JPH0587811A - Manufacture of carrier for fixing - Google Patents

Abstract

PURPOSE:To enable accurate and reproduciable quantitative measurement of specific components in a fluid sample by providing a process in which carriers are immersed in antibody (or antigen) solution and an operation process in which a container where the antibody (or antigen) solution is contained is moved at an approximate rate where the carriers are not substantially be deviated. CONSTITUTION:For example, a specified antibody solution 1000ml is put in a plastic container 3. Next, polystyrene beads 2 are put in a mesh basket 1, and the basket 1 is suspended in the container 3 to immerse the polystyrene beads 2 in the antibody solution. Then the basket 1 is, for example, rotated at a rate of 3 rpm at a temperature of 4 deg.C or below for one full day to fix the antibody. After that, they are washed by phosphoric buffer, immersed in a specified solution, incubated while being agitated at 37 deg.C for 24 hours, freezed at -40 deg.C, and dried. In an immunity measurement method, in which a carrier on which the antibody (or antigen) thus obtained is fixed is used, specific components in a fluid sample can be quantified accurately.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an immobilization carrier used for measuring a trace component in a fluid sample, particularly a specific trace component in a biological fluid sample.

[0002]

BACKGROUND OF THE INVENTION Various analytical methods have been developed as a method for detecting a substance contained in a trace amount in a biological fluid sample. As one of the analysis methods, there is one that uses an immune reaction as its principle. Various measuring methods using this principle have been developed and are known to be highly accurate.

That is, Berson and Y in 1958
Since allow succeeded in measuring serum insulin using bovine insulin labeled with radioisotope I and anti-insulin antibody in serum of diabetic patients, immunoassays using radioisotopes have been widely used. I've been taken. Since then, various substances other than radioisotopes have been developed as labeling substances. Examples include enzymes, enzyme substrates, coenzymes, enzyme inhibitors, bacteriophages, circulating reactants, metal and organometallic complexes, organic prosthetic groups, chemiluminescent reactants and fluorescent molecules.

By the way, when immunoassay is performed, a carrier on which an antibody (or an antigen) is immobilized is used. This immobilized carrier is produced by putting the carrier in an antibody (or antigen) solution and adsorbing the antibody (or antigen) on the carrier. However, in immunoassays using the thus obtained immobilized carrier, it has been found that there are problems in that the measured values vary greatly and reproducibility is low.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a technique capable of accurately and precisely quantifying a specific component in a fluid sample. The object of the present invention is to immerse a carrier in an antibody (or antigen) solution, and to operate a container containing the antibody (or antigen) solution at a speed such that the carrier does not substantially shift. It is achieved by a method for producing an immobilization carrier, which comprises:

[0006] Further, the step of immersing the carrier in the basket in the antibody (or antigen) solution and the step of immersing the basket in the basket at the speed at which the carrier in the basket does not substantially shift
And an actuating step that can be moved relative to the solution. The carrier to which the antibody (or antigen) is physically and / or chemically bound is preferably porous, and the material for such carrier is diatomaceous earth, titanium dioxide, barium sulfate, zinc oxide, Examples thereof include various synthetic resins such as lead oxide, microcrystalline cellulose, silica sand, glass, silica gel, crosslinked dextrin, crosslinked polyacrylamide, agarose, crosslinked agarose, and polystyrene. And if it is a porous carrier of such a material, an antibody (or an antigen)
The primary shape on which is immobilized may be particles (beads), rods, or plates,
Particles (beads) are preferable.

The antibody or antigen can be bound chemically or / and physically directly or indirectly to these porous insolubilized carriers. For example, by immersing a carrier such as polystyrene beads in a container containing an antibody (or antigen) solution, and then rotating the container at a low speed so that the carrier hardly moves, or as shown in FIG. The polystyrene beads 2 contained in the mesh basket 1 are suspended and dipped in the container 3 containing the antibody or antigen solution, and the basket 1 or container 3 is rotated at such a speed that the polystyrene beads 2 are not displaced. As a result, the antibody (or antigen) can be uniformly bound to the polystyrene beads 2. At this time, flow force may be applied only to the antibody (or antigen) solution without applying displacement force to the polystyrene beads 2, for example, stirring means by a stirrer, jet force applying means, convection by heat is applied. May be. Regarding the binding technology in other respects, 1976, published by Kodansha, Ichiro Senbata et al., "Experiment and Application Affinity Chromatography" (No. 1)
(Printed), published in 1975 by Kodansha, Makoto Yamazaki et al., "Affinity Chromatography" (1st edition), edited by 2 members. After the binding reaction, a protein that does not participate in the specific reaction to be measured can be loaded for the purpose of eliminating the nonspecific reaction of the labeled antibody (or antigen). Typical examples thereof include normal mammalian and avian serum proteins, albumin, skim milk, lactic acid fermentation products, collagen, and degradation products thereof.

In the immunoassay, any form of solution, colloidal solution and the like can be used as a sample, but preferably a biological fluid sample such as blood, plasma, serum, cerebrospinal fluid, saliva, amniotic fluid, milk, urine , Sweat, gravy, etc. A specific component in a measurable fluid sample is a substance (substance group) in which a substance that specifically binds to the specific component may exist.
Is. That is, examples thereof include polypeptides, proteins, complex proteins, polysaccharides, lipids, complex lipids, nucleic acids, hormones, vitamins, drugs, antibiotics, pesticides and the like. Specifically, JP-A-62-90539 and JP-A-63-1
The substances (substance groups) described in Japanese Patent No. 31062 can be mentioned, but the substances are not limited thereto.

As the labeling substance used in the immunoassay, those generally used in ordinary immunoassays can be used, and examples thereof include radioactive substances, luminescent substances and fluorescent substances, and also enzymes, enzyme substrates, enzymes and enzymes. Substances that change the activity of precursors (enzyme inhibitors, prosthetic groups, coenzymes), enzyme precursors, apoenzymes, etc. can also be used. Specific examples of the substance include those described in JP-A-62-90539, preferably an enzyme or a fluorescent substance, more preferably β-D-galactosidase, alkaline phosphatase, Peroxidase, glucose oxidase, glutamate dehydrogenase,
It is an enzyme such as amylase. When these enzymes are used as the labeling substance, known ones can be used for the enzyme reaction system and the color development system. Specifically, JP-A-61-292060,
JP-A-62-90539, JP-A-63-1310
62, JP-A-63-45562, Japanese Patent Application No. 6
The substances (substance groups) described in the specification of JP-A-3-219893 are included, but the present invention is not limited thereto. Then, the binding of these labeling substances to the antibody (antigen) can be performed by known reagents and methods known to those skilled in the art,
For example, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, "Enzyme Immunoassay (3rd Edition), Medical Institute, 1987" and the Japanese Society for Clinical Pathology, "Clinical Pathology" Special Issue No. 53, "Immunoassays for clinical tests" -Technology and application-, Clinical Pathology Society,
The method described in “1983” can be referred to.

The origin of the antibody used in the present invention is not particularly limited, and the antigen is administered to mammals,
The antiserum and ascites fluid obtained by immunization may be used as they are, or may be a conventionally known method such as sodium sulfate precipitation method, ammonium sulfate precipitation method, gel filtration method using Sephadex gel, ion exchange cellulose chromatography method, electrophoresis method, etc. Shunsuke Bipolar "Immunochemistry" Nakayama Shoten pp74-8
8) and can be used after purification. Alternatively, hybrid cells (hybridomas) can be obtained from spleen cells or myeloma cells (myeloma) of mammals (eg, mice) sensitized with an antigen to prepare monoclonal antibodies, which can specifically bind to specific components. Use as a substance is preferable because the specificity is improved. In addition, these antibodies are IgG, Ig
Fractions of M, IgA, IgD, and IgE can be used, or Fab and Fa can be obtained by treating these antibodies with an enzyme.
It may be used as an active antibody fragment such as b ′ or F (ab ′) ₂ . Furthermore, these antibodies may be used alone or in combination of a plurality of antibodies.

The antigen used in the present invention reacts with a specific antibody and contains a hapten and its derivative. The reaction type by the immunoassay method includes, but is not particularly limited to, the competitive method, the antibody method, the sandwich method and the like. Further, an immunoassay method using another bioactive substance (for example, biotin, avidin) can also be applied. In the present specification, an immune reaction is mentioned as a reaction type for measuring a specific component in a fluid sample, but a specific reaction of a substance showing biological activity according to the immune reaction (this specific reaction is also referred to in the present specification). It is also possible to utilize (included in immune reaction).

The signal caused by the label can be detected by an absorbance method (colorimetric method), a fluorescence method, a luminescence method or a radioactivity measuring method. As a measuring method, a rate measurement for measuring a change with time of the signal is carried out. Method or an endpoint measurement method of measuring a signal after a certain period of time. The absorbance method is preferred, and the absorbance method (colorimetric method) can utilize ultraviolet light, visible light, and near infrared light.

[0013]

[Example 1]

[Preparation of immobilized beads by rotating basket] Monoclonal antibody against cancer-associated galactosyltransferase (GAT) prepared by the method described in Japanese Patent Application No. 2-54567 was dissolved in 1M NaCl 0.1M. Was dissolved in the carbonate buffer (pH 9.5) of 10 μg / ml, and 1000 ml of this antibody solution was placed in a plastic container 3 as shown in FIG.

Next, polystyrene beads (Sekisui Chemical # 80) 2 were placed in the mesh-shaped basket 1, the basket 1 was suspended in the plastic container 3, and the polystyrene beads 2 were dipped in the antibody solution. And basket 1
Was rotated at, for example, 3 rpm to immobilize the antibody at 4 ° C. overnight. At this rotation speed, the polystyrene beads 2 do not move in the basket 1,
In the rotating coordinate system, it can be regarded as stationary, that is, in the rotating coordinate system, only the antibody solution can be regarded as flowing.

Then, after washing with a phosphate buffer (PBS), 0.002% of p-hydroxybenzoic acid-n was added.
-Immerse in 1% BSA-PBS solution containing -butyl, 3
Incubate at 7 ° C for 24 hours with agitation, then-
It was frozen at 40 ° C. and freeze-dried. [Example 2] [Preparation of immobilized beads by rotating container at low speed] 10 µg of a monoclonal antibody against cancer-associated galactosyltransferase (GAT) in 0.1 M carbonate buffer (pH 9.5) in which 1 M NaCl was dissolved. / Ml, and 1000 ml of this antibody solution was placed in a plastic container.

Polystyrene beads (Sekisui Chemical # 8
0) was also put in a plastic container, and this plastic container was rotated at low speed (0.03 to 0.5 rpm). The antibody was immobilized overnight at 4 ° C.
0.002 after washing with phosphate buffer (PBS)
% -N-butyl p-hydroxybenzoate 1
% BSA-PBS solution, incubated at 37 ° C. for 24 hours with stirring, then frozen at −40 ° C. and lyophilized.

[Comparative Example 1] [Preparation of immobilized beads by standing] The above-mentioned Example 1 was not carried out, that is, standing was carried out, and the antibody solution and BSA were prepared.
It was prepared by dipping polystyrene beads (Sekisui Chemical # 80) in the solution. [Comparative Example 2] [Preparation of immobilized beads by rotating the entire container at high speed] The same procedure as in Example 2 was carried out at a rotation speed of 1 rpm.

[Immunoassay] The antibody-immobilized beads prepared in each of the above-mentioned examples were used in an amount of 10 U / ml, 30 U / ml, 90 U / m.
0.05 ml of GAT standard solution and 50 mM phosphate buffer solution (pH 6.5) in which 1 M sodium chloride was dissolved.
2 ml was put into a mixed solution and incubated at 37 ° C. for 2 hours.

After washing with PBS, an anti-GAT monoclonal antibody that recognizes a site different from the immobilized antibody was analyzed by Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, “Enzyme Immunoassay (3rd
Ed.), Igakushoin, 1987 ”, 1% BSA-PBS solution of peroxidase-labeled anti-GAT monoclonal antibody labeled by the method described in pp 108, 109 (0.5 μg /
0.25 ml) was added and incubated at room temperature for 1 hour.

After washing with PBS, a citrate-phosphate buffer solution containing 3 mg / ml o-phenylenediamine dissolved therein (p
0.3 ml of H5.0, containing 0.02% hydrogen peroxide) was added, and color was developed for 30 minutes at room temperature. And 1N sulfuric acid 1
The color reaction was stopped with ml and the absorbance at 492 nm was measured. The standard solution of each concentration was measured at n = 50 and the coefficient of variation CV was determined. The results are shown in the table below.

Table 10 U / ml 30 U / ml 90 U / ml Rotational speed Example 1 3.1% 2.2% 2.1% Example 2 4.3% 3.1% 2.8% 0.5 rpm 3. 6% 3.3% 3.3% 0.2 rpm 3.3% 3.5% 2.5% 0.1 rpm 4.4% 3.3% 3.6% 0.03 rpm Comparative Example 1 10.7% 8.3% 6.4% Comparative Example 2 8.2% 5.7% 5.1% 1.0 rpm According to this, the antibody (or antigen) obtained by the present invention
It can be seen that the immunoassay method using a carrier on which is immobilized has a remarkably small coefficient of variation, and a specific component in a fluid sample can be accurately, accurately and reproducibly quantified.

[Brief description of drawings]

FIG. 1 is a schematic view of an apparatus used in the method for producing an immobilization carrier of the present invention.

[Explanation of symbols]

 1 Basket 2 Polystyrene beads 3 Container

Claims (2)

[Claims] 1. A step of immersing a carrier in an antibody (or antigen) solution, and an operation step of moving a container containing the antibody (or antigen) solution at a speed at which the carrier does not substantially shift. A method for producing an immobilization carrier, comprising: 2. The carrier in the basket is an antibody (or antigen)
An immobilization carrier comprising: a step of immersing in a solution; and an operation step of moving the basket relative to the antibody (or antigen) solution at a speed at which the carrier in the basket does not substantially shift. Production method.