JPH0453579Y2 - - Google Patents

Description

[Detailed description of the invention] [Field of industrial application] The present invention is a kit for qualitatively or semi-quantitatively and extremely easily measuring biological substances.
In particular, the present invention relates to a kit for carrying out the above-mentioned measurement using a color reaction on a sheet of paper on which antibodies are immobilized.

[Prior Art] In order to accurately understand the pathological condition, it is necessary to know how the values of specific components (eg, hormones, etc.) in body fluid samples such as serum, urine, or lymph fluid vary.

Furthermore, in order to treat diseases before they become serious, it is important to periodically detect the above-mentioned components in a healthy state, constantly observe fluctuations, and detect abnormalities at an early stage.

In recent years, methods that utilize specific binding reactions between substances bound to enzyme labeling substances and other biological substances have been developed as methods capable of measuring such specific components.

What is generally called enzyme immunoassay is a typical example of the above measurement method, and is extremely superior in terms of safety, especially compared to previous methods that use radioactive isotopes as labeling substances.

Among the enzyme immunoassay methods mentioned above, in a common method called the heterogeneous method, a substance that specifically reacts with the test substance (e.g., an antigen or an antibody) is immobilized on a substance called a solid phase. By washing operations after each reaction step, the so-called B (bound)
F (free) separation is performed. Materials conventionally used as this solid phase include polystyrene beads, glass beads, dextran, silicon disks, silicon rubber, agarose, and paper.

[Problems to be solved by the invention] However, the conventional enzyme immunoassays mentioned above were mainly designed to be used in well-equipped laboratories and examination rooms. It is something that Therefore, these devices are relatively expensive and require complicated cleaning operations.

Therefore, in order to detect diseases at an early stage, there is a strong desire for a method and an inexpensive and simple device that can be easily used by anyone at home and that can easily detect the above-mentioned specific components on a daily basis. It is something that

In order to solve the above-mentioned problems, the inventor of the present invention has developed a method that can be used by any ordinary person at home, etc., to easily develop a biological system using a specific binding reaction between substances and a coloring reaction catalyzed by an enzyme, which is a labeling substance. The present invention was achieved by developing an inexpensive and simple kit that can qualitatively or semi-quantitatively measure derived substances.

[Means for Solving the Problems] That is, the present invention is a kit for measuring biological substances, which includes a foldable container and a substance that can specifically bind to the biological substances fixed on a piece of paper. a region containing a solution containing a substance capable of specifically binding to the biological substance and labeled with an enzyme, and a coloring reagent capable of reacting with the labeled enzyme. The present invention relates to the kit, characterized in that the kit comprises a film-like support having a region in which a solution is enclosed, and the film-like support is joined to the bottom of the container.

The collapsible container used in the kit of the present invention must have sufficient capacity and strength to contain and hold the amount of urine, blood, or other specimen required for the reaction for the time required for the reaction. It does not matter what size, shape, and material it has, but from the viewpoint of economy, transportability, stockability, etc.
For example, a folding cup made of paperboard, which is a non-thermal adhesive base material, disclosed in Utility Model Application Publication No. 52545/1980 is suitable.

The inner surface of the collapsible container is preferably coated with a water-repellent and heat-adhesive film such as polyethylene.

The film-like support of the present invention, such as plastic or paper, can be used to support the specimen (urine, blood, etc.).
It is preferably made of an inert material and is in the form of a thin slip.

Preferably, a paper on which a substance capable of specifically binding to a biological substance is immobilized is bonded onto the film-like support by an appropriate means. It is also possible for this paper to function as the film-like support of the present invention as it is.

The region in which the solution is enclosed is preferably formed in the form of a bag, and the bag is bonded onto the film-like support by suitable means.

A plurality of the above-mentioned strips of paper may be bonded onto a film-like support depending on the type of desired analyte.

This bag can stably hold a normal aqueous solution in the neutral range, and can be easily torn by being hit by the corners of the paperboard, for example, the corners of the folding container in the kit of the present invention. The bag may be made of any material, but a plastic bag is particularly suitable.

Any ordinary paper used in the field of chemistry can be suitably used as the paper in the present invention, but Toyo paper from No. 2 to No. 8 can be used in the kit of the present invention. It is particularly preferable for the paper to be used.
The paper may have any size as long as the color reaction can be visually confirmed.

Examples of test substances include HCG, FSH,
Hormones such as LH, T ₃ , T ₄ , ETR and TSH,
Tumor markers such as CEA and AFP and HBs antigen,
Further examples include biologically derived substances such as various antibodies or receptors for each of these substances.

Therefore, the substance to be immobilized on the paper of the kit of the present invention is a substance that can specifically bind to the above-mentioned biological substance, that is, for example, when the test substance is HCG, the substance to be immobilized on the paper is Anti-HCG antibodies are preferred. Furthermore, when the substance that can specifically bind to the test substance is an antibody, the antibody is preferably a monoclonal antibody.

In addition to the above-mentioned antigen-antibody reaction, the specific binding reaction between the test substance and the substance immobilized on the paper includes, for example, a ligand (hormone)-receptor reaction.
Examples include reactions between.

Desired substance (e.g. antibody against test substance)
have, for example, activated Ro paper with BrCN and then immobilized the substance on Ro paper by the method of Porath, J. (Porath, J., Aspberg, K., Drevin, H.,
& Axen, R., Prepetion of cyanogen
bromide activated agarose gels.J.
Chromatography, 86: 53 , 1973).

The film-like support of the present invention is fixed directly to the bottom of the collapsible container, for example with an adhesive such as double-sided tape, or bonded to the bottom by other suitable means.

As the enzyme that can be used as a labeling substance, any enzyme commonly used in the field of enzyme immunoassay may be used, but horseradish peroxidase (HRP) is preferred.

The enzyme can be crosslinked, for example, by the glutaraldehyde crosslinking method (AVRAMEAS, S., Immunochemistry, 6:
43-52, 1969) and periodate cross-linking method (NAKANE, P., K., & KAWAOI, A., J.
Histochm. Cytochem. 22: 1084-1091, 1974
The target substance (e.g., the above-mentioned antibody) is detected by a method commonly used in the field of the above-mentioned enzyme immunoassay.
The enzyme-labeled substance is preferably dissolved in a buffer solution, such as, for example, phosphate-buffered saline.

Furthermore, the measurement sensitivity of the above reaction can be increased by using biotin and avidin by a method commonly used in enzyme immunoassay (see Heggeness and Ash (1977)).

The coloring reagent is one that can cause a coloring reaction using the labeled enzyme as a catalyst, for example, when the enzyme is HRP, 2,2-azinobis(3-
ethylbenzothiazoline-6 sulfonic acid)
(ABPC) or OPT, and H ₂ O ₂ . In this case, ABPC or OPT is oxidized by H ₂ O ₂ and exhibits a blue or red color reaction, respectively.

These coloring reagents are, for example, in the form of a solution dissolved in phosphate buffered saline or water, and H ₂
Preferably, O ₂ and ABPC or H ₂ O ₂ and OPT are each sealed in separate bags.

The kit of the present invention can further include a color table used as a standard for color reaction, an operation manual, and the like.

[Operation/Effect] In the kit of the present invention, first, a collapsible container is assembled, a sufficient amount of a sample such as urine or blood is received in this container, and the sample is brought into contact with the paper bonded to the bottom of the container. Between the substance fixed on this paper and the test substance contained in the sample,
A specific binding reaction is allowed to occur for a certain period of time, and then the sample is discarded from the container. After that, the bag containing the solution of the enzyme-labeled substance is torn, and the solution is poured into the bottom of the container. contacting with a paper for a certain period of time to cause a specific binding reaction for a certain period of time between the test substance specifically bound to the paper by the reaction and the enzyme-labeled substance;
Next, the bag in which the solution containing the coloring reagent was sealed is torn, and the solution is brought into contact with the paper for a certain period of time in the same manner as before, and the labeled enzyme immobilized on the paper via the bound analyte is catalyzed. A color reaction is caused on the paper, and the target test substance is measured qualitatively or semi-quantitatively based on the color reaction.

Incidentally, during this operation, it is also possible to tear the bag at the same time and bring the enclosed solutions into contact with the paper at the same time.

When the target test substance is present in a sample such as urine or blood, the test substance specifically binds to the paper, and furthermore, the enzyme-labeled substance to the test substance specifically binds to the test substance. Upon binding, a coloring reaction occurs on the paper due to the catalytic action of the labeled enzyme.

The feature of this kit is that the solid phase in the above reaction is bonded to the bottom of the container, eliminating the need for the troublesome cleaning step that was previously required with this type of measuring device. It's summer. That is, after each reaction is completed, by simply tilting the container or simply discarding each reaction solution out of the container, the analyte unbound to the paper and the colored reaction solution are removed from the paper, and as a result, each reaction is completed. The so-called (bound)/F (free) separation of components can now be performed extremely easily without the troublesome operation of washing that was previously required.

As a result, by using the kit of the present invention, biological substances can be measured extremely easily and economically at home, etc., and this kit can be used to carry out such measurements on a daily basis. By performing this on a regular basis, it has become possible to detect abnormalities in the body at an early stage.

The present invention will be explained below based on the accompanying drawings. The kit of the present invention shown in the accompanying drawings embodies the present invention, and the content of the present invention is not limited thereto.

FIG. 1 is an enlarged plan view of a plastic piece 1 which is the film-like support of the kit of the present invention. A piece of paper 2 on which a substance that can specifically bind to the test substance is immobilized is bonded to the center of the plastic piece.

A bag 3 encloses a solution containing a substance capable of specifically binding to a test substance and labeled with an enzyme, and a solution containing a coloring reagent capable of reacting with the labeled enzyme. bag 4,
4' are also attached to one side of the above paper 2.
It is similarly bonded onto the plastic piece 1.

FIG. 2 is a developed view of the foldable container 5 of the kit for measuring biological substances of the present invention. A polyethylene film 8 is laminated to the entire inner surface of a paperboard 7 having a cutout hole 6 in a part thereof, and heat-bonded parts 9 of a constant width are provided along the edges of the four sides of the paperboard 7. A central part 10 and a folded part 1 parallel to it
Perforations are formed at 13 and 14 along lines 1 and 12. As a result, the foldable container is folded into a W-shape and sealed by the heat bonding part 9, so that it takes the form of a flat bag.

The plastic piece 1 is bonded to the inner surfaces of the folded portions 11 and 12, which form the bottom of the foldable container 5 when assembled, as shown by dotted lines.

The upper part of the foldable container 5 is cut off along the perforations 15 formed in the upper part to open the container. Next, pinch the heat-adhesive parts 9 with your fingers,
By pressing lightly from the left and right toward the center, the folded parts 11 and 12 open, and the perforations 16 and 1 formed vertically with respect to the folded container are opened.
7, the folding container is assembled as a whole into a rectangular box-shaped pot having folded portions 11 and 12 as the bottom.

The inner surface of this tip is made of polyethylene film 8.
Because it is uniformly covered with water, the liquid sample placed inside the cup is retained without leaking.

An example of the production of the kit of the present invention and the measurements used therein will be shown below.

[Example] I Pretreatment of Ro paper 100 g of Toyo Ro paper (No. 6) was infiltrated with 7 ml of distilled water for 20 to 60 minutes at room temperature, and a concentration of 3 to 10% by weight was added to the Ro paper.
of distilled water. Next, 7 ml of a 3-10% by weight BrCN aqueous solution was added to this with stirring. Immediately after this, add 1M NaOH aqueous solution and
The BrCN solution was adjusted to pH 10-10.5. After 2-5 minutes, add 0.005M NaHCO ₃ aqueous solution cooled to 4℃ to 140℃.
ml was added and stirred, and finally the solution was filtered through a Buchner funnel to remove the reaction solution.
The washing operation with _NaHCO3 was repeated 3-4 times.

Next, place each piece of paper on this Buchner funnel, about approx.
It was washed sequentially with 50 ml of cold acetone aqueous solutions (4° C.) of 30%, 70%, 100% and 100% by weight. Place the washed and pre-treated paper in the refrigerator for 1-2 minutes.
It took a while to dry. This paper is then heated to -30℃.
It is stable and retains its activity for about 2 years if stored at room temperature.

Immobilization of antibody Target amount of HCG antigen (800IU/l ~
20-30 ml of an aqueous solution containing enough anti-HCG antibody (manufactured by UCB (Belgium)) to detect 1000 IU/l or more
100 g of the pretreated paper was placed inside and reacted at 4° C. for 36 to 72 hours with stirring using a magnetic stirrer. Thereafter, excess of the antibody solution was removed, 70 to 100 ml of aqueous sodium bicarbonate (0.1M) was added and mixed, and the aqueous sodium bicarbonate was removed by suction filtration. Next, 70 to 100 ml of a 0.1M NaHCO ₃ aqueous solution of β-ethanolamine (0.05M) was added and stirred with a magnetic stirrer for 2.5 to 3.5 hours at room temperature. Thereafter, the β-ethanolamine solution was removed by suction filtration, and the filter paper was then washed with about 100 ml of 0.1 M NaHCO ₃ aqueous solution, followed by 0.1 M acetate buffer (pH 3.5 ~
4.5) Repeated washing 3 to 4 times with approximately 100 ml.

After that, the paper was soaked in phosphate buffered saline containing 0.5-0.2 wt% BSA (0.05-0.1 wt% Tween20,
PH7.4-PH7.6) Washed 2-3 times with about 100 ml and stored in the refrigerator (can be stored in dry state or in phosphate buffered saline).

Preparation of a complex between antibody and labeled enzyme Target amount of HCG antigen (800 IU/l ~
Approximately equal volume of 50 ml of anti-HCG antibody (same as above) aqueous solution capable of detecting 1000 IU/l or more)
HRP aqueous solution (2-5% by weight) was incubated at 4° C. for 2-3 hours using a magnetic stirrer.

Preparation of coloring reagent 20-30 mg/ml of 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and 5-30 mg/ml
Water-soluble color reagent containing 10% _by weight _H2O2 0.5-1
ml was sealed in a plastic bag and placed in a foldable container.

The amount of HCG in a urine specimen was measured as follows using the kit of the present invention comprising the above-mentioned reaction reagents.

First, a collapsible container was assembled as described above, and approximately 5 ml of each urine sample was placed in this container.
The urine was discarded after several tens of seconds.

Next, the plastic bag containing the HRP-anti-HCG antibody complex solution and the plastic bag containing the coloring reagent were simultaneously torn to bring these solutions into contact with the paper.

After about 2 minutes, the container was tilted, the reaction solution was collected in the corner of the container, and the filter paper and the reaction product were separated.

If the test was positive, the paper turned blue.

The above test results were compared with results obtained for the same specimen using a commercially available testing device.

Correlation with commercial products Specimen No. Kit of the present invention Mochida Latex Device using reagent 1 to 10 Positive Positive 11 to 20 Negative Negative Out of n = 20 cases, 10 positive cases and 10 negative cases were both tested. There was a 100% correlation between the two.

[Brief explanation of the drawing]

FIG. 1 is an enlarged plan view of a plastic piece 1 of the present invention. FIG. 2 is an external development view of the foldable container 5 of the present invention. 1... Plastic piece, 2... Paper, 3, 4,
4'...Bag, 5...Folding container, 6...Notch hole, 7...Paperboard, 8...Polyethylene film,
9...Thermal bonding part, 10...Central part, 11, 12...
...folding part, 13, 14, 15, 16, 17...
…Perforation.

Claims (1)

[Claims for Utility Model Registration] (1) A kit for measuring biological substances, comprising a foldable container, an area on which a substance capable of specifically binding to the biological substances is immobilized on a piece of paper, and a kit for measuring biological substances. A region in which a solution containing a substance that can specifically bind to a biological substance and is labeled with an enzyme is enclosed, and a solution containing a coloring reagent that can react with the labeled enzyme is enclosed. and a film-like support having a region in which the kit is formed, the film-like support being joined to the bottom of the container. (2) The kit according to claim 1, wherein the substance that can specifically bind to a biological substance is an antibody. (3) The kit according to claim 2, wherein the antibody is an anti-HCG antibody. (4) Utility model registration claim No. 1 characterized in that the labeling enzyme is horseradish peroxidase and the coloring reagents are 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and hydrogen peroxide. The kit according to item 2 or 3. (5) The kit according to claim 2 or 3, wherein the labeling enzyme is horseradish peroxidase, and the coloring reagents are OPT and hydrogen peroxide. (6) The kit according to any one of claims 1 to 5 for utility model registration, which includes a color tone table as a standard for color reaction. (7) Utility model registration claims 1 to 5, characterized in that the solution containing the substance, which is labeled with an enzyme and is capable of specifically binding to a biological substance, further contains a buffering agent. The kit according to any of paragraph 6.