JP3490560B2 - Method for detecting human hemoglobin in feces - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting human hemoglobin in feces.

[0002]

2. Description of the Related Art As a method for examining diseases of the lower digestive organs such as colorectal cancer, there is known a method of estimating the disease by examining the presence or absence of occult blood components caused by bleeding from digestive organs, particularly human hemoglobin, in feces. Has been. Above all, an immunological detection method using an anti-human hemoglobin antibody that does not require restriction of food intake or drug administration has been proposed. Such a method for detecting human hemoglobin in feces includes
For example, the reverse passive hemagglutination method, which detects by utilizing the precipitation phenomenon that occurs when animal blood cells are sensitized with anti-human hemoglobin antibody and fecal lysate, high molecular latex particles are sensitized with anti-human hemoglobin antibody. There are a latex agglutination method in which detection is performed by using an agglutination image produced by mixing the prepared one and a fecal lysate, and an enzyme immunoassay in which an enzyme-labeled anti-human hemoglobin antibody is used.

An immunochromatography method is known as a method for more easily detecting human hemoglobin in feces. The immunochromatographic method comprises, on a water-absorbent substrate, providing a region of a stationary phase on which an anti-human hemoglobin antibody is immobilized, and binding a complex of human hemoglobin and a colored particle-labeled anti-human hemoglobin antibody in the region to bind to human. It is a method of detecting hemoglobin by trapping it on a water-absorbent substrate.

[0004]

In the conventional immunochromatographic method, the collected feces were once dissolved in a buffer solution or the like, and the fecal solution was spread on a water-absorbent substrate for measurement. As a specific detection procedure, for example, a small amount of stool is collected with a stool collection stick, and this is put into an inspection container with a nozzle containing a buffer solution to make a stool dissolution solution, and then the tip of the nozzle is opened. The procedure includes dropping a fecal solution on a water-absorbent substrate, developing the solution, and inspecting the solution.

Therefore, when inspecting a large number of samples,
Since it is necessary to have as many test containers as the number of samples, and it is necessary to dissolve the feces in each container one by one and repeat the operation of opening and dropping each container, the operation is complicated for the inspection operator. There was a risk that the samples would be mixed up.

It is also known that the three-dimensional structure of human hemoglobin is gradually denatured in the fecal lysate by intestinal bacteria, digestive enzymes and the like. However, in the actual fecal occult blood test, the subject himself / herself often collects feces at home or the like, puts the feces in a container containing a buffer solution, and leaves the feces dissolved solution for several days. In such a case, human hemoglobin is denatured for the above-mentioned reason, and it becomes difficult to bind to the anti-human hemoglobin antibody, which may result in false negative.

The object of the present invention is to improve the complexity of mistaking the sample when detecting human hemoglobin in the feces according to the immunochromatographic method, and with a simple operation, and
It is an object of the present invention to provide a method for detecting human hemoglobin in feces, which can suppress degeneration of human hemoglobin.

[0008]

The present invention has the following features. (1) A sheet-like water-absorbent substrate having a region to which a first anti-human hemoglobin antibody (hereinafter referred to as “first antibody”) is immobilized is prepared. Separate sample application member
After applying the sample feces to the sample application member,
This is brought into contact with the outside of the area on the water-absorbent substrate and transferred.
By doing so, the feces of the sample were arranged to have a thickness of 3 mm or less.
Then, according to the immunochromatography method, the labeled second anti-human hemoglobin antibody (hereinafter, the second anti-human hemoglobin antibody is referred to as the "second antibody") is bound to the human hemoglobin in the sample, A second antibody labeled with human hemoglobin in the sample (hereinafter, human hemoglobin is referred to as “human Hb”) and the first antibody by being developed to a region where the first antibody is immobilized by a developing solution A method for detecting human Hb in feces, which comprises detecting a human Hb in a sample by forming a complex with and capturing it in the first region.

(2) On the above-mentioned sheet-shaped water-absorbent substrate,
This non-water-absorbing layer is laminated with a non-water-absorbing layer made of limmer film
Of the water-absorbent substrate of the lower layer by partially forming through holes in the hydrophilic layer.
Water absorption exposed inside the through hole by exposing the surface outside the area
Attach the sample application member coated with the sample feces to the substrate.
By touching and transferring the sample feces,
The method for detecting human Hb in feces according to (1) above , which is quantitatively arranged outside .

[0010]

( 3 ) The binding of human Hb in the sample with the labeled second antibody is carried out by incorporating the labeled second antibody into the developing solution. ) Or (2), the method for detecting human Hb in feces.

( 4 ) The binding of human Hb in the sample to the labeled second antibody is labeled between the feces of the arranged sample and the region where the first antibody is immobilized. The method for detecting human Hb in feces according to (1) or (2) above, which is performed by providing a region in which the immobilized second antibody is immobilized.

( 5 ) The binding between the human Hb in the sample and the labeled second antibody is on the opposite side of the region where the first antibody is immobilized, with the feces of the sample placed in between. In the area of, a labeled second antibody is immobilized, a developing solution is first added to the labeled second antibody, and this is developed in the feces of the sample in which it is placed. A certain method for detecting human Hb in feces according to the above (1) or (2) .

[0014]

In the detection method of the present invention, arranging the feces of the sample means that the feces of the sample are not dissolved in a buffer solution as in the conventional method, but that the feces of the excreted composition are developed or developed. Means to place the feces of the sample in a composition provided with unintended additives. However, the presence or absence of evaporation due to drying or the like does not matter. When arranging the feces of the sample, it is particularly preferable to apply it so as to have a small thickness by the method described below. By arranging the sample as described above, the sample can be developed on the water-absorbent substrate simply by adding the developing solution to the arranged sample without previously dissolving the sample in the buffer solution. Therefore, the work process of dissolving the sample in the buffer is eliminated, and the inspection work is simplified. Further, along with this, the problems peculiar to the working process are solved. In particular, in the conventional method of dissolving a sample in a buffer solution, denaturation of human Hb due to intestinal bacteria, digestive enzymes, etc. has been a problem, Feces are gradually dried, and the denaturation of human Hb by intestinal bacteria, digestive enzymes, etc. is suppressed. Therefore, the storage stability of human Hb in the sample is further improved.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below with reference to the drawings. FIG. 1 is a diagram schematically showing a state in which human Hb in feces is being detected according to the detection method of the present invention. As shown in the figure, the first antibody 1 is immobilized in the region A1 on the water-absorbent substrate 4. On this water absorbent substrate 4, the sample 3 (feces) is placed in the area A3 other than the area A1. Human Hb is assumed to be present in the sample. In addition, the example of the same figure is an example in which the labeled second antibody 2 is contained in the developing solution 5. In this state, the immunochromatography method is applied to the sample 3 arranged in the area A3. That is, a developing solution is added to the sample, and the labeled second antibody is bound to human Hb in the sample, and as shown by the direction of arrow 6 in the figure, the developing solution reaches the region A1. Deploy. As a result, in the area A1, a complex of human Hb in the sample, the first antibody and the labeled second antibody is formed, and the human Hb in the sample is captured by the area A1 by the first antibody. It The captured human Hb can be detected by the label of the second antibody.

The water-absorbent substrate may be any material to which the immunochromatographic method can be applied to the sample. That is, it is not particularly limited as long as it can well absorb the developing solution and develop the human Hb in the sample and the labeled second antibody. In addition, the water-absorbent substrate has an appropriate water-absorbing property in order to ensure sufficient time for human Hb in feces to sufficiently react with the labeled second antibody and the immobilized first antibody. Those having properties are preferable. When the water absorption is low, the development is slow and rapid measurement cannot be performed. On the other hand, if the water absorption is too high, the development is rapid and the time for human Hb to react sufficiently with the second and first antibodies is insufficient, so sufficient labeling effect cannot be obtained, and determination is difficult. become.

Examples of preferable materials for the water-absorbent substrate include non-woven fabrics such as rayon and polyester, filter paper, glass fiber cloth, glass filters, nitrocellulose filters, and porous materials. Further, in order to adjust the water absorption property of the water absorbent substrate, the water absorbent substrate may be coated or impregnated with a hydrophilic polymer, protein or emulsifier. Further, the water-absorbent substrate, even if made of a single material,
It may be made of plural kinds of materials. When the water-absorbent substrate is composed of a plurality of materials, examples include a mode in which different materials are joined in the surface direction and a mode in which different materials are laminated.
The shape of the water-absorbent substrate is not particularly limited as long as the sample can be spread with the developing solution. For example, a rectangular sheet shape or a rod shape is preferable.

The first antibody is not particularly limited as long as it is an antibody that can specifically bind to human Hb. Examples thereof include anti-human Hb rabbit polyclonal antibody, anti-human Hb goat polyclonal antibody, anti-human Hb sheep polyclonal antibody, anti-human Hb monoclonal antibody and the like.

The method for immobilizing the first antibody on the water-absorbent substrate is not particularly limited, but a known adsorption method or covalent bond method is a preferable method. In the case of the covalent bonding method, when the water-absorbent substrate does not have a functional group for the covalent bonding method, for example, a polymer having an appropriate functional group may be attached to the substrate. Further, the first antibody is immobilized by applying a mixed solution of the first antibody and the hydrophilic polymer onto a water-absorbent substrate, and then immersing the mixture in a coagulation bath agent that coagulates the hydrophilic polymer. be able to. Hydroxypropylmethyl cellulose, polyvinyl alcohol, hydroxyethyl cellulose and the like are used as the hydrophilic polymer, and acetone, methanol, ethanol and the like are used as the coagulation bath.

When the sample is placed, as described in the explanation of the above-mentioned action, the sample is not dissolved in the buffer solution as in the conventional method, but is placed in the excreted composition.
The composition in the excreted state does not cause a change in the content of water and the like due to evaporation from excretion to placement. In addition, in some cases, for the purpose other than developing the sample, such as the purpose of softening the sample for easier placement and the purpose of storage,
The necessary amount of additive may be added to the sample within a range in which the sample is not developed. For example, a diluent such as water may be used as an additive for softening the sample and facilitating its application to the water-absorbent substrate. Add an amount that cannot be obtained.

The amount of the sample placed is 0.05 mg to 1
About 0 mg is preferable. Moreover, the thickness of the sample to be placed is preferably 3 mm or less. If it is too thick, it takes time to dry the feces described below, and human Hb in the feces may be denatured during this time.

As a method for arranging the sample on the water-absorbent substrate, coating is a preferable method because it is preferable that the sample is thin. The coating method is not particularly limited, but a method for preparing a sample coating member separately from the water-absorbent substrate, arranging the sample on it, and transferring it by bringing it into contact with the sample-arranged position on the water-absorbent substrate. Is a preferred method. Examples of the sample application member include filter paper, non-woven fabric, glass filter and the like.

Further, there is a method in which a sample is taken with a stick having a recess or groove near the tip, and the sample contained in the recess or groove of the stick is brought into contact with the sample placement position of the water-absorbent substrate to transfer the sample. Can be mentioned. When such a stick is used, the sample can be quantitatively taken in the concave portion and the groove portion having a constant volume, so that the measurement accuracy is improved.

Regardless of the sample arranging method, a sample arranging member having the same developability as that of the water absorbing base material may be installed on the water absorbing base material, and the sample may be arranged thereon. Preferable examples of the sample placement member include filter paper, non-woven fabric, and glass filter. Further, by laminating a non-water-absorbent layer made of a polymer film such as PET on the water-absorbent substrate and partially forming through holes in the non-water-absorbent layer to expose the surface of the lower water-absorbent substrate. The through holes may be used for quantitatively arranging the sample.

The immunochromatographic method can be applied to the stool of the sample, whether it is undried immediately after being placed on the water-absorbent substrate or dried after a long time has passed due to storage or the like. In the case of the sample immediately after being placed, since human Hb in the sample is hardly denatured, accurate detection can be performed. In addition, the sample that has been placed on the water-absorbent substrate for a certain period of time is different from the one dissolved in a conventional buffer solution, unlike the sample dissolved in a conventional buffer solution, which denatures human Hb due to digestive enzymes and intestinal bacteria. Since the reaction that occurs is suppressed by drying, it is possible to prevent a positive sample from becoming a false negative. The placed sample may be dried in a well-ventilated state, but it is preferable to put it in a closed bag or a container containing a desiccant because the sample dries faster.

The sample is placed on the water-absorbent substrate by the method having the above characteristics, and the immunochromatography method is applied to the sample by the method having the following characteristics. That is, a developing solution is added to the sample placed on the water-absorbent substrate, the labeled second antibody is bound to human Hb in the sample, and this is transferred to the region where the first antibody is immobilized. It is developed and human Hb is trapped and detected in that region.

The developing solution is not particularly limited as long as it can carry out the immunochromatographic method such as a buffer solution which has been conventionally used. As the buffer solution, a phosphate buffer solution, a glycine buffer solution, a borate buffer solution and the like are known. Also,
Sodium chloride may be added at a physiological saline concentration in order to improve the reactivity between human Hb and the antibody. The components and pH of the buffer solution are preferably such that the labeled second antibody described below does not spontaneously aggregate. This is because when the labeled second antibody spontaneously aggregates, a labeling substance such as colored particles causes clogging in the water-absorbent substrate, resulting in non-specific color development. The pH is usually around 5 to 9 which is near neutral.

As for the method of adding the developing solution to the sample, the method of directly dropping and adding the developing solution to the sample can also be used by dropping and adding the developing solution to another region (water absorbing part) of the water-absorbent substrate. It may be a method of reaching and adding. Further, a method in which the developing solution is put in a container with a nozzle or the like that can take out a fixed amount of liquid droplets, and a predetermined number of drops is added to the water-absorbing base material / sample, conversely, A method of simplifying the operation procedure is, for example, a method of immersing the sample in a well containing a developing solution.

The binding of human Hb in the sample to the labeled second antibody may be performed at any time before the human Hb in the sample reaches the region of the first antibody. In general, the following methods can be used for binding. (A) A method in which a developing solution contains a labeled second antibody as shown in FIG. By adding this developing solution to the water-absorbent substrate so that the sample and the first antibody pass in this order, the binding between the human Hb in the sample and the labeled second antibody is performed first. It (B) A method of immobilizing a labeled second antibody on a water-absorbent substrate. The position of the immobilization may be before or after the position where the sample is arranged, but in each case,
It is important to add the developing solution to the water-absorbent substrate so that the developing solution passes through in the following order. That is, as shown in FIG. 2A, the labeled second antibody 2, the sample 3, and the first antibody 1 are in this order. Alternatively, as shown in FIG. 2B, the sample 3, the labeled second antibody 2, and the first antibody 1 are arranged in this order.

The second antibody is not particularly limited as long as it is an antibody that specifically binds to human Hb. For example, anti-human Hb rabbit polyclonal antibody, anti-human Hb goat polyclonal antibody, anti-human Hb sheep polyclonal antibody,
Anti-human Hb monoclonal antibody etc. are mentioned.

Any label may be used as long as it can bind to the second antibody, and colored particles are particularly preferable. As the colored particles, colloidal metal particles of gold, silver, copper or the like, colored polymer latex particles, pigment particles and the like are used, but gold colloid particles and colored polymer latex particles are particularly preferable. The particle size of the colored particles is preferably in a range that does not cause clogging of the water-absorbent substrate used, and is preferably about 0.01 μm to 3 μm.

The method of binding the colored particles to the second antibody is as follows:
The conventionally known physical adsorption method or chemical bonding method is used. In particular, a method of using colored particles having a functional group on the particle surface and binding the colored particles to the second antibody by a covalent bond method is preferable. For example, a method of covalently binding an antibody to a colored particle having an amino group or a carboxyl group by using a water-soluble carbodiimide as a binding reagent is exemplified.

In the method of immobilizing the labeled second antibody on the water-absorbent substrate as described in (b) above, the labeled second antibody is brought into contact with the developing solution, It is important to immobilize on the water absorbent substrate so that it can be detached from the water absorbent substrate. For example, a method of applying a labeled second antibody to a water-absorbent substrate and then drying and immobilizing it under appropriate conditions can be mentioned. Specifically, a method in which a labeled second antibody is dispersed in a solution of a water-soluble polymer or sucrose, the solution is applied to a water-absorbent substrate, and then dried is exemplified. By immobilizing the second antibody by this method, when the developing solution comes into contact with the immobilized part, the water-soluble polymer or saccharose easily becomes water-soluble, and the labeled second antibody becomes Detach from the substrate immediately. In addition, in this method, aggregation and modification of the second antibody are unlikely to occur during drying, and further, the second antibody is less likely to be released from the water-absorbent substrate after drying. Further, in order to allow the labeled second antibody to be suitably contained in a predetermined region of the water-absorbent substrate,
It is preferable to adjust the concentration of the water-soluble polymer or saccharose so that the solution has an appropriate viscosity. Alternatively, the labeled second antibody may be immobilized on another water-absorbent substrate, processed into an appropriate shape, and attached to the main water-absorbent substrate.

Examples of the water-soluble polymer include polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, cellulose ethers (eg, methyl cellulose, ethyl cellulose, benzyl cellulose, trityl cellulose, cyanethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, oxyethyl cellulose), Gelatin or the like is preferably used.

Next, the procedure of the detection method according to the present invention will be described. It is assumed that the sample feces contained human Hb. By the method described above, the feces of the sample are placed on the water-absorbent substrate directly or via the sample placement member. When the detection is not performed immediately after placing the sample, the sample is dried by the above-described drying method to suppress the denaturation of human Hb in the sample.

By adding the developing solution, the arranged sample is dissolved and human Hb in the sample is extracted. This human H
b and the labeled second antibody are bound by the method described above to form a complex. By flowing the developing solution through the water-absorbent substrate, the complex also moves and reaches the region where the first antibody is immobilized. In this region the complex is captured by binding to the immobilized first antibody. As a result, when a certain amount or more of human Hb is present in the sample, the label is immobilized on the region where the first antibody is immobilized, and the human is observed by visual observation or optical measurement of this label. The existence or amount of Hb can be known. In the above, it is difficult to combine all human Hb in the sample with the second antibody to form a complex,
Further, since it is difficult to bind all of the complex to the first antibody, when the amount of human Hb in the sample is less than a certain amount, the labeled second antibody immobilized in the region is used. Are smaller, and are difficult to observe visually or to measure optically.

[0037]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 In this example, colored particles are used as a substance for labeling, a second antibody labeled by the colored particles is contained in a developing solution, and the developing solution is added to a sample. Is.

(1) Preparation of labeled second antibody Blue carboxylated polystyrene latex (average particle diameter 0.22 μm) was used as the colored particles, and
It was dispersed in a 1 M borate buffer (pH 7.5) so that the solid content concentration was 1%. To 10 ml of this dispersion was added 0.2 ml of an aqueous solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (5 mg / ml), and 10
After stirring for 10 minutes at ℃, anti-human H as the second antibody
b Rabbit polyclonal antibody (10mg / ml) 2ml
Was added and stirred at 10 ° C. for 24 hours. This was centrifugally washed with the same buffer as above and redispersed to a solid concentration of 1% to obtain a second antibody labeled with blue latex particles.

(2) Immobilization of the first antibody on the water-absorbent substrate Using an anti-human Hb rabbit polyclonal antibody as the first antibody, 0.1 M phosphate buffer (NaCl; 0.9% content, pH 7. It diluted with 4) and adjusted to the solution of 1 mg / ml. As a water-absorbent substrate, a nitrocellulose membrane (tissue pore diameter 1 μm, outer shape 5 mm × 100 mm, thickness 0.15
mm), and 1 μl of the above-mentioned solution containing the first antibody was applied to a site 20 mm from one end thereof and dried overnight at room temperature. Next, this was immersed in a 1% bovine serum albumin aqueous solution for 1 hour and then dried overnight at room temperature to obtain a water-absorbent substrate having a region where the first antibody was immobilized.

(3) Application of incidental parts to the water-absorbent substrate: A PET film (thickness: 25 μm, 5 × 100 mm) was provided with a through hole of φ2 mm at a site 10 mm from one end. The PET film and the water-absorbent substrate were bonded together so that the distance from the position of the first antibody on the material was 50 mm to form a nitrocellulose / PET film. Next, a rayon nonwoven fabric (thickness: 2.4 mm, 5 × 5 mm) was attached to one end of the water-absorbent substrate opposite to the position of the first antibody as a water-absorbing portion.

(4) Preparation of sample The sample had a human Hb content of 1 μg / g in 5 g of feces.
μg, 15 μg, 30 μg, 60 μg, 120 μg, 1
Seven kinds of 000 μg were used. Samples with these 7 kinds of contents were prepared by newly adding an appropriate amount of human Hb to the stool of a healthy person and mixing them. Further, in the preparation, the content of human Hb in 1 g of feces was quantified using an EIA kit (Wakamoto Pharmaceutical Co., Checkmate Haemo, etc.), and confirmed and prepared. In addition, the sample was frozen and stored until the start of the following human Hb detection experiment so that the human Hb in the sample is not inactivated.

(5) Arrangement of Samples Feces (7 types) containing human Hb, which is the sample prepared in (4) above, were applied in the through holes of the nitrocellulose / PET film prepared in (3) above. However, the excess stool that had protruded from the hole was wiped off with a spatula-shaped jig. (Approximately 0.08 mg of stool enters the hole)

(6) Detection of human Hb in sample 0.1% of the labeled second antibody prepared in (1) above is used.
Dilute with M phosphate buffer (NaCl; 0.9%, sodium azide; 0.1% content, pH 7.4) and add 0.01
% Developing solution (dispersion) was prepared. This developing solution 100μ
Immediately after applying the sample, 1 is added to the water-absorbent substrate from the water-absorbing part to allow the sample to reach the region of the first antibody,
Coloring of the fixed part after 10 minutes was visually observed to detect human Hb. In addition, a water-absorbent substrate in which the same sample is arranged in the same manner is separately prepared, and silica gel (tablet-like, 1-
g) Put in a zipper bag (6 × 12 cm) and sealed, store at 37 ° C. for 3 days and 6 days to evaporate water, and then add the dispersion liquid to each in the same manner as above to add human Hb.
Was detected.

The following Table 1 shows the detection results of the above 7 kinds of human Hb content samples immediately after application of the samples, 37
It shows the detection results after evaporating water by storing at 3 ° C. for 3 days and 6 days. As is clear from Table 1 below, there is no difference in the detection results over time, and human Hb in the sample
It can be seen that the denaturation with time is suppressed.

[0045]

[Table 1]

Example 2 This example is an example in which the labeled second antibody is immobilized on the water-absorbent substrate in advance and only the developing solution is added to the sample. The type of sample and the manufacturing method are the same as in Example 1.

(1) Immobilization of the labeled second antibody on the water-absorbent substrate The immobilization of the water-absorbent substrate and the first antibody was the same as in Example 1,
Further, a nitrocellulose / PET film was formed.
As the labeled second antibody, an anti-human Hb rabbit polyclonal antibody labeled with blue latex particles was used as in Example 1 above. A mixed solution of 1 μl of the labeled second antibody (solid content 1%) and 4 μl of a 10% sucrose aqueous solution was mixed with a rayon non-woven fabric (thickness 0.4 mm,
5 × 3 mm) and dried overnight in a desiccator at room temperature. This non-woven fabric is 20 m from the edge of the water absorbing part of the water absorbent base material.
It was applied to the site m and the labeled second antibody was immobilized on the water-absorbent substrate.

(2) Detection of Human Hb in Sample A sample containing human Hb was applied and filled in the PET through holes of this nitrocellulose / PET film in the same manner as in Example 1. Immediately after applying the sample, 100 μl of 0.1 M phosphate buffer (NaCl; 0.9%, containing sodium azide; 0.1%, pH 7.4) was used as a developing solution for the water-absorbent substrate immediately after the application. The sample was allowed to reach the area of the first antibody, and the color of the fixed part was observed with the naked eye after 10 minutes. In addition, a water-absorbent substrate in which the same sample is arranged by the same method is separately prepared, and the water-absorbent substrate is prepared under the same conditions as in Example 1 to
After storing at 7 ° C. for 3 days and 6 days to evaporate the water content, the dispersion liquid was added and detection was performed as described above.

The following Table 2 shows the detection results in this Example. Similar to Table 1 above, there is no difference in the detection results over time, and the denaturation of human Hb in the sample over time is suppressed. You can see that

[0050]

[Table 2]

Example 3 This example is an example in which the sample is applied to a separately prepared sample placement member when the feces of the sample are placed, and the sample is attached to the water-absorbent substrate. The type of sample and the manufacturing method are the same as in Example 1.

(1) Arrangement of sample Filter paper having an outer diameter of 2 mm as a sample arrangement member (No. 1)
Was used. This sample placement member was immersed in a 1% bovine serum albumin aqueous solution for 1 hour, then dried, and the sample was applied to a thickness of about 25 μm.

(2) Detection of human Hb in a sample A nitrocellulose / PET film (provided that the second antibody and the first antibody were immobilized in the same manner as in Example 2) on the sample placement member (however, (PET does not have a through hole) is attached to a portion 10 mm from the end of the water absorbing portion, and
Human Hb was detected in the same manner as in. In addition, a water-absorbent substrate was separately prepared by attaching the same filter paper coated with the same sample in the same manner, and the conditions were the same as those in Examples 1 and 2, and 37 ° C.
After storing for 3 days and 6 days to evaporate the water content, the dispersion liquid was added and detection was performed in the same manner as above. The detection results of these Examples are exactly the same as those of Examples 1 and 2 described above, and there is no difference in the detection results over time.
It was found that the denaturation of the above was suppressed.

Comparative Example This comparative example is an example in which the sample was dissolved and stored in a buffer solution according to a conventional method when the feces of the sample were placed, and the sample was attached to a water-absorbent substrate and developed. is there. The type of sample and the manufacturing method are the same as in Example 1.

In order to make the amount of feces per one detection the same in Example 3 and Comparative Example, 10 sheets of φ2 mm filter paper coated with the sample prepared in Example 3 were used in 0.1 M phosphate buffer solution. (NaCl; 0.9%, sodium azide; 0.1%
The sample was completely dissolved by immersing in 1 ml of the solution (pH 7.4). 100 μl of this lysate was used in Example 3.
Add to the same nitrocellulose / PET film (the second antibody and the first antibody are fixed), human Hb
Was detected. Further, the same lysate was stored at 37 ° C. for 3 days and 6 days, and then the same detection as described above was performed.

Table 3 below shows the detection results in this comparative example. As is clear from the table, the samples near the detection sensitivity became negative after the third day of storage of the sample, and in the conventional immunochromatographic method using the fecal lysate, human Hb in the sample was denatured with time. It was confirmed that it was easy.

[0057]

[Table 3]

[0058]

EFFECTS OF THE INVENTION According to the present invention, the operational procedure for detecting human Hb in feces by applying the immunochromatography method becomes simple.
Particularly when the number of samples is large, simplification of the operation procedure appears as a more remarkable effect of shortening the inspection time. That is, even if there are many water-absorbent substrates on which the sample is arranged, it is only necessary to apply a common developing solution to them. In addition, the setup required for setting to the detection device is shortened, which brings the merit of putting it on the line of the automatic detection device. In addition, the number of containers in which individual samples have been dissolved is eliminated by one stage, and thus the probability of an accident in which samples are mixed up is significantly reduced. Further, even if the sample is left for several days, it is possible to suppress the false negative reaction and obtain reliable results.

[Brief description of drawings]

FIG. 1 is a diagram schematically showing a state of detecting human Hb in feces according to the detection method of the present invention.

FIG. 2 is a diagram schematically showing an embodiment in which a second antibody is immobilized on a water-absorbent substrate.

[Explanation of symbols]

1 first antibody 2 Second antibody 3 samples 4 Water-absorbent substrate 5 developer

Front page continued (58) Fields surveyed (Int.Cl. ⁷ , DB name) G01N 33/48 G01N 33/53 G01N 33/543 G01N 33/72

Claims (5)

(57) [Claims] 1. A sheet-shaped water-absorbent substrate having a region to which a first anti-human hemoglobin antibody is immobilized is prepared, and a sample application member is prepared separately from the water-absorbent substrate, and the feces of the sample are prepared.
On the water-absorbent substrate after applying to the sample coating member
By contacting and transferring outside the area,
After placing the feces to a thickness of 3 mm or less, the human hemoglobin in the sample was
A labeled second anti-human hemoglobin antibody is bound, and this is developed to a region where the first anti-human hemoglobin antibody is immobilized by a developing solution, and the human hemoglobin and the first anti-human in the sample. By forming a complex between a hemoglobin antibody and a labeled second anti-human hemoglobin antibody and capturing the complex in the first region, the detection of human hemoglobin in the sample is characterized by the detection of human hemoglobin in feces. Detection method. 2. A polymer is provided on the sheet-shaped water-absorbent substrate.
-By laminating a non-water-absorbing layer of film, this non-water-absorbing layer
Through holes are partially formed in the above-mentioned region of the lower water-absorbent substrate.
A water-absorbent substrate whose outer surface is exposed and which is exposed in the through hole
Contact the sample application member coated with the sample feces.
And transfer the sample feces to the outside of the above area.
The method for detecting human hemoglobin in feces according to claim 1 , which is arranged quantitatively . 3. Binding of human hemoglobin in a sample to a labeled second anti-human hemoglobin antibody by incorporating a labeled second anti-human hemoglobin antibody in a developing solution. Claim 1 or 2 which is
A method for detecting human hemoglobin in feces according to the description. 4. The binding between the human hemoglobin in the sample and the labeled second anti-human hemoglobin antibody is caused by the feces of the arranged sample and the region where the first anti-human hemoglobin antibody is immobilized. The method for detecting human hemoglobin in feces according to claim 1 or 2, wherein a region to which the labeled second anti-human hemoglobin antibody is immobilized is provided between the two . 5. The binding between the human hemoglobin in the sample and the labeled second anti-human hemoglobin antibody is such that the first anti-human hemoglobin antibody is immobilized with the feces of the sample placed therebetween. In a region opposite to the region, a labeled second anti-human hemoglobin antibody is immobilized, a developing solution is first added to the labeled second anti-human hemoglobin antibody, and a sample in which this is arranged The method for detecting human hemoglobin in feces according to claim 1 or 2, wherein the method is carried out by developing into human feces.