JPH0510952A - Preparation of carrier containing immobilized substance - Google Patents

Abstract

PURPOSE:To enable a specific constituent within a fluid sample to be determined with improved accuracy and reproducibility by allowing a carrier where an antibody (or antigen) is immobilized to be frozen and dried. CONSTITUTION:A fixation carrier is prepared by enabling a carrier where an antibody (or an antigen) is immobilized to be frozen and dried. Namely, after freezing with an inactive and extremely low-temperature refrigerant, pressure-reduction and drying are performed. It is desirable that a carrier where the antibody (or antigen) is connected physically/chemically is porous. The antibody or antigen can be directly or indirectly connected to these porous insolubilized carrier chemically/physically. Then, after an insolubilized finely divided antibody (or antigen) where the obtained antibody (or antigen) is connected physically/chemically and the antibody (antigen) which is labeled by an enzyme, etc., as well as a sample are subjected to contact and immunity reaction and then B/F separation, enzyme activation of an enzyme labeled body which exists in liberated state without being connected to the insolubilization finely divided antibody (antigen) is measured.

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 immobilization carrier is prepared by placing the carrier in an antibody (or antigen) solution and stirring it so that the antibody (or antigen) is adsorbed on the carrier.
It is then manufactured by freeze-drying. However, in the immunoassay using the thus obtained immobilization carrier, the measured value may vary greatly,
It turns out that there are problems with low reproducibility.

[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. This object of the present invention is to provide an antibody (or antigen)
Is a method for producing an immobilized carrier by freeze-drying the immobilized carrier, which is achieved by a method for producing an immobilized carrier, which comprises freezing with an inert cryogenic refrigerant and then drying under reduced pressure. It

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, or the like. Examples thereof include various synthetic resins such as zinc oxide, lead oxide, microcrystalline cellulose, silica sand, glass, silica gel, crosslinked dextrin, crosslinked polyacrylamide, agarose, crosslinked agarose and polystyrene.
If the porous carrier of such a material is used, the primary shape on which the antibody (or antigen) is immobilized may be particles (beads), rods or plates, but The shape of (beads) is preferable.

The antibody or antigen can be bound chemically or / and physically directly or indirectly to these porous insolubilized carriers. Although conventional means may be directly adopted as the binding method, for example, a mesh-shaped (basket-shaped) container with legs in which a carrier such as polystyrene beads is roughly contained in a container containing a solution of an antibody or an antigen is used. Put, soak the carrier such as polystyrene beads in the antibody or antigen solution, and keep the magnetic stirrer at the bottom of the container.In this state, rotate the magnetic stirrer and rotate the carrier such as polystyrene beads with a rotating force. A means for uniformly binding the antibody (or antigen) to a carrier such as polystyrene beads by flowing an antibody (or antigen) solution in a stationary state in which no antibody is added is preferable.
The flow of the antibody (or antigen) solution is not limited to stirring means by a stirrer, for example, a jet force may be applied, and any means may be used. Then, the carrier such as polystyrene beads is allowed to stand, and after the antibody or antigen is bound to the carrier by applying a fluid force to the antibody or antigen solution, the immobilized carrier is freeze-dried. , Take out the mesh-shaped (basket-shaped) container in which the immobilization carrier is placed, and immerse the taken-out mesh-shaped (basket-shaped) container as it is in an inert cryogenic refrigerant such as liquid nitrogen to immobilize it. The immobilized carrier according to the present invention can be obtained by flash-freezing the carrier and drying it under reduced pressure. The inert cryogenic refrigerant preferably has a cooling temperature of about −150 ° C. or lower, and liquid nitrogen is most practically used because of this.

[0008] As for the combining technique regarding other points,
Published by Kodansha in 1976, Ichiro Chibata and 2 others, "Experimentation and Application Affinity Chromatography" (first edition),
In 1975, you can refer to "Affinity Chromatography" (1st edition) published by Kodansha and edited by Makoto Yamazaki and 2 others. 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.

Then, the antibody (or antigen) obtained as described above is physically and / or chemically bound to the insolubilized fine particle antibody (or the insolubilized fine particle antigen) and the antibody (or antigen) labeled with an enzyme or the like. After contacting with the sample, immunoreacting, and performing B / F separation, the enzyme activity of the enzyme-labeled substance existing in a free state without binding to the insolubilized fine particle antibody (or insolubilized fine particle antigen) is measured by, for example, a dry analytical element Thus, the antigen (or antibody) in the sample can be accurately measured by a simple operation with good sensitivity, accuracy, precision and reproducibility.

In the immunoassay, any form of solution, colloidal solution and the like can be used, but preferably a biological fluid sample such as blood, plasma, serum, cerebrospinal fluid, saliva, amniotic fluid, milk and 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.

Examples of labeling substances used in immunoassay include enzymes, enzyme substrates, substances that change the activity of enzymes and enzyme precursors (enzyme inhibitors, prosthetic groups, coenzymes), enzyme precursors, apoenzymes. , Fluorescent substances, radioisotopes, etc. Specific substances include those described in JP-A-62-90539 and the like, preferably an enzyme or a fluorescent substance, more preferably β-D-galactosidase, alkaline phosphatase, peroxidase, Enzymes such as glucose oxidase, glutamate dehydrogenase and 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 and JP-A-62-9053.
9, JP-A-63-131062, JP-A-6-6
Examples thereof include substances (substance groups) described in JP-A-3-45562 and Japanese Patent Application No. 63-219893, 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 and Kawai.
Tadashi, Kiyoshi Miyai, "Enzyme-linked immunosorbent assay (2nd edition), Medical Shoin, 1978" and Japan Society for Clinical Pathology, "Clinical Pathology" Extra Edition Special Issue No. 53, "Immunoassays for Clinical Testing-
Techniques and applications-, Clinical Pathology Publication Society, 1983 ”, etc. 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, a substance capable of specifically binding to a specific component by preparing a hybrid antibody (hybridoma) from spleen cells or myeloma cells (myeloma) of a mammal (eg, mouse) infected with an antigen, and producing a monoclonal antibody When used as, the specificity is improved, which is preferable. 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 or a luminescence method. As a measuring method, a rate measuring method for measuring a change with time of the signal or after a certain period of time is used. Can be measured by an endpoint measurement method that measures the signal of. The absorbance method is preferred,
The absorbance method (colorimetric method) can utilize ultraviolet light, visible light, and near infrared light.

[0015]

[Example 1]

[Freeze-drying using liquid nitrogen] A mouse monoclonal antibody against cancer-associated human galactosyltransferase (GAT) was dissolved in 1M sodium chloride to give 0.1M.
It was dissolved in a carbonate buffer solution (pH 9.5) at 10 μg / ml, and polystyrene beads (Sekisui Chemical # 8) were added to the solution.
0) was immersed and fixed overnight at 4 ° C. After washing this with phosphate buffered saline (PBS), 1% B
It was immersed in the SA-PBS solution at 37 ° C. for 24 hours.

1000 pieces of the antibody-immobilized polystyrene beads thus obtained were placed in a basket-like container (bottom 1
0 cm × 10 cm, height 10 cm), the container was immersed in liquid nitrogen for 3 minutes, and then cooled and dried under reduced pressure.
In addition, the mouse anti-GAT antibody is Japanese Patent Application No. 2-54567 (the title of the invention "a monoclonal antibody specific to a cancer-related human-derived galactosyltransferase, a hybridoma producing the same, and a cancer-related human-derived human in a specimen using the same. Method for measuring galactosyltransferase ”(filed on Mar. 6, 1990). That is, Cancer Rese
In accordance with the method described in Arch 48, 5325 (1988), anti-GAT monoclonal antibody is produced by cell fusion according to a conventional method using spleen cells obtained by immunizing mice with GAT purified from ascites of cancer patients as an antigen. A hybridoma was obtained and obtained from this.

[Comparative cooling 1] [Freeze-drying by freezer cooling at -20 ° C] Example 1
1% of antibody-immobilized polystyrene beads obtained in the same manner as
It was immersed in a BSA-PBS solution at 37 ° C. for 24 hours. After removing 1% BSA-PBS from 1000 polystyrene beads, the mixture was allowed to stand in a freezer at -20 ° C for 30 minutes, and then cooled and dried under reduced pressure.

[Immunoassay GAT measurement] The polystyrene beads obtained in each of the above-mentioned examples were used in an amount of 10 U / ml, 30 U / ml,
0.05 ml of 90 U / ml GAT standard solution and 50 mM phosphate buffer solution (pH 6.
5) Place in a mixed solution of 0.2 ml and 2 at 37 ° C.
Incubated for hours. After washing with PBS, 0.25 ml of a 1% BSA-PBS solution (0.5 μg / ml) of a peroxidase-labeled anti-GAT monoclonal antibody that recognizes a site different from the immobilized antibody was added, and the mixture was incubated at room temperature for 1 hour. 0.02% after washing with PBS
Citrate-phosphate buffer (pH 5.0) 0.3 ml containing H ₂ O ₂ and 3 mg / ml o-phenylenediamine
Was added and color was developed for 30 minutes at room temperature. Then, the color reaction was stopped with 1 ml of 1N sulfuric acid, and the absorbance at 492 nm was measured.

The coefficient of variation CV was obtained by measuring each of the samples at n = 25, and the results are shown in the table below. Table 10 U / ml 30 U / ml 90 U / ml Example 1 5.2% 4.8% 4.2% Comparative Example 1 8.7% 6.6% 5.7% According to the present invention Antibody (or antigen)
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.

Claims (1)

Claim: What is claimed is: 1. A method for producing an immobilized carrier by freeze-drying a carrier on which an antibody (or an antigen) is immobilized, which comprises decompressing after freezing with an inert cryogenic refrigerant. A method for producing an immobilized carrier, which comprises drying.