JPH0894618A - Simple measuring method and simple measuring device - Google Patents

Abstract

(57) [Summary] [Purpose] The present invention uses a plurality of colored latex particles having different color tones for the purpose of avoiding misjudgment of judgments on test results, and a single latex is used at different positions on an immunochromatographic support. (EN) An immunological diagnostic method indicated by color tone and a simple measuring device that can be performed by anyone, anytime, anywhere. According to the present invention, a plurality of immunologically different substances (including an indicator substance) are simply and quickly used by using colored latex particles having two or more kinds of different color tones, at different positions on the same support. It is composed of a method of detecting by coloring with a color tone, a test piece prepared according to this method, and a detection section equipped with the test piece, a sample dissolving section, and a sample collecting section, which can be integrated or connected and have an immunological reaction. After completion, it consists of a simple measuring device that can separate the detection part.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a simple measuring method and a simple measuring device, and more specifically, it applies an analytical method using a specific binding reaction for determining the presence or absence of a target in a sample to be analyzed. The present invention relates to a method and a device for easily and quickly measuring a. Examples of samples to which the present invention is applicable include all biological fluids such as serum, plasma, whole blood, urine, and feces, and products thereof.

[0002]

2. Description of the Related Art Conventionally, colloidal metal particles, colloidal metal oxide particles and colloidal non-metal particles have been widely used as labeling particles in immunochromatography using fine particles. It is already known as described in JP 15100 and JP-A-64-35372. Further, in recent years, there has been a remarkable technological development of dyed synthetic polymer latex particles with the development of the field of synthetic polymer chemistry, and an immunochromatographic method using these fine particles has also been developed, which is disclosed in JP-A-6-18032 and JP-A-6-18032. The method is already known as described in -160388. The products using this method are represented by pregnancy diagnostic agents (Clear Blue One Step <registered trademark> Unipass).

In the conventional detection method using immunochromatography, human gonadotropin (hC) as an antigen is prepared by using one kind of colored colloidal particles and one kind of colored latex particles as seen in the above-mentioned pregnancy diagnostics.
G) was detected to achieve the intended purpose. However, one of the most important patent requirements of the present invention, "method of using colored latexes having different color tones and simultaneously, simply, rapidly and clearly measuring on the same support", has been disclosed as an example. Absent.

Further, the simple measuring device of the present invention is characterized in that it has three functions of a sample dissolving section, a sample collecting section and a detecting section, and is composed of parts which can be divided into respective functions. (Fig. 3) When measuring, each part can be integrated or connected, and after measurement, only the detection part can be removed.
There is no disclosed example of a simple measuring device that achieves the purpose with an extremely simple operation, is hygienic, and has excellent characteristics such as storage of test results.

[0005]

Conventionally, as an application to the field of in vitro diagnostics using immunochromatography, it can be found in pregnancy diagnostics (Clear Blue One Step <registered trademark> Unipass) currently on the market. As described above, as an important marker of pregnancy, a method of determining the presence or absence of pregnancy or assisting the diagnosis by detecting human gonadotropin peptide hormone in urine is easy to operate, quick, and accurate in the field of in vitro diagnostics. It is an epoch-making product in that information can be obtained. However, a pregnancy diagnostic drug using a commercially available immunochromatographic method such as Clear Blue One Step is shown by a line for detecting human gonadotropin in urine and two lines indicating the end of reaction, and the color tone indicating these lines is, for example, Since it is the same color such as blue, if the user does not fully understand the sufficient explanations or written descriptions by the pharmacy, drugstore, or doctor, it may cause misidentification or the judgment result to be a big social problem. It can be.

Especially in Japan, since the pregnancy diagnostic drug can be purchased as an in-vitro diagnostic drug for general use at pharmacies and drug stores, this product can be operated by anyone anytime, anywhere, and the result is wrong. It is the most important thing to be able to make a clear decision without any. The present invention attaches particular importance to this point,
Using colored latex particles of two or more different colors,
It discloses a method of devising a simple measuring device that can be operated by anyone at any time, anywhere, and by clearly distinguishing the negative side and the positive side in the determination of results. For example, according to one aspect of Example 2, when the test result is negative, it is indicated by one blue indicator line, when it is positive, the indicator line is blue and, for example, the red color that appears when hemoglobin is detected. Indicated by two of the lines.

[0007]

[Means for Solving the Problems] The background to the completion of the present invention and the means for solving the problems will be described in detail below. In recent years, the detection of a substance having a desired immunological ability by utilizing an antigen-antibody reaction has made great progress mainly in the medical field. In the first place, the antigen-antibody reaction itself is a reaction occurring in the living body or the natural world according to necessity, and it is greatly benefited by the fact that human beings are elucidating the principle and mechanism. Particularly in the medical field, it has great significance as a prediction of diagnosis or as an aid to diagnosis, and since blood, urine, feces, body fluids, etc. are used as test liquids, it is advantageous that the test can be performed without causing pain to the patient.

Originally, the antigen-antibody reaction is "lock and keyhole"
As is similar to the relationship of, a specific binding substance is formed by the binding between a substance that is involved in an immunological reaction and a corresponding substance, most of which are not strong bonds such as covalent bonds, It holds with a very weak bond. From this, it is possible to artificially recover the antigen or the antibody individually from the antigen-antibody reaction complex. Therefore, in the antigen-antibody reaction system, just like the relationship of the substrate to the enzyme, the concept of affinity is established. That is, when you want to isolate and purify the target enzyme, the substrate of the target enzyme is immobilized on the resin (carrier) in advance, and only the target enzyme binds to the substrate, so after washing with water, the eluent High-purity enzyme can be recovered all at once by elution with. Such an isolation / purification means is called affinity chromatography. The immunochromatography used as the detection means of this method should be included in the affinity chromatography in view of the chromatographic system, and the partition coefficient between the solute and the solvent, which is generally described in the name of chromatography, is used. It is different from the means of separating and purifying substances by the difference of.

That is, an antigen or antibody immobilized in advance on a support is present, and colored labeled fine particles at the lower end of the support are sequentially diffused by the action of capillarity of the support using a solvent such as water as a medium. Upon migration, the complex-colored labeled particles immunologically formed reach the position where the previous antigen or antibody was immobilized, and the complex-fine particles specifically reach the position. -An antibody reaction occurs, which can be visually observed as a colored signal.

Based on the above-mentioned principle of affinity chromatography, the present method was further developed and earnestly studied, and as a result, the present invention was completed. It is possible to use different colloidal metal particles having different colors, or different colloidal metal oxide particles having different colors, and further different colloidal non-metal particles having different colors as the fine particles used in the immunochromatography described in this patent. However, synthetic polymer latex particles are preferable in consideration of the variety of color tones and the sharpness after development by immunochromatography. Commercially available synthetic polymer latex particles used for immunochromatography are designed so that those skilled in the art can carry out the chromatography under the optimal conditions, and also in view of the current synthesis technology level of polymer chemistry, it is of a high standard. It is a high-quality product that makes full use of technology. By the way,
From the results of use in the examples described in the present patent by the inventors, preparation of labeled particles with an antigen or antibody, stability by a drying step after labeling, and further diffusion on a support
It does not cause any problems in terms of mobility and sharpness of coloring at the solid-phased position. Such high quality latex particles can be easily obtained by those skilled in the art.

For example, latex particles having different color tones of blue latex particle dispersion, green latex particle dispersion and red latex particle dispersion can be easily obtained from Polymer Laboratories. In addition, it is also available from Rhone Poulin, Ceradaine, International Dynamics Corporation and Sekisui Chemical Co., Ltd. The size of latex particles for immunochromatography is 0.05
It is commercially available in the range of up to 5 μm, and it can be used in any size within this range, but it is preferable to select the particle size appropriately depending on the antigen or antibody to be labeled. Also important from the top, usually 0.45
A size of μm is suitable.

Next, the support used in the immunochromatography used in the present invention will be described. The support used in the immunochromatography useful in the present invention has an action of causing a capillary phenomenon, and a complex of a synthetic polymer labeled latex particle and a substance to be detected is a developing agent for immunochromatography, for example, water. Any support can be used so long as it can be diffused and moved quickly with or buffer solution. Generally, a glass fiber sheet, a filter paper, a nylon sheet, a nitrocellulose sheet or the like can be used as a carrier used for immunochromatography, and the sheet suitable for the present invention is a nitrocellulose sheet. Nitrocellulose sheet is BAS-85 (Schleicher
& Schuell), HAHY (Millip
ore), and can be easily obtained by those skilled in the art.

Next, a method for immobilizing a plurality of antigens or antibodies on a support will be described. A nitrocellulose sheet can be selected as a support useful in completing the invention,
The immobilization of a plurality of antigens or antibodies on the present support can be well understood by specifically explaining it by taking the embodiment described in Examples as an example.

The indicator substance of the present invention is used in the present invention as long as it is a substance present in a test liquid to be measured and a substance that does not cross-react with a substance capable of immunologically reacting with the substance. However, it is not particularly limited, and immunoglobulins of other animals are usually preferable. Further, the red line has the same interpretation as the traffic signal color of the traffic sign, and indicates a warning or abnormality, and the blue line alone means that no abnormality is recognized. Therefore, since it is easy for anyone to distinguish negative or positive by colors having different color tones in this way, erroneous determination can be avoided and it can be used with confidence.

That is, usually, a nitrocellulose support prepared in an arbitrary size suitable for carrying out immunochromatography is prepared in advance, and the first antigen or antibody (in the predetermined zone of the support is arbitrarily selected). In Example 2, an anti-rabbit IgG polyclonal antibody) is applied or sprayed, and the second antigen or antibody (anti-human hemoglobin antibody in Example 2) is applied or sprayed in a zone other than the above to solidify. To be compatible. Also, the solid phase immobilization of the third, fourth, fifth, etc. antigens or antibodies can be carried out in the same manner. Incidentally, here, the antigen or antibody immobilized on the support is subjected to the subsequent immunochromatographic development, and the antigen-antibody is immobilized on the immobilized zone at different positions.
It is desirable to set the coloring shape in advance because it causes an antibody reaction and causes coloring.

However, in the present invention, since the coloring is different in each zone, any shape of coloring, such as ●, −, +, ⊚, ×, Δ, ... Is acceptable. Preferably, the spot-shaped (●) or line-shaped (-) display is simple and clear.

Next, the drying process will be described. Normal drying may be carried out at room temperature, but if necessary, drying with hot air at 30 to 50 ° C. or vacuum drying may be appropriately performed. After drying, masking with another protein to inactivate areas other than the solidified zone of the first and second or third, fourth, fifth, etc. antigens or antibodies on the nitrocellulose support. It is usually done. Generally, as a masking agent, a casein solution prepared by dissolving 0.2% in physiological saline or 1%
Skim milk (manufactured by DIFCO), 4% Block Ace (manufactured by Meiji Dairy Co., Ltd.) and the like are used, but it is necessary to select an appropriate masking agent each time depending on the properties of the antigen or antibody or the support to be used.

Next, a method for preparing and attaching the synthetic polymer-labeled colored latex particles will be described. In labeling different latex particles having respective colors with a plurality of antigens or antibodies, for example, red latex particles have a first antigen or antibody, blue latex particles have a second antigen or antibody, Furthermore, the yellow or green latex particles are individually labeled with the third and fourth antigens or antibodies. The labeling method is not particularly limited as long as it is a commonly used method, but generally, the latex particles and the substance to be labeled are kept at room temperature at about 1
It can be achieved by mixing the time. Thereafter, each labeled colored latex particle is individually collected by a method such as centrifugation or a suction filtration method using a Millipore filter, washed well with a commonly used buffer solution, for example, a phosphate buffer solution, etc., and then each masked with a masking agent. The labeled latex particles of are individually masked. The masking agent may be the same as the masking agent used after immobilization of the antigen or antibody on the support.

However, it is necessary to judge whether the masking agent is suitable or not depending on the properties of the antigen or antibody used for labeling and the support. The colored latex particles that have been individually labeled in this way are stored in a storage solution. A predetermined amount of each labeled colored latex particle is taken out from the stored solution, mixed well, thoroughly washed, impregnated with a porous polymer such as a nonwoven fabric, and freeze-dried. By mounting this on a fixed place of the support, the detectable test piece of the present invention is completed. In addition,
The attachment of the labeled latex particles to the support is not particularly limited, and a method of directly applying the labeled latex particles to the support can also be used. The mounting position is determined by the immunochromatography deployment method.For example, if the deployment method is the ascending method, it is at the lower end, if it is the descending method, it is at the upper end, and if it is the horizontal method, it is at either end. In the case of the circular system, it is attached to the center of the concentric circles.

In the present invention, the developing method is not particularly limited, but an ascending method which is generally performed is desirable. In this case, it is advantageous that the tester can develop the support by only immersing the mounted support portion in the test liquid.

Next, the test piece will be described. (See Fig. 1) Regarding the test pieces for immunochromatography,
Since many inventions have already been disclosed, it is not necessary to particularly refer to the present invention in detail. However, one example will be given as a "proof" that the method of the present invention can be carried out reliably and accurately by the parties concerned. The test pieces shown in the examples are made of nitrocellulose or the like and cut into an arbitrary rectangular size in consideration of ease of use to prepare a support. Dip the lower end of the support into the test solution and use a capillary phenomenon to absorb a small amount of an antigen or antibody dissolved in a solvent such as water as a medium, and attach a part selected from an inert material that can move to the top of the nitrocellulose support. To do. As the inert material, filter paper, glass fiber sheet, membrane filter sheet, etc. are generally used. At the lower end of the nitrocellulose support, a lyophilized product of the labeled mixed colored latex particles (colored latex particle labeling substance lyophilized carrier) placed on the above-mentioned nonwoven fabric is cut into a certain size, for example, There is no problem even if it is lightly fixed with tape, etc., or pressure-bonded with cellophane tape etc. at the stage when it is finally assembled as a test piece. In addition, as shown in FIG. 1, a plurality of antigens or antibodies are immobilized in advance at different positions on nitrocellulose.

On the other hand, by mounting a water-absorbing carrier on the upper end portion of the nitrocellulose support as shown in FIG. 1, the immunochromatography can be developed accurately and quickly, so that it can be further developed. It is desirable to mount this carrier for the purpose of preventing liquid leakage after finishing. Such a water-absorbent carrier is a filter paper No. manufactured by Advantech Toyo Co., Ltd. 526 and Whatman 17Cr, 3MM
Are commercially available and can be easily obtained. As described above, the method of the present invention can be verified or implemented with such a simple test piece.

For the sake of convenience described in the present specification, when the substance to be detected is an antigen, for example, an antibody capable of recognizing a certain site of the substance to be detected is immobilized on a nitrocellulose support, while The method of the present invention can be achieved by labeling the synthetic polymer latex particles as long as the antibody can recognize an antigen molecule at a position different from the above antibody. In addition, it is easily understood by those skilled in the art that, contrary to the above, the present invention can be carried out in the case of detecting an antibody by a system composed of immobilized antigen-substance to be detected-antibody of substance to be detected. See. Therefore, the present invention is not limited to the case where the substance to be detected is an antigen, and also includes an antibody, and the expression is described as "antigen or antibody", but if either one is determined, Naturally, the other corresponding will be determined, and the method of the present invention can be carried out by appropriately using the letters "antigen" and "antibody".

Next, the simple measuring device will be described. An example of this device is shown in FIG. 3, but if the subject is semi-solid such as feces or fluid such as urine or blood, it is necessary to change the shape of the sampling portion as appropriate according to the physical properties of the fluid. That is self-evident. When the subject is semi-solid, for example,
This object can be achieved by providing a spiral groove, a circle-shaped depression, a spot-shaped depression, or the like. On the other hand, in the case of a fluid such as urine or blood, the object can be achieved by, for example, forming the sampling portion into a spoon shape or sandwiching an inert sheet such as a filter paper or a glass fiber sheet capable of absorbing a certain amount of liquid. . The apparatus shown in FIG. 3 will be described as one shape for measuring human hemoglobin in feces when the subject is feces, but the apparatus of the present invention is not necessarily limited to this. When collecting feces, the sample collection component and the detection component can be connected to each other and the tip portion can be pierced into the stool at several places to easily insert the stool into the sample collection hole. At that time, the stool attached to the other part can be wiped off with a toilet paper or the like to collect a certain amount of the stool. Gently press the tip of the collection part where the stool was collected to break the seal of the sample dissolution part, and push it in as it is to bring it into close contact, and then shake the integrated simple measuring device vigorously up and down to dissolve the stool.

After that, by keeping the integrated simple measuring device horizontal, a fixed amount of the lysing solution enters the sample intake port, and is developed and moved by this lysing solution amount, and after 3 to 5 minutes, a blue line and a red line. It is possible to easily judge as positive when two of the lines are observed and negative when only the blue line is present. Further, regarding the handling of the inspection result, since the detection unit can be removed, there are the following merits. That is, since the simple type device achieves the purpose by an extremely simple operation and is easy to store, mail or carry the test results, conventionally, considering that the subject directly submits a stool to an outpatient, The device is extremely hygienic, eliminates mental distress to the subject, and is extremely advantageous in terms of biohazard to medical personnel.

[0026]

The present invention will be described in more detail and concretely with reference to the following examples, but the method and apparatus of the present invention are not limited to these examples. Example 1 Method for detecting hemoglobin and transferrin

(1) Anti-human hemoglobin monoclonal
Preparation of Antibody Double-crystal human hemoglobin (manufactured by Sigma) was dissolved in sterile distilled water to prepare a 10 mg / ml human hemoglobin solution. Next, human hemoglobin solution and Freund
An equal amount of the complete adjuvant of 1. was mixed to obtain an oil emulsion. This is a BALB / cA mouse (BALB
/ CA jcl, 7 weeks old, female) 0.2 ml subcutaneously on the back
Each was administered. After the first immunization, booster immunization was performed on the 7th and 16th days, and further 3 days before cell fusion, a human hemoglobin solution was intraperitoneally administered at 0.25 mg / 0.2 ml.
Mouse spleen cells and myeloma cells (P3x63-Ag-6,5,3) on the third day after the final immunization were fused at a ratio of 10: 1 with 50% polyethylene glycol 4000 and selected by HAT medium. On the 14th day after cell fusion, the antibody activity against human hemoglobin in the culture supernatant was measured by EI.
It was measured by Method A. The measurement method is human hemoglobin 10
A 96-well EIA plate (manufactured by Coaster) immobilized at 0 μg / ml was used, and 200 μl of a culture solution of the fused cells was added. Then, after reacting at 37 ° C. for 1 hour, washing was performed and peroxidase-labeled anti-mouse IgG (manufactured by Cappel,
200 μl of 1: 500) was added. After washing, a substrate solution (0.1 M O -phenylenediamine and 0.012% hydrogen peroxide solution) was added to each well in an amount of 200 μl and reacted at room temperature for 5 minutes. After the reaction, add 50 N of 4N sulfuric acid to each well.
The enzyme reaction was stopped by adding μl each. Next, the absorbance value at 492 nm was measured, and the clone reacting with human hemoglobin was cloned twice by the limiting dilution method.
As a result, 10 monoclonal antibodies were obtained as ascites fluid after cloning. 1 ml of each of these obtained ascites was diluted 2-fold with 1 ml of PBS, 2 ml of saturated ammonium sulfate was added dropwise, and the mixture was left at 4 ° C. for 4 hours. Then, the mixture was centrifuged at 3,000 rpm for 20 minutes, and the precipitate was suspended in 2 ml of PBS (phosphate buffered saline) and dialyzed. Of these, two clones with high reactivity (K-
6, K-8) was selected and used in the following tests. The immunoglobulin subclasses of 2 clones are IgG ₁ , L
The chains were kappa type.

(2) Preparation of antibody-immobilized support Nitrocellulose sheet (BAS-85, Schlei)
cher & Schuell) 5mm x 50m
m, and at the position 10 mm from the lower end, 0.5 mg / ml of anti-human hemoglobin monoclonal antibody solution (K
-6), 0.5 mg / ml anti-human transferrin monoclonal antibody solution (Chemicon) at 15 mm position, 0.5 mg / ml anti-rabbit IgG polyclonal antibody solution (Cappel) at 20 mm position, respectively. (Manufactured by Olympus Co., Ltd.) to apply a line of anti-human hemoglobin antibody, anti-human transferrin antibody, and anti-rabbit IgG antibody. After drying at room temperature for 2 hours, 1% skim milk (manufactured by DIFCO) -0.
Masking was performed by immersing in PBS containing 1% Tween 20 at 37 ° C. for 2 hours. Then, it was sufficiently dried to prepare an antibody-immobilized support.

(3) Preparation of colored latex particle label a. Red latex particle-labeled anti-human hemoglobin antibody Red latex particle dispersion (PL-Latex, 10
%, 450 nm, Polymer Laborator
1.2 ml of PBS was added to 300 μl of 1)
Centrifugation was performed at 3,000 rpm for 5 minutes. Anti-human hemoglobin monoclonal antibody solution (0.5 mg
/ Ml) (K-8) (1 ml) was added, mixed well, and reacted at room temperature for 1 hour. To remove unreacted anti-human hemoglobin monoclonal antibody, 13,000 rpm,
After centrifugation for 5 minutes, the precipitate was suspended in 1.5 ml of PBS and centrifuged again. 1 ml of 4% Block Ace (manufactured by Meiji Dairy Co., Ltd.) was added, and the reaction was carried out at room temperature for 60 minutes for masking. After that, centrifugation was performed at 13,000 rpm for 5 minutes, and sedimentation was performed with 1% skim milk-0.01%.
The cells were suspended in 1.5 ml of PBS containing sodium azide and stored under refrigeration. b. Green latex particle labeled anti-human transfer
Antibody green latex particle dispersion (PL-Latex, 1
0%, 450nm, Polymer Laborato
RIE) and anti-human transferrin monoclonal antibody (0.5 mg / ml, manufactured by Advance Immunochemical). c. Blue Latex Particle Labeled Rabbit IgG Blue Latex Particle Dispersion (PL-Latex, 10
%, 450 nm, Polymer Laborator
ies) and rabbit IgG (0.5 mg / ml,
(Manufactured by Cappel) was prepared in the same manner as above. d. Colored latex particles labeled freeze-dried carrier These three types of colored latex particles labeled are mixed in equal amounts, and Benliese (registered trademark) non-woven fabric (manufactured by Asahi Kasei Corporation) 5 m
It was prepared by impregnating 10 μl of m × 5 mm and freeze-drying.

(4) Preparation of test piece A colored latex labeled lyophilized carrier was piled up to a position 2.5 mm from the lower end of the antibody-immobilized support. Further, a carrier for dipping the test liquid was placed on the freeze-dried carrier for colored latex label up to a position 2.5 mm from the lower end. In addition, a water-absorbent carrier 5 mm x 20 m up to a position 5 mm from the upper end of the antibody-immobilized support
m (No. 526, manufactured by Advantech Toyo Co., Ltd.),
Finally, a transparent tape was attached to the upper part and fixed to obtain a test piece. (Fig. 1)

(5) Reactivity test using a standard solution Human hemoglobin (double crystal, Sigma) and human transferrin (Chemicon) 100 μg / each
Two kinds of standard solutions were prepared by dissolving with PBS containing 0.1% BSA so as to make up to ml. 200 μl of the standard solution was added dropwise to the carrier for immersing the test solution in the test piece, and then developed. 5 minutes later,
Judgment was made based on the presence / absence of coloring of the solid-phased line portion. As a control, P containing 0.1% BSA
The same operation was performed using only BS. When two kinds of standard solutions were developed, a red line (human hemoglobin), a green line (human transferrin) and a blue line (indicator substance) were detected. In addition, only red and blue lines were detected in the human hemoglobin standard solution, green and blue lines were detected in the human transferrin standard solution, and only the blue line was detected in the control. (Fig. 2)

Example 2 Method for detecting human hemoglobin in feces (test piece) (1) Preparation of antibody-immobilized support Nitrocellulose sheet (BAS-85, Schlei)
cher & Schuell) 5mm x 50m
m, and at the position 10 mm from the lower end, 0.5 mg / ml of anti-human hemoglobin monoclonal antibody solution (K
-6), 0.5 mg / ml of anti-rabbit IgG polyclonal antibody solution (manufactured by Cappel) was applied to the position of 20 mm using an airbrush (manufactured by Olympos), and a line of anti-human hemoglobin antibody and anti-rabbit IgG antibody was applied. Was produced. After drying at room temperature for 2 hours, PB containing 1% skim milk (manufactured by DIFCO) -0.1% Tween 20.
Masking was performed by immersing in S at 37 ° C. for 2 hours. Then, it was sufficiently dried to prepare an antibody-immobilized support.

(2) Preparation of Colored Latex Particle Labeled Product Red latex particle labeled anti-human hemoglobin antibody and blue latex particle labeled rabbit IgG used each labeled latex particle prepared in Example 1. Colored latex particle labeled material Lyophilized carrier These two types of colored latex particle labeled materials were mixed in equal amounts, and Benliese (registered trademark) non-woven fabric (manufactured by Asahi Kasei) 5 m
It was prepared by impregnating 10 μl of m × 5 mm and freeze-drying. (3) Preparation of test piece It was prepared in the same manner as in Example 1.

(4) Reactivity test using a standard solution Human hemoglobin (2 times crystal, manufactured by Sigma) was 1 mg / ml, 1 μg / ml, 100 ng / ml, 10 respectively.
A standard solution was prepared by dissolving with PBS containing 0.1% BSA so that the concentration became ng / ml. 200 μl of the standard solution was added dropwise to the carrier for immersing the test solution in the test piece, and then developed. The same operation was performed using PBS containing 0.1% BSA as a control. A red line was detected up to a human hemoglobin concentration of 100 ng / ml. In addition, the line of rabbit IgG, which is an indicator substance, was detected in all cases. The test results are shown in Table 1.

[0035]

[Table 1]

(5) Human Hemoglobin Detection Test in Feces Human hemoglobin was added to fecal occult blood negative stools at 1 / g of stool.
0.1, 0.05 and 0.01 mg were added and mixed well. Next, 10 mg of each human hemoglobin-containing stool was collected, and 0.005% Tween 20-0.1% BSA was collected.
The suspension was suspended in 2 ml of PBS containing the above, mixed well, and then quickly filtered with a sponge to give a fecal suspension. Stool suspension 200μ
1 was added dropwise to the carrier for immersing the test liquid and then developed. The same measurement was performed for human hemoglobin-free stool as a control.
In human hemoglobin-added feces, a red line was detected up to a concentration of 0.05 mg / g. In addition, the blue line of rabbit IgG, which is the indicator substance, was detected in all cases. The test results are shown in Table 2.

[0037]

[Table 2]

Example 3 Method of Detecting Human Hemoglobin in Feces (Measurement by Using Integrated Simple Measuring Device) (1) Preparation of Integrated Simple Measuring Device This device is a sample dissolving part (31) and a sample collecting part (32). , A detection unit (33). Specimen dissolution section (31)
The solution (34) (PBS containing 0.1% BSA-0.005% Tween 20) (2 ml) was added to and the opening was completely adhered by a seal of an aluminum base material so as to prevent liquid leakage. The detection part (33) has a structure that can be divided into two parts up and down, and the test piece prepared in Example 1 was mounted therein. Next, at the opening of the sample collecting part (32) on the side of the detecting part (33), 5 mm × as a sample solution absorbing carrier (37)
A filter paper (No. 526, manufactured by Advantech Toyo Co., Ltd.) cut into 30 mm was inserted. The detection unit (33) and the sample collection unit (32) were connected via the connection unit (38) to provide a device capable of collecting a sample.

(2) Test Method Feces were pierced several times on the side of the specimen collecting part (32) of the specimen collecting specification, and the feces were put into the specimen collecting hole (35). Sample collection unit (3
Wipe off excess stool attached to the tip of 2) with toilet paper, tear the seal of the sample dissolving part (31) at the tip of the sample collecting part, and connect the sample dissolving part (31) with the sample collection specification, and then A simple body shape measuring device was used, and the device was vigorously shaken up and down to dissolve feces. Immediately after the judgment part was turned up, the integrated simple measuring device was left horizontally horizontally. By this operation, a fixed amount (200 μl) of the sample solution entered through the intake port, developed and moved, and the reaction was completed after 5 minutes. Then, the detection part (33) was removed from the connection part of the sample collection part (32) and the detection part (33), and the result was determined.

(3) Human Hemoglobin Detection Test in Feces Human hemoglobin was added to fecal occult blood negative stools at 1, 0.1, 0.05 and 0.01 mg per 1 g of stool, and mixed well to add human hemoglobin Stool was prepared. This added stool was measured by the test method of Example 3 (2). As shown in Table 3, even when using the integrated simple measuring device, the same result as in the case of only the test piece was shown.

[0041]

[Table 3]

(4) Measurement of stools of patients with colorectal cancer Ten stools of patients diagnosed with colorectal cancer were measured using an integrated simple measuring device. As a control, 10 healthy stools were also used. As a result, red lines (human hemoglobin) and blue lines (rabbit IgG) were detected in all patients with colorectal cancer, and it was determined that fecal occult blood was positive. In all the stools of healthy subjects, only the blue line (rabbit IgG) was detected, and the fecal occult blood was determined to be negative.

Comparative Example 1 Comparison with the latex method Using 140 cases of fecal samples, a reagent "OC-hemodia" for measuring hemoglobin in stool based on the latex agglutination method.
(Registered trademark) (manufactured by Eiken Chemical Co., Ltd.) and a fecal occult blood measurement method using an integrated simple measuring device were compared. 14 as shown in Table 4
131 cases out of 0 cases were coincident in both cases, and the concordance rate was 93.6%, which was a good result.

[0044]

[Table 4]

Comparative Example 2 Comparison of Difficulty of Judgment Result by Conventional Method and Present Invention For the case where the indicator line and the line of the substance to be detected have different color tones (the method of the present invention) and the same color tone (the conventional method) Regarding the difficulty of the judgment result, 12 panelists were randomly selected and compared. (1) Preparation of test piece by conventional method (same color tone) The antibody-immobilized support was prepared by using an anti-mouse IgG antibody (Cap) instead of the anti-rabbit IgG polyclonal antibody solution of Example 2.
(manufactured by pel) was similarly prepared. On the other hand, a blue latex particle-labeled anti-human hemoglobin antibody was prepared in the same manner as in Example 1. The particles were prepared by impregnating 10 μl of Benlyse (registered trademark) non-woven fabric (5 mm × 5 mm, manufactured by Asahi Kasei Co., Ltd.) and freeze-drying. A test piece was prepared according to the method of Example 1 using the above support and carrier, and a reactivity test was performed using the standard solution of Example 2. As a result, a blue line was obtained up to a human hemoglobin concentration of 100 ng / ml. Detected. In addition, the indicator line was detected as a blue line in all cases, and it was found that this test piece operates normally.

(2) Preparation of Colored Specimen by Method According to the Present Invention and Conventional Method The test pieces of the present invention and the conventional method prepared in Example 2 were prepared with human hemoglobin standard solution (1 μg / ml, 0.1, respectively). %
(Dissolved in PBS containing BSA) was reacted according to the method of Example 2 (4) to prepare colored test pieces by both methods.

(3) Comparison of Difficulty of Judgment Results by the Method According to the Present Invention and Conventional Method In order to compare the difficulty of judgment results by both methods, 12 panelists (including 5 females) were arbitrarily selected and 2 The judgment was made in accordance with the sensory test by the point preference test method ("Sensory Test Handbook", Nikkan Giren Sensory Test Committee (ed.), 1985). As shown in Table 5, from the methods conforming to the present inspection, all 12 persons answered that the method according to the present invention was “easier to judge”, and the method of the present invention was clearly superior.

[0048]

[Table 5]

[0049]

INDUSTRIAL APPLICABILITY According to the present invention, a plurality of substances can be visually detected simultaneously in different colors by immunochromatography using labeled latex particles having different colors. By providing the simplified measuring device according to the present invention, for example, when it is applied to an in-vitro diagnostic drug as an over-the-counter drug, it can be easily performed by anyone, anytime, anywhere. Further, since latex particles having different color tones are used for the determination of the inspection result, it is possible to make a clear determination without making a mistake. In addition, since this simple measuring device was devised as a unitary device that can be connected, it is possible to disconnect only the detection parts promptly after the inspection, collect them, and mail or deliver them to the inspection organization or hospital. it can. As described above, the present device is extremely hygienic and provides an extremely excellent device for medical personnel in terms of biohazard.

[Brief description of drawings]

1 shows one embodiment of a test piece of the present invention,
It is the test piece top view and sectional drawing.

FIG. 2 shows one embodiment of measurement results using the method of the present invention, which is the result of simultaneous detection and measurement of human hemoglobin and human transferrin.

FIG. 3 is a view showing one embodiment of a connection-integrated type simple measurement device, which is an integrated type (overall view), sample collection specifications (cross-sectional view, upper view), and measurement specifications (upper view).

[Explanation of symbols]

1 Absorbable carrier 2 Antibody-immobilized support 3 Colored latex particle label Freeze-dried carrier 4 Carrier for immersion of test liquid 5 Anti-rabbit IgG antibody-immobilized zone 6 Anti-human transferrin antibody-immobilized zone 7 Anti-human hemoglobin antibody Immobilization zone 21 Chromatogram developed with a mixture of human hemoglobin standard solution and human transferrin standard solution 22 Chromatogram developed with human hemoglobin standard solution 23 Chromatogram developed with human transferrin standard solution 24 Chromatogram developed with control 31 Specimen dissolution part 32 Specimen sampling part 33 Detection part 34 Lysis solution 35 Specimen collection hole 36 Specimen dissolution solution intake port 37 Specimen dissolution solution absorbent carrier 38 Coupling part 39 Test piece 40 Judgment window

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masahiko Wakasugi 1-5-3 Nihombashi Muromachi, Chuo-ku, Tokyo Inside Wakamoto Pharmaceutical Co., Ltd. (72) Inventor Eriko Matsuyama 1-5-3 Nihonbashi Muromachi, Chuo-ku, Tokyo Inside Wakamoto Pharmaceutical Co., Ltd. (72) Inventor Takeshi Maeda 1-5-3 Nihombashi Muromachi, Chuo-ku, Tokyo Inside Wakamoto Pharmaceutical Co., Ltd.

Claims (5)

[Claims] 1. An immunological measuring method using two or more kinds of colored latex particles having different color tones, comprising: a. In advance, two or more types of colored latex particles having different color tones are individually labeled with an indicator substance and one or more types of substances that are involved in immunological reaction, respectively. B. On the other hand, at least two kinds of substances capable of immunologically reacting at different positions on the same support,
Immobilized in advance, c. Then, a mixture of labeled colored latex particles comprising an indicator substance and one or more substances that are involved in an immunological reaction and at least one specific immunological substance in a test liquid are reacted, and d. Then, the test solution is developed and moved to a mixture of the colored latex particles labeled with the indicator substance and the labeled colored latex complex immunologically formed, and e. Colored latex particles labeled with corresponding indicator substances at a plurality of positions on which solidified substances having two or more immunological reactivities have been immobilized in advance and labeled colored latex immunologically formed. An immunological measuring method, characterized in that the complex is colored in different colors. 2. The method according to claim 1, wherein the specific immunological substance in the test liquid is hemoglobin. 3. A test piece according to the method of claims 1 and 2. 4. A simple measuring device comprising three functional parts including a sample dissolving part, a sample collecting part, and a detecting part, and each functional part can be integrated or connected as necessary, comprising: a. Harvesting is accomplished by coupling the harvesting component and the sensing component, b. Dissolution of the sample after collection and a fixed amount of sampling and immunological reaction are achieved by integrating or linking the sample dissolution component, the collection component, and the detection component, c. A simple measuring device that can separate the detection parts after completion of immunological reaction. 5. A simple measuring device incorporating the test piece according to claim 3 by the method according to claim 1 and claim 2.