JPS60173471A - Detection of antigen - Google Patents

Abstract

PURPOSE:To detect an antigen with good accuracy by bringing a specimen into reaction with an analyzing element formed by providing a porous layer immobilized with an antibody onto a liquid impermeable base then dropping a cleaning liquid thereto to diffuse the unreacted component to the periphery, acting a reactive liquid to the dropped part and measuring the optical change. CONSTITUTION:An analyzing element formed with a porous layer coated thereon with a coating liquid contg. filter paper powder, etc. immobilized with an antibody together with a binder such as gelatin and a suitable crosslinking polymer and nonionic surface active agent on a impermeable base of polyester, etc. is manufactured. A specimen contg. the antigen of which the antigen of an antibody-antigen conjugate such as enzyme, hemoglobin has a catalytic effect on the reactive liquid is coated on such element and after incubation, a detergent such as a buffer contg. a nonionic surface active agent of the like is dropped to the coated part to diffuse the unreacted component to the periphery. The reactive liquid which colors, discolors or emits fluorescence by the catalytic effect of the antigen is then dropped to the part where the detergent is dropped and the optical change thereof is measured. The easy detection of the antigen is made possible with good accuracy without receiving the influence of the disturbing component in the specimen.

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.

[Prior art]

In order to quantitatively detect an organic compound that has a catalytic effect on a specific chemical reaction in a sample in which various compounds coexist, the target organic compound 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 catalytic method is the most used because it is easy to operate. The catalytic method uses the peroxidahose-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, phenolphthaleino method, 0-tolidine method, guaiac method, The benzidine method, the viramidon method, etc. are known. These color reactions occur not only with human hemoglobin and its decomposition products, but also with red meat, pig blood, hemoglobin and its decomposition products, certain vegetable therapeutic drugs that have oxidase activity, and inorganic salts. False-positive reactions often occur because the molecule is also catalyzed. Commercially available tests for the detection of occult blood in feces using a catalytic method available in the form of test slides For example, in pressure tests, the test subject must eat poultry meat and processed meat products, seafood and processed products for 2 to 5 days before the test. 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 bogitib reactions.For diagnosis of colon cancer, rectal cancer, etc., colon Time-consuming and expensive tests such as mirrors or rectal bells 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 P paper, and a sample fluid extracted from the adsorbent is bound to a specific antibody against human hemoglobin. Incubate in contact with the solid phase and decant the sample solution.

Either try enzyme immunoassay (hereinafter abbreviated as E-A) on the same phase using the sandwich method, or perform competitive EIA using enzyme-labeled human hemoglobin at the same time as the sample solution, or perform guaiac. Hemoglobin is detected by performing a reaction. In this method, polyethylene cuvettes, balls, rings, disks, etc. are used as the same phase to which specific antibodies against human hemoglobin are bound, but they require complicated operations such as incubation in the sample solution and washing after decantation. The same thing applies.

JP-A No. 58-23796 discloses a method for immunologically detecting hemoglobin using a material such as paper that has adsorbed blood-derived hemoglobin contained in feces after being used in a catalytic method. 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 sample solution in which a small amount of feces is suspended, incubated, taken out, and washed with physiological saline. Obtain a sample. The sample is colored using a catalytic method.

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

[Purpose of the invention]

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.

[Structure of the invention]

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 provided by the specimen, and then by dropping a cleaning solution onto the area to which the specimen has been applied, unreacted components are removed from the area to which the specimen has been applied. 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 has been applied, causing a change in spectral characteristics on the analytical element. The feature is that the change is measured.

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

The origin of the antibodies used in the present invention is not particularly limited, and can be obtained from serum containing antibodies obtained by administering and immunizing mammals with antigens, or by conventionally known methods for purifying antibodies. Shunsuke Migita ed. “Immunochemistry” Nakayama Shoten No. 74-8
(see page 8), sodium sulfate precipitation method, ammonium sulfate precipitation method, gel filtration method using Sephadex gel, ion exchange cellulose chromatography method, electrophoresis method, etc.

Monoclonal antibodies may be produced by obtaining hybrid cells (hybridoma) from spleen cells of a mammal (for example, mouse) sensitized with a human antigen and bone marrow lung cells (myeloma).

In the present invention, the carrier refers to a solid that is insoluble in a sample and maintains its structural state when in contact with the sample, and is not limited to any material as long as the antibody can be immobilized 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, JP-A-49-558
No. 88, No. 55-90859, No. 57-67860, No. 57-125847, No. 57-197466,
It is also possible to use the spreading layer constituent materials described in JP-A No. 58-70161 and the like.

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 phase in the layer.

It is preferable to use pores with a diameter of 5 μm to 300 μm, and the method for forming the phase material into a layer is as follows:
Unexamined Japanese Patent Publication 1973. -53888, 55-90859,
No. 57-67860, No. 57-125847, No. 5
7-197466 and 58-70161 can be used to form the spreading layer. It is possible to use the particle conjugate described in JP-A-58-21485△4 as a phase, and the method of the present invention can be carried out by the method described in JP-A-58-214854. It is suitable for use in

It is possible to form a porous layer containing the carrier on which the antibody is 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 at this time, an appropriate buffer of about 1101 to 1M can be used. The concentration of the shredded substance to be adsorbed is in the range of 0001 to 1.0%, and the carrier whose surface has been sufficiently cleaned is immersed in the substance to be adsorbed.

The temperature for the above adsorption is preferably room temperature or lower, and the time is preferably 10 to 100 hours. The resulting particle unit or particle conjugate is preferably washed with 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 Koeisha). −
Examples include the diazotization method, the amide method, the alkylation method, and the glutaraldehyde hexamethylene diisocyanate method.

Naturally, the antibody can be bound to the carrier according to the present invention before the particle conjugate is prepared, or the antibody can be bound after a layer has been prepared in advance. Furthermore, the carrier according to the present invention can carry proteins that are not involved in the immune reaction to be measured, if necessary, in order to eliminate non-specific reactions in the immune reaction. Examples include normal serum proteins of mammalian animals, albumin, gelatin, and decomposition products thereof.

As the method of adhering them, the physical adsorption method and the chemical bonding method can be appropriately used as described above.

In the present invention, the reaction solution 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
(a) o-dianisidine or its acid salt, (b) o-dianisidine or its acid salt, (C) O-phenylenediamine or its acid salt, (d) disclosed in JP-A-50991. Guaiac, (θ) adrenaline, (f
) phenolphthalein, (g) 2,2'-azinodi(5-ethylbenzothiazoline-6-sulfonic acid) ammonium salt, (h) ferrocyanide, (→-4-aminoantipyrine, its substituted derivatives or their substituted derivatives) Combinations of acid salts and phenol or naphthol or derivatives thereof, disclosed in JP-A-50-137192 (1) Aniline and derivatives thereof, o-toluidine, p
-) Monoaminos such as luidine, (2) o-phenylenediamine, 1q, N'-dimethyl-p-phenylenediamine, 1q, N'-diethylphenylenediamine, benzidine (appears tel or brown), dianisidine (green) (3) Phenol itself (yellow color), thymol, o-, m+, and p-cresol (greenish-yellow, pink, and milky suspension, respectively), alpha-naphthol (deep red color) ), beta-naphthol (causes scuttling)
(4) Catechol, guaiacol (orange color),
Osinol, pyrogallol (appears red or yellow)
, Rp-dihydroxydiphenyl, and polyphenols such as phloroglucinol; (5) aromatic acids such as salicylic acid, pyrocatechinic acid, and gallic acid; (6) leucomalachite green (presenting malachite green); leuco dyes such as phenoltskurein (preferably used in an alkaline medium), (7) 2.6-cyclophenolindofe/
(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, and potassium iodide. , sodium iodide and other water-soluble iodides, and substances such as bilirubin (which gives a greenish color); salts, and special dyes such as 3.3'-diaminobenzidine, 0η 4-aminoantipyrine, its substituted derivatives or its acid salts and 1.7-
A combination with cyhydroxynaphthalene or a derivative thereof.

The composition of the cleaning liquid is not limited as long as it can diffuse unreacted components to the periphery of the area to which the specimen has been applied. Basically, water is fine. Preferably, a surfactant is added to increase cleaning properties. Advantageously used surfactants include nonionic surfactants such as Triton X-100 (octylphenoxypolyethoxyethanol manufactured by Rohm and Haas) and Surf Actane 10G (nonylphenoxy polyglycidol manufactured by Olean). You can praise the medicine.

It is known that a certain fim enzyme is increased in the serum and urine of patients, and this is becoming increasingly important in the diagnosis and prognosis of diseases such as bruxism. The method of the present invention is also useful as a method for detecting these enzymes. As these enzymes,
γ-glutamyl transferase isoenzyme,
Examples include aldolase isoenzyme, alkaline phosphatase, acid phosphatase aldolase, pyruvate kinase, and terminal deoxynucleotidyl transferase.

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

Example 1 (1) Immobilization of anti-human hemoglobin on cellulose fibers Filter paper powder 0 (300 mesh or more, manufactured by Toyo P Paper ■) 5f
100d of 2.5M potassium phosphate buffer (I) HI
3.1) While gently stirring with a stirrer, gradually add 50d of 10T BrGN aqueous solution,
Activation was performed.

Activated filter paper powder C was mixed with 1,500 g of distilled water and 0.0 g of activated filter paper powder C on a glass strainer. After washing with I M NaHCOl aqueous solution, 100 d of Q, I M NaHC
It was reacted with 1.5 f of rabbit anti-human hemoglobin dissolved in O3,0, 5M Na06 solution for 8 hours at room temperature.

After further removing the reaction solution, add IM to IJ-HCl buffer 200#+7! (Reacted with pI(ELO) for 3 hours at room temperature.

After washing with [15M acetate buffer, [+, 5M NaHCO3 solution, and pBS (phosphate buffered saline)], freeze-drying was performed to obtain anti-human hemoglobin-immobilized filter paper powder 0. Change in absorption side of reaction solution (28゜nut)
It was found that anti-human hemoglobin in 150 mylftf paper powder C was immobilized.

(2) Preparation of occult blood detection element A mixture having the following composition was coated on a transparent undercoated polyethylene terephthalate support-F having a thickness of 180 μm.

The obtained coated material was cut into 2.5ω square pieces, and 2.5Ω
Filter papers cut into cm pieces (Toyo Oki Shiyo 1) were layered and housed in a mount as shown in Figure @1 to serve as a detection element. Note that FIG. 1 is a perspective view of the slide, and shows (a) an li base,
(b) shows the lid.

(3) Odl 1ml per 100y of fresh stool,
2 ml, 4 wtl, 8 ml, 16 - of human blood, and 101 nIj of horseradish peroxidase, each treated with 4 - of goat blood, bovine blood, rabbit blood, horse blood and homogenized.

These fecal samples were taken with the tip of a spatula, applied to the paper of the detection element, incubated at 37°C for 10 minutes, separated from the filter paper, and 20μt of 5titritonX was placed in the center.
The -100 solution was added dropwise, and the unreacted substances were diffused into the surrounding area.

Furthermore, a 0-tolidine-ethanol-water mixture containing 3% H,02 was added dropwise, and the change in color tone was visually observed after 2 minutes (judgment 0 to 0).
4+).

Further, as a comparative example, a test paper as disclosed in Japanese Patent Application Laid-Open No. 54-24697 was prepared, the same sample as above was applied, and the results were observed.

0 Preparation of Comparative Example Element 1.0M citrate buffer (pH 4,5) After dissolving sodium alginate 202 and diocter sodium sulfosuccinate 0.059 in 100me, add cumene hydroberoside 1.5-. Emulsify. A filter paper is impregnated with this emulsion and dried under vacuum overnight. Then 05%
O-) A filter paper after drying overnight is impregnated with a chloroform solution containing 1.0% of U-gin, and dried under reduced pressure in the dark.

The results obtained are shown in Table 1 below.

Table 1 As is clear from the results shown in Table 1, by using the detection method of the present invention, only hemoglobin in human occult blood can be specifically detected.

Example 2 A detection element similar to Example 1 was produced except that the filter papers were not overlapped.

1 ? each of the stool samples used in Example 1? 5rnt of pBs (pu z6) was added to the sample, mixed and stirred for 5 minutes, allowed to stand, the supernatant was collected, and 20μt of the same sample was applied to the device three times.

Incubation, washing, and coloring solution as in Example 1.
After dropping, the reflection density was measured from the support side using a reflection densitometer (Kura PDA65, manufactured by Konishiroku Photo Industry Co., Ltd.). The results are shown in FIG.

In other words, Figure 2 shows the amount of blood in 100v of stool.
(horizontal axis) and reflection density (relative value) (vertical axis). As is clear from FIG. 2, there is a linear relationship between the human serum needle in the stool sample and the reflected concentration in this concentration range, and it was also found that it was quantitative. Furthermore, it can be seen that the sample mixed with blood other than human blood is slightly larger than the blank, and is specific only to human occult blood hemoglobin.

Fruit can example 3 (1) Introduction of polymer into polyethylene fibers Polyethylene fibers cut into pieces of 300 mesh or less 51] IF is styrene 40t1 glycidyl methacrylate.
) 10f, divinylbenzene sr, 2f-azobis-(
2,4-dimethylvaleronitrile) 1. It was impregnated with a mixed solution of o y.

After stirring vigorously using a homomixer, polyethylene fibers impregnated with the above monomers were added to 500ml of an aqueous solution containing 20t1 of tricalcium phosphate and 0.11ml of sodium dodecylbenzenesulfonate, and the mixture was stirred for 200ml under a nitrogen stream.
After stirring for 30 minutes at a stirring speed of rpm, 1L
Transfer the contents to a turning flask and incubate at 60°C under a nitrogen stream for 8 hours.
Allowed time to react. The contents were cooled to room temperature, tricalcium phosphate was decomposed and removed with dilute hydrochloric acid, washed with water repeatedly, separated from the oven, and then dried.

(2) Introduction of antibody into the fiber-polymer 20 μl of the polyethylene fiber-polymer was treated with ajM NaHCO containing 50 μm of anti-human alkaline phosphatase antibody.
2. Dispersed in 100 d of α5 M NaO4 solution and heated at 5°C.
After stirring for 24 hours to bind the anti-alkaline phosphatase antibody, the mixture was separated in a furnace, washed repeatedly with α15M Na0t aqueous solution, and then freeze-dried.

(3) Preparation of serum alkaline phosphatase (hereinafter abbreviated as AtP) detection element A detection element was prepared by applying a mixture having the following composition onto a transparent subbed polyethylene terephthalate support having a thickness of 180 μm.

(4) Detection The detection element includes Be5sey-L
owry) units of 0.1.3.5.7 and 1° units of AtP (measured by the Be5sey-Lowry method) standard solution was added dropwise. After incubation at 37°C for 15 minutes, 5% Tritone was added to the center. X-100 aqueous solution 2
At the same time, 20 μt of glycine buffer containing 1)-2) disodium rophosphate and having a pH of 1α5 was added dropwise. (Incubation at 57° C. for 10 minutes) Reflection density at 410 nm was measured from the support side. The results are shown in FIG.

That is, FIG. 3 is a graph showing the relationship between the amount of AtP (Betsey-Lowry units) in the standard solution (horizontal axis) and the reflection density (relative value) (vertical axis).

As is clear from Figure 3, a correlation was observed between the AtP interval and the reflected density.

[Effects of the Invention] As explained in detail above, the method of the present invention has the remarkable effect of being able to detect a specific substance in a sample simply and with high precision without the influence of interfering substances. .

[Brief explanation of drawings]

Fig. 1 is a perspective view of a slide, Fig. 2 is a graph showing the relationship between blood volume in stool i oar and reflection concentration measured by the method of the present invention, and Fig. 3 is a graph measured by the method of the present invention. It is a graph showing the relationship between alkaline phosphatase concentration in a standard solution and reflection concentration. Patent Applicant Roku Konishi Photo Industry Co., Ltd. Agent Hirodo Nakamoto Shodo Inoue Katsura Yoshimine Figure 1 Figure 2 Stool: Amount of blood in 100g and m) Procedure Amendment (voluntary amendment) Showa March 27, 1959 Kazuo Wakasugi, Commissioner of the Patent Office Indication of Case 1 Patent Application No. 28571 of 1983, Title of Invention Relationship with Case of Person Who Amends Antigen Detection Method Five Amendments Address of Patent Applicant Shinjuku, Tokyo Ward Nishi-Shinjuku 1-26-2 Name
(127) Konishiroku Photo Industry Co., Ltd. Representative Ide
Megumi Iku Nishi-Shinbashi Chuo Building 502 Telephone (437)-3467 ゛"""3°""""" K 6 Column for detailed explanation of the invention in the specification subject to the amendment l Details of the invention in the description of the contents of the amendment Please correct the explanation column as below. (1) Amend “Pseudo-1” to “Doubt” on page 4, line 5 of the specification,
Zo (2) Correct "Pressure" in line 7 of the same page to "fueka" (2) Correct "of the present invention...antibody" in lines 2 to 4 of page 11 as follows: [In the method of the present invention] It is possible to form a porous layer containing a carrier on which antibodies which are suitable for use are immobilized. Regarding antibodies to carriers, J (3) "Particle unit or particle conjugate" in line 4 from the bottom of the 11th page of the same document is corrected to "carrier." (4) In lines 1209-10 of the same, "antibody...conjugate" should be corrected as follows: "A porous layer is formed using a carrier to which antibodies are bound."

Claims (1)

[Claims] 1. A sample is applied to an analytical element having at least one porous layer containing a carrier on which an antibody is immobilized on a liquid-impermeable support, and a sample is obtained from the immobilized antibody in the analytical element and the sample. By reacting with the antigen that has been applied, and then dropping a washing solution onto the area to which the specimen has been applied, unreacted components are diffused to the periphery of the area to which the specimen has been applied, and the antigen bound to the antibody is then reacted with the antigen bound to the antibody. A reaction solution containing a component that causes a change in spectral characteristics as a catalyst is applied to an area to which the specimen is applied to cause a change in spectral characteristics on an analytical element, and the change is measured. Detection method.