JPH09318623A - Sampling and testing method for organic sample and simple testing device therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect trace of material to be tested with a good accuracy by sampling a predetermined quantity of a suspension in a measuring cup provided in a vessel, after organic sample is suspended in a dispersant and contacting an analyzing tool with the sampled suspension to be tested through an opening formed in a wall portion of the vessel opposed to the measuring cup. SOLUTION: A body 2 of the vessel can accommodates the dispersant of a predetermined quantity, can be inserted by an organic sample sampler 3 and has an opening as an insert section 4. A recess 14, receiving a predetermined quantity of an objective liquid to be tested while a volume is measured, is formed on the inside of a vessel wall of the body 2 of the vessel. The recess 14 is positioned so that when a immuno- chromatography test paper is contacted to the recess 14, the test paper is not blocked by a device which is already inserted. A means for producing an opening by a human hand or a device 3 is provided on the wall portion of the body 2 of the vessel opposed to the recess 14. A specimen preparation and sampling of a predetermined quantity of an objective liquid are conducted, the analytical tool is fittingly inserted into the opening and a predetermined quantity of the objective liquid is supplied to the analytical tool.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for easily carrying out a biological sample collection, a sample transportation, a sample preparation, and a sample test using an in vitro diagnostic agent for performing a clinical test, and a simple method for use in the method. The present invention relates to an inspection apparatus, for example, an inspection method for various test substances such as enzymes, occult blood, toxins and viruses using an immunological inspection method such as immunochromatography, and a simple inspection apparatus therefor.

[0002]

2. Description of the Related Art Using an in-vitro diagnostic agent to examine the presence of enzymes, occult blood, toxins, viruses, etc. in biological samples such as feces, sputum, vomiting and pus, which are collected from the human body,
It is extremely important in the diagnosis of cancer, ulcers, infectious diseases and various other diseases. To perform these tests, without going to a medical institution, a biological sample is collected at home or in a place such as a group medical examination, and transported to an inspection institution to detect the test substance hygienically, easily and with high sensitivity. A method and a simple inspection device used therefor have been proposed for early detection of these diseases.

For example, hemoglobin appearing in human feces has long been known to be a marker for digestive system diseases, and its examination has been conducted. Regarding the detection of hemoglobin, there are a chemical method utilizing a chemical reaction between a reagent such as the guaiac method and hemoglobin, and an immunological method utilizing an antigen-antibody reaction with an immunological reagent containing an anti-human hemoglobin antibody. However, the chemical reaction is a reaction with low specificity that also reacts with hemoglobin other than humans, and dietary restrictions and drug restrictions are required, and thus immunological methods have been widely used in recent years.

From such a background, a simple test method based on biological sample collection and immunological principles, which is excellent in hygiene in handling, simple operation, and detection sensitivity, or a simple test apparatus therefor is provided. Several proposals have been made in the past. For example, in Japanese Utility Model Publication No. 5-17653, a biological sample for testing, particularly a non-invasive testing sample such as stool, is prepared, a suspension is quantitatively prepared, filtered, and a sample for an immunological test. As described above. However, since an analytical tool such as immunochromatography is not bound to this sample preparation container, it is not possible to perform a series of operations from sample collection to sample preparation to completion of the test with one device. When testing is performed in a facility that does not have automatic testing equipment, or when using an automatic testing equipment because the number of specimens is small, the time for warming up the equipment and the cost of reagents that cannot be collected and remain in the equipment may increase. is there. In such a case, it is necessary to provide a simple device for performing the analysis operation manually with a simple mechanism that can combine the container for collecting the sample and preparing the sample and the analysis tool in a simple and hygienic manner, In addition, a simple device is required for self-inspection at home.

As a technique for responding to this, JP-A-6-32
No. 4037 discloses a container for collecting stool and preparing a sample, and a stool occult blood immunochromatographic determination unit.
Disclosed is a fecal occult blood determination device coupled via a partition wall and a spring. In this device, all the steps from stool collection to determination are performed by one device, so the operation is simplified. However, in this device, a container for preparing and storing a sample (hereinafter,
The bottom partition wall of the sample container (abbreviated as sample container) is destroyed by a flow path forming means such as a dropping needle, and the sample is made to flow out to the developing part that constitutes the determination part by immunochromatography in a single stroke. It is not possible to control the amount and flow rate of the sample that comes into contact with the biological reagent. As a result of the excessive supply of the sample in this way, the following problems occur and the detection accuracy becomes unsatisfactory.

The analytical principle of immunochromatography is
A labeled antibody to the test substance (hereinafter, abbreviated as labeled first antibody) is installed at one end of a developing portion, which is a columnar or thin strip-shaped plate-shaped stationary phase made of a porous material, and the labeled first A second antibody that recognizes an epitope different from the first antibody is immobilized at a position distant from the site where the antibody is set up. When a liquid sample containing a substance is developed, the first epitope of the test substance undergoes a specific antigen-antibody reaction with the first antibody to bind and generate while the sample moves as the mobile phase on the stationary phase. When the test substance-labeled first antibody complex further moves and reaches the second antibody immobilization site, an antigen-antibody reaction occurs between the second epitope of the test substance portion in the complex and the second antibody, and the test Substance
Since the labeled first antibody complex is captured by the second antibody immobilization site, the presence of the test substance can be visually recognized by using a labeling substance having a color tone that can be clearly distinguished from the developed part. Further, practically, the antibody against the first antibody is immobilized on the opposite side of the labeled first antibody installation site on the stationary phase with the second antibody immobilization site sandwiched therebetween. The labeled first antibody that has migrated to this point forms an immune complex regardless of the presence or absence of the test substance, which causes coloration, and this coloration indicates the end point of immunochromatography and the immune reaction is normally performed. It is used as an indicator to show that.

Here, if the sample is supplied in excess,
(A) The test substance has a low concentration, and the specimen that should be determined to be negative shows a false-positive determination result, and (b) the prozone phenomenon that the specimen that should be determined to be positive is due to excess antigen. For example, it shows a negative judgment result. (C) Further, the excess labeled first antibody is dispersed in the stationary phase, and the contrast of the color tone in the second antibody fixed part and the analysis end point indicator part is lowered. (D) If the second antibody is immobilized on the stationary phase by physical adsorption, the immunocomplex with the labeled first antibody once formed diffuses into the stationary phase and is analyzed by immunochromatography. There is a possibility that the end point becomes unclear, or the judgment result becomes unclear, and re-examination becomes necessary.

If the supply of the sample is insufficient, the sample cannot be developed on the stationary phase due to the lack of the amount of liquid as the mobile phase, and the analysis operation itself does not proceed. Therefore, in order to improve the inspection accuracy of the prior art utilizing immunochromatography, it is necessary to solve the above problems.

Furthermore, Japanese Patent Laid-Open No. 7-49347 discloses that
An apparatus for detecting fecal occult blood using immunochromatography using antibody-sensitized gold colloid as a carrier is disclosed. Even in this detection device, no structural measure is taken against the above problem, and therefore the device user is instructed to drop the amount of the sample so that the supply amount of the sample falls within a certain range. However, maintaining accurate sample dropping amount requires skill, and therefore, a general person is demanded to have a simpler method. In addition, the structure of the device is delicate and complicated, which is a problem in practical use for generalization.

On the other hand, Japanese Patent Laid-Open No. 7-63754 discloses that
A fecal occult blood test apparatus is disclosed which utilizes an immunological fecal occult blood reaction and performs a test by a latex agglutination method using antibody-sensitized latex. In this inspection apparatus, the side wall of the sample container for preparing the sample is pressed to drop the sample onto the analysis tool. Therefore, if the amount of drop is too large, the sample becomes excessive and retesting is required. In addition, since an indicator showing the end point of the reaction cannot be provided because it depends on a detection method different from immunochromatography, an experienced measurer must manage the reaction time and judge the detection result. The inspection is not easy for the person.

[0011]

Therefore, according to the present invention, a sample containing a biological sample collected for conducting a clinical test can be prepared in a hygienic and convenient manner, and subsequently the sample can be tested by immunochromatography. When performing, to provide a simple method that can determine the presence or absence of the test substance with high accuracy even without causing the problems of the prior art as described above, and also to collect the sample from biological sample collection It is an object to provide a simple device that can consistently carry out the transportation of the container as it is stored to the inspection agency, the mailing as a fixed-sized mail if desired, the preparation of the sample, and the determination of the presence or absence of the test substance. is there.

[0012]

Means for Solving the Problems The present inventor has studied in detail the problems of the above-mentioned prior art in immunochromatography, and has provided an analytical tool based on the immunochromatographic principle, particularly a fixed amount of antibody sensitizing reagent. It has been found that the above problems can be remarkably improved by controlling the amount of the sample liquid flowing to the developing part of the analytical device having a stationary phase (hereinafter, abbreviated as analytical device), and the invention of claim 1 was completed. Further, a simple mechanism for measuring a predetermined amount of the sample in the same container as the sample container for preparing the sample and subsequently supplying the sample to the analytical tool was found, and the invention of claim 2 was completed.

The present invention according to claim 1 relating to an inspection method, collects a biological sample using a biological sample collecting instrument, introduces the biological sample into a container containing a predetermined amount of dispersion medium in advance, and After suspending or dissolving in the dispersion medium, the following operation is carried out: (1) An aliquot of the suspension or the solution is dispensed into a measuring cup provided in the container to obtain a sample, (2) An opening is made in the vessel wall portion of the container facing the measuring cup, and (3) the analysis tool is brought into contact with the separated sample through the opening.
The invention is an invention of a simple method for collecting a biological sample, preparing a sample, and testing, which is characterized by performing each of the operations.

The present invention relating to the inspection apparatus according to the second aspect of the present invention, the container body 2 capable of accommodating a predetermined amount of the dispersion medium 1, the biological sample collecting instrument 3, and the sampling instrument insertion portion provided in the container body 2. An apparatus for preparing and testing a specimen, which comprises (4) a) a concave portion (14) for collecting a fixed amount of specimen on the inner surface of the container body (2). The invention is a simple biological sample testing device, characterized in that means for easily forming an opening is formed in the vessel wall portion of the container body 2 facing 14 of the invention.

The test method of the present invention comprises, for example, immobilization of immunochromatography using a labeled first antibody sensitized with a known first antibody that causes a specific antigen-antibody reaction with respect to a test substance of interest, and a second antibody. It is a method that can be applied to an inspection using a phase (hereinafter, abbreviated as immunochromatographic test piece) as an analytical tool. It is a method that can be widely used for fecal occult blood test for gastrointestinal disease screening that needs to efficiently process a large number of specimens, sputum test for respiratory disease screening, toxin test for group food poisoning, etc. For convenience, only the fecal occult blood test will be described in detail, but it can be applied if the operation procedure of the analytical tool is similar to immunochromatography, and is not limited thereto.

In the method of the present invention as set forth in claim 1, a sample is prepared by using the collected biological sample, a fixed amount of the sample is sampled, and the sampled sample is contacted with an analytical tool such as an immunochromatographic test strip. A container having a structure capable of performing the series of operations up to the same container is used.

First, a predetermined amount of the dispersion medium is stored in such a predetermined container in advance. In general, this dispersion medium can dissolve or disperse the test substance in the collected biological sample, can be stably stored without deterioration during the period from dissolution to completion of the test, and the antigen of the antibody and the test substance to be used. It should not interfere with the antibody reaction.

In the case of fecal occult blood test, a salt solution in the vicinity of neutrality such as physiological saline, phosphate buffer, Tris buffer containing tris (hydroxymethyl) aminomethane as a buffer is used. An antiseptic agent or a deodorant may be added to this, if necessary. Although the amount of the buffer solution is adjusted depending on the amount of the biological sample collected, generally 0.5 ml-10 ml is preferable.

In the method of the present invention, when collecting a biological sample, when it is desired to mix a fixed amount of the collected biological sample with the dispersion medium in the container body, the sliding of the container with the sampling tool insertion port is performed. Use a sampling device equipped with a mechanism that removes excess amount by interaction such as cutting action and leaves only a predetermined amount. In order to prevent bias in collection, two collection devices may be used so as to collect from the surface and inside of the stool sample, and in this case, the container is provided with two collection device insertion ports.

Then, the biological sample collecting instrument is inserted into the insertion port of the container, the insertion port is sealed with a lid, and the container is shaken to dissolve the biological sample in the dispersion medium. Rotate the container so that the pre-installed measuring cup as described above is inverted with the solution at the top and bottom at the top. Next, as shown in FIG. 2 (b), the container is rotated in the direction of the arrow in FIG. Meet and inset.

If it is necessary to remove the insoluble matter in the solution, the insoluble matter is allowed to settle by allowing the insoluble matter to settle in the state of FIG. 2 (a), and then the container is gently moved in the direction of the arrow in FIG. While spinning, dispense the supernatant of the solution into a measuring cup. In addition,
As another method for filling the sample in the measuring cup, first place the container in a horizontal position, then tilt the container in the direction in which the measuring cup is installed, and then fill the sample in the measuring cup. It is also possible to return the container to the horizontal position again. The method of the present invention is not limited to the manner of tilting or rotating the container as long as the sample is quantitatively collected using the measuring cup provided in the container.

At this time, the dispersion medium initially contained in the container is adjusted to a predetermined liquid amount so that the liquid level of the solution remaining outside the measuring cup is lower than the full liquid level in the measuring cup. By such a simple operation, it is possible to surely dispense the liquid amount suitable for use as the sample into the measuring cup.

Next, an opening is made in a required portion of the container wall facing the measuring cup by using a hand or an instrument, and the development starting end of the immunochromatographic test piece is formed from this opening as shown in FIG. 1 or in FIG. In the case of a test piece equipped with a sample absorbing part as shown, the sample absorbing part is inserted or fitted into the container, and the total amount of the sample held in the measuring cup is absorbed by capillary action by itself, and the test is carried out. Deploy on one side. In particular, in the test piece shown in FIG. 3, the entire developing portion except the sample absorbing portion is covered with a film made of a material that does not allow moisture or a dispersion medium component to escape by evaporation. If this is used, there will be no difference in evaporation of the dispersion medium due to changes in temperature and humidity during deployment,
It is particularly preferable because the influence on the analysis result can be suppressed.

The labeled first antibody that recognizes the first epitope of the test substance is applied as a immunological detection reagent to a portion of the immunochromatographic test strip that is exposed above the sample surface near the development start end by means such as coating. A second antibody that recognizes a second epitope different from the first epitope is immobilized as an immunological detection reagent at a position further apart from this placement site by a certain distance, that is, the activity is not impaired. Fix the test piece on the porous nitrocellulose membrane.

The test substance in the sample reaches the site where the labeled first antibody is located above the sample liquid surface due to the capillary phenomenon. Therefore, if the test substance is present, it binds to the reagent and becomes
Generate labeled first antibody complex. The generated complex is the second
It moves to the antibody immobilization site, is captured by the second antibody, and accumulates at the immobilization site.

Here, when an immunochromatographic test strip is used as the analytical tool, it is desirable that at least a part of the portion of the test strip where the labeled first antibody is arranged is always exposed above the sample liquid surface. If the entire site is submerged below the surface of the liquid, that is, completely submerged in the sample, the test substance-labeled first antibody complex does not move due to the capillary phenomenon, and the analysis itself cannot be performed. Therefore, the adjustment of the sample liquid volume is not only important for avoiding the prozone phenomenon, but also the mutual positional relationship between the immunological reagent placement site of the immunochromatographic test strip and the liquid surface at the moment when the sample comes into contact is controlled. In order to do so, it is a major factor to enhance the detection accuracy of this inspection method that a predetermined amount of the sample is always taken out by the measuring cup fixed in the sample container and brought into contact with the test piece.

Known immunochromatographic test strips can be used depending on the test substance. For example, in the case of fecal occult blood test, an anti-human hemoglobin murine monoclonal antibody is used as the first antibody, which is a label. Anti-human / hemoglobin / mouse monoclonal antibody bound to colloidal gold was applied to the development start end of the test piece, and anti-human / hemoglobin / mouse monoclonal antibody was immobilized as the second antibody near the center of the test piece. In other words, if human hemoglobin is present, this immobilization site will turn reddish purple. In another example, an anti-human hemoglobin A ₀ monoclonal antibody was used as the first antibody, and a colored latex labeled monoclonal antibody sensitized with a label styrene-vinyltoluene emulsion copolymer blue latex was used as a test piece. When the anti-human hemoglobin A ₀ polyclonal antibody as the second antibody is immobilized on the side of the development start end of the test piece in the vicinity of the center of the test piece,
If human hemoglobin is present, this immobilization site will turn blue.

Next, the present invention according to claim 2 will be described with reference to FIG. Container body 2 used in the device of the present invention
Is a device in which a predetermined amount of the dispersion medium can be accommodated and the insertion portion 4 of the biological sample collecting instrument 3 is inserted so as to be inserted therein. If desired, the interior of the container is divided into two chambers, and a stocker portion 8 is provided so as to be inscribed in the insertion port, a partition wall 7 for forming the stocker portion, and the collection device 3 on the partition wall.
It is also possible to provide an insertion hole 11 or the like for inserting the.

If desired, forming the container body 2 into a flat shape is convenient for transporting the container of the present invention containing a biological sample, and the flat surface may be a flat surface or an elliptic cylinder side surface. It may be a curved surface. For practical use, it is particularly preferable that the thickness and width are such that it can be put in an envelope and mailed as a fixed-size mail for convenience of transportation from a home or the like to an inspection agency. As will be described later, as a secondary effect of the flat shape, an optical character reader can be applied by attaching a label in which information such as inspection items is described on a flat surface.

A sampling instrument insertion portion 4 for inserting the biological sample sampling instrument 3 is bored in the container body 2. If the container body 2 is flat, the collection instrument 3 can be inserted in parallel with the flat instrument wall so that the insertion axis direction of the collection instrument 3 is parallel to the flat plane. When drilled,
The overall shape is compact, which is preferable. For example, when the container body 2 is a flat rectangular parallelepiped, the container main body 2 is formed in a side wall of the flat rectangular parallelepiped, that is, a side wall of the flat rectangular parallelepiped. The device wall to be bored may be flat or curved.

If necessary, two sampling instrument insertion sections 4 may be provided. In addition, the inside of the insertion portion 4 can be sealed by a lid 9 that is integrally formed with the sampling instrument 3,
A screw portion protruding outside the container body 2 may be provided.

When it is necessary to form a stocker portion 8 inside the container body 2 following the insertion portion 4 depending on the properties of the biological sample, a partition wall 7 that divides the interior of the container into two chambers is provided.
This partition wall 7 can be provided with an insertion hole 11 that fits with the sampling device 3. In order to ensure the liquid tightness of the dispersion medium 1 housed in the container body 2, a known liquid tight means such as rubber O-ring may be provided in the insertion hole 11.

The stocker section 8 is a fixed-quantity sorting mechanism for the sampling instrument 3 when the sampling instrument 3 equipped with the conventionally-known quantitative sorting mechanism 10 is inserted from the insertion section 4 while being fitted into the insertion hole 11. The sliding action of 10 and the insertion hole 11 has a function of accommodating an excess biological sample in an amount equal to or larger than that required for sample preparation.

Next, as the characteristic elements of the present invention,
Inside the container wall of the container body 2, there is provided a recess 14 capable of measuring and separating a fixed amount of sample (a). The position where the recess 14 is provided must be a position where the sampling instrument 3 that has already been inserted does not hinder the subsequent contact of the immunochromatographic test strip with the recess 14.

The concave portion 14 has a function of a measuring cup for measuring and dispensing a fixed amount of the sample contained in the sample container, and its size allows the analytical tool to be connected later to properly contact the sample. Therefore, it suffices to have an opening surface sufficient for insertion or fitting. The depth of the recess 14 is such that when the container is rotated and the sample is filled in the recess, the recess 14 has a volume capable of holding the liquid amount of the sample to be supplied to the immunochromatographic test strip.
Calculate and set considering the relationship with the size of the opening surface. A typical sample requirement for immunochromatography is 25
Since it is .mu.l-500 .mu.l (0.025 ml-0.5 ml), it must be manufactured by adjusting it according to the opening surface of the above-mentioned concave portion so as to secure the volume in this range. It is desirable that the bottom of the recess 14 be substantially horizontal so that the contained sample can be completely contacted with the immunochromatographic test piece when the sample container is left in a state where the analysis work is normally performed.

The concave portion 14 may be provided so that the concave portion directly projects from the wall surface of the container described above, or a step portion 12 is provided on a part of the vessel wall and is provided on the step. May be. Alternatively, it may be provided so as to partition one corner inside the container body 2. In short, when the container is left with the container wall provided with the recess 14 facing downward, the recess 14 is formed as shown in FIG. 2B. As long as the upper end of the constituting side wall 13 is located at a high position so as to be exposed from the liquid surface of the dispersion medium 1 which is housed in the container body 2 in advance, it is not limited to these installation modes.

As another component of the present invention, a means 15 (hereinafter, simply referred to as opening means) for forming an opening easily by a hand or an instrument is formed on the container wall surface of the container body 2 facing the recess 14. Yes (I). As a specific opening means, the following can be exemplified. (A) A part of the vessel wall is thinned and manually broken. (B) The thin-walled portion is further provided with line markings such as cross-shaped cuts. (C) The device wall of (a) and (b) above is broken using an instrument such as a punch. (D) Make a hole in the desired shape in advance on the vessel wall, and close it with a seal such as aluminum foil until the sample preparation is completed.
It is manually peeled off at the time of inspection. (E) A hole having a desired shape is preliminarily formed in the vessel wall, and the hole is plugged. However, it is not practical due to the size limitation of the entire container.

The shape of the opening formed by the opening means 15 must be such that it is large enough to bring the analytical tool to be used into contact with the sample in the facing recess 14. The immunochromatographic test piece used as an analytical tool is a strip-shaped one, or a test piece in which a thin plate-like developed portion is covered with a non-moisture permeable membrane, a test piece with a porous sample absorption portion at one end, etc. Therefore, when a strip-shaped test piece is used, the shape of this means 15 is practically a slit shape corresponding to the width of the immunochromatographic test piece used as shown in FIG. Are preferred. Alternatively, as shown in FIG. 3, when the test piece is provided with a pad-shaped sample-absorbing part made of porous polyethylene or the like, an opening means that gives an opening having a shape suitable for fitting the sample-absorbing part, for example, The above (d) is preferable.

Biological sample collecting device 3 used in the apparatus of the present invention
According to the property of the biological sample, the known quantitative dispensing mechanism 10 is provided so that a certain amount of the biological sample can be attached and introduced into the container body 2 by the sliding action with the insertion hole 11 as described above. And practically convenient. For example, a tool having concave grooves, ridges, through-holes, mesh-like irregularities or the like processed near the tip portion of the sampling instrument, or one having a coarse porous body near the tip portion is exemplified. be able to.

In addition to the basic configuration described above, the apparatus of the present invention according to claim 2 includes the following embodiments. [Embodiment 1] The simple biological sample testing apparatus according to claim 2, wherein the concave portion 14 is provided on the step portion 12 provided on the inner surface of the container wall of the container body 2.

[Embodiment 2] The simple biological sample testing device according to claim 2, wherein the concave portion 14 is formed by using a side wall or a side wall forming one corner of the container body 2 and provided inside the container body 2.

[Embodiment 3] The simple biological sample testing apparatus according to claim 2, wherein the opening means 15 closes a previously opened opening by sealing a sealing foil.

[Embodiment 4] The opening means 15 is formed so that the container wall of the container main body 2 is preliminarily marked according to the shape of the opening so as to be easily broken by human power. Simple biological sample inspection device.

[0044]

EXAMPLE An inspection method of the present invention according to claim 1
An example and a comparative example are shown. [Example 1 and Comparative Example 1] (1) Preparation of Anti-Human Hemoglobin A ₀ Polyclonal Antibody From human hemoglobin hemolysate, Journal of Biochemistry (J. Biol. Chem., No. 235, p 2327 (197).
8) A human hemoglobin A ₀ solution purified according to the method described above was mixed with an equal amount of Freund's complete adjuvant to prepare an emulsion. After 1 ml of this emulsion was immunized with New Zealand White Rabbit species, booster immunization was performed 3 times every 2 weeks, and 1 week after the final immunization, blood was collected from the ear vein and centrifuged to obtain antiserum. The .gamma.-globulin fraction was salted out from the obtained antiserum by a 40% saturated ammonium sulfate salting-out method, and the precipitate obtained by centrifugation was mixed with 0.02M phosphate buffer (pH 7.2) containing 0.15M sodium chloride. Dissolved in PBS), dialyzed against the same buffer for 24 hours,
1% protein concentration based on absorbance at 280 nm
Was prepared as an anti-human hemoglobin A ₀ polyclonal antibody (second antibody).

(2) Preparation of Anti-Human Hemoglobin A ₀ Monoclonal Antibody BALB / c mice were immunized with the same emulsion as in step 1, and booster immunization was performed 3 times every 2 weeks, and 3 days after the final immunization, the spleen was removed. Take out and read Nature Magazine (Nature No. 25
6, p 495 (1975)) and fused with P3-NS1 / 1-Ag4-1 myeloma according to the method of Keller and Milstein. Antibody-producing hybridomas were selected by the ELISA method and cloned by the limiting dilution method. Among several antibody-producing clones obtained as a result of the screening, those which did not rapidly aggregate when reacted with hemoglobin in a state sensitized to latex particles as a label were selected and used in the following experiments. BALB obtained by administrating pristane intraperitoneally to the obtained clone
/ C mouse was intraperitoneally administered, and ascites was collected 3 weeks later. The .gamma.-globulin fraction was salted out from this ascites by the 40% saturated ammonium sulfate salting-out method, the precipitate obtained by centrifugation was dissolved in PBS, dialyzed against the same buffer for 24 hours, and then at 280 nm. The protein concentration was adjusted to 1% based on the absorbance to give an anti-human hemoglobin A ₀ monoclonal antibody (first antibody).

(3) Labeling of anti-human hemoglobin A ₀ · monoclonal antibody with colored latex The monoclonal antibody obtained in step 2 was added to 1 part of PBS in an equal amount.
Mix with blue polystyrene latex (average particle size 0.303 μm) dispersed to 0% w / v, 37 ° C.
It was physically adsorbed. The precipitate obtained by centrifugal washing with the same PBS was dispersed in PBS containing 1% bovine serum albumin (hereinafter abbreviated as BSA), and allowed to stand at room temperature for blocking. This was again centrifuged and washed with PBS, and finally dispersed in the same buffer solution so that the latex concentration was 2% w / v to obtain a colored latex labeled monoclonal antibody (hereinafter, abbreviated as labeled first antibody). 100 μl of the obtained labeled first antibody was slowly added dropwise to a porous polyethylene sheet (1 mm thick, 10 mm width × 15 mm length) which had been coated with PBS containing sucrose and BSA and dried, and then dried and labeled. A site where the first antibody was provided (hereinafter, abbreviated as a labeled first antibody region) was created.

(4) Creation of development zone (chromatographic stationary phase) A nitrocellulose membrane filter (made by Toyo Roshi Kaisha, Ltd.) having a pore size of 5 μm was cut into a width of 10 mm and a length of 50 mm,
0.6 mm at the position 25 mm from one end, that is, in the center in the longitudinal direction
3 μl of anti-human hemoglobin A ₀ polyclonal antibody (second antibody) diluted to 10%, and 3 μl of anti-mouse IgG antibody (rabbit) diluted to 0.6% at a position 10 mm from one end are spotted as analysis end point indicators. After drying, it was immersed in PBS containing 2% BSA. The surface of this membrane was washed with water and dried to form a development area.

(5) Preparation of immunochromatographic test strip Anti-mouse I at the end of the labeled first antibody region and the developed region
The end portion on the side where the gG antibody was not spotted was overlapped by 5 mm and joined. In this state, the overlapped portion was wrapped with Parafilm (trade name Parafilm, American Can Company) so that the end of the labeled first antibody region was exposed, and fixed to obtain a test piece.

(6) Preparation of biological sample collecting tool and specimen container An apparatus similar to the structural example shown in FIG. 1 was prepared. The biological sample collecting tool 3 and the lid 9 are made of polystyrene, and the container body 2 and its parts are made of polyethylene. The volume of the recess 14 was formed by molding so as to be 100 μl when the container body 2 was formed. The opening means 15 provided on the flat surface of the container main body 2 is provided with the first label for the immunochromatographic test strip in advance.
The flattened wall was excised according to the shape of the antibody region, and the resulting opening was sealed with an aluminum laminar seal. The capacity of the container body 2 is 5 ml, and PB as the dispersion medium 1
S2 ml was injected.

(7) Volumetric test of the measuring cup of sample container After injecting 2 ml of purified water into the sample container prepared in step 6, the sample container is rotated as shown in FIG. After that, the entire amount of purified water measured in this recess was collected from the opening and weighed. Repeat this operation 10 times,
The average, maximum value, minimum value, and standard deviation were calculated. Table 1 shows the results
Shown in It was confirmed by the method of the present invention that a fixed amount of dispersion medium, and thus a fixed amount of sample, can be collected in the recess 14.

(8) Detection of human hemoglobin 1 (comparison of sensitivity of the method of the present invention) Human hemoglobin hemolyzed blood was diluted with PBS containing 0.1% of BSA to obtain 0 to 160 ng of human hemoglobin, respectively.
A sample containing / ml was prepared. Hemoglobin concentration is measured by Medical Physics, vol.
2, p1072 (1950)) and the cyanmethemoglobin method. After injecting 2 ml of the above-mentioned sample into the sample container prepared in step 6 and rotating the container to fill the concave portion 14 with the sample, the seal of the opening means 15 is manually peeled off to make an opening, and the immunochromatographic test is carried out at this opening. I inserted a piece. As a control, 2 ml of the above-mentioned sample was taken in a 24-well plate for cell culture, and the labeled first antibody region of the immunochromatographic test piece was dipped and held, and at the same time, immunochromatography was started. Table 2 shows the determination results when the test piece inserted into the sample container of the present invention absorbed the entire amount of the sample in the recess 14. The judgment symbols are +, in order of strong coloration.
± and −. From Table 2, it was found that the method of the present invention had the same sensitivity as the control. Further, when the immunochromatographic test piece used in the method of the present invention has absorbed all the amount of the sample in the measuring cup, the development of immunochromatography is completed, and the tip of the dispersion medium reaches the spot where the anti-mouse IgG antibody is spotted. And it was colored.

[Example 2 and Comparative Example 2] The test piece according to the method of the present invention and the control test piece, which had been developed as described above, were allowed to stand for 1 hour to show a change in coloration when an excessive amount of the sample was absorbed. I observed. Table 3 shows the results. Immunochromatography by the method of the present invention, the development reaches the end point at the time when the test piece absorbs the total amount of the sample in the measuring cup, and the color change even when left standing in the state where the test piece is inserted in the sample container. I couldn't see it. On the other hand, in the control, the sample continued to be absorbed, and after standing for 1 hour, coloration was also observed in the sample having a hemoglobin concentration of 10 ng / ml, which was negative in the determination. However, conversely, the hemoglobin concentration is 40 ng / m
For samples with a high value of 1, the coloration weakened due to the diffusion of the antigen-antibody complex in the developed part. Diffusion of the antigen-antibody complex was also observed in the spots with the other concentrations of the sample and the anti-mouse IgG antibody. As described above, according to the method of the present invention, by controlling the amount of the sample to be constant, it is possible to effectively prevent the influence of the excess sample while maintaining the same sensitivity as the conventional method.

[0053]

[Table 1]

[0054]

[Table 2]

[0055]

[Table 3]

[0056]

According to the inspection method and the inspection apparatus of the present invention, when the sample is inspected by the immunochromatographic test piece which is an analytical tool, a certain amount of the sample is surely brought into contact with the test piece from the sample container and is supplied by the capillary phenomenon. Therefore, it is possible to prevent the problem of false positive of a negative sample due to excess sample and the problem of unclear detection judgment and analysis end point as in the case of the conventional technique. Therefore, the detection accuracy of a small amount of test substance is good.

The apparatus of the present invention is provided with a concave portion 14 which is a mechanism for collecting a fixed amount of liquid of a sample so as to project from the vessel wall in the container body 2. Therefore, the mechanism for preventing the excessive supply or the insufficient supply of the sample is simple, and the configuration of the entire apparatus can be remarkably simplified as compared with the conventional technique.

In the method of the present invention, a method is used in which a mechanism for collecting a fixed amount of a sample is provided inside a sample container for collecting a biological sample and preparing a sample. Therefore, there is no operation that requires skill, and even an unskilled person can perform highly accurate inspection.

In the method of the present invention, a fixed amount of sample can be collected in the closed space of the sample container by an extremely simple operation, so that an accurate amount of the prepared sample can be transferred from the sample container to another analysis cup or the like. Compared with the conventional technique that involves the operation of sorting into an open-type weighing container, no skill is required and the inspection can be performed by a simple operation. Therefore, it is a hygienic and simple inspection method and has a large practical effect.

The method of the present invention is simple in that the sample is easily weighed, and that the immunochromatographic operation is not required to monitor the analysis end point. As described above, the method of the present invention allows an unskilled person to simply and hygienically carry out sample collection having a significant significance as described above in a container used for biological sample collection from the idea of self-care. It is extremely effective in promoting the voluntary inspection of the general public.

In the apparatus of the present invention, since the container body 2 can be configured in a form suitable for mailing as a fixed-size mail, a new container can be mailed between each home and an inspection agency at the time of group medical examination and the like. It is convenient for practical use because it is possible to mail the container containing the sample after collection. Further, since the apparatus of the present invention can attach a label to the flat surface of the container body 2, a label having higher visibility can be used than the conventional cylindrical container and the like, and the label can be described as needed. The information can be automatically read by an optical character reader or a bar code reader, which is practically convenient.

[0062]

[Brief description of drawings]

FIG. 1 is a perspective view conceptually showing the basic structure of an apparatus of the present invention.

[0063]

FIG. 2 is a cross-sectional view illustrating one embodiment of the method of the present invention.

[0064]

FIG. 3 is a sectional view showing an example of an immunochromatographic test piece used in the present invention.

[0065]

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Dispersion medium 2 ... Container main body 3 ... Biological sample collection tool 4 ... Collection tool insertion part 5 ... Flat surface wall 6 ... Precipitation and deposition surface 7 ... Partition wall 8 ...・ ・ Stocker 9 ・ ・ ・ Lid 10 ・ ・ Quantitative collection mechanism 11 ・ ・ Insertion hole 12 ・ ・ Step 13 ・ ・ Concave side wall 14 ・ ・ Concave 15 ・ ・ Opening means 16 ・ ・ Immunochromatographic test piece 20 ・ ・Specimen absorption part of immunochromatographic test piece 21 ... Stationary phase of immunochromatographic test piece 22 ... Insertion part of immunochromatographic test piece 23 ... Stationary phase cover 24 ... Second antibody immobilization site (coloring site) 25 ... Observation window Department

Claims (2)

[Claims] 1. A biological sample is collected using a biological sample collecting tool, the biological sample is introduced into a container containing a predetermined amount of a dispersion medium, and the biological sample is suspended or dissolved in the dispersion medium. The following operations are performed: (1) An aliquot of the suspension or the solution is dispensed into a measuring cup provided in the container as a sample, and (2) the container wall of the container facing the measuring cup. A simple method for collecting and examining a biological sample, which comprises performing an operation in which an opening is formed in a portion and (3) an analytical tool is brought into contact with the sampled sample through the opening. 2. A sample body for preparing and testing, which comprises a container body 2 capable of accommodating a predetermined amount of dispersion medium 1, a biological sample collecting instrument 3, and a sample instrument inserting section 4 formed in the container body 2. An apparatus comprising: (a) providing a concave portion 14 on the inner surface of the container wall of the container body 2 for collecting a fixed amount of sample.
(A) A simple biological sample testing device, characterized in that a means for easily forming an opening is formed in the vessel wall portion of the container body 2 facing the recess 14.