JPH0980051A - Solution for stabilizing solid phase immunological reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To preserve a solid phase immunological reagent stably for a long term under dry condition without lowering the immunological activity by adding any one of casein, whey protein or a decomposed product of casein into the solid phase immunological reagent. SOLUTION: The solution for stabilizing a solid phase immunological reagent contains casein and/or whey protein or a decomposed product of casein. Casein in milk, including α-casein, β-casein, γ-casein, κ-casein and λ-casein, is added into the solution for stabilizing the reagent, for example. Concentration of casein is normally in the range of 0.05-3.0w/v%, preferably in the range of 0.2-2.0w/v%. The whey protein includes that in milk. Concentration of whey protein is normally in the range of 0.05-3.0w/v%, preferably in the range of 0.2-1.5w/v%. The decomposed product of casein includes enzyme decomposed casein and acid decomposed casein and the concentration thereof is normally in the range of 0.05-3.0w/v%.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a stabilizing solution for stably storing a solid-phased immunoreagent in a dry state for a long period of time without lowering immune activity.

[0002]

2. Description of the Related Art A solid-phased immunoreagent is a reagent in which an antigen or antibody used for immunochemical measurement is physically or chemically bound to an insoluble solid-phase carrier to insolubilize it. Usually, these solid-phase immunizing reagents are preserved in the immersion state and retain the immunological activity, but they need to be in a dry state for easy handling. Japanese Patent Application Laid-Open No. 54-1 for stabilizing the immobilized immunoreagent.
28396, saccharides of JP-A-60-35263 and JP-A-6-35263
2-34509 pyrrolidonecarboxylates, dextrin and polyvinyl alcohol. On the other hand, Japanese Patent Laid-Open No. 19766/1990 describes a casein degradation product used in an immunoreaction solution to remove non-specific adsorption. This is used in an immunoreaction system to stabilize a solid-phase immunoreagent. Is separate from the use of.

[0003]

However, the conventional stabilizing solution has a problem that the solid-phased immunoreagent cannot maintain its immune activity in a dry state for a long period of one year or more.

[0004]

Means for Solving the Problems As a result of intensive studies to solve these problems, the present inventors have found that casein and / or casein which can be stably stored in a dry state for a long period of one year or more The present invention has been reached by finding a solution consisting of a whey protein or a casein degradation product.

That is, the present invention is a stabilized solution of a solid-phased immunoreagent containing casein and / or whey protein or a casein degradation product.

Examples of casein in the stabilizing solution used in the present invention include casein in milk. For example, casein described in the “Fifth Edition Food Additive Official Standard Manual (issued by Hirokawa Shoten)” can be used. Specific examples include α-casein, β-casein, γ-casein, κ-casein, and λ-casein. The concentration of casein is usually 0.05 to 3.0 w / v%, preferably 0.2 to 2.0 w / v%.

[0007] Whey proteins include whey proteins in milk. Examples of whey proteins include concentrated whey and whey powder described in “Ministry Ordinance on Ingredients for Milk and Dairy Products in the Food Sanitation Law”. The concentration of whey protein is usually 0.05 to 3.0 w / v%, preferably 0.2 to 1.5 w / v%.

As a casein decomposition product, Japanese Patent Application Laid-Open No. 2-36 is known.
The enzyme-decomposed casein described in Japanese Patent No. 353 publication is mentioned. As acid-decomposed casein, casein-produced by Kanto Chemical Co., Inc.
An acid hydrolyzate can be used. The concentration of casein degradation products is usually 0.05 to 3.0 w / v%, preferably 0.5 to 2.
It is 0 w / v%.

By using a solution in which casein and whey protein coexist, the stability of the solid-phase immunoreagent in a dry state can be further improved. The concentration at this time was 0.05 to 3.0 w / v% casein and 0.05 whey protein.
Is preferably 2.0 to 2.0%, particularly preferably casein 0.
2 to 2.0 w / v%, whey protein 0.2 to 1.5 w / v
%.

Examples of the insoluble carrier used for the immobilized immunoreagent include all insoluble carriers that can be used for immunochemical measurement, for example, the insoluble carrier described in JP-A-2-205774. Of these, preferred are synthetic polymer compounds such as polystyrene, polypropylene, polyethylene and polyvinyl chloride, glass, alumina, silica gel, metal and paper. The forms of the insoluble carrier include tubes, plates, microtiter plates,
Balls, particles, etc. are used.

The method for binding the antigen or antibody to the insoluble carrier may be either physical adsorption or chemical adsorption, and for example, it may be the same as the method described in JP-A-2-205774. That is, a method for binding glass to a monoclonal antibody (for example, US Pat. Nos. 4,280,992 and 365,2761) and a method for physically adsorbing an antibody on a plastic (for example, E. Engvar, J. Johnson; Biokim). By Office Actor (E. Engvall, J. Jonson, P. Parlmann; Biochim.B
iophys.Acta), 251 (1971) 427-434).

The immersion treatment time of the solid-phased immunoreagent bound with the antigen or antibody in the stabilizing solution is about 2 hours to 2 weeks, preferably 12 to 24 hours. The drying conditions after the solid phase reagent immersion treatment may be any of natural drying, aeration drying, vacuum drying and freeze drying.

[0013]

EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

Example 1 Preparation of Anti-Insulin Antibody-Binding Glass Beads An anti-insulin antibody (guinea pig) was bound to ground glass beads having an average particle size of 6.8 mm according to the predetermined binding method described above to prepare a solid phase immunoreagent. did.

Preparation of Stabilizing Solution Phosphate buffer solution containing 0.85 w / v% sodium chloride, 0.5 w / v% bovine serum albumin at 0.02 mol / L, pH 7.0 (hereinafter abbreviated as (a)). To prepare. Next, a stabilizing solution (b) prepared by adding 2.0 w / v% casein to (a), a stabilizing solution prepared by adding 1.0 w / v% whey protein (c), 2.0 w / v% casein-acid. A stabilizing solution (d) containing a hydrolyzate and a stabilizing solution (e) containing 2.0 w / v% casein and 1.0 w / v% whey protein were prepared. Further, for comparison, a stabilizing solution (f) obtained by adding 5.0 w / v% sucrose to (a),
Stabilization solution (g) to which 5.0 w / v% lactose was added, 2.
A stabilizing solution (h) to which 0 w / v% sodium pyrrolidonecarboxylate was added was prepared.

Preparation of Dried Anti-Insulin Antibody-Bearing Glass Beads The anti-insulin antibody-binding glass beads were dipped at 2 to 8 ° C. for 24 hours using each of the stabilizing solutions (a) to (h), and then the stabilizing solution was added. It was removed and dried by vacuum drying to prepare dried beads.

Preparation of buffer solution for immune reaction (R1) A phosphate buffer solution containing 0.02 M, pH 7.2 containing 1% bovine serum albumin and 0.9% sodium chloride was prepared.

Preparation of enzyme-labeled antibody solution (R2) 0.5% of peroxidase-labeled anti-insulin antibody (guinea pig) was added to a 0.02M phosphate buffer containing 1% bovine serum albumin and 0.9% sodium chloride at pH 7.2.
It was added and prepared so that it might become g / ml.

EIA-Insulin Assay Method After adding R1-300 μl to 50 μl of a test sample (standard insulin solution etc.) in a test tube for assay to make a uniform solution, anti-insulin antibody (guinea pig) -bound glass beads were added, and the mixture was incubated at 37 ° C. Incubate for 60 minutes. After completion of the reaction, the unreacted material is removed by an aspirator, and then suction-washed three times with 3 ml each of physiological saline. Next, R2-300μ
1 and incubate at 37 ° C. for 60 minutes. After completion of the reaction, the unreacted material is removed by an aspirator, and then suction-washed three times with 3 ml each of physiological saline. Thereafter, a substrate solution containing 0.3% o-phenylenediamine and 0.04% hydrogen peroxide is added, and the mixture is incubated at 37 ° C for 30 minutes. After quenching the reaction with 3 ml of 1N sulfuric acid, 492n for each
Measure absorbance at m.

Comparative Example 1 Evaluation of daily stability of dried glass beads After each dried glass bead prepared was allowed to stand at 4,40 ° C. for a predetermined period, the immunoreactivity of each dried glass bead was 250 μU / m.
It was evaluated from the assay absorbance of 1 standard insulin, and the daily stability was compared and evaluated. The assay absorbance was measured according to the EIA-insulin measuring method. The evaluation results are shown in Table 1.

[0021]

[Table 1]

Stabilizing solutions (b), (c), (d),
It was possible to confirm good stability in the long-term stability and thermal stability tests of the product subjected to the immersion treatment in (e).

Example 2 Preparation of Anti-Insulin Antibody-Binding Polystyrene Beads An anti-insulin antibody (guinea pig) was bound to polystyrene beads having an average particle size of 6.4 mm according to the above-described binding method to prepare a solid-phased immunoreagent. did.

Preparation of Dried Anti-Insulin Antibody-Binding Polystyrene Beads Using each of the stabilizing solutions (a) to (h), the anti-insulin antibody-binding polystyrene beads were immersed in the stabilizing solution at 2 to 8 ° C. for 24 hours, and then the stabilizing solution was added. It was removed and dried by vacuum drying to prepare dried beads.

Comparative Example 2 Evaluation of daily stability of dried polystyrene beads After each prepared dried polystyrene bead was allowed to stand at 4,40 ° C. for a predetermined period of time, the immunoreactivity of each dried polystyrene bead was evaluated as 2
It was evaluated from the assay absorbance of 50 μU / ml of standard insulin, and the daily stability was compared and evaluated. The assay absorbance was measured according to the EIA-insulin measuring method. The evaluation results are shown in Table 2.

[0026]

[Table 2]

Stabilizing solutions (b), (c), (d),
It was possible to confirm good stability in the long-term stability and thermal stability tests of the product subjected to the immersion treatment in (e).

[0028]

Industrial Applicability According to the present invention, it is possible to improve the stability of the immune activity by drying the solid-phased immunoreagent, and it becomes possible to store it in a dried state for a long time and use it as it is.
Therefore, it is suitable for improving the operability in the immunoassay.

Claims (3)

[Claims] 1. A stabilizing solution of a solid-phased immunoreagent, which contains any of casein, whey protein, and a casein degradation product. 2. The stabilizing solution according to claim 1, which contains any of casein, whey protein, and a casein degradation product in an amount not exceeding 3.0 w / v%. 3. The stabilizing solution according to claim 1, which contains both casein and whey proteins in an amount not exceeding 5.0 w / v%.