JPS6162864A - Detection of antigen - Google Patents

Abstract

PURPOSE:To achieve an efficient disffusion of unreacted components outside the sample application range in the reaction between a sample and an immobilized antibody, by developing a cleaning liquid in an analysis element through a capillary in a detection method of an organic compound having a catalytic action for a specified chemical reaction. CONSTITUTION:A sample is applied to an analysis element 1 having at least one of porous layers containing a carrier having an antibody immobilized on a non-liquid-permeable support and the antibody immobilized in the analysis element is made to react with an antigen in the sample. Then, unreacted components are diffused outside the area where the sample is applied by cleaning. Then, a reaction liquid containing a component prone to change in the spectroscopic characteristic is applied to the area 2 where the sample is applied to cause a change in the spectroscopic characteristic on the analysis element with the antigen bonded to the antibody as catalyst and the change is measured. In this detection method of the antigen, a cleaning liquid 3 is developed in the analysis element through a capillary to clean.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for detecting an organic compound that has a catalytic effect on a specific chemical reaction, and in particular involves using an immune reaction to perform complicated separation and purification methods. The present invention relates to a method for detecting an organic compound that has a catalytic effect on a specific chemical reaction on an analytical element.

[Conventional technology]

To quantitatively detect an organic compound that catalyzes a specific chemical reaction in a sample in which various compounds coexist, the organic compound of interest is usually detected by chromatography, centrifugation, salting out, or gel analysis. After separation and purification by ultrafiltration, electrophoresis, etc., quantitative measurements are performed using methods such as spectroscopic absorption measurement, fluorescence measurement, nuclear magnetic resonance, and colorimetry.

Quantitative detection of organic compounds contained in blood, body fluids, urine, feces, etc. is important for pathological diagnosis, and it is required to be performed easily and quickly. Or efforts have been made to make it unnecessary.

It is common practice to select a reaction in which only the target organic compound is a factor, allow the reaction to occur in the coexistence of various compounds, and physically measure the components consumed or produced by the reaction. However, it is difficult to distinguish between coexisting compounds that cause similar reactions, and improvements are desired. An example of this is the measurement of hemoglobin in feces.

Detecting occult blood in feces is important for the diagnosis and treatment of diseases that cause ulcers in the gastrointestinal tract, such as ulcers, cancers, tuberculosis, and typhoid fever. , hemin crystal test method, etc.

Among them, the paper catalyst method is the most used because it is easy to operate. The catalytic method uses the peroxidase-like catalytic activity of hemoglobin and its decomposed products to cause a color reaction in the presence of hydrogen peroxide. Depending on the reagent used, there are phenolphthalein method, 0-tolidine method, guaiac method, The benzidine method, the pyramidon method, etc. are known. These color reactions occur not only in human hemoglobin and its decomposition products, but also in hemoglobin and its decomposition products derived from animal and fish meat contained in red meat, sausages mixed with pig blood, and certain vegetable treatments. Among the products, mediastinal reactions often occur because they are also catalyzed by those with oxidase activity and inorganic salts. A commercially available test for detecting occult blood in feces using a catalytic method available in the form of Testos and Ride. It is prohibited to eat fresh vegetables, fruits such as melons, pears, citrus peels, etc. These prohibitions are troublesome for subjects, and even if they are followed, it is not possible to completely exclude false Bositep reactions.For diagnosis of colon cancer, rectal cancer, etc., colon Time-consuming and expensive tests such as speculum or proctoscopy are required.

JP-A-54-158995 describes a hemoglobin measurement kit in which immobilized anti-hemoglobin antibodies are set on a flat plate, and an example using a slide glass as the flat plate is described, but separation from coexisting substances is difficult. It is difficult and the quantitative accuracy is also poor. Furthermore, this method is not intended to measure hemoglobin under conditions where a wide variety of coexisting substances exist, such as measurement of hemoglobin in feces.

JP-A-56-106154 discloses that a specific antibody against human hemoglobin is used bound to a solid phase, and human hemoglobin in a stool sample is bound to the antibody by an immunological antigen-antibody reaction and isolated. , a method for detecting hemoglobin or hemoglobin degradation products in feces is described, which is characterized by measuring human hemoglobin bound to the antibody.

According to this method, blood-derived hemoglobin and/or decomposition products of hemoglobin contained in feces are adsorbed onto an adsorbent material such as filter paper, and then the extracted sample fluid and a solid bonded with a specific antibody against human hemoglobin are adsorbed. Decant the ink and sample liquid while in contact with the phase.

Either try an enzyme immunoassay (hereinafter abbreviated as E-A) on the solid phase using the Sand-Deutsch method, or perform a competitive method E'IA using enzyme-labeled human hemoglobin at the same time as the sample solution. Hemoglobin is detected by performing the guaiac reaction. In this method, polyethylene cuvettes, balls, rings, disks, etc. are described as the in-phase material bound with a specific antibody against human hemoglobin, but these require cumbersome operations such as incubation in the sample solution and washing after decantation. are similar.

JP-A No. 58-23796 describes a method for immunologically detecting hemoglobin using a material such as a filter paper that has adsorbed blood-derived hemoglobin contained in feces after being used in a catalytic method. is disclosed. The method is based on Japanese Patent Application Laid-Open No. 106-1983, except that the adsorption material used in the catalytic method was used.
This method is the same as the method described in No. 154. The solid phase bound to a specific antibody against hemoglobin is a bead, but it requires incubation in a sample solution, washing after decantation, and complicated operations.

JP-A-58-205854 describes immobilized antibodies for occult blood reaction tests in which specific antibodies for human hemoglobin with different degrees of denaturation are immobilized. According to the publication, a plate, sheet, or fiber made of polyvinyl alcohol or cellulose on which a specific antibody has been immobilized is immersed in a specimen solution in which a small amount of feces is suspended, incubated, taken out, and washed with physiological saline. 7. Obtain a sample. The sample is colored using a catalytic method.

This method also requires incubation in a sample solution, washing after decantation, and cumbersome operations.

[Problem that the invention seeks to solve]

In the invention of Japanese Patent Application No. 59-28571, which improves these, testing operations and false positive and negative reactions are improved. In addition, the area near the center where the cleaning solution is applied is not sufficiently cleaned, and false positive and negative substances remain in that area, making it difficult to make accurate judgments.

An object of the present invention is to provide a simple method for detecting organic compounds (antigens) that have a catalytic effect on specific reactions, such as enzymes and hemoglobin. It is an object of the present invention to provide a cleaning method that efficiently diffuses unreacted components out of a sample application area in a reaction with a chemically modified antibody.

[Failure to solve the problem]

To summarize the present invention, the present invention relates to a method for detecting an antigen, and the present invention relates to a method for detecting an antigen. is applied to cause the immobilized antibody in the analytical element to react with the antigen in the specimen, and then the unreacted components are washed to diffuse the specimen outside the application area, and then the antigen bound to the antibody is catalyzed. In an antigen detection method in which a reaction solution containing a component that causes a change in spectral properties is applied to an area to which the specimen is applied to cause a change in spectral properties on an analytical element, and the change is measured, the washing solution is It is characterized by cleaning by spreading it into the analytical element by capillary action.

According to the present invention, unreacted components of the reaction between the antigen brought from the specimen and the immobilized antibody in the analytical element can be efficiently diffused outside the region to which the specimen is applied.

The cleaning method of the present invention is based on the same principle as in thin layer chromatography and paper chromatography, which are techniques for separating mixtures of organic compounds, in which a developing solvent is developed and a mixture is separated. , the developing solvent is a cleaning solution, a part of the analytical element is immersed in the cleaning solution tank, and the cleaning liquid moves and spreads inside the analytical element due to capillary action, removing unreacted components outside the sample application area. The method is to spread it to The cleaning solution tank may be within the analytical element, or the analytical element may be placed in the cleaning solution tank. Further, although the direction of expansion may be either direction, it is preferable that the expansion distance is longer.

In terms of operation, it is preferable to wash the analytical element to obtain the maximum cleaning effect with the minimum amount of washing.

When dropping the cleaning liquid onto the center where the specimen has been applied, the amount of cleaning liquid seems to be at least -h, but as mentioned earlier,
The drawback is that unreacted substances remain in the center, causing false positive and negative reactions. Although it is possible to increase the amount of cleaning liquid,
The permissible amount of cleaning liquid for analytical elements varies depending on the nature of the carrier and other materials that make up the element, the material used, and the thickness of the membrane of the analytical element. do not have.

The cleaning method by spreading the cleaning liquid in this manner obtains the maximum cleaning effect within the allowable amount of the cleaning liquid for the analytical element. Furthermore, since these methods utilize the same principle as paper chromatography and thin layer chromatography, unreacted components are developed simultaneously with the development of the cleaning solution, so it is presumed that the cleaning effect is large.

The liquid-impermeable support in the present invention means a water-impermeable membrane, and there are no particular limitations on the material. Mention may be made, for example, of polymeric materials such as cellulose acetate, polyethylene terephthalate, polycarbonate and polyvinyl compounds (eg polystyrene), or glass, ceramics, metals and resin-coated papers. It is preferable that the support is light-transmissive because spectroscopic changes can be measured from the support side.

The origin of the antibodies used in the present invention is not particularly limited, and the antibodies can be purified using conventionally known methods for purifying antibodies from serum containing antibodies obtained by administering and immunizing mammals with antigens. Shunsuke Migita ed. “Immunochemistry” Nakayama Shoten No. 74~
(see page 88), sodium sulfate precipitation method, ammonium sulfate precipitation method, gel V filtration method using Sephadex gel, ion exchange cellulose chromatography method, electrophoresis method, etc.

Alternatively, monoclonal antibodies may be produced by obtaining hybridomas from lung cells of a mammal (eg, a mouse) sensitized with an antigen and myeloma cells (myeloma).

In the present invention, the carrier refers to a solid that is insoluble in the sample and maintains its structural state when in contact with the sample, and is not limited to any material as long as it can immobilize the antibody by a physical or chemical method. Examples of carriers include paper, cellulose, glass fibers, nonwoven and woven fabrics, various organic polymer fibers (polyamide, polyethylene, polypropylene, etc.), particulate organic polymers, and other film-forming materials.

Although it partially overlaps with the examples of these carriers,
No. 88, No. 55-90859, No. 57-67860, No. 57-125847, No. 57-197466,
5B-70161 and the like may be used as the carrier.

The layer containing the carrier is a porous layer in which the test substance, such as an enzyme or hemoglobin, can freely contact the antibody immobilized on the surface of the carrier in the layer.

It is preferable to use pores with a short diameter of 5 μm to 300 μm, and the method of forming the carrier into a layer is described in Japanese Patent Application Laid-open Nos. 49-53888, 55-90859, and 5
No. 7-67860, No. 57-j25847, No. 57
No. 197466, No. 58-7 [1161] The methods described in each publication can be used to form the spreading layer. It is also possible to use the particle conjugate described in JP-A No. 58-214854 as a carrier, and the method of the present invention can be carried out by the method described in JP-A-58-214854. It is possible to form a porous layer containing a carrier on which antibodies which are suitable for use are immobilized. The antibody may be immobilized on the carrier by a physical method or by forming a chemical bond.

As for the physical adsorption method, the antibody can be dissolved in water or an appropriate buffer solution, and the carrier can be immersed in the solution for adsorption.

As the buffer solution in this case, an appropriate buffer solution of about 0.001 to 1M can be used. Further, the concentration of the substance to be adsorbed is in the range of 0.0 O1 to 1.0%, and the carrier whose surface has been sufficiently cleaned is immersed to adsorb it.

The temperature for the adsorption is preferably room temperature or lower, and the adsorption time is preferably 10 to 100 hours. The obtained carrier is preferably washed with separated water or a buffer solution to remove antibodies that have not participated in adsorption.

As a method using chemical bonding, it is possible to bond the antibody to the functional group on the carrier according to the present invention directly or using a polyfunctional reagent. For these methods, it is possible to apply, for example, the enzyme immobilization technology described in "Immobilized Enzymes" (edited by Chibata, 1975, published by Kodansha). - Examples include diazotization method, amide method, alkylation method, glutaraldehyde method and hexamethylene diisocyanate method.

Naturally, a porous layer can be prepared using a carrier to which an antibody is bound, or a layer can be prepared in advance and then the antibody can be bound thereto. Furthermore, the carrier according to the present invention can support proteins that are likely to be involved in the immune reaction to be measured, if necessary, for the purpose of eliminating non-specific reactions in the immune reaction. Typical examples of these include mammalian normal serum proteins, albumin, gelatin, and decomposition products thereof.

As the supporting method for these, the physical adsorption method and the chemical bonding method can be appropriately used as described above.

The reaction solution in the present invention refers to a solution containing a component that causes a change in spectral characteristics using an antigen bound to an antibody as a catalyst. A change in spectral characteristics means not only a change in absorbance for visible light, but also a change in absorbance for ultraviolet light and infrared light, as well as the emission of light different from the absorbance wavelength of fluorescence and emission without absorption. . It also means quenching. For example, when hemoglobin is to be detected, the reaction solution basically only needs to contain hydrogen peroxide and a compound that generates a colored substance or changes color in the presence of hydrogen peroxide and peroxidase.

Examples of such compounds or compositions include JP-A-49
-50991, (a) O-dianisidine or its acid salt, lblo o-) Lysine or its acid salt, (C) o-phenylenediamine or its acid salt, (
d) Guaiac, (θ)adrenaline, (flphenolphthalein, (ω2,2'-azinodi(3--114lupenzotheazoline-6-sulfonic acid) ammonium salt,
(6) Combination of ferrocyanide, +114-aminoantipyrine, substituted derivatives thereof, or acid salts thereof with phenol or naphthol or derivatives thereof, JP-A-1977
(1) Aniline and its derivatives, 0-toluidine, p-
) Monoamines such as luidine, (2) o-phenylenediamine, N,N'-dimethyl-p-phenylenediamine, N,N'-diethylphenylenediamine, benzidine (appears ill or brown), dianisidine (greenish or brownish), (3) Phenol itself (appears yellow), thymol, n-, m-, and p-cresol (shows green-yellow, pink, and opalescent suspensions, respectively), alpha-naphthol (
(4) Catechol, guaiacol (Orange color)
(5) Aromatic acids such as salicylic acid, pyrocatechinic acid, and gallic acid. , (6) leuco dyes such as leucomalachite green (presenting malachite green), leucophenolphthalein (preferably used in an alkaline medium), (7) 2,6-cyclophenol indophenol, etc. colored dyes, (8) various biochemicals such as epinephrine, flavones, tyrosine, dihydroxyphenylalanine (which gives an orange-red color) and tryptophan; (9) others such as guaya gum, guaiaconic acid, potassium iodide, sodium iodide and Other water-based iodides, as well as substances such as bilirubin (greenish in color), 2,2'-di-3-ethyl-6-su-11, benzothiazoline) or salts thereof. , and special dyes such as 3,37-diamitubenzidine, a combination of 4-aminoantipyrine, a substituted derivative thereof or an acid salt thereof with 1,7-cyhydroxynafucrene or a derivative thereof. .

The composition of the cleaning solution is not limited as long as it can diffuse unreacted components to the periphery of the area to which the specimen is applied. Basically, water is fine. Preferably, a surfactant is added to increase cleaning properties. Advantageously used surfactants include Tri-I-N@x-1no (octylphenoxypolyethoxyethanol manufactured by Rohm and Haas), Surfactant 101■ (1
, nonylphenoxy polyglycidol) and the like.

It is known that certain enzymes increase in the serum and urine of patients, and these enzymes are becoming increasingly important in diagnosing cancer and determining the course of prognosis. The method of the present invention is also useful as a method for detecting these enzymes. Examples of these enzymes include γ-glutamyl transferase isoenzyme, aldolase isoenzyme, alkaline phosphatase, acid phosphatase, pyruvate kinase, terminal deoxynucleotedyl transferase, and the like.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these examples.

Note that FIG. 1 is a schematic diagram showing an example of the cleaning method of the present invention. In FIG. 1, 1 means a detection element, 2 means a region to which a specimen is applied, and 6 means a cleaning solution.

Example 1 (1) Immobilization of anti-human hemoglobin to cellulose fibers ν paper powder C! (More than 300 mesh, made of Toyo ν paper ■)
so t, 1,4-butanediol diglycidyl ether [manufactured by Kanto Chemical Co., Ltd.] 75 at, NaBH4
[Manufactured by Wako Tsumugi Co., Ltd.] Contains 22m9/[1,6M
'NaOH solution 75- was mixed and stirred at 40°C for 5.5 hours to perform activation.

On a glass filter, the activated filter paper powder C was washed with 5,000 ml of distilled water, and then 300 ml of 0.5 M Na
HOOl-Na, 00. Rabbit anti-human) moglobin antibody [manufactured by Miles Co., Ltd.] dissolved in a buffer solution (pH 1 p, o) 0. The mixture was reacted with T at 40°C for 14 hours.

Furthermore, after removing the reaction solution, add 1M) + 7 Souc hydrochloric acid buffer
It was reacted with pHao at 40°C for 14 hours. 0.
5M acetate buffer, 0.5M NaHCJO, solution, P
After washing with BS (IJ acid buffered saline), freeze-drying was performed to obtain anti-human hemoglobin-immobilized filter paper powder C. From the change in absorption amount of the reaction solution (280nm), 70yn
It was found that anti-human hemoglobin in gltF paper powder c was immobilized.

(II) Preparation of Occult Blood Detection Element A mixture having the following composition was coated on a transparent undercoated polyethylene terephthalate support having a thickness of 180 μm.

The anti-human hemoglobin-immobilized filter paper powder CI9/10h
- Triton X-100 (manufactured by Rohm and Haas)
5 ov/100m polyvinyl alcohol (NM-44 manufactured by Nippon Gosei Kagaku Kogyo Co., Ltd.)
209/100 down 2 copoly (styrene)
glycidyl methacrylate) monomer weight ratio 90:1
0 20■/1ooω The obtained coating material was 2.0
It was cut into a rectangle of fi X 4.0 ('Ill), and a filter paper (Toyo Roshi A1) cut into a 2.0α angle was layered thereon to serve as a detection element.

(DI) detection Fresh human feces only (sample number 1), 1o.

2 fresh human feces, 21 ml each of human blood (2)
, 10 In9 myoglobin (3), 10 mg hematin (4), 2 ml human blood, and 200 μl ascorbic acid (5) to prepare a homogenized sample.

These stool samples were taken with the tip of a spatula, applied to the filter paper of the detection element prepared in (■), incubated at 37°C for 10 minutes, separated from the filter paper 17, and applied as a washing solution as shown in Figure 1. 200 μl 5% Triton X-10
A phosphate buffer solution containing 0 (pH 7, 6) was developed to diffuse unreacted substances outside the sample application area. Further, an o-tolidine-ethanol-acetic acid mixture containing 5% &Oz was dropped near the center of the area to which the stool sample was applied, and a Δ color change after 2 minutes was visually observed. In addition, as a comparative example, a cleaning liquid was dropped near the center of the area to which the stool sample was applied, and the same coloring liquid was added thereto. The results are shown in Table 1 below.
Shown below.

As is clear from these results, the cleaning method of the present invention
It can be seen that the unreacted substances can be reliably diffused outside the area to which the stool sample is applied, and that the detection method performed according to this method is unlikely to make an error in judgment.

〔Effect of the invention〕

As explained above, according to the method of the present invention, it becomes possible to sufficiently wash away specific substances in a specimen, and the remarkable effect that the influence of interfering substances can be easily and accurately determined can be achieved.

[Brief explanation of the drawing]

FIG. 1 shows an example of the cleaning method of the present invention. Schematic 1: Detection element, 2: Application of specimen 1. Area 5: Cleaning liquid

Claims (1)

[Claims] 1. An analytical element having at least one porous layer containing a carrier on which an antibody is immobilized on a liquid-impermeable support,
After applying the sample and allowing the immobilized antibody in the analytical element to react with the antigen in the sample, unreacted components are removed by washing.
The specimen is diffused outside the application area, and then a reaction solution containing a component that causes a change in spectral characteristics using the antigen bound to the antibody as a catalyst is applied to the area to which the specimen is applied to change the spectral characteristics on the analytical element. 1. A method for detecting an antigen in which a change is caused and the change is measured, the method comprising washing by spreading a washing liquid into an analytical element by capillary action.