JPH04357458A - Immunoassay method - Google Patents

Abstract

PURPOSE:To obtain an immunoassay method where information to be obtained is much even if the number of measurements of a specific minute amount of constituent within a fluid sample is small. CONSTITUTION:A title item allows a specific constituent within a fluid sample to be analyzed by enabling it to be subjected to immunity reaction using an insoluble carrier where a substance which can be bonded to the specific constituent peculiarly is fixed. A reaction container where immunity reaction is performed and an insoluble substance which is loaded into this reaction container are used as an insoluble carrier.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method for 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. That is, in 1958 Berson and Y.
Immunoassay methods using radioisotopes have been widely used since ``allow'' succeeded in measuring insulin in serum using bovine insulin labeled with radioisotope I and anti-insulin antibodies in serum of diabetic patients. I came here.

[0003] 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. By the way, until now, for example, if you wanted to obtain information about two items, you had to repeat the immune reaction twice, that is, you had to do it in the same way for each item. , it took a lot of time.

[0004] Furthermore, when a sample is measured and the range is exceeded, the sample must be diluted and remeasured, which is time-consuming.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide an immunoassay method that provides a large amount of information even when a specific trace component in a fluid sample is measured a small number of times. The purpose of the present invention is to analyze a specific component in a fluid sample by immunoreacting the specific component in a fluid sample using an insoluble carrier immobilized with a substance that can specifically bind to the specific component. This is achieved by an immunoassay method characterized in that, as the insoluble carrier, both a reaction container in which an immune reaction is performed and an insoluble substance placed in the reaction container are used.

[0006] If the substance that can specifically bind to the specific component immobilized on the reaction container and the substance that can specifically bind to the specific component immobilized on the insoluble substance are different substances, It is possible to measure two items with a single immune reaction operation, which saves time and effort.In addition, a substance that can specifically bind to the specific component immobilized in the reaction container and an insoluble substance immobilized If the substance that can specifically bind to the specific component is the same substance, but the immobilized concentration of both is different, for example, if the substance is immobilized at a low concentration, a high value of the analyte can be measured, and when the substance is immobilized at a high concentration, With this method, it is possible to measure analytes with low values, which allows for a wider range of measurements.

[0007] In the present invention, any form of solution, colloidal solution, etc. can be used as the 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. The specific components in a fluid sample that can be measured by the present invention are:
A substance (substance group) in which there may be a substance that specifically binds to that particular component. That is, polypeptides, proteins,
Examples include complex proteins, polysaccharides, lipids, complex lipids, nucleic acids, hormones, vitamins, drugs, antibiotics, agricultural chemicals, and the like. Specifically, the substances (substance groups) described in JP-A-62-90539 and JP-A-63-131062 can be mentioned, but are not limited thereto.

As the labeling substance used in the present invention, those commonly used in ordinary immunoassay methods can be used, such as radioactive substances, luminescent substances, fluorescent substances, enzymes, etc. , substances that change the activity of enzymes and enzyme precursors (enzyme inhibitors, prosthetic groups, coenzymes), enzyme precursors, apoenzymes, etc. can also be used. Specific substances include those described in JP-A No. 62-90539, but enzymes such as β-D-galactosidase, alkaline phosphodase, peroxidase, glucose oxidase, glutamate dehydrogenase, and amylase are preferred. , or a fluorescent substance. When these enzymes are used as labeling substances, known enzyme reaction systems and coloring systems can be used. Specifically, JP-A-6
1-292060, JP 62-90539, JP 63-131062, JP 63-4
Examples include substances (substance groups) described in Japanese Patent Application No. 5562 and Japanese Patent Application No. 63-219893, but are not limited thereto.

[0009] Binding of these labeling substances to a substance that specifically binds to a specific component in a sample, such as an antibody (or antigen), can be carried out using known reagents and methods known to those skilled in the art. For example, Eiji Ishikawa, Tadashi Kawai, and Kiyoshi Miyai (eds. Enzyme Immunoassay (3rd edition), Igaku Shoin, 1978) and Japanese Society of Clinical Pathology (edited), "Clinical Pathology," Special Issue No. 53, "For Clinical Testing. The method described in ``Immunoassay - Techniques and Applications'', Clinical Pathological Publishing Society, 1983 can be referred to.

[0010] 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.

[0011] Reaction types for the immunoassay of the present invention include competitive methods, two-antibody methods, sandwich methods, etc., with sandwich methods being preferred. Furthermore, immunoassay methods using other biologically active substances (eg, biotin, avidin) can also be applied. In the present invention, an immune reaction is mentioned as a reaction type to measure a specific component in a fluid sample, but a specific reaction of a substance that exhibits biological activity similar to an immune reaction (in this specification, this specific reaction is also referred to as an immune reaction) It is also possible to use the reaction (inclusion in the reaction). Examples of combinations of substances that specifically bind include the following.

Enzyme and Substrate (Product) Enzyme and Inhibitor Enzyme and Prosthetic Group Enzyme and Coenzyme Enzyme and Allosteric Effector Antibody and Antigen Antibody and Protein A Lectin and Polysaccharide Lectin and Glycoprotein Nucleic Acid and Complementary Base Sequence Nucleic Acid and Histone Nucleic Acids and Nucleic Acids Nucleic Acids and Polymerases Hormones and Receptors Pyotin and Avidin (Streptavidin) Bioticin and Avidin (Streptavidin) Desthiobiotin and Avidin (Streptavidin) Oxybiotin and Avidin (Streptavidin) The antigens used in the present invention are It reacts with specific antibodies and contains haptens and their derivatives.

[0013] The insoluble carrier on which a substance that specifically binds to a specific component in a sample, such as an antibody (or antigen), is immobilized is a reaction vessel in which an immune reaction is carried out, an insoluble substance placed in this reaction vessel, and A major feature is that both are used. A test tube or microplate made of inorganic glass or plastic is usually used as a reaction vessel. The insoluble substance is usually in the form of particles (beads), sheets, rods, or fibers. Among them, granules are preferably used. Insoluble materials include agarose, cellulose, cross-linked dextran, polyacrylamide, cellulose, microcrystalline cellulose, cross-linked agarose, cross-linked polyacrylamide, glass, silica gel, diatomaceous earth, titanium dioxide, barium sulfate, zinc oxide, lead oxide, Silica sand, various synthetic resins such as polystyrene, and magnetic fine particles can be used. Preferably agarose, crosslinked agarose, crosslinked dextran, polyacrylamide, crosslinked polyacrylamide, glass, silica gel, polystyrene,
Cellulose, microcrystalline cellulose, etc., and more preferably polyacrylamide, crosslinked polyacrylamide, polystyrene, microcrystalline cellulose, etc.

[0014] Antibodies (or antigens) can be chemically and/or physically bound to these insoluble carriers directly or indirectly by methods known to those skilled in the art. Regarding binding methods, in 1976, published by Kodansha, edited by Ichiro Chibata and two others, "Experiments and Applications Affinity Chromatography" (
1st printing), 1975, published by Kodansha, Makoto Yamazaki et al.2
You can refer to the famous book "Affinity Chromatography" (1st edition). After the binding reaction, proteins that are not involved in the specific reaction to be measured can be supported in order to eliminate non-specific reactions of the labeled antibody (or antigen). Typical examples thereof include normal serum proteins of mammals and birds, albumin, skim milk, lactic acid fermentation products, collagen, and their decomposed substances.

[0015] Furthermore, the above-mentioned non-specific adsorption-inhibiting protein is not only supported on an insolubilized carrier, but also added in a certain amount to the immune reaction solution during the immune reaction, thereby further increasing the effect of suppressing non-specific adsorption. . The signal due to the label can be detected by absorbance (colorimetry), fluorescence, luminescence or radioactivity assays, including rate assays that measure changes in the signal over time or constant assays. It can be measured using an endpoint measurement method that measures the signal after a certain period of time. Preferably the absorbance method, the absorbance method (colorimetric method)
It can use ultraviolet light, visible light, and near-infrared light.

[0016]

EXAMPLES The present invention will be explained in more detail below with reference to Examples, but the present invention is not limited to these Examples. [Example 1] One of two commercially available mouse monoclonal carcinoembryonic antigen (CEA) (Funakoshi)
Dissolved in M carbonate buffer (pH 9.5) to a concentration of 10 μg/ml, immersed polystyrene beads (#80 from Sekisui Chemical Co., Ltd.) with a diameter of 6.35 mm in this solution and fixed it overnight at 4°C. It became. Subsequently, the beads were washed with phosphate buffered saline (PBS) and then immersed in a 1% BSA-PBS solution at 37° C. for 24 hours to obtain anti-CEA antibody-immobilized beads.

[0017] The other is Eiji Ishikawa, Tadashi Kawai, and Kiyoshi Miyai (eds.), Enzyme Immunoassay (3rd edition), Igakushoin, 1987.
A labeled antibody was obtained by labeling peroxidase by the method described in p109 to p111. Similarly, one of two mouse monoclonal antibodies against α-fetoprotein (AFP) (Cosmo Bio) was added to 0.1M carbonate buffer (
(pH 9.5) to a concentration of 10 μg/ml, 250 μl was added to a polystyrene test tube with a diameter of 11 mm, and the mixture was fixed at 4° C. overnight. Subsequently, after washing the test tube with PBS, 500 μl of 1% BSA-PBS solution was added.
A test tube immobilized with an anti-AFP antibody was obtained.

[0018] The other antibody was labeled with peroxidase to obtain a labeled antibody. 50 μl of the beads and sample in the test tube
A mixed solution of 200 μl of PBS and PBS was added, and the mixture was reacted at 37° C. for 2 hours. The samples used were standard solutions of CEA and AFP, and five types of samples were used, each of which was contained in a 1% BSA-PBS solution at the concentrations shown in Table 1 below.

[0019]
Table-1
No1 No2 No3 No4
No5 CEA (ng/ml) 0
5 10 30 50
AFP (ng/ml) 0 12.
5 25 50 100 then PBS2m
After washing three times with l, anti-CEA (10 μg/ml) and anti-A
Labeled antibody of FP (5 μg/ml) in 1% BSA-PBS
250 μl of the solution was added and reacted at 37° C. for 1 hour. After washing three times with 2 ml of PBS, the beads were removed from the test tube and transferred to a test tube in which no antibody was immobilized.

[0020] Thereafter, citric acid-phosphate buffer (pH 5.0) in which 3 mg/ml o-phenylenediamine was dissolved was added.
, containing 0.02% hydrogen peroxide) was added to each of the antibody-immobilized test tube and the non-antibody-immobilized test tube, and
Color was allowed to develop for 1 hour at room temperature. After that, 1 ml of 1N sulfuric acid each
was added to stop the color reaction, and the absorbance at 492 nm was measured.

The results are shown in the graphs of FIGS. 1 and 2, and it can be seen that both CEA and AFP could be measured by a single immunoreaction procedure. [Example 2] AFP monoclonal antibody was added to test tubes and polystyrene beads at 0.5 μg/ml and 10 μg/ml, respectively.
Immobilization was carried out in the same manner as in Example 1 at a concentration of
Anti-AFP immobilized test tubes and anti-AFP immobilized beads were obtained. Using these test tubes and beads, AFP solutions of known concentrations (0, 12.5, 25, 50, 100,
200, 400, 800, 1600 μg/ml) were measured in the same manner as in Example 1.

The results are shown in Figure 3; samples with high values can be measured when immobilized at low concentrations, and samples with low values can be measured when immobilized at high concentrations, thereby widening the range. I am able to take it.

[0023]

[Effect] In the present invention, when the substance that can specifically bind to the specific component immobilized on the reaction container and the substance that can specifically bind to the specific component immobilized on the insoluble substance are different substances. This allows two items to be measured by performing a single immune reaction operation, which saves time and effort.

[0024] Further, the substance capable of specifically binding to the specific component immobilized on the reaction container and the substance capable of specifically binding to the specific component immobilized on the insoluble substance are the same substance,
If the immobilization concentrations of the two are different, for example, the one immobilized at a lower concentration can measure an analyte with a higher value, and the one immobilized at a higher concentration can measure an analyte with a lower value, thereby allowing a wider range. Can be done.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the absorbance of CEA.

FIG. 2 is a graph showing the absorbance of AFP.

FIG. 3 is a graph showing the absorbance of AFP.

Claims (3)

[Claims] Claim 1: An immunoassay in which a specific component in a fluid sample is analyzed by immunoreacting the specific component in a fluid sample using an insoluble carrier immobilized with a substance that can specifically bind to the specific component. An immunoassay method, characterized in that, as the insoluble carrier, both a reaction vessel in which an immune reaction is carried out and an insoluble substance placed in the reaction vessel are used. 2. A substance capable of specifically binding to the specific component immobilized on the reaction container and a substance capable of specifically binding to the specific component immobilized on an insoluble substance are different substances. The immunoassay method according to claim 1, wherein: 3. The substance that can specifically bind to the specific component immobilized on the reaction container and the substance that can specifically bind to the specific component that is immobilized on the insoluble substance are the same substance, and both Claim 1 characterized in that the immobilized concentrations of are different.
immunoassay.