WO2021200916A1 - Method for detecting hemoglobin, solution, feces reservoir, and method for evaluating feces-containing liquid - Google Patents

Abstract

This method for detecting hemoglobin includes: preparing a feces-containing liquid that contains feces and at least one selected from the group consisting of sulfurous acid, sulfite, disulfurous acid, disulfites, dithionous acid, and dithionites, and that has been stored for an arbitrary time; and detecting the hemoglobin contained in the feces-containing liquid by immunochromatography.

Description

Hemoglobin detection method, solution, stool preservative, and stool-containing liquid evaluation method

The present disclosure relates to a method for detecting hemoglobin, a solution, a stool preservative, and a method for evaluating a stool-containing liquid.
The present application claims priority based on Japanese Patent Application No. 2020-063212 filed in Japan on March 31, 2020, the contents of which are incorporated herein by reference.

Fecal occult blood test is known as a useful test method for diagnosing tumors in the lower gastrointestinal tract such as the large intestine. In fecal occult blood test, hemoglobin in feces is generally detected. On the other hand, as a method for detecting hemoglobin in stool, various methods such as a chemical measurement method, an immunoassay method by a latex agglutination method, and an immunoassay method by an immunochromatography method are known.

In any of the above detection methods, stool that has been stored for a certain period of time after stool collection may be used for the inspection. In Patent Document 1, the stool sample after stool collection is brought into contact with a desiccant to dry the stool sample, the dried stool sample is stored in the desiccant, and then the dried stool sample is stored. A method for measuring a component in a stool sample, which comprises adding an aqueous medium to the agent, dissolving the component in the stool sample in the aqueous medium, and measuring the component in the stool sample dissolved in the aqueous medium. Is disclosed.

International Publication No. 2017/104132

From the viewpoint of using the fecal occult blood test for diagnosing diseases, in clinical practice, there is a demand for a method that can detect hemoglobin contained in stool with high accuracy and without taking a long time by simple operation. ..

Therefore, the present disclosure provides a method for detecting hemoglobin, which is simple, stable and can detect hemoglobin quickly. The present disclosure also provides solutions and stool storage devices suitable for a simple, stable and rapid method for detecting hemoglobin. Furthermore, the present disclosure provides a method for evaluating a stool-containing liquid, which is simple and can evaluate a stool-containing liquid in a stable and rapid manner.

An example of the embodiment is given below. The present invention is not limited to the following embodiments.

One embodiment contains stool and at least one selected from the group consisting of sulfites, sulfites, disulfurous acid, disulfurous acid, dithionous acid, and dithionate, and has been stored for any time. The present invention relates to a method for detecting hemoglobin, which comprises preparing a subsequent stool-containing liquid and detecting hemoglobin contained in the stool-containing liquid by an immunochromatography method.

Another embodiment contains at least one selected from the group consisting of sulfite, sulfites, disulfurous acid, disulfurous acid, dithionous acid, and dithionous acid, as a stool preservation solution, and. It relates to a solution used as a developing solution for an immunochromatography method capable of detecting hemoglobin.

The other embodiment includes a stool collection container and the solution of the above-described embodiment contained in the stool collection container, and the stool collection container has a stool collection rod and a container body, and the stool collection container has a stool collection rod and a container body. The rod relates to a stool container having a grip portion on one side and a rod portion on the other side, and the rod portion has a stool collecting portion at or near the tip of the other side.

Another embodiment relates to a method for evaluating a stool-containing liquid, which comprises evaluating whether or not the stool-containing liquid contains hemoglobin using the hemoglobin detection method of the above-mentioned one embodiment.

According to the present disclosure, there is provided a method for detecting hemoglobin that is simple, stable and can detect hemoglobin quickly. Further, the present disclosure provides a solution and a stool storage device suitable for a simple, stable and rapid method for detecting hemoglobin. Further, according to the present disclosure, there is provided a method for evaluating a stool-containing liquid, which is simple, stable and can quickly evaluate a stool-containing liquid.

FIG. 1 is a schematic perspective view showing an example of an immunochromatographic apparatus. FIG. 2 is a front schematic view showing an example of a stool storage device.

An embodiment of the present invention will be described. The present invention is not limited to the following embodiments. The following embodiments can be implemented alone or in combination.

<Hemoglobin detection method>
The method for detecting hemoglobin according to an embodiment of the present invention contains at least one selected from the group consisting of sulfite, sulfite, disulfurous acid, disulfurous acid, dithionous acid, and dithionate and stool. It includes at least preparing a stool-containing liquid that has been stored for an arbitrary time and detecting hemoglobin contained in the stool-containing liquid by an immunochromatography method. The method for detecting hemoglobin may further include any step. In the detection method according to the embodiment of the present invention, the immunochromatography method is used for the detection of hemoglobin. The immunochromatography method is a simple method capable of detecting hemoglobin.

In general, in the fecal occult blood test, a certain amount of time elapses from the collection of stool to the start of the operation for detecting hemoglobin contained in the stool. The stool will be stored between collection and detection. According to the detection method according to the embodiment of the present invention, stable and rapid detection of hemoglobin can be realized. In the detection method according to the embodiment of the present invention, a stool-containing liquid containing the above-mentioned acid and / or salt is used. In the stool-containing liquid, it is presumed that the acid and / or salt acts as a stabilizer for hemoglobin, so that hemoglobin can be kept in a state of suppressing decomposition and denaturation even after storage. Therefore, it is considered that hemoglobin can be stably detected even when the stool-containing liquid is used after a certain period of time has passed since the preparation.

Further, the acid and / or salt can prevent the viscosity of the stool-containing liquid from increasing as compared with the case where a saccharide or the like generally known as a stabilizer is used. As a result, it is considered that the development time of the stool-containing liquid in the immunochromatography method, that is, the detection time of hemoglobin by the immunochromatographic method can be shortened, and rapid examination of hemoglobin can be performed. However, the present invention is not limited by these speculations.

In the method for detecting hemoglobin, the stool-containing liquid may be a liquid containing a solution (developing liquid) described later and stool. The method for detecting hemoglobin may further include providing a developing solution and receiving a stool-containing solution containing the developing solution and stool. Examples of providing the developing solution include the delivery of the developing solution to the patient by a medical worker such as a doctor or a nurse, and the delivery of the developing solution to the subject by an examination worker who detects hemoglobin. Be done. The patient or the subject who received the developing solution can collect stool and mix the collected stool with the developing solution to obtain a stool-containing solution. Examples of receiving the stool-containing liquid include the medical worker receiving the stool-containing liquid from the patient, the inspection worker receiving the stool-containing liquid from the subject, and the like.

[Stool-containing liquid]
The stool-containing liquid contains at least one selected from the group consisting of sulfite, sulfite, disulfurous acid, disulfurous acid, dithionous acid, and dithionate, and stool. The stool-containing liquid preferably contains water as a liquid capable of dissolving and / or dispersing the above-mentioned acid and / or salt (sulfur-containing acid) and stool. The stool-containing liquid may further contain any component other than water. Optional components consist of, for example, buffers, proteins, antibiotics, preservatives, sugars, ferrocyan compounds, chelating agents, protease inhibitors, surfactants, inorganic acids and salts thereof, and organic acids and salts thereof. At least one selected from the group is mentioned.

Each component that can be contained in the stool-containing liquid will be described later. The stool-containing liquid can be obtained, for example, by mixing at least a solution (developing liquid) described later with stool. Examples of the content of each component contained in the stool-containing liquid include the content exemplified for each component in the developing liquid described later. In this case, the standard of the content may be the volume of the stool-containing liquid instead of the volume of the developing liquid.

The stool is not particularly limited, and examples thereof include stool excreted or collected from mammals. Mammals include, for example, humans, monkeys, gorillas, orangutans, pandas, dogs, cats, horses, pigs, sheep, wild boars, rabbits, rats, squirrels, hamsters, tigers, lions and the like. The method for detecting hemoglobin is suitable for humans.

The total content of stool-derived components contained in the stool-containing liquid is, for example, 0.001% (w / v) or more and 0.005% (w / v) or more based on the volume of the stool-containing liquid. , 0.01% (w / v) or more, 0.03% (w / v) or more, or 0.05% (w / v) or more. The total content of stool-derived components contained in the stool-containing liquid is, for example, 5% (w / v) or less, 3% (w / v) or less, and 2% (2%) based on the volume of the stool-containing liquid. It may be w / v) or less, 1% (w / v) or less, or 0.5% (w / v) or less.

In the present disclosure, "% (w / v)" means a percentage of mass (g) based on volume (100 mL). For example, "the content of the component X is x% (w / v) based on the volume of the stool-containing liquid" means that the component X is contained in 100 mL of the stool-containing liquid (x (g)). Means. Further, in the present disclosure, for a certain content, an arbitrary numerical value is selected from an example of an upper limit value and an example of a lower limit value, and a numerical range obtained by combining them is also considered to be exemplified in the present disclosure. .. The same applies to other numerical values such as pH, viscosity, temperature, and time.

For the method of detecting hemoglobin, use the stool-containing liquid after being stored for an arbitrary time. The stool-containing liquid after storage contains at least one selected from the group consisting of sulfite, sulfite, disulfurous acid, disulfurous acid, dithionous acid, and dithionate, and at least both stool. It may be a stool-containing liquid after an arbitrary time has elapsed after preparing the stool-containing liquid. The state in which the stool-containing liquid being stored is placed is not particularly limited, and examples thereof include standing, transporting, and shaking. The stool-containing liquid may be a stool-containing liquid stored in a temperature-controlled atmosphere. Alternatively, the stool-containing liquid may be, for example, a stool-containing liquid stored under a normal living environment without any particular temperature control.

The lower limit of the arbitrary time is not particularly limited, but considering the time required from the acquisition of the stool-containing liquid to the inspection by the immunochromatography method, for example, 30 minutes or more, 1 hour or more, 5 hours or more, 10 hours. It may be 1 day or more, 3 days or more, or 5 days or more. The arbitrary time may be, for example, 21 days or less, 14 days or less, 10 days or less, 8 days or less, 7 days or less, 5 days or less, or 3 days or less. If it is 21 days or less, it tends to prevent hemoglobin from being denatured or decomposed. The starting point for any time is when the stool is in contact with at least one selected from the group consisting of sulfite, sulfites, disulfurous acid, disulfurous acid, dithionous acid, and dithionate, i.e. both. It may be when a stool-containing liquid containing at least is obtained. The end point of any time may be when the stool-containing solution is fed to the apparatus used for the immunochromatography method.

The storage temperature may be, for example, 0 ° C. or higher, 2 ° C. or higher, 4 ° C. or higher, 8 ° C. or higher, 10 ° C. or higher, or 15 ° C. or higher. The storage temperature may be, for example, 50 ° C. or lower, 48 ° C. or lower, 45 ° C. or lower, 40 ° C. or lower, or 38 ° C. When the temperature is 50 ° C. or lower, hemoglobin tends to be easily prevented from being denatured or decomposed. For example, it is preferable that the temperature of the atmosphere during storage is included in the above range. For example, it is preferable that the temperature of the stool-containing liquid during storage is within the above range.

The pH of the stool-containing liquid may be, for example, 6.0 to 7.5. Examples of the pH of the stool-containing liquid include the pH values exemplified for the developing liquid described later. The viscosity of the stool-containing liquid may be, for example, 1.00 to 1.80 cP. Examples of the viscosity of the stool-containing liquid include the viscosity values exemplified for the developing liquid described later. The pH and viscosity of the stool-containing liquid can be measured by the same method as the pH and viscosity of the developing liquid.

[Immunochromatography method]
In the method for detecting hemoglobin, hemoglobin is detected by an immunochromatography method. The immunochromatography method is not particularly limited, and a general method utilizing an immune reaction and a capillary phenomenon can be used. Since the immunochromatography method is a method that does not require the use of a large-scale device or a complicated process like the latex agglutination method, hemoglobin can be easily detected. The method for detecting hemoglobin by the immunochromatography method is a method suitable for so-called POCT (Point of Care Test).

The immunochromatography method uses, for example, a membrane having a region in which a first capture substance capable of capturing hemoglobin is immobilized, and a second capture substance labeled with a labeled substance and capable of capturing hemoglobin. To prepare; to form a complex in the region comprising a first trapping agent, hemoglobin, and a second trapping substance; and at least to detect the complex. The immunochromatography method may further include any step.

In the immunochromatographic method, the above-mentioned preparation includes, for example, a sample pad, a conjugation pad, a membrane having a region in which a first trapping substance capable of capturing hemoglobin is immobilized, and at least a second capturing substance. An immunochromatographic apparatus having the same may be prepared. The immunochromatographic apparatus may further include any site such as an absorption pad.

The immunochromatographic method may further include supplying the stool-containing liquid to the sample pad. In the immunochromatography method, the detection of the complex may be to detect the complex after an arbitrary time has elapsed since the stool-containing liquid was supplied to the sample pad. The arbitrary time is, for example, 10 seconds to 30 minutes. The longer the arbitrary time, the greater the effect of speeding up tends to be obtained in one detection. Even if the arbitrary time is short, in places such as hospitals and laboratories where tests are performed on many stool-containing liquids (for example, specimens), the speed of one detection makes it large as a whole. The effect of time saving can be obtained.

Examples of the capture substance include antibodies, antibody fragments, antigens, antigen fragments and the like. One or both of the first capture substance and the second capture substance may contain at least one selected from the group consisting of antibodies and antibody fragments. Examples of the label include metal colloidal particles such as gold colloidal particles, silver colloidal particles, and platinum colloidal particles; colored latex particles such as poly (meth) acrylic polymer particles and polystyrene particles containing a coloring agent; fluorescent particles and the like. Can be mentioned. The label may contain at least one selected from the group consisting of metal colloidal particles and colored latex particles. The membrane, sample pad, conjugation pad, and absorbent pad may be, for example, filter paper or non-woven fabric. Examples of the material of the filter paper or the non-woven fabric include cellulose, polyester, polyolefin, cotton and the like.

FIG. 1 is a schematic perspective view showing an example of an immunochromatographic apparatus. The immunochromatographic apparatus 10 comprises a sample pad 11, a conjugation pad 12, a membrane 14 having a region 13 on which the first capture substance A1 is immobilized, and a second capture substance A2 contained in the conjugation pad 12. Have. The second trapping substance A2 is a substance labeled by the labeled substance M and capable of trapping hemoglobin. The immunochromatographic apparatus 10 further has a region 15 on which the third capture substance A3 is immobilized, and an absorption pad 16. The third trapping substance A3 is a substance capable of trapping the second trapping substance A2.

In the hemoglobin detection method using the immunochromatography apparatus 10, the stool-containing liquid 17 can be supplied to the sample pad 11. The complex of hemoglobin Hb contained in the supplied stool-containing liquid 17, the second trapping substance A2, and the first trapping substance A1 is formed in the region 13.

In the present disclosure, the hemoglobin detection method can be used for diagnosing or predicting a disease accompanied by bleeding from the lower gastrointestinal tract such as the large intestine. Examples of diseases associated with bleeding from the lower gastrointestinal tract include colorectal cancer, ischemic enteritis, drug-induced enteritis, infectious enteritis, ulcerative colitis, Crohn's disease and the like.

<Solution>
The solution according to an embodiment of the present invention is a solution for use as a stool storage solution and as a developing solution for an immunochromatography method capable of detecting hemoglobin. The solution contains at least one selected from the group consisting of sulfites, sulfites, disulfurous acid, disulfurous acid, dithionous acid, and dithionate. In the present disclosure, this solution may be referred to as a "developing solution". In the present disclosure, at least one selected from the group consisting of sulfite, sulfite, disulfurous acid, disulfurous acid, dithionous acid, and dithionous acid may be referred to as "sulfur-containing acid". In the present disclosure, sulfur-containing acids (eg, sulfuric acid) other than sulfite, sulfite, disulfate, disulfate, dithioic acid, and subdithionate are not included in the "sulfur-containing acid". Since the stool-containing liquid contains a sulfur-containing acid, it is caused by the passage of time from the acquisition of the stool-containing liquid to the start of the inspection while suppressing the increase in viscosity of the stool-containing liquid and shortening the time required for development. It is presumed that the fluctuation of the obtained detection result can be suppressed. According to the developing solution according to the embodiment of the present invention, rapid and stable hemoglobin inspection can be realized.

The developing liquid preferably contains water as a liquid capable of dissolving a sulfur-containing acid. The developing solution may further contain any component other than water. Optional components consist of, for example, buffers, proteins, antibiotics, preservatives, sugars, ferrocyan compounds, chelating agents, protease inhibitors, surfactants, inorganic acids and salts thereof, and organic acids and salts thereof. At least one selected from the group is mentioned. Specific examples of the developing solution include developing solutions containing sulfur-containing acids, buffers, preservatives, and water; developing solutions containing sulfur-containing acids, buffers, proteins, antibiotics, preservatives, and water. Be done.

The stool, stool storage, and immunochromatography method are as described above. The developing liquid can be preferably used to obtain the stool-containing liquid in the method for detecting hemoglobin of the above-described embodiment. The developing solution can function as a mobile phase in the immunochromatography method.

(Sulfur-containing acid)
The developing solution contains at least one selected from the group consisting of sulfite, sulfite, disulfurous acid, disulfurous acid, dithionous acid, and dithionate. The developing solution may contain two or more kinds selected from the group consisting of sulfite, sulfite, disulfurous acid, disulfurous acid, dithionous acid, and dithionate.

From the viewpoint of stable detection, the developing solution may contain at least one selected from the group consisting of, for example, sulfites, disulfurous acid salts, and dithionous acid salts. From the viewpoint of stable detection, the developing solution may contain, for example, at least one selected from the group consisting of disulfurous acid and disulfurous acid. From the viewpoint of stable detection, the developing solution may contain, for example, disulfurous acid salt.

Examples of the cations contained in the salt include alkali metal ions such as lithium ion, sodium ion and potassium ion; alkaline earth metal ions such as magnesium ion and calcium ion; inorganic cations such as ammonium ion, and trimethylammonium and triethyl. Organic cations such as organic ammonium such as ammonium can be mentioned. The cation may include, for example, an inorganic cation, an alkali metal ion, or a sodium ion.

Specifically, the developing solution is sulfite, lithium sulfite, sodium sulfite, potassium sulfite, ammonium sulfite, disulfurous acid, lithium disulfite, sodium disulfite, potassium disulfate, ammonium disulfate, dithionous acid, lithium dithionate. , Sodium sulfite, potassium dithionate, and ammonium dithionate may contain at least one selected from the group.

The content of the sulfur-containing acid is, for example, 0.0001% (w / v) or more, 0.0005% (w / v) or more, 0.001% (w / v) or more, based on the volume of the developing solution. , 0.002% (w / v) or more, 0.003% (w / v) or more, 0.004% (w / v) or more, 0.005% (w / v) or more, 0.006% ( w / v) or more, 0.007% (w / v) or more, 0.008% (w / v) or more, 0.009% (w / v) or more, 0.01% (w / v) or more, It may be 0.02% (w / v) or more, 0.03% (w / v) or more, 0.04% (w / v) or more, or 0.05% (w / v) or more. The content of the sulfur-containing acid is, for example, 5% (w / v) or less, 1% (w / v) or less, 0.5% (w / v) or less, 0.1, based on the volume of the developing solution. % (W / v) or less, 0.09% (w / v) or less, 0.08% (w / v) or less, 0.07% (w / v) or less, 0.06% (w / v) Below, 0.05% (w / v) or less, 0.04% (w / v) or less, 0.03% (w / v) or less, 0.02% (w / v) or less, 0.01% (W / v) or less, 0.009% (w / v) or less, 0.008% (w / v) or less, 0.007% (w / v) or less, 0.006% (w / v) or less , Or 0.005% (w / v) or less. Within the above range, the effect of sufficient stabilization of hemoglobin tends to be easily obtained.

(water)
The water contained in the developing liquid is not particularly limited, and examples thereof include deionized water and distilled water. The water content may be the balance of other components.

(Buffer)
The developing solution may contain a buffer. When dissolved in water, the buffer is, for example, phosphate buffer, carbon dioxide buffer, ammonia buffer, acetate buffer, lactic acid buffer, citrate buffer, tartrate buffer, borate buffer, glycine buffer. It may be a buffer solution that can obtain a buffer solution such as a liquid, a Tris buffer solution, or a Good buffer solution. Examples of the buffer for obtaining a good buffer include 2-morpholinoetan sulfonic acid (MES), piperazine-N, N'-bis (2-ethane sulfonic acid) (PIPES), and N- (2-). Acetamide) -2-aminoethanesulfonic acid (ACES), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Biz) -Tris), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid [(H) EPPS], 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 3- [4- (2-hydroxyethyl) -1-piperazinyl] -2-hydroxy Propane sulfonic acid (HEPPSO), 3- (morpholino) propane sulfonic acid (MOPS), 3- (morpholino) -2-hydroxypropane sulfonic acid (MOPSO), piperazin-N, N'-bis (2-hydroxypropane sulfonic acid) ) (POPSO), N- [Tris (hydroxymethyl) methyl] -2-hydroxy-3-aminopropanesulfonic acid (TAPSO), N- [Tris (hydroxymethyl) methyl] -2-aminoethanesulfonic acid (TES) , N- (2-acetamide) iminodiacetic acid (ADA), N, N-bis (2-hydroxyethyl) glycine (Bicine), N- [tris (hydroxymethyl) methyl] glycine (Tricine), N-tris ( Hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-3-amino -2-Hydroxypropanesulfonic acid (CAPSO) and the like can be mentioned. The buffer may contain N- (2-acetamide) iminodiacetic acid (ADA), particularly from the viewpoint of stable detection. The developing solution may contain a buffering agent alone or in combination of two or more.

When the developing solution contains a buffer, the content of the buffer is, for example, 0.01% (w / v) or more, 0.05% (w / v) or more, 0, based on the volume of the developing solution. It may be 1% (w / v) or more, 0.3% (w / v) or more, or 0.5% (w / v) or more. The content of the buffer is, for example, 10% (w / v) or less, 5% (w / v) or less, 3% (w / v) or less, and 2% (w / v) based on the volume of the developing solution. ) Or less, or 1% (w / v) or less. Within the above range, fluctuations in the pH of the stool-containing liquid tend to be suppressed, and decomposition and denaturation of hemoglobin tend to be easily prevented.

(protein)
The developing solution may contain a protein. Examples of the protein include albumin, iron protein and the like. Examples of albumin include serum albumin, egg white albumin, lactalbumin and the like, and serum albumin is preferable. As the serum albumin, for example, serum albumin prepared from the serum of mammals such as humans, cows and horses according to a conventional method; commercially available serum albumin and the like can be used. Suitable serum albumin includes, for example, bovine serum albumin (BSA), human serum albumin and the like. The developing solution may contain one type of protein alone or a combination of two or more types.

When the developing solution contains a protein, the protein content is, for example, 0.001% (w / v) or more, 0.005% (w / v) or more, 0.01 based on the volume of the developing solution. It may be% (w / v) or more, 0.05% (w / v) or more, or 0.1% (w / v) or more. The protein content is, for example, 10% (w / v) or less, 5% (w / v) or less, 1% (w / v) or less, 0.5% (w / v) or less based on the volume of the developing solution. It may be v) or less, or 0.3% (w / v) or less. Within the above range, hemoglobin tends to be detected more stably and quickly.

(Antibiotic)
The developing solution may contain an antibiotic. Examples of antibiotics include β-lactam antibiotics, tetracycline antibiotics, macrolide antibiotics, aminoglycoside antibiotics, nucleoside antibiotics, ansamycin antibiotics, polypeptide antibiotics, and other antibiotics. Examples include substances. The antibiotic may include an aminoglycoside antibiotic, and examples of the aminoglycoside antibiotic include streptomycin, streptomycin B, dehydrostreptomycin, oxystreptomycin, canamycin, kasgamycin, gentamicin A, gentamicin C, lincomycin, bleomycin, man. Examples thereof include noside hydroxyoside and streptomycin.

The antibiotic may be salt. Examples of the salt include acid addition salts (salts, sulfates, phosphates, acetates, fumarates, oxalates, tartrates, etc.), ammonium salts, organic amine addition salts (triethylamine salts, etc.), metals. Examples thereof include salts (lithium salt, sodium salt, potassium salt, magnesium salt, calcium salt, etc.). The developing solution may contain one type of antibiotic alone or a combination of two or more types.

When the developing solution contains an antibiotic, the content of the antibiotic is, for example, 0.005% (w / v) or more, 0.01% (w / v) or more, 0, based on the volume of the developing solution. It may be 0.05% (w / v) or more, 0.1% (w / v) or more, or 0.2% (w / v) or more. The content of the antibiotic is, for example, 5% (w / v) or less, 3% (w / v) or less, 1% (w / v) or less, 0.5% (w) based on the volume of the developing solution. / V) or less, or 0.4% (w / v) or less. Within the above range, it tends to be easy to obtain a sufficient effect by containing an antibiotic in the developing solution.

(Preservative)
The developing solution may contain a preservative. Examples of the preservative include azides and chelating agents. The preservative may contain an azide, and examples of the azide include sodium azide and the like. The preservative may contain a chelating agent, and examples of the chelating agent include a chelating agent described later. The developing liquid may contain one type of preservative alone or in combination of two or more types.

When the developing solution contains an antiseptic, the content of the preservative is, for example, 0.001% (w / v) or more, 0.005% (w / v) or more, 0, based on the volume of the developing solution. It may be 0.01% (w / v) or more, 0.03% (w / v) or more, or 0.05% (w / v) or more. The content of the preservative is, for example, 2% (w / v) or less, 1% (w / v) or less, 0.5% (w / v) or less, 0.3% based on the volume of the developing solution. It may be (w / v) or less, or 0.1% (w / v) or less. Within the above range, it tends to be easy to obtain a sufficient effect by including the preservative in the developing liquid.

(Sugar)
The developing solution may contain sugars. Examples of saccharides include glucose, sucrose, maltose, cyclodextrins, fructose, sorbose, saccharose, lactose, trehalose, galacturonic acid, mannitol, D-glucosamine, mannose, cellobiose, glycidol, inositol and the like. The developing solution may contain saccharides alone or in combination of two or more.

When the developing solution contains saccharides, the saccharide content is, for example, 0.005% (w / v) or more, 0.01% (w / v) or more, 0.03 based on the volume of the developing solution. It may be% (w / v) or more, 0.05% (w / v) or more, or 0.1% (w / v) or more. The saccharide content is, for example, 5% (w / v) or less, 3% (w / v) or less, 1% (w / v) or less, 0.5% (w / v) or less, based on the volume of the developing solution. It may be v) or less, or 0.3% (w / v) or less. When it is 0.005% (w / v) or more, stable detection of hemoglobin tends to be easy. When it is 5% (w / v) or less, it tends to be easy to prevent the developing liquid from becoming highly viscous. From the viewpoint of performing a quicker inspection, the developing solution does not contain saccharides, or when it contains saccharides, the content is preferably small. The sugar content is, for example, 0.1% (w / v) or less, 0.01% (w / v) or less, 0.001% (w / v) or less, or 0.000% (w / v). v) may be.

(Ferocyanide)
The developing solution may contain a ferrocyanide compound. Examples of the ferrocyanide compound include sodium ferrocyanide, potassium ferrocyanide, 11-ferrocenyl-1-undecanethiol, 8-ferrocenyl-1-octanethiol, 6-ferrocenyl-1-hexanethiol, and 11-ferrocyanylundecyl-. Examples thereof include polyoxyethylene ether and 11-ferrocyanyltrimethylundecylammonium bromide. The developing solution may contain one type of ferrocyanide compound alone or a combination of two or more types.

(Chelating agent)
The developing solution may contain a chelating agent. Examples of the chelating agent include ethylenediaminetetraacetic acid, O, O'-bis (2-aminophenyl) ethylene glycol-N, N, N', N'-tetraacetic acid, and diethylenetriamine-N, N, N', N'. ', N''-pentaacetic acid, ethylenediamine-N, N'-diacetic acid, ethylenediamine-N, N'-bis (methylenephosphonic acid) and the like can be mentioned. The chelating agent may be a salt. Examples of the salt include ammonium salt, sodium salt, potassium salt, magnesium salt, calcium salt and the like. The developing solution may contain a chelating agent alone or in combination of two or more.

(Protease inhibitor)
The developing solution may contain a protease inhibitor. Protease inhibitors include, for example, clonete (trademark, manufactured by Roche), antipapain dihydrochloride, aprotinin, chymostatin, E-64, leupeptin, pefabloc (trademark, manufactured by Roche), bepstatin, phosphoramidone, and the like. Examples thereof include antithrombin III, (4-amidinofenyl) methanesulfonyl fluoride, carbain inhibitor 1,3,4-dichloroisocoumarin, α-macroglobulin, bestatin and the like. The developing solution may contain a protease inhibitor alone or in combination of two or more.

(Surfactant)
The developing solution may contain a surfactant. Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, amphoteric surfactants and the like. The developing solution may contain one type of surfactant alone or in combination of two or more types.

(Inorganic acid and its salt)
The developing solution may contain an inorganic acid and / or a salt of the inorganic acid. Examples of the inorganic acid and the salt of the inorganic acid include an inorganic acid other than the optional components described above and a salt of the inorganic acid (hereinafter, may be referred to as “another inorganic acid and a salt thereof”). Examples of the inorganic acid include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and the like. Examples of the salt include ammonium salt, sodium salt, potassium salt, calcium salt, magnesium salt and the like. The developing solution may contain other inorganic acids and salts thereof, one type alone or in combination of two or more types.

The contents of other inorganic acids and salts thereof are, for example, 0.001% (w / v) or more, 0.005% (w / v) or more, and 0.01% (w) based on the volume of the developing solution. / V) or more, 0.03% (w / v) or more, or 0.05% (w / v) or more. The content of other inorganic acids and salts thereof is, for example, 10% (w / v) or less, 5% (w / v) or less, 1% (w / v) or less, 0, based on the volume of the developing solution. It may be 5.5% (w / v) or less, or 0.1% (w / v) or less. Within the above range, hemoglobin tends to be detected more stably and quickly. The content of other inorganic acids and salts thereof is, for example, 0.05% (w / v) or less, 0.01% (w / v) or less, 0.001% (w / v) or less, or 0. It may be .000% (w / v).

In particular, when the developing solution contains a halogenated alkali metal salt, the content of the halogenated alkali metal salt is, for example, 0.001% (w / v) or more, 0.005% based on the volume of the developing solution. It may be (w / v) or more, 0.01% (w / v) or more, 0.03% (w / v) or more, or 0.05% (w / v) or more. The content of the alkali metal halide is, for example, 10% (w / v) or less, 5% (w / v) or less, 1% (w / v) or less, 0.5, based on the volume of the developing solution. It may be% (w / v) or less, or 0.1% (w / v) or less. Within the above range, hemoglobin tends to be detected more stably and quickly. The alkali metal halide can be represented by X ^- M ⁺ (X represents a halogen ion and M represents an alkali metal ion). Examples of halogenated alkali metal salts include sodium chloride, potassium chloride and the like. For example, it is preferable that sodium chloride satisfies the above range of contents. From the viewpoint of performing a more stable inspection, the developing solution does not contain an alkali metal halide, or when it does, the content is preferably small, for example, 1% (w / v) or less. It may be there. The content of the alkali metal halide is, for example, 0.05% (w / v) or less, 0.01% (w / v) or less, 0.001% (w / v) or less, or 0.000. It may be% (w / v).

The total content of all the inorganic acids and the salts of the inorganic acids contained in the developing solution is, for example, 0.001% (w / v) or more and 0.005% (w / v) based on the volume of the developing solution. ) Or more, 0.01% (w / v) or more, 0.03% (w / v) or more, or 0.05% (w / v) or more. The total content of all the inorganic acids and salts of the inorganic acids contained in the developing solution is, for example, 15% (w / v) or less, 10% (w / v) or less, 5 based on the volume of the developing solution. It may be% (w / v) or less, 1% (w / v) or less, 0.5% (w / v) or less, or 0.1% (w / v) or less. Within the above range, it tends to be easy to prevent the decomposition and denaturation of hemoglobin.

The content of the sulfur-containing acid is, for example, 5% by mass or more, 10% by mass or more, 15% by mass or more, and 20% by mass based on the total mass of all the inorganic acids and the salts of the inorganic acids contained in the developing solution. As mentioned above, it may be 25% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, or 90% by mass or more. The content of the sulfur-containing acid is, for example, 100% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass, based on the total mass of all the inorganic acids and the salts of the inorganic acids contained in the developing solution. Hereinafter, it may be 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 25% by mass or less, or 20% by mass or less. Within the above range, it tends to be easy to prevent the decomposition and denaturation of hemoglobin.

(Organic acid and its salt)
The developing solution may contain an organic acid and / or a salt of the organic acid. Examples of the organic acid and the salt of the organic acid include organic acids other than the above-mentioned optional components and salts of the organic acid. Examples of the organic acid include malic acid, succinic acid, fumaric acid, glycolic acid, 2-ketoglutaric acid, isocitric acid, lactic acid, pyruvate, uric acid, oxalacetic acid and the like. Examples of the salt include ammonium salt, sodium salt, potassium salt, calcium salt, magnesium salt and the like. The developing solution may contain an organic acid and / or a salt of an organic acid, either alone or in combination of two or more.

The developing solution may be a solution containing a sulfur-containing acid, a buffer, a protein, an antibiotic, a preservative, and water. In this case, the total content of the sulfur-containing acid, buffer, protein, antibiotic, preservative, and water contained in the developing solution is, for example, 90% (w / v) or more and 92% (w / v). 95% (w / v) or more, 98% (w / v) or more, 99% (w / v) or more, 99.3% (w / v) or more, 99.5% (w / v) or more , 99.8% (w / v) or more, or 100.0% (w / v).

[PH]
The pH of the developing solution may be, for example, 6.0 or higher, 6.3 or higher, 6.5 or higher, 6.6 or higher, 6.7 or higher, or 6.8 or higher. The pH of the developing solution may be, for example, 7.5 or less, 7.2 or less, 7.0 or less, 6.9 or less, 6.8 or less, or 6.7 or less. Within the above range, it tends to be easy to prevent the decomposition and denaturation of hemoglobin. The pH of the developing solution can be measured with a general pH meter. Examples of the pH measuring device include "pH / ion meter HM-42X" manufactured by DKK-TOA CORPORATION. The temperature of the developing solution at the time of measurement is 25 ° C.

[viscosity]
The viscosities of the developing solution are, for example, 1.00 cP or higher, 1.05 cP or higher, 1.10 cP or higher, 1.15 cP or higher, 1.20 cP or higher, 1.25 cP or higher, 1.30 cP or higher, 1.35 cP or higher, 1. It may be 40 cP or more, 1.45 cP or more, or 1.50 cP or more. The viscosities of the developing solution are, for example, 1.80 cP or less, 1.75 cP or less, 1.70 cP or less, 1.65 cP or less, 1.60 cP or less, 1.55 cP or less, 1.50 cP or less, 1.45 cP or less, 1. It may be 40 cP or less, 1.35 cP or less, or 1.30 cP or less. Within the above range, hemoglobin tends to be detected more quickly. From the viewpoint of rapid detection, the viscosity is preferably low. The viscosity of the developing solution can be measured with a vibrating viscometer. Examples of the vibration viscometer include "Viscomate VM-1G" manufactured by SEKONIC CORPORATION. The temperature of the developing solution at the time of measurement is 25 ° C.

[Use]
The solution (developing solution) according to the embodiment of the present invention is used as a preservative solution for stool and as a developing solution for an immunochromatography method capable of detecting hemoglobin. A stool-containing liquid is obtained by mixing the solution and stool. The stool can be stored for any time in a state of being dissolved or dispersed in a solution as a preservative solution. In addition, the stool-containing liquid can be used in an immunochromatography method as a sample sample containing a solution as a developing liquid and stool.

A stool collection container described later can be used for mixing the solution and stool and / or storing the stool, but without particular limitation, a known container that can be used for a fecal occult blood test is used. May be good. The immunochromatographic method in which the solution is used may be the method in the detection method of the above-described embodiment, but may be a known method without particular limitation.

<Stool storage device>
The stool storage device according to the embodiment of the present invention includes a stool collection container and a solution (developing liquid) contained in the stool collection container. The stool collection container has a stool collection rod and a container body. The stool collection rod has a grip portion on one side and a rod portion on the other side, and the rod portion has a stool collection portion at or near the tip of the other side. The “near the tip” is, for example, a portion included from the tip on the other side of the rod portion to the center of the rod portion. The stool storage device may further include any part.

FIG. 2 is a front schematic view showing an example of a stool storage device. The stool storage device 20 includes a container body 21, a solution (developing liquid) 22, and a stool collection rod 23. Using the stool storage device shown in FIG. 2, stool is collected with a stool collection stick, and the stool-containing liquid is obtained in the stool collection container by inserting the stool collection rod with stool attached into the stool collection container. be able to. The stool-containing liquid can be stored in the stool storage device for any time.

<Evaluation method of stool-containing liquid>
The method for evaluating a stool-containing liquid according to an embodiment of the present invention includes evaluating whether or not the stool-containing liquid contains hemoglobin using the method for detecting hemoglobin according to the above-described embodiment. The method for evaluating the stool-containing liquid may further include any step.

If hemoglobin is detected by the immunochromatography method according to the hemoglobin detection method, it can be determined that the stool-containing liquid contains hemoglobin. On the other hand, when hemoglobin is not detected by the immunochromatography method, it can be determined that the stool-containing liquid does not contain hemoglobin.

<Example of Embodiment>
Preferred examples of embodiments of the present invention are listed below. Embodiments of the present invention are not limited to the following examples.
[1] After containing stool and at least one selected from the group consisting of sulfite, sulfite, disulfurous acid, disulfurous acid, dithionous acid, and dithionous acid, and storing for any time. Prepare stool-containing liquid and
A method for detecting hemoglobin, which comprises detecting hemoglobin contained in the stool-containing liquid by an immunochromatography method.
[2] The immunochromatography method is
To prepare a membrane having a region in which the first capture substance capable of capturing the hemoglobin is immobilized, and a second capture substance labeled with a labeled substance and capable of capturing the hemoglobin.
The detection method according to the above [1], which comprises forming a complex containing a first trapping substance, hemoglobin, and a second trapping substance in the region, and detecting the complex.
[3] The detection method according to the above [2], wherein the first capture substance contains at least one selected from the group consisting of an antibody and an antibody fragment.
[4] The detection method according to the above [2] or [3], wherein the second capture substance contains at least one selected from the group consisting of an antibody and an antibody fragment.
[5] The detection method according to any one of [2] to [4] above, wherein the labeled substance contains at least one selected from the group consisting of metal colloidal particles and colored latex particles.
[6] The detection method according to any one of the above [1] to [5], wherein the arbitrary time is 1 hour to 10 days.
[7] The detection method according to any one of the above [1] to [6], wherein the storage temperature is 4 to 45 ° C.
[8] Any one of the above [2] to [7], wherein the preparation includes a sample pad, a conjugation pad, the membrane, and an immunochromatographic apparatus having the second capture substance. The detection method described in the section.
[9] The immunochromatography method further includes supplying the stool-containing liquid to the sample pad.
The detection method according to the above [8], wherein detecting the complex includes detecting the complex after an arbitrary time has elapsed since the stool-containing liquid was supplied to the sample pad.
[10] Containing at least one selected from the group consisting of sulfite, sulfite, disulfurous acid, disulfurous acid, dithionous acid, and dithionous acid, as a stool storage solution and detecting hemoglobin. A solution used as a developing solution for the obtained immunochromatography method. Alternatively, a solution containing at least one selected from the group consisting of [10'] sulfite, sulfite, disulfate, dithionous acid, dithionous acid, and dithionate, as a stool preservation solution. And use as a developing solution of an immunochromatography method capable of detecting hemoglobin.
[11] The solution according to the above [10], which has a pH of 6.5 to 6.9. Alternatively, [11'] the use according to the above [10'], wherein the pH of the solution is 6.5 to 6.9.
[12] The solution according to the above [10] or [11], which has a viscosity at 25 ° C. of 1.65 cP or less. Alternatively, [12'] The use according to the above [10'] or [11'], wherein the viscosity of the solution at 25 ° C. is 1.65 cP or less.
[13] The solution according to any one of [10] to [12] above, further containing a buffer, a preservative, and water. Alternatively, [13'] The use according to any one of the above [10'] to [12'], wherein the solution further contains a buffer, a preservative, and water.
[14] The solution according to [13] above, wherein the buffer contains N- (2-acetamide) iminodiacetic acid. Alternatively, [14'] the use according to [13'] above, wherein the buffer contains N- (2-acetamide) iminodiacetic acid.
[15] The above [10] to the above [10], wherein the alkali metal halide is not contained, or when the alkali metal halide is contained, the content of the alkali metal halide is 1% (w / v) or less. The solution according to any one of [14]. Alternatively, [15'] When the solution does not contain an alkali metal halide or contains an alkali metal halide, the content of the alkali metal halide is 1% (w / v) or less. The use according to any one of the above [10'] to [14'].
[16] The solution according to any one of [10] to [15] above, wherein the content of the inorganic acid and the salt of the inorganic acid is 0.1 to 1% (w / v). Alternatively, [16'] any of the above [10'] to [15'], wherein the content of the inorganic acid and the salt of the inorganic acid in the solution is 0.1 to 1% (w / v). Use as described in item 1.
[17] The solution according to any one of the above [10] to [16], which is used for the detection method according to any one of the above [1] to [9]. Alternatively, [17'] The use according to any one of the above [10'] to [16'] in the detection method according to any one of the above [1] to [9].
[18] The stool-containing liquid according to any one of the above [10] to [16], wherein the stool-containing liquid contains the solution according to any one of the above [10] to [16] and the stool. Detection method.
[19] The detection method according to the above [18], further comprising providing the solution according to any one of the above [10] to [16] and receiving the stool-containing liquid.
[20] The stool collection container and the solution according to any one of the above [10 to [17] contained in the stool collection container are included.
The stool collection container has a stool collection rod and a container body, and has a stool collection rod.
The stool collection rod has a grip portion on one side and a rod portion on the other side.
The rod portion is a stool storage device having a stool collecting portion at or near the tip of the other side.
[21] Whether or not the stool-containing liquid contains hemoglobin is evaluated by using the hemoglobin detection method according to any one of the above [1] to [9], [18] and [19]. A method for evaluating a stool-containing liquid, which comprises.

The embodiment of the present invention will be specifically described with reference to Examples. Embodiments of the present invention are not limited to the following examples.

<Example A: Preparation and evaluation of solution (developing liquid)>
A developing solution containing a sulfur-containing acid and a developing solution not containing a sulfur-containing acid were prepared, and the stability of hemoglobin with respect to each developing solution was evaluated.

[Preparation of developing solution]
(Preparation of developing solution A1)
N- (2-acetamide) iminodiacetic acid (ADA) 30 mmol / L, bovine serum albumin (BSA) 0.2% (w / v), streptomycin sulfate 0.38% (w / v) (3,000 kU /) A developing solution A1 containing L), sodium azide 0.1% (w / v), sodium sulfite 1 mM (0.013% (w / v)), sodium hydroxide for pH adjustment, and water was prepared. ..

(Preparation of developing solutions A2 to A7)
Sodium sulfite and its developing solution A2 to the developing solution A2 to the same method as the developing solution A1 except that the concentration of 1 mM (“ mM” is 10 ^-3 mol / L) in the developing solution was changed to the additives and concentrations shown in Table 1. I got A7.

The reagents used to prepare the developing liquids A1 to A7 are as follows.
N- (2-acetamide) iminodiacetic acid (ADA, manufactured by Dojin Chemical Research Institute)
Bovine serum albumin (BSA, manufactured by Boval)
Streptomycin sulfate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
Sodium azide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
Sodium sulfite (manufactured by Junsei Chemical Co., Ltd.)
Sodium hydroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
Sodium sulfite (manufactured by Junsei Chemical Co., Ltd.)
Sodium dithionite (manufactured by Tokyo Chemical Industry Co., Ltd.)
Sodium lactate 70% solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
Trehalose (manufactured by Hayashibara Co., Ltd.)
α-Ketoglutaric acid (2-oxoglutaric acid, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
Iron (II) Sulfate (7hydrate, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[Measurement of pH]
The pH of the developing liquids A1 to A7 prepared above was measured at a liquid temperature of 25 ° C. A pH / ion meter HM-42X (manufactured by DKK-TOA CORPORATION) was used as a pH measuring instrument. The results are shown in Table 1.

[Evaluation of developing solution]
The stability of hemoglobin to the developing solution was evaluated by calculating the residual rate of hemoglobin after storing the hemoglobin in the developing solution.

(Evaluation of developing solution A1)
(1) Preparation of sample solution 3 mg of hemoglobin was added to 100 mL of the developing solution A1 prepared above to prepare a sample solution A1.
(2) Measurement of hemoglobin in sample solution immediately after preparation Using Extel "Hemo Auto" HS antibody-sensitized latex suspension and Extel "Hemo Auto" HS hemoglobin buffer, humans in fully automatic stool The hemoglobin concentration in the sample solution A1 prepared in (1) above was measured by the hemoglobin analyzer HM-JACKarc.

(2-1) Preparation of calibration curve According to the method described in the package insert of Extel "Hemo Auto" HS, a calibration curve showing the relationship between hemoglobin concentration and turbidity is created using Extel hemoglobin standard HS. bottom.
(2-2) Measurement of hemoglobin concentration in sample solution immediately after preparation Extel "Hemo Auto" HS antibody sensitized latex suspension (90 μL) and Extel "Hemo Auto" HS hemoglobin buffer (190 μL) Was added to the HM-JACKarc dedicated cup, and then the sample solution A1 (20 μL) prepared in (1) above was added, and the reaction was carried out at 25 ° C. The turbidity 72 seconds after the addition of the sample solution A1 and the turbidity 288 seconds later were measured, and the turbidity 72 seconds after 288 seconds was subtracted from the turbidity to obtain the measured values. The obtained measured value was compared with the calibration curve prepared in (2-1) to determine the hemoglobin concentration in the sample solution A1.

(3) Preparation of sample solution for evaluation of storage stability The sample solution A1 prepared in (1) above was stored at 30 ° C. for 7 days to prepare a sample solution A1 (after storage).

(4) Measurement of hemoglobinometry in sample solution (after storage) With the above (2) except that the sample solution A1 (after storage) prepared in (3) above was used instead of the sample solution A1 immediately after preparation. The hemoglobin concentration in the sample solution A1 (after storage) was determined by the same method.

(5) Calculation of hemoglobin residual rate in sample solution (after storage) The hemoglobin concentration in the sample solution A1 immediately after preparation determined in (2) above is set as the reference 100%, and the sample determined in (4) above with respect to the concentration. The percentage of hemoglobin concentration in solution A1 (after storage) (hemoglobin residual rate (%)) was calculated. The results are shown in Table 1.

(Evaluation of developing liquids A2 to A7)
Sample solutions A2 to A7 were prepared in the same manner as the sample solution A1 except that the developing solutions A2 to A7 prepared above were used instead of the developing solution A1.

The hemoglobin residual ratio (%) in the sample solutions A2 to A7 was calculated by the same method as in (2) to (5) except that the sample solutions A2 to A7 were used instead of the sample solution A1. The results are shown in Table 1.

The reagents, equipment, etc. used for the evaluation of the developing liquids A1 to A7 are as follows.
Hemoglobin (Human hemoglobin lyophilized powder, manufactured by Sigma)
Fully automatic fecal human hemoglobin analyzer HM-JACKarc (manufactured by Hitachi Chemical Diagnostics Systems Co., Ltd.)
Extel "Hemo Auto" HS antibody sensitized latex suspension, Extel "Hemo Auto" HS hemoglobin buffer, Extel hemoglobin standard HS (above, manufactured by Hitachi Chemical Diagnostics Systems)
pH / ion meter HM-42X (manufactured by DKK-TOA CORPORATION)

As shown in Table 1, hemoglobin showed good stability with respect to the developing solution containing a sulfur-containing acid. Hemoglobin can be stably detected by using a developing solution containing a sulfur-containing acid.

<Example B: Detection of hemoglobin by lateral chromatography method>
As the developing solution of the immunochromatography method, a developing solution containing a sulfur-containing acid or a developing solution not containing a sulfur-containing acid was used, and the rapidness of hemoglobin detection was evaluated.

[Preparation of developing solution]
(Development liquid B1)
The developing liquid B1 (pH 6.8) was prepared by the same method as the developing liquid A1.

(Development liquid B2)
A 10 mM phosphate buffer (PBS) (pH 7.4) containing 140 mM sodium chloride was prepared. The obtained PBS was used as a developing solution B2.

(Development liquid B3)
N- (2-acetamide) iminodiacetic acid (ADA) 30 mmol / L, bovine serum albumin (BSA) 0.2% (w / v), streptomycin sulfate 0.38% (w / v) (3,000 kU /) A buffer solution containing L), sodium azide 0.1% (w / v), sodium lactate 3.5% (w / v), sodium hydroxide for pH adjustment, and water was prepared. The obtained buffer solution was used as a developing solution B3 (pH 7.2).

(Development liquid B4)
Further, a developing solution B4 (pH 7.2) was obtained by the same method as that of the developing solution B3 except that it contained 1% (w / v) of trehalose.

(Development liquids B5 to B8)
By the same method as the developing solution B4 except that the concentration of trehalose in the developing solution was changed from 3% (w / v) to 10% (w / v), the developing solutions B5 to B8 (both have a pH of 7.2). ) Was obtained.

The reagents used to prepare the developing liquids B1 and B3 to B8 are as described above. The reagents used to prepare the developing solution B2 (PBS) are as follows.
Sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
Disodium hydrogen phosphate (manufactured by Kanto Chemical Co., Inc.)
Sodium dihydrogen phosphate (manufactured by Kanto Chemical Co., Inc.)

[Measurement of viscosity]
The viscosities of the developing liquids B1 to B8 and purified water prepared above were measured at a liquid temperature of 25 ° C. A viscometer Viscomate VM-1G (manufactured by SEKONIC CORPORATION) was used for measuring the viscosity. The results are shown in Table 2.

[Detection of hemoglobin]
(Preparation of evaluation half strip and preparation of antibody solution)
The following anti-human hemoglobin antibody-immobilized half-strip and gold colloid-labeled anti-human hemoglobin antibody solution were prepared.

(1) Preparation of anti-human hemoglobin antibody-immobilized half strip As a membrane, Hi-Flow Plus HFC070504 having a size of 50 mm in the vertical direction and 250 mm in the horizontal direction was prepared. Next, at a position 21 mm from the lower end of the membrane, using a Biojet Quanti dispensing module, an anti-human hemoglobin antibody solution (1 mg / mL anti-human hemoglobin antibody-containing 5 mM phosphate buffer pH 6. 0) was linearly applied to form a detection region (the coated surface is used as the surface of the membrane). After application, the membrane was dried at 25 ° C. for 2 hours. After drying, the membrane is immersed in blocking buffer (50 mM borate buffer pH 8.5 containing 0.5% (w / v) casein) for 30 minutes, followed by wash solution (0.5% (w / v) sucrose and 0. Soaked in 50 mM Tris-HCl buffer pH 7.5) containing 0.05% (w / v) sodium borate for 30 minutes. The washed membrane was dried overnight in a desiccator. Next, the back surface of the membrane was attached to the adhesive surface of the precut backing sheet. Then, CELLULOSE FIBER SAMPLE PADS was attached as an absorption pad on the surface of the membrane so as to overlap the region above the position 20 mm from the upper end of the membrane. Next, the membrane was cut in the vertical direction so as to have a size of 50 mm in the vertical direction and 5 mm in the horizontal direction to obtain an anti-human hemoglobin antibody-immobilized half strip.

(2) Preparation of gold colloid-labeled anti-human hemoglobin antibody solution Gold Colloid: 40 nm (manufactured by BBI solution) was prepared as a gold colloid solution. Next, 900 μL of a colloidal gold solution, 100 μL of 20 mM borate buffer pH 8.5, and 100 μL of a 30 μg / mL anti-human hemoglobin antibody aqueous solution were mixed and allowed to stand at 25 ° C. for 30 minutes. Then, 55 μL of 1% (w / v) polyethylene glycol 20,000 aqueous solution and 110 μL BSA aqueous solution (BSA aqueous solution adjusted to pH 8.0 with sodium hydroxide) were further added to the obtained mixed solution. After mixing, the obtained mixed solution was centrifuged at 8,000 g at 10 ° C. for 10 minutes. Then, after removing the centrifugation supernatant, a buffer solution for storing gold colloid (0.05% (w / v) polyethylene glycol 20,000, 150 mM sodium chloride, 1% (w / v) BSA and 0.1) was added to the precipitate. 2 mL of 20 mM Tris-HCl buffer pH 8.2) containing% (w / v) sodium azide was added and the precipitate was dispersed in the buffer using a tabletop ultrasonic washer W-113Mk II. Then, the treatment from centrifugation to dispersion of the precipitate was repeated twice more under the same conditions, and then a buffer solution for storing gold colloid was added to adjust the concentration to obtain a gold colloid-labeled anti-human hemoglobin antibody solution. The concentration of the gold colloid-labeled anti-human hemoglobin antibody solution was adjusted so that the turbidity at 525 nm when diluted to 5% (v / v) with a buffer solution for storing gold colloid was 0.3.

(Measurement of hemoglobin detection time)
The developing solutions B1 to B8 were used as the developing solution of the immunochromatography method, and the time required for the detection of hemoglobin was measured. For the immunochromatography method, the anti-human hemoglobin antibody-immobilized half strip prepared above and the gold colloid-labeled anti-human hemoglobin antibody solution were used.

(Evaluation of developing solution B1)
(1) Preparation of mixed solution To one well of a 96-well microplate, 90 μL of developing solution B1, 5 μL of 50 μg / mL hemoglobin aqueous solution, and 5 μL of gold colloid-labeled anti-human hemoglobin antibody solution were added to 100 μL. A mixture was prepared.

(2) Penetration of mixed solution into half strip The lower end (CELLULOSE FIBER SAMPLE PADS) of the anti-human hemoglobin antibody-immobilized half strip is attached to 100 μL of the mixed solution prepared in (1) above. The non-side edge) was soaked and the mixture was allowed to penetrate the half strip.

(3) Detection of hemoglobin Hemoglobin was detected by confirming the pink coloration derived from colloidal gold in the detection region on the anti-human hemoglobin antibody-immobilized half strip. The time from immersion of the lower end of the anti-human hemoglobin antibody-immobilized half-strip to detection of hemoglobin was measured. The results are shown in Table 2.

(Evaluation of developing liquids B2 to B8)
The time until hemoglobin was detected was measured by the same method as that of the developing solution B1 except that the developing solutions B2 to B8 were used instead of the developing solution B1. The results are shown in Table 2.

The reagents, equipment, etc. used for the evaluation of the developing liquids B1 to B8 are as follows.
Anti-human hemoglobin antibody (anti-human hemoglobin sheep polyclonal antibody, 4870-3979G, manufactured by Bio-Rad Laboratories)
Casein (manufactured by Snow Brand Megmilk)
Boric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
Sucrose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
Sodium cholic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
Tris hydroxymethylaminomethane (Tris, manufactured by Tokyo Chemical Industry Co., Ltd.)
Polyethylene glycol 20,000 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
Hi-Flow Plus HFC070504 (Membrane, manufactured by Merck Millipore)
Pre-cut backing sheet (manufactured by Nippon Engineering)
CELLULOSE FIBER SAMPLE PADS (manufactured by Merck Millipore) Gold Colloid: 40 nm (gold colloid solution, manufactured by BBI solution)
Biojet Quanti dispensing module (coating machine, manufactured by BioDot)
Desktop ultrasonic cleaner W-113Mk II (manufactured by Honda Electronics Co., Ltd.)
Viscometer Viscomate VM-1G (manufactured by SEKONIC)

As shown in Table 2, hemoglobin could be detected in a short time when a developing solution containing a sulfur-containing acid was used. Further, from Table 2, it can be seen that hemoglobin can be detected in a short time when a developing solution having a low viscosity is used.

10 Immunochromatography device 11 Sample pad 12 Conjugation pad 13 Region where the first trapping substance is immobilized 14 Membrane 15 Region where the third trapping substance is immobilized 16 Absorption pad 17 Stool-containing liquid A1 First Capture substance A2 Second capture substance A3 Third capture substance M Labeled substance Hb Hemoglobin 20 Stool storage 21 Container body 22 Solution (developing liquid)
23 Stool collection rod 23a Gripping part 23b Rod part 23c Stool collection part 24 Stool collection container

Claims (21)

1. A stool-containing liquid containing stool and at least one selected from the group consisting of sulfite, sulfite, disulfurous acid, disulfurous acid, dithionous acid, and dithionate, and stored for an arbitrary period of time. And prepare
   A method for detecting hemoglobin, which comprises detecting hemoglobin contained in the stool-containing liquid by an immunochromatography method.
2. The immunochromatography method
   To prepare a membrane having a region in which the first capture substance capable of capturing the hemoglobin is immobilized, and a second capture substance labeled with a labeled substance and capable of capturing the hemoglobin.
   The detection method according to claim 1, wherein a complex containing a first trapping substance, hemoglobin, and a second trapping substance is formed in the region, and the complex is detected.
3. The detection method according to claim 2, wherein the first capture substance contains at least one selected from the group consisting of an antibody and an antibody fragment.
4. The detection method according to claim 2 or 3, wherein the second capture substance contains at least one selected from the group consisting of an antibody and an antibody fragment.
5. The detection method according to any one of claims 2 to 4, wherein the labeled body contains at least one selected from the group consisting of metal colloid particles and colored latex particles.
6. The detection method according to any one of claims 1 to 5, wherein the arbitrary time is 1 hour to 10 days.
7. The detection method according to any one of claims 1 to 6, wherein the storage temperature is 4 to 45 ° C.
8. The detection method according to any one of claims 2 to 7, wherein the preparation comprises preparing an immunochromatographic apparatus having a sample pad, a conjugation pad, the membrane, and the second capture substance. ..
9. The immunochromatographic method further comprises supplying the stool-containing liquid to the sample pad.
   The detection method according to claim 8, wherein detecting the complex includes detecting the complex after an arbitrary time has elapsed since the stool-containing liquid was supplied to the sample pad.
10. Immunochromatography containing at least one selected from the group consisting of sulfite, sulfite, disulfurous acid, dithionous acid, dithionous acid, and dithionate, which can be used as a stool preservation solution and can detect hemoglobin. A solution used as a developing solution for the method.
11. The solution according to claim 10, which has a pH of 6.5 to 6.9.
12. The solution according to claim 10 or 11, which has a viscosity at 25 ° C. of 1.65 cP or less.
13. The solution according to any one of claims 10 to 12, further containing a buffer, a preservative, and water.
14. The solution according to claim 13, wherein the buffer contains N- (2-acetamide) iminodiacetic acid.
15. Any of claims 10 to 14, wherein the alkali metal halide is not contained, or when the alkali metal halide is contained, the content of the alkali metal halide is 1% (w / v) or less. The solution according to item 1.
16. The solution according to any one of claims 10 to 15, wherein the content of the inorganic acid and the salt of the inorganic acid is 0.1 to 1% (w / v).
17. The solution according to any one of claims 10 to 16, which is used in the detection method according to any one of claims 1 to 9.
18. The detection method according to any one of claims 1 to 9, wherein the stool-containing liquid contains the solution according to any one of claims 10 to 16 and the stool.
19. The detection method according to claim 18, further comprising providing the solution according to any one of claims 10 to 16 and receiving the stool-containing liquid.
20. The stool collection container and the solution according to any one of claims 10 to 17 contained in the stool collection container are included.
    The stool collection container has a stool collection rod and a container body, and has a stool collection rod.
    The stool collection rod has a grip portion on one side and a rod portion on the other side.
    The rod portion is a stool storage device having a stool collecting portion at or near the tip of the other side.
21. A method for evaluating a stool-containing liquid, which comprises evaluating whether or not the stool-containing liquid contains hemoglobin using the hemoglobin detection method according to any one of claims 1 to 9, 18 and 19.

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