.2) containing en20. Thereafter, a solution prepared by preparing α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin (all manufactured by Nacalai Tesque, Inc.) with phosphate buffer (pH 7.0) to the concentrations shown in Table 1, Table 2, and Table 3 was applied to the plate. To 200
μl / well was added. After reacting at 25 ° C. for 60 minutes, the solution was removed and air-dried in a desiccator.

[0013] Save these plates at 40 ° C., immediately after drying (day 0), after 7 days, the patient serum of microscopic mirrors manner nodosa as a sample in each plate after 14 days (anti myeloperoxidase antibody-containing) With phosphate buffer (pH
The sample diluted 50-fold in 7.3) was dispensed at 200 μl / well.

After reacting at 25 ° C. for 60 minutes, 0.05% Tw
above with phosphate buffer (pH 7.2) containing een-20
Was washed plate, phosphate buffer (pH 7.3)
Alkaline Phosphatase Labeled Anti-Human I Prepared Using
A gG antibody (manufactured by Sigma) was dispensed at 200 μl / well.

After the reaction at 25 ° C. for 60 minutes, 0.05% Tw
above with phosphate buffer (pH 7.2) containing een-20
The plate was washed, and 200 μl of disodium p-nitrophenylphosphate (manufactured by Sigma) prepared with diethanolamine-HCL buffer (pH 9.8) was used as a coloring agent.
After adding the color former , the absorbance difference between 0 minute and 60 minutes was measured. The absorbance was measured at 405 nm using a microplate reader Type MTP 120 (manufactured by Corona).

7 days, taking the absorbance immediately after drying as 100%
Value (residual activity ) of the absorbance difference after 14 days and 14 days
And the change with time at 40 ° C.
The stability of the sidase was investigated. The results are shown in Table 1 for α-cyclodextrin, Table 2 for β-cyclodextrin, and Table 3 for γ-cyclodextrin.

[0017] As a comparative example Comparative Example 1, to create a plate immobilized with similarly myeloperoxidase Example 1, without adding cyclodextrin, perform the measurement in the same manner myeloperoxidase peroxidasin
The stability of the enzyme was examined. The results are shown in Tables 1, 2, and 3.

[0018] than the results in Table 1, a material obtained by adding α-cyclodextrin from day 7 after drying, was not intended and initially observed value of residual antigenicity addition, the value of the additive-free at the time of day 14 Although significantly reduced, those added at effective concentrations show high residual antigenicity values. Table 2
From the results, it can be seen that when β-cyclodextrin is added, the antigen stability is good at 0.05 to 2 W / V% on the 7th and 14th days. Further, Table 3 shows that the effect is high when the addition of γ-cyclodextrin is performed in the range of 0.05 to 4 W / V%. From this, it can be said that it is effective to add cyclodextrin effectively for the stability of myeloperoxidase.

[0019]

[Table 1]

[0020]

[Table 2]

[0021]

[Table 3]

[0022]

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

Claims (2)

(57) [Claims] (1) 0.0000 myeloperoxidase
A method for stabilizing the antigenicity of myeloperoxidase, comprising adding a 5 to 14 W / V% cyclodextrin solution . 2. The method according to claim 1, wherein the myeloperoxidase is immobilized on a myeloperoxidase.
2. The method according to claim 1, which is peroxidase.