JP2003215127A - Method for stabilizing solid phase immunological reagent and stabilizing solution used therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stabilizing a solid phase immunological reagent and a stabilizing solution used therefor capable of maintaining high stability in a dry state over a long period by combining the saccharide of a specific concentration with the buffer solution of a specific concentration. <P>SOLUTION: The method for stabilizing solid phase immunological reagent is characterized in that antigen or antibody fixedly retained on the solid phase is immersed in the saccharide which ranges from 20 to 80% and the buffer solution in saccharide which ranges from 0.5 to 2 M, then, the antigen or the antibody is dried, and the stabilying solution used therefor is also provided. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for stabilizing a solid-phased immunoreagent, and in particular, a solid-phased antigen or antibody can be stably stored in a dry state for a long period of time without lowering immunological activity. The present invention relates to a method for stabilizing a solid-phased immunoreagent.

[0002]

2. Description of the Related Art Conventionally, a solid-phased immunoreagent in which an antigen or antibody is bound to an insoluble carrier has been used as many immunological measurement reagents. For example, a solid-phased immunoreagent in which an antibody is bound to an insoluble carrier such as polystyrene is effective for sandwich enzyme immunoassay.

Such a solid-phased immunoreagent has been dipped in an aqueous solution containing a serum protein such as bovine serum albumin, but it is known that the activity is lowered and a precipitate is produced. . It is also known that this solid-phased immunoreagent is advantageous if it can be freeze-dried for handling, but it cannot retain the activity when freeze-dried.

Under such circumstances, in the stabilization method by freeze-drying, there is also a method of freeze-drying the immobilized immunoreagent together with a phosphate buffer containing a protein such as bovine serum albumin as a stabilizer. It is also known (Japanese Patent Laid-Open No. 59-206761).

Further, a solid-phase immunoreagent has been proposed in which an antibody immobilized on a solid phase is immersed in a solution containing a saccharide, bovine serum albumin and a lower polyhydric alcohol, and then dried (see (Kaisho 60-35263).

However, in these methods,
Since a white powder derived from a protein such as bovine serum albumin coexists, the powder is difficult to dissolve, and the dried protein and the like are easily scattered.

In order to solve this problem, a solid-phased immunoreagent stabilized by treatment with a stabilizing solution containing a saccharide, a chelating agent, etc. has been proposed (JP-A-61).
No. 241665). Further, a treatment with a stabilizing solution containing dextran, pyrrolidonecarboxylic acid and polyvinyl alcohol has been proposed (Japanese Patent Laid-Open No. 34350/1987).

However, the above-mentioned stabilizing solution has a problem that the immobilized immunoreagent cannot be stably maintained in a dry state for a long period of time. Therefore, a stabilizing solution of a solid-phased immunoreagent containing casein, whey protein, and a casein degradation product has been proposed (JP-A-9-80051).

[0009]

However, even with the above solid-phased immunoreagent, a satisfactory stabilized solid-phased immunoreagent has not yet been obtained, and a solid-phased immunoreagent capable of maintaining higher stability can be obtained. The advent of immunological reagents has been strongly desired.

Therefore, the present invention has been made in view of such conventional problems, and a method for stabilizing a solid-phased immunoreagent capable of maintaining high stability in a dry state for a long period of time, and the same. It is intended to provide a stabilizing solution for use in.

[0011]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a combination of a sugar having a specific concentration and a buffer solution having a specific concentration can be used in a dry state for a long period of time. The present invention has been completed by finding that a solid-phased immunoreagent that can maintain high stability can be obtained.

The method for stabilizing the immobilized immunoreagent of the present invention comprises:
20-80% of the antigen or antibody fixedly retained on the solid phase
In the range of 0.5 to 2 mol / L (hereinafter,
It is characterized in that it is immersed in a buffer solution in the range of (abbreviated as M) and then dried. Hereinafter, the present invention will be described in more detail.

The saccharides used in the present invention include monosaccharides such as glucose, galactose, xylose and fructose, disaccharides such as trehalose, sucrose, lactose and maltose, trisaccharides (oligosaccharides) such as raffinose, dextran and dextrin. , And polysaccharides such as gluconic acid. These saccharides may be used alone or in combination of two or more. Among these sugars, disaccharides such as trehalose and sucrose are particularly preferable.

The concentration of this saccharide is 20 to 80%, preferably 30 to 60%, and most preferably 45%. If the concentration is less than 20%, a sufficient stabilizing effect cannot be obtained, while if it exceeds 80%, the viscosity becomes high, which is not preferable. .

The buffer used in the present invention can be appropriately selected from buffers generally used in biochemistry such as phosphate buffer, Tris buffer, Good's buffer, etc. Liquids are preferred.

The concentration of this buffer solution is 0.5 to 2M, preferably 0.8 to 1.5M, and most preferably 1M. If the concentration is less than 0.5M, the stabilizing effect is significantly reduced, and conversely, if it exceeds 2M, the stabilizing effect cannot be further improved. The pH of this buffer is 4.0-1
It is 0, preferably 6.0 to 8.0.

The insoluble carrier used for the solid-phased immunoreagent may be appropriately selected from those used for immunoassay. Specific examples of the insoluble carrier include synthetic polymer compounds such as polystyrene, polyethylene and polypropylene, and inorganic substances such as porous glass, glass beads and magnetic particles. The form of the insoluble carrier includes tubes, plates, microtiter plates, fine particles and the like.

The binding of the antigen or antibody to these insoluble carrier particles can be carried out according to a known method. Specific examples thereof include, for example, glutaraldehyde, bisdiazobenzidine, tolylene diisocyanate, difluoronitrobenzene, carbodiimides, quinones, chromium chloride, a chemical coupling method using a so-called coupling agent such as tannic acid, an antigen or A physical adsorption method in which the antibody and the carrier are brought into contact with each other in a water-soluble solvent (for example, water, physiological saline, various buffers, etc.) can be mentioned.

The immersion treatment time of the solid-phased immunoreagent bound with the antigen or antibody in the stabilizing solution may be about 10 minutes to 10 hours, preferably about 30 minutes to 2 hours. Further, the drying after the immersion of the solid-phased immunoreagent may be any of natural drying, aeration drying, vacuum drying, and freeze drying.

The present invention provides a stabilizing solution of a solid-phased immunoreagent, which comprises a saccharide in the range of 20 to 80% and a buffer solution in the range of 0.5 to 2M. it can. In this case, the sugar and the buffer may be appropriately selected from those mentioned above.

According to the present invention, it is possible to provide an immunological measurement method to which the stabilization technique for the solid phase immunoreagent is applied. Examples of the immunological measurement method include a latex agglutination reaction method, a gold colloid agglutination reaction method, an immunochromatography method, an ELISA method, and the like. In either measurement method, the stabilization solution of the immobilized immunoreagent contains a specific concentration of saccharide and a buffer of a specific concentration to highly stabilize the immobilized immunoreagent and suppress the decrease in the measured value. To be done.

[0022]

INDUSTRIAL APPLICABILITY The present invention can provide a solid-phased immunoreagent which can maintain high stability for a long period of time even in a dry state by combining a saccharide having a specific concentration and a buffer solution having a specific concentration. . Therefore, according to the present invention, since it can be used in a dry state, operability in the immunoassay can be improved.

[0023]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited thereto.

Example Stabilizing effect of immobilized immunoreagent (1) Preparation of antibody for solid phase Ascites fluid containing mouse monoclonal antibody against prostate-specific antigen (PSA) was fractionated 3 times with 40% saturated ammonium sulfate and 10
Dialyze overnight against mM phosphate buffer (PBS: pH 7.2) for 1
Centrifuge at 0000 rpm for 10 minutes to remove the precipitate,
1% IgG after filtration with 0.45 μm filter
It was adjusted to the concentration and stored at -80 ° C.

(2) Immobilization of antibody Using 10 mM PBS (pH 7.2), 5 μg / mL of I
0.2 mL of an antibody solution having a GG concentration was prepared for each solid phase of polystyrene beads, and the beads and the antibody solution were mixed. After standing and heating at 37 ℃ for 4 hours, 10 mM PB
It was washed 5 times with S (pH 7.2). Antibody-immobilized beads and blocking solution (1% sodium casein-100 mM PBpH
7.2) and were mixed and allowed to stand overnight at 4 ° C.

(3) Discard all the solid phase dry block solution, and dry buffer (1M PB-45).
% Trehalose) was added and left at room temperature for 30 minutes or longer. As the solid phase floats, stir every 10 minutes and dry
The entire amount of Buffer was discarded, water was absorbed with filter paper, and then dried with a freeze dryer overnight.

(4) Method for measuring residual activity The solid phase immunoreagent obtained above was used at a temperature of 30 ° C. and a humidity of 90.
It was stored in a thermo-hygrostat set to%, and taken out after 1 week. Next, using the solid phase before and after the humidification test,
A chemiluminescent enzyme immunoassay was used to react with a known concentration of the antigenic substance. Based on the obtained measured value (luminescence value), the ratio of the luminescence value after humidification to the luminescence value before the humidification test was determined, and this was taken as the residual activity. The result is shown in FIG. As shown in FIG. 1, the residual activity is extremely high at 98%, and it can be seen that the stability can be maintained for a long period of time.

Comparative Example In place of the dry buffer (1M PB-45% trehalose) used in the examples, 1M PB-10% trehalose and 0.1M PB-45% were used as dry buffers.
Trehalose, 0.1 M PB-10% trehalose, 0.01 M PB-45% trehalose, 0.0
The residual activity was measured in the same manner as in Example except that 1M PB-10% trehalose was used. The result is shown in FIG. As shown in FIG. 1, it can be seen that the stability of the comparative example is significantly lower than that of the example.

[Brief description of drawings]

FIG. 1 is a graph showing changes in stability depending on the composition of dried buffer.

Claims (10)

[Claims] 1. A saccharide in the range of 20 to 80% and 0.5 to 2 mol of an antigen or an antibody immobilized and retained on a solid phase.
A method for stabilizing a solid-phased immunoreagent, comprising immersing in a buffer solution in the range of / L and then drying. 2. The method for stabilizing a solid-phased immunoreagent according to claim 1, wherein the saccharide is at least one selected from the group consisting of monosaccharides, disaccharides, trisaccharides and polysaccharides. 3. The disaccharide is trehalose, sucrose,
The method for stabilizing a solid-phased immunoreagent according to claim 2, which is at least one selected from the group consisting of lactose and maltose. 4. The method for stabilizing a solid phase immunoreagent according to claim 1, wherein the buffer solution is at least one selected from the group consisting of a phosphate buffer solution, a Tris buffer solution, and a Good's buffer solution. 5. The method for stabilizing a solid phase immunoreagent according to claim 1, wherein the solid phase is an insoluble carrier. 6. The method for stabilizing a solid phase immunoreagent according to claim 5, wherein the insoluble carrier is polystyrene, polyethylene, porous glass, glass beads, or magnetic particles. 7. Sugars in the range of 20-80% and 0.
A stabilized solution of a solid-phased immunoreagent, which contains a buffer solution in the range of 5 to 2 mol / L. 8. The stabilized solution of a solid phase immunoreagent according to claim 7, wherein the saccharide is at least one selected from the group consisting of monosaccharides, disaccharides and polysaccharides. 9. The disaccharide is trehalose, sucrose,
9. The stabilized solution of the immobilized immunoreagent according to claim 8, which is at least one selected from the group consisting of lactose and maltose. 10. A buffer solution is a phosphate buffer solution, a Tris buffer solution,
The stabilizing solution of the immobilized immunoreagent according to claim 7, which is at least one selected from the group consisting of: and Good's buffer.