JP2001201506A - Solid phase carrier for solid phase immunoassay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide solid phase material having excellent adsorption capacity relative to binder material, especially lectin. SOLUTION: This solid phase carrier is manufactured by using plastic material selected from a group comprising polybutadiene, polystyrene butadiene, polycarbonate, nitrocellulose, HIPS, polyallylate and a cumarone resin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a solid phase carrier for an immunosolid phase assay.

[0002]

The immunoassays such as the EIA, RIA, and MPHA methods are methods for specifically detecting and quantifying a substance in a sample using an antigen-antibody reaction. In particular, an immuno-solid phase assay using a solid phase to which an antibody or an antigen is adsorbed is widely used in the field of clinical tests, mainly in ELISA.

[0003] Polystyrene is used as the solid phase for the immunosolid phase assay.
(PS) and polyvinyl chloride (PVC) are frequently used, and the surface of the solid-phase plate is usually subjected to a coating treatment in order to improve the adsorption ability.

Conventionally, it has been usual to immobilize an antibody directly on a solid phase. In recent years, however, substances capable of specifically or non-specifically binding a certain substance such as lectin or adhesion protein ( A method of adsorbing an antibody or an antigen to a solid phase via a binder substance (hereinafter referred to as a binder substance) has come to be used, and thereby, it has become possible to adsorb various substances to the solid phase. In particular, lectins have a characteristic of binding a specific sugar chain to each type, and are therefore extremely useful as a binder substance for adsorbing erythrocyte antigens and the like to a solid phase (Japanese Patent Laid-Open No. 2-124464). ), And has been used in many immunosolid phase assays.

[0005] As described above, recently, a binder substance is often adsorbed and used on a solid phase plate. However, a conventional solid phase plate has been prepared so that antibodies can be adsorbed efficiently. Therefore, few substances have excellent adsorption ability to substances other than antibodies.

[0006] For this reason, the development and search of solid phase plate materials having an excellent ability to adsorb to a binder substance, particularly lectin, and the development of a method of adsorbing to a solid phase plate material have been actively carried out, but not always good results. Has not been obtained. In addition, despite the fact that solid-phase plates are difficult to reuse, conventional solid-phase plates have been subjected to a special surface treatment to enhance the adsorption capacity, so the solid-phase plates themselves are expensive, Costly use is forced.

[0007] For example, the example of JP-A-2-124464 which discloses an immunological measurement method using magnetic marker particles includes preparation of a plate on which erythrocytes are immobilized via lectin,
And a blood type determination method using the plate.

In this embodiment, the lectin is adsorbed on a polystyrene U-shaped microplate (Nunc.), But neither the material nor the shape is suitable for lectin adsorption.

As a method for immobilizing a lectin on a solid phase plate, Japanese Patent Application Laid-Open No. 8-319300 discloses a method for immobilizing a lectin using a sugar chain polymer. In this method, first, a sugar chain polymer is physically adsorbed to a solid phase plate, and a lectin is bound to a solid phase substrate via the sugar chain polymer. As a method of adsorbing a substance on a solid support, there is a method of chemically covalent bonding with a method by physical adsorption, but in this method, without using a covalent bonding method that is complicated in operation, using only physical adsorption. I have. In addition, since sugar chains can strongly adsorb lectins, the general drawback of physical adsorption, which has a weak binding force, has been overcome.

[0010] However, this method requires one step of physically adsorbing the sugar chain polymer to the solid phase substrate, and does not provide a solid phase substrate capable of directly and strongly adsorbing lectin.

[0011]

SUMMARY OF THE INVENTION The present invention has been made to overcome such disadvantages of the prior art, and has been developed for use in an immunosolid phase assay for directly physically adsorbing a binder substance. It is intended to provide a phase carrier.

In particular, an object of the present invention is to provide a solid support for directly adsorbing lectin.

[0013]

In order to solve the above-mentioned problems, the present invention uses a plastic material selected from the group consisting of polybutadiene, polystyrene butadiene, polycarbonate, nitrocellulose, HIPS, polyarylate, and cumarone resin. The present invention provides a solid phase carrier for an immunosolid phase assay prepared as described above, for directly adsorbing a binder substance for immobilizing an antibody or an antigen on the solid phase carrier.

[0014]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a solid phase carrier for an immunosolid phase assay for directly physically adsorbing a binder substance.

As used herein, the term "immuno-solid phase assay" means that a specific affinity substance having a specific affinity for a desired test substance to be detected in a sample is adsorbed on a solid phase,
In the solid phase assay for detecting a test substance based on the specific affinity with the solid phase, the adsorption to the solid phase is enhanced through the binding of the substance which can be an immunological antibody or antigen by an antigen-antibody reaction. This is what we did. Here, the specific affinity substance can be an antibody or an antigen, and can be directly adsorbed by an antigen-antibody reaction when adsorbed on a solid phase,
For example, a particle aggregation method, a mixed aggregation method, and an enzyme-linked immunosorbent assay (ELISA) can be mentioned. The specific affinity substance may have a non-immunological activity such as a genetic activity or a biochemical activity. In this case, the specific affinity substance is applied to the solid phase by an antigen-antibody reaction utilizing the binding property as an antigen. Adsorb or
Alternatively, an antibody corresponding to a specific affinity substance can be adsorbed on a solid phase, and then can be secondarily adsorbed on the solid phase by an antigen-antibody reaction via the adsorbed antibody.
For example, a biochemical or genetic assay by enzyme activity or nucleic acid hybridization can be performed.

The solid support of the present invention is prepared by a well-known method using a plastic material selected from the group consisting of polybutadiene, polystyrene butadiene, polycarbonate, nitrocellulose, HIPS, polyarylate, and coumarone resin.

The shape of the solid phase carrier of the present invention depends on the type of immunoassay to be performed, such as a petri dish, a microplate,
It can be a cuvette, film, bead, porous filter, small diameter tube, rod, needle, and the like.

The substance to be adsorbed on the solid phase carrier of the present invention is a binder substance.

Here, the term "binder substance" refers to a substance interposed between the antibody or antigen and the solid phase carrier and having an affinity for the antibody or antigen for immobilizing the antibody or antigen on the solid phase carrier. Means a substance having, for example, tannin, lectin,
Plant-based adhesive substances such as balsam.

The preferred binder substance in the present invention is a lectin as a plant agglutinin obtained from wheat, soybean, etc., and wheat germ agglutinin (WGA) is particularly preferred.

An antibody or an antigen is bound to the binder substance. When lectin is used as the binder substance, it is preferable to bind erythrocytes.

The binder substance is physically adsorbed on the solid support of the present invention. In order to cause the binder substance to be adsorbed on the solid phase carrier, a solution containing the binder substance is applied onto the carrier material, and then the mixture is incubated for an appropriate time.

Hereinafter, the present invention will be described in detail with reference to examples.

Example 1 In this example, the WGA adsorption ability of various plastic materials was examined.

The following table shows the plastic materials used in this example.

[0026]

[Table 1]

In this embodiment, the ability of each plastic material to adsorb WGA is evaluated by examining the binding state of red blood cells by utilizing the ability of red blood cells to bind WGA.

The adsorption of WGA on each of the above plastic materials was performed as follows.

Among the plastic materials, those dissolved in tetrahydrofuran (THF) are dissolved to a concentration of about 5%,
100 μL was dropped on a slide glass and quickly stretched to the glass surface using a cover glass to form a plastic thin film on the slide glass.

The plate-like material insoluble in THF and the microplate were used as they were.

A donut-shaped perforated vinyl patch (Kokuyo Vinyl Patch Clear) is attached to the formed plastic thin film and the plate-like material, and 50 μL of 10 μG / mL W
GA (dissolved in 0.01M carbonate buffer pH 9.6) was dispensed therein. Then incubate for 1 hour at room temperature,
Washed with 0.05% Tween 80 / pure water.

For a microplate, 50 μL of 10 μG
/ mL WGA (dissolved in 0.01 M carbonate buffer pH 9.6) was dispensed into the wells and treated similarly.

Washed O-type erythrocytes diluted to 1.0% with physiological saline were dispensed to each plastic material to which WGA was adsorbed in such a manner as to have a concentration of 25 μL / well, and allowed to stand at room temperature for 10 minutes. Thereafter, the cells were washed with physiological saline, and the binding of red blood cells was observed.

Table 2 shows the results.

[0035]

[Table 2]

As is apparent from Table 2, the plastic materials in which erythrocytes were favorably solid-phased were polybutadiene (Aldrich), polystyrene-butadiene (Aldrich), polycarbonate (Aldrich), nitrocellulose (Aldrich), and polycarbonate (Teijin Kasei Pan). Light), HIPS (Asahi Kasei Asaflex AFX80)
5), polyarylate (Unitika polymer U-100), and cumarone resin.

Among these, polycarbonate, polyarylate, and coumarone resin were particularly excellent in binding of red blood cells.

From the above results, it was shown that these plastic materials have excellent WGA adsorption ability and can bind erythrocytes firmly through WGA. These plastic materials would be suitable for blood typing tests using red blood cells.

Example 2 In this example, a U-bottom microplate was prepared using polycarbonate (Teijin Kasei Panlite) and polyarylate (Unitika Polymer U-100), which had good WGA binding in Example 1. Molded and W
The solid state of GA was compared.

As controls, Nunc Maxisorp (Cat. No. 475078), which has been conventionally used for adsorbing WGA, Nunc
Polysorp (Cat. No. 475086) and Nunc Microwell plane (Cat. No. 262170) were used.

First, 5, 2.5, 1.25, 0.625, 0.31, 0.15
μg / well in 0.01M carbonate buffer pH 9.6,
WGA (Seikagaku Cat. No. 300191, Lot. No. 62404)
Was diluted and incubated for 1 hour at room temperature.

Thereafter, the substrate was washed with 0.05% Tween 80 / pure water.

After washing, commercially available O-type screening erythrocytes (Ortho Selectogen) were added to the wells with physiological saline at about 1%.
, And dispensed, and allowed to stand to immobilize erythrocytes. Excess red blood cells were washed away with physiological saline.

After the erythrocyte binding, the erythrocyte binding was visually observed,
Thereafter, 100 μL / well of purified water was dispensed to lyse hemolysate, and O
Measure hemolysis at D410nm. The obtained hemolysis value was defined as the amount of red blood cells bound.

The results are shown in FIG.

[0046]

[Table 3]

As shown in FIG. 1, polycarbonate (PC) and polyacrylate (PAr) have been conventionally used as solid supports for adsorbing lectins, despite no special surface treatment. Nunc has been
It had the same erythrocyte binding ability as Maxisorp.

Nunc.'S Microwell is clearly inferior in erythrocyte binding ability to these plastic materials,
Polysorp hardly bound red blood cells.

Table 3 shows the results of the determination of the erythrocyte binding state of each plastic material at each concentration by visual and microscopic determination.

The results in Table 3 are the same as the results shown in FIG.
At a concentration of 1.25 μg / mL or more, erythrocytes were solid-phased very well.

Note that the present invention is not limited to the above-described example.
The present invention also includes a case where a genetic or biochemical substance such as a nucleic acid or an enzyme is immobilized via an antibody to perform solid-phase assay.

[0052]

According to the present invention, polybutadiene, polystyrene butadiene, polycarbonate, nitrocellulose, HIPS, polyarylate, and coumarone resin can adsorb lectin well and are suitable as a carrier for immobilizing lectin. It became clear that. These resins are the only solid-phase supports that are suitable for immobilizing lectins.
It has the same lectin adsorption capacity as owell, does not require special surface treatment, and can be used for inexpensive solid-phase immunoassay.

[Brief description of the drawings]

FIG. 1 is a view showing the results of comparing the lectin adsorption capacities of various plastic materials.

Claims (5)

[Claims] 1. A solid phase carrier for an immunosolid phase assay prepared using a plastic material selected from the group consisting of polybutadiene, polystyrene butadiene, polycarbonate, nitrocellulose, HIPS, polyarylate, and coumarone resin. A solid support capable of directly adsorbing a binder substance for immobilizing an antibody or an antigen to the solid support without substantially performing surface treatment. 2. The solid phase carrier according to claim 1, wherein the binder substance is a lectin. 3. The solid support according to claim 2, wherein the lectin is wheat germ agglutinin. 4. The solid phase carrier according to claim 1, wherein red blood cells are bound via the binder substance. 5. The solid phase carrier according to claim 1, wherein the solid phase carrier is in the form of a petri dish, a microplate, a cuvette, a film, or a bead.