JPH04363662A - Manufacture of immobilizing carrier - Google Patents

Abstract

PURPOSE:To accurately quantify the specific component in a fluid sample with good reproducibility by a method wherein a carrier is immersed in an antibody (antigen) solution to be allowed to stand and the solution is fluidized to manufacture an immobilizing carrier. CONSTITUTION:A carrier to which an antibody (or antigen) is physically and/or chemically bonded is pref. porous and, as the material thereof, diatomaceous earth, titanium dioxide or a synthetic resin such as polystyrene is designated and said carrier pref. has a granular (bead like) shape. For example, a mesh-like container 3 having legs in which polyethylene beads 2 are received is inserted in a container 1 in which a solution of an antibody or an antigen is received and the beads 2 are immersed in the solution. A magnetic stirrer 3 is rotated in this state and the antibody or antigen solution is fluidized in a stationary state imparting no rotatory power to the beads 2 to uniformly bond the antibody or antigen to the beads 2.

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 for use in measuring trace components in fluid samples, particularly specific trace components in biological fluid samples.

0002

BACKGROUND OF THE INVENTION Various analytical methods have been developed for detecting trace amounts of substances contained in biological fluid samples. One of these analytical methods is based on immune reactions. Various measurement methods using this principle have been developed and are known to be highly accurate.

[0003] That is, in 1958, Berson and Y.
Since ``allow'' succeeded in measuring insulin in serum using bovine insulin labeled with radioisotope I and anti-insulin antibodies in the serum of diabetic patients, immunoassay methods using radioisotopes have been widely used. I came here. Since then, various labeling substances other than radioactive isotopes have been developed. Examples include enzymes, enzyme substrates, coenzymes, enzyme inhibitors, bacteriophages, cycling reactants, metal and organometallic complexes, organic prosthetic groups, chemiluminescent reactants, fluorescent molecules, and the like.

[0004] When immunoassays are performed, a carrier on which antibodies (or antigens) are immobilized is used. This immobilization carrier is produced by placing the carrier and a stirrer in an antibody (or antigen) solution, stirring by applying stirring force to the stirrer, and adsorbing the antibody (or antigen) onto the carrier. There is. However, it has been found that immunoassays using immobilized carriers obtained in this manner have the problem of large fluctuations in the measured values and low reproducibility.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a technique that can accurately quantify specific components in a fluid sample with high precision and reproducibility. The purpose of this invention is to obtain antibodies (or antigens)
This is achieved by a method for producing an immobilized carrier, which is characterized by comprising the steps of immersing the carrier in a solution, and allowing the carrier to stand substantially while applying a fluid force to the antibody (or antigen) solution.

[0006] The carrier to which the antibody (or antigen) is physically and/or chemically bonded is preferably porous, and examples of such carrier materials include diatomaceous earth, titanium dioxide, barium sulfate, Examples 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 is made of such a material, antibodies (
The primary shape in which the particles (or antigen) are immobilized may be in the form of particles (beads), rods, or plates, but particles (beads) are preferable.

[0007] Antibodies or antigens can be chemically and/or physically bound to these porous insolubilized carriers directly or indirectly. For example, as shown in Figure 1,
A mesh-like (basket-like) legged container 3 in which polystyrene beads 2 are roughly placed is placed in a container 1 containing an antibody or antigen solution, and the polystyrene beads 2 are immersed in the antibody or antigen solution. By placing a magnetic stirrer 4 at the bottom of the beads 1 and rotating the magnetic stirrer 4 in this state, the antibody (or antigen) solution is made to flow in a stationary state without applying rotational force to the polystyrene beads 2. , the antibody (or antigen) can be uniformly bound to the polystyrene beads 2. still,
The flow of the antibody (or antigen) solution is not limited to stirring means using a stirrer, but may also be achieved by, for example, applying a jet force or convection by heat; any means may be used. Regarding bonding techniques related to other points, see "Experiments and Applied Affinity Chromatography" (1st printing) published by Kodansha in 1976, edited by Ichiro Chibata and two others, and "Affinity Chromatography" published by Kodansha in 1975, edited by Makoto Yamazaki and others. You can refer to "Graphics" (1st edition). After the binding reaction, a protein that is not involved in the specific reaction to be measured can be supported for the purpose of eliminating non-specific reactions of the labeled antibody (or antigen). Typical examples include normal serum proteins of mammals and birds, albumin, skim milk, lactic acid fermentation products,
Examples include collagen and decomposed substances thereof.

[0008] Then, the antibody obtained as described above (
The insolubilized particulate antibody (or insolubilized particulate antigen) physically and/or chemically bound to the antibody (or antigen) labeled with an enzyme, etc., and the sample are brought into contact, subjected to an immunoreaction, and subjected to B/F separation. After that, by measuring the enzyme activity of the enzyme label that exists in a free state without binding to the insolubilized particulate antibody (or insolubilized particulate antigen) using, for example, a dry analytical element, the antigen (or antibody) in the sample can be easily analyzed. So, the sensitivity,
Accurate measurements are made with high accuracy, precision and reproducibility.

[0009] In immunoassays, any form of solution, colloidal solution, etc. can be used as a sample, but fluid samples of biological origin, such as blood, plasma, serum, cerebrospinal fluid, saliva, amniotic fluid, milk, and urine, are preferably used. , sweat, meat juices, etc. A specific component in a fluid sample that can be measured is a substance (substance group) for which there may be a substance that specifically binds to the specific component. That is, polypeptides, proteins, complex proteins,
Examples include polysaccharides, lipids, complex lipids, nucleic acids, hormones, vitamins, drugs, antibiotics, agricultural chemicals, and the like. Specifically, Japanese Patent Application Laid-open No. 62-90539 and Japanese Patent Application Laid-Open No. 63-131
Examples include the substances (substance group) described in Publication No. 062, but are not limited thereto.

Labeling substances used in immunoassays include, for example, enzymes, enzyme substrates, substances that change the activity of enzymes and enzyme precursors (enzyme inhibitors, prosthetic groups, coenzymes), enzyme precursors, and apoenzymes. , fluorescent substances, etc. As a specific substance, JP-A No. 62-90539
Examples include those described in Japanese Patent Publication No. 2003-100003, preferably enzymes or fluorescent substances, and more preferably enzymes such as β-D-galactosidase, alkaline phosphodase, peroxidase, glucose oxidase, glutamate dehydrogenase, and amylase. be. When these enzymes are used as labeling substances, known enzyme reaction systems and coloring systems can be used. Specifically, Japanese Patent Publication No. 61-2920
No. 60, JP-A-62-90539, JP-A-6
Substances described in Publication No. 3-131062, Japanese Unexamined Patent Application Publication No. 63-45562, and Japanese Patent Application No. 219893-1983 (
(group of substances), but is not limited to these. Binding of these labeling substances to antibodies (antigens) can be carried out using known reagents and methods known to those skilled in the art. 2nd edition), Igakushoin, 1978.”
``Clinical Pathology'' Special Issue No. 53 edited by the Japanese Society of Clinical Pathology
Reference may be made to the method described in "Immunoassay for Clinical Testing - Techniques and Applications -" published by Clinical Pathology Publishing Society, 1983.

[0011] The origin of the antibody used in the present invention is not particularly limited.
Antiserum obtained by immunization, ascites fluid may be used directly, or conventionally known methods such as sodium sulfate precipitation, ammonium sulfate precipitation, gel filtration with Sephadex gel, ion exchange cellulose chromatography, electrophoresis, etc. It can be used after purification using Shunsuke's "Immunochemistry" Nakayama Shoten pp. 74-88). Alternatively, hybrid cells (hybridoma) are obtained from spleen cells or myeloma cells (myeloma) of a mammal (e.g. mouse) infected with an antigen, and a monoclonal antibody is created, which is then used as a substance that can specifically bind to a specific component. It is preferable to use it as the specificity improves. In addition, these antibodies are IgG,
IgM, IgA, IgD, and IgE fractions can be used, or these antibodies can be treated with enzymes to produce Fab, Fab.
It may also be used in the form of active antibody fragments such as b' or F(ab')2. Furthermore, these antibodies may be used alone or in combination.

The antigen used in the present invention reacts with specific antibodies and contains haptens and derivatives thereof. Examples of reaction types by immunoassay methods include competitive methods, two-antibody methods, sandwich methods, etc., but are not particularly limited. Furthermore, immunoassay methods using other biologically active substances (eg, biotin, avidin) can also be applied. In this specification, an immune reaction is mentioned as a reaction type for measuring a specific component in a fluid sample. It is also possible to utilize the immune response (inclusive of immune response).

[0013] The signal caused by the label can be detected by an absorbance method (colorimetric method), a fluorescence method, or a luminescence method, and measurement methods include a rate measurement method that measures changes in the signal over time, or a detection method that measures changes in the signal over time. can be measured using an endpoint measurement method that measures the signal. Preferably, the absorbance method is used,
The absorbance method (colorimetric method) can use ultraviolet light, visible light, and near-infrared light. For example, when using serum and plasma as fluid samples, green light is used to reduce the influence of light absorption by serum and plasma. Preferably, red light or near-infrared light is used.

[0014]

[Example 1]

[Preparation of immobilized beads with stirring using a stirrer] A monoclonal antibody against cancer-related galactose transferase (GAT) was dissolved in 0.1 M carbonate buffer (pH 9.5) containing 1 M NaCl to a concentration of 10 μg/ml. 1000 ml of this antibody solution was placed in a plastic container 1 as shown in FIG.

Next, polystyrene beads (Sekisui Chemical #80) 2 were placed in a basket-shaped container 3 with legs, the container 3 with legs was immersed in an antibody solution, and the polystyrene beads 2 were immersed in the antibody solution. Then, stirring was performed using a stirrer bar 4 placed at the bottom of the plastic container 1 (in this stirring, the polystyrene beads 2 remained stationary and only the antibody solution was stirred and flowing), and the mixture was heated at 4°C. Antibodies were immobilized over a day and night.

[0016] Thereafter, after washing with phosphate buffer (PBS), it was immersed in a 1% BSA-PBS solution, incubated at 37°C for 24 hours with stirring in the same manner as in the above stirring process, and then stored at 4°C. . [Example 2] [Preparation of immobilized beads using thermal convection] 10 μg/ml of a monoclonal antibody against cancer-related galactose transferase (GAT) was added to 0.1 M carbonate buffer (pH 9.5) in which 1 M NaCl was dissolved. 1000 ml of this antibody solution was put into a plastic container.

Next, polystyrene beads (Sekisui Chemical #80) were placed in a donut-shaped basket-like container with legs, and the container was immersed in an antibody solution, and the polystyrene beads were immersed in the antibody solution. This container is equipped with a heat source in the center, and when the power switch is turned on and the temperature is set to, for example, 10°C, the solution in the plastic container is subjected to convection that flows in the vertical direction, that is, an upward flow on the center side. However, the structure is such that a downward flow is caused on the outer peripheral wall side of the plastic container.

[0018] Then, the power switch was turned on and the antibody was immobilized overnight at 4°C while causing convection to flow in the antibody solution in the plastic container. Note that in this state, the polystyrene beads remain stationary, and only the antibody solution is flowing. After this, after washing with phosphate buffer (PBS), 1% BSA-PB
The cells were immersed in S solution, incubated at 37°C for 24 hours with stirring, and then stored at 4°C.

[Comparative Example 1] [Preparation of immobilized beads by standing] Polystyrene beads (Sekisui Chemical #80) were immersed in the antibody solution and BSA solution without stirring as in Example 1, that is, by standing still. It was made by [Comparative Example 2] [Preparation of immobilized beads by rotating the entire container] Polystyrene beads (Sekisui Chemical #80), antibody solution, and BSA solution were incubated by rotating the entire container using a sealable container. Stir (both polystyrene beads and antibody solution flow during this stirring),
Created.

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

After washing with PBS, a 1% BSA-PBS solution (0.5 μg/ml) of a peroxidase-labeled anti-GAT monoclonal antibody that recognizes a site different from that of the immobilized antibody was added.
) was added and incubated for 1 hour at room temperature. After washing with PBS, add 3 m of 0.02% H2O2.
g/ml and 0.3 ml of o-phenylenediamine solution were added, and the color was allowed to develop at room temperature for 30 minutes. Then, the color reaction was stopped with 1 ml of 1N sulfuric acid, and the absorbance at 492 nm was measured.

[0022] Standard solutions of each concentration were measured with n=25, and the coefficient of variation CV was determined, and the results are shown in the table below.
Table 10U/m
l 30U/ml 90U/ml
Example 1 3.2% 3.0
% 2.6% Example 2
3.9% 2.8%
3.0% Comparative Example 1 10.7
% 8.3% 6.4%
Comparative example 2 8.6%
5.0% 4.7% According to this, the immunoassay method using the carrier on which the antibody (or antigen) obtained by the present invention is immobilized has a significantly small coefficient of variation, and it is possible to identify It can be seen that the components can be accurately quantified with good precision and reproducibility.

[Brief explanation of the drawing]

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

[Explanation of symbols]

1 Container 2 Polystyrene beads 3 Container with legs 4 Magnetic stirrer

Claims (1)

[Claims] 1. Immobilization characterized by comprising the steps of immersing a carrier in an antibody (or antigen) solution, and allowing the carrier to remain substantially still while applying a fluid force to the antibody (or antigen) solution. Method for manufacturing carrier.