JP3448071B2 - Fecal hemoglobin detection device - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an apparatus for detecting human hemoglobin (hereinafter referred to as human Hb) in feces. More specifically, the present invention relates to a device for detecting fecal occult blood, which is important for diagnosis and treatment of diseases causing a neoplastic mechanism of the digestive tract.

[0002]

2. Description of the Related Art Previously, as a method of detecting occult blood in feces, a non-specific detection method utilizing the peroxidase-like action of Hb was used. However, this method has a positive reaction due to hemoglobin and other substances in meat, so that it requires strict dietary restrictions before the test and has a problem in accuracy.

In recent years, an immunological assay for human Hb by anti-human Hb antibody (Barrows, American Journal of Clinical Pathology (Am. J. Cli. P
athol.) 69 342-346, 1978), a method for detecting occult blood in feces that does not require dietary restriction has been successively developed.

Japanese Unexamined Patent Publication No. 56-106154 describes a method for detecting Hb by sandwich EIA or competitive EIA. These methods have the advantage of being very sensitive and specific. But,
On the other hand, there are many operation procedures, which are complicated and require a long time for the measurement, so that the skill of the user is required and the use scene is limited.

JP-A-59-125064 describes a method for detecting Hb by an immunological latex agglutination reaction using an anti-Hb antibody-sensitized latex. This method has the advantage that the operation is simple. But,
If the reaction time is not strictly adhered to, there is a problem in accuracy, and the determination is confusing because it is performed by confirming the presence or absence of latex agglutination with the naked eye, and it is not suitable for simultaneous treatment of multiple specimens such as group examination. In addition, since the stool suspension must be reacted on the slide plate, there is a drawback in that the user feels unpleasant such as odor and uncleanness.

In this regard, Japanese Patent Application Laid-Open No. 1-161152 describes an automated machine for optically detecting an agglutination reaction by a latex sensitized with an anti-Hb antibody. According to this method, Hb can be quantitatively measured and the ambiguity of the determination is eliminated, and automation has improved the operability and the operator's discomfort. However, the machine itself becomes very expensive and suitable for processing a large number of samples for mass screening, but is not suitable for measuring a small number of samples at home and in a clinic.

JP-A-59-182367 and JP-A-61-228351 disclose a method in which a stick or a saji is directly contacted with stool to adsorb human Hb for washing and then subjected to a specific reaction. Have been described. Further, JP-A-60-173471 discloses a method of applying feces on a filter paper on which an anti-Hb antibody is immobilized and washing. However, all of these methods require operations such as washing and coloring, and have the drawback of being complicated.

Japanese Patent Application Laid-Open No. 2-141665 discloses an anti-H
It describes a method of detecting a monoclonal antibody by a change in color tone due to aggregation of gold colloid particles sensitized. This method is excellent in stability of results, easiness of judgment, and easiness of operation, but has a drawback that the reaction time is as long as several tens of minutes.

[0009]

DISCLOSURE OF THE INVENTION The present invention has a combination of operation, easiness of judgment and stability of judgment result so that occult blood in feces can be measured even at home, and further the operator's discomfort is eliminated. It is an object of the present invention to provide an inexpensive device that can be easily operated and can detect human Hb in a short time.

[0010]

Means for Solving the Problems As a result of intensive studies in view of the above-mentioned objects, the present inventors have found that in the detection of human Hb,
A sandwich immunoassay in which an antibody that specifically binds to human Hb in a sample is incorporated into a chromatographic medium,
It has been found that the object can be achieved by an apparatus for performing an immunochromatographic method in which a chromatograph for separating reaction and unreacted components together is performed under a constant condition that is not hindered by an agglutination reaction between human Hb and an antibody. The present invention has been completed.

The present invention is labeled with colored particles,
Porous matrix having site (1) impregnated with a first antibody that specifically binds to human hemoglobin in a sample
And a second that specifically binds to human hemoglobin in the sample
Site antibodies of immobilized reaction (2) have a, the site
Porosity in contact with (1) but having the site (1)
The matrix a human hemoglobin detector in a sample consisting chroma <br/> chromatograph medium and (3), another porous matrix, the site (1) and reactive site (2) or
The device has a distance of 0.5 cm or more and 4 cm or less .

Next, the device of the present invention will be described.

[0013] device of the present invention, possess a porous matrix having a site (1) a first antibody impregnated, a reactive site that the second antibody is immobilized (2), the site (1 )When
Porous matrix which is in contact with but has the site (1)
And a chromatographic medium (3) , which is a porous matrix other than the glass .

A monoclonal antibody against human Hb is used as the first antibody of the present invention. As the second antibody, if it is an antibody that can specifically bind to human Hb,
There is no particular limitation.

The first antibody is labeled with colored particles. The colored particles used for this labeling are not particularly limited as long as they are particles that can be detected by the naked eye, but metal colloid such as gold colloid or colored latex is preferably used. The size of the particles is usually in the range of about 20 to 300 nm in diameter, but particles of other sizes can be used.

Labeling of the first antibody with such particles can be carried out by adsorption of the antibody onto the surface of the colored particles. For example, when gold colloid is used as the colored particles, the antibody and gold colloid are mixed immediately and incubated for 1 to 2 minutes, and then polyethylene glycol or bovine serum albumin is added thereto to prevent non-specific aggregation. Then, it is centrifuged at high speed to remove the supernatant by suction. The precipitate thus obtained is resuspended in a buffer solution containing polyethylene glycol or bovine serum albumin, and the unlabeled antibody remaining after centrifugation is removed to obtain an antibody labeled with colored particles. (Georghegan et al.,
Journal of Immunological Methods (JI
mmunol Methods) 34, 11-21, 1
980, etc.).

The first antibody which is labeled with colored particles, the click chromatograph medium is impregnated and dried on a porous matrix of another fiber, the matrix Ru is contacted with the chromatographic medium. As such a matrix, non-woven fabric or felt is preferably used.

The second antibody is immobilized on a certain site of the chromatographic medium.

The method of immobilization varies depending on the type of chromatographic medium, but it is usually a filter paper, glass filter,
In the case of nylon membranes, the antibody is first bound to the latex particles. As the latex particles to be used, those having a particle diameter larger than the size of the mesh of the filter (retention particle diameter) are used, or those having a small particle diameter are aggregated and used.

The solid phase latex in which the second antibody and the latex particles are bound is prepared by an ordinary method. For example, the antibody and the latex emulsion are incubated at room temperature for about 2 hours, then a buffer solution containing bovine serum albumin or the like is added, and the mixture is centrifuged. The obtained precipitate is washed with the same buffer solution by suspending and centrifuging 1 to 3 times to obtain an antibody bound to the latex particles, that is, a solid phase latex.

The antibody bound to the latex particles is fixed in the state of being suspended in the buffer solution by spotting or direct jet printing.

On the other hand, in a nitrocellulose membrane or some kind of activated membrane, the antibody solution is immobilized as it is by spotting or direct jet printing.

The site on which the second antibody is immobilized is 0.5 cm or more and 4 cm or less, preferably 0.5 cm or more, on the chromatographic medium in the downstream direction from the site impregnated with the first antibody for the reason described below. Must be less than 2 cm. That is, since human Hb has a special structure of being a tetramer composed of two subunits each of α and β, two identical antigenic determinants per hemoglobin are present.
You will have one by one. Therefore, even if a monoclonal antibody is used as the first antibody in the chromatographic medium,
The human Hb-labeled antibody conjugate forms an aggregate and becomes undeployable. However, when the distance between the labeled antibody and the reaction site is 4 cm or less, particularly 2 cm or less, the human Hb / labeled antibody conjugate is rapidly expanded before the expansion becomes impossible due to the growth of aggregates. It was found to be captured by the immobilized antibody. On the other hand, when the distance is shorter than 0.5 cm, the time required for the first antibody to contact the antigen (human Hb) and reach the second antibody is short, that is, the sufficient time required for the antigen-antibody reaction. I can't get enough sensitivity. Therefore, a distance of 0.5 cm or more is required.

The chromatographic medium used in the apparatus of the present invention is not particularly limited as long as it can develop a fecal suspension sample. However, since the sample is not adsorbed and a uniform and fast developing speed is obtained, a glass filter is used. Is particularly preferable. The porous matrix of this chromatographic medium is molded into a strip and used.

The device of the present invention may have a sample pad (4) for temporarily absorbing and filtering the stool suspension sample when it is applied to the device. As such a sample pad, a porous matrix of fibers, preferably a non-woven fabric, felt or the like, which is sized to hold a sample amount necessary for chromatographic development, is used. The sample pad is positioned on the chromatographic medium by partially overlapping the first antibody-impregnated site and the downstream side of the site. Since the stool suspension is filtered by this sample pad, it is not necessary to previously remove the insoluble matter such as fibers contained in the stool.

The device of the present invention may have a water absorbing pad (6) for holding the sample which has been developed. This water absorbent pad is composed of a highly water-absorbing porous matrix. The water absorbent pad is large enough to hold all the developed sample and is located overlapping the downstream end of the chromatographic medium.

The device of the invention may have a housing (5). The housing is made of a non-water-immersible and moldable material, such as polyvinyl chloride, polystyrene, polyethylene, calp or polypropylene, and covers the entire apparatus including a sample pad, a chromatographic medium and a water absorption pad, and a sample application site and The second antibody immobilization site has a window. By providing this housing, leakage of odor and fecal suspension can be prevented.

Human H in stool suspension using the device of the invention
The detection of b is performed as follows. That is, a test sample is brought into contact with the side impregnated with the first antibody (1), and human Hb is reacted with the labeled first antibody,
In the sample, the formed human Hb / first antibody / colored particle complex is further moved on the chromatographic medium (3) to react with the immobilized second antibody (2) to capture the complex. The presence or absence of human Hb is expressed as a coloring signal. The presence or absence of human Hb in the sample can be determined by whether or not a colored signal appears at the reaction site.
Here, the solvent for suspending the feces is not particularly limited, but for example, 0.1 wt% bovine serum albumin-containing phosphate buffered saline or the like is used.

[0029]

The present invention will be described in more detail based on the following examples, but the invention is by no means limited to these.

Example (assay by immunochromatography of human Hb) (a) Preparation of solid phase latex Commercially available latex emulsion (N-450 manufactured by Sekisui Chemical Co., Ltd.) was added with phosphoric acid to a solid content concentration of 0.6% by weight. It was diluted with buffered saline (hereinafter referred to as "PBS"). 1 ml thereof and 1 ml of a rabbit antibody against human Hb at a concentration of 100 μg / ml were placed in an Eppendorf centrifuge tube and incubated at room temperature for 2 hours to sensitize the latex particles with the rabbit antibody. Then the concentration 0.1
Weight% bovine serum albumin (hereinafter referred to as "BSA")
A solid phase latex was prepared by washing 3 times with PBS containing PBS by centrifugation at 8000 G for 10 minutes and finally resuspending it to 2 ml.

(B) Preparation of Gold Colloidal Particles The gold colloidal particles are prepared by the method of G. Frens (Nature Physical Science).
ICALSCIENCE) 241 Jan.1 1973).
That is, 200 ml of a 0.01 wt% gold chloride aqueous solution was boiled, and 2 ml of a 1 wt% sodium citrate aqueous solution was added thereto. A gold colloidal dispersion having an average particle diameter of 0.04 μm was prepared by heating and boiling until the color of the solution changed from pale yellow to purple or red.

(C) Preparation of antibody labeled with gold colloidal particles 1 part of the above gold colloidal dispersion having a gold concentration of 0.01% by weight
The pH was adjusted to 6.7 with M potassium carbonate solution. To this, a monoclonal antibody against human Hb was added at a rate of 5 μg per 1 ml of gold colloidal dispersion. 1-2
After 10 minutes, 0.1 ml of 10% by weight BSA was added to the 10 ml,
Remove the supernatant by centrifugation at 10,200 G for 1 hour
10 times with PBS containing A at a concentration of 0.1% by weight, 10 times
After washing by centrifugation at 200 G for 1 hour, the first antibody labeled with colloidal gold particles was prepared. This is the P
It was redispersed in 10 ml of BS, 0.1 ml was impregnated with a 5 × 10 mm Benlyse nonwoven fabric (trade name, manufactured by Asahi Kasei Kogyo Co., Ltd.) and freeze-dried.

(D) Preparation of chromatographic medium Commercially available glass filter (Advantech Toyo, GC-
From 50), a 10 × 100 mm membrane strip was cut, placed on a glass plate and fixed with adhesive tape for easy handling. 0.2, 0.5, 1. from the edge of the strip.
At the position of 0, 2.0, 3.0, 4.0 or 5.0 cm, an acurajetter liquid jetting device (trade name, manufactured by Nordson Co.) and an XY table aero stage (trade name, manufactured by THK Co., Ltd.) are used, perpendicular to the deployment direction. That is, 1 μl of solid phase latex was jet-printed to a length of 10 mm in parallel with the short side of the strip.

(E) Assembly of the device The chromatographic strip was placed on a 10 × 120 mm adhesive sheet so that the width of the chromatograph strip was 10 mm, and the end farthest from the area where the latex was jet-printed was aligned, and 3 was placed on top of it.
10x40mm filter paper (made by Whatman, 17C
hr) were overlaid and taped. On the other end of the chromatographic strip, the gold colloid-labeled antibody-containing nonwoven fabric was placed so as to overlap by 2.5 mm. Further, a 10 × 20 mm Benlyse non-woven fabric was placed as a sample pad so as to overlap the non-woven fabric containing the gold colloid labeled antibody by 2.5 mm.

(F) Preparation of Human Hb-Containing Sample Human Hb (manufactured by Sigma) was diluted with PBS containing 0.1% by weight of BSA to obtain human Hb concentrations of
Samples containing human Hb at 0.1, 1.0, 10 and 100 μg / ml were prepared.

(G) Assay 200 μl of the above-mentioned human Hb-containing sample or 200 μl of PBS containing 0.1% by weight of BSA as a blank was dropped on the sample pad of the device obtained in (e) above to develop the sample. Let After 5 minutes, the presence or absence of a line at the reaction site was observed.

When nothing appeared at the reaction site, it was evaluated as-, when a slight line was recognized as ±, when a line was recognized as + and when a clear line was recognized as ++. . The results are shown in Table 1.

[0038]

[Table 1]

The following can be seen from Table 1. That is,
If the distance between the labeled antibody-impregnated site and the reaction site is 5.0 cm, and the Hb concentration in the sample becomes 10 μg / ml or more, the line at the reaction site becomes ambiguous and the Hb concentration becomes 100 μm.
If it is more than g / ml, a negative result will be obtained. On the other hand, when the distance between the labeled antibody-impregnated site and the reaction site is 0.2 cm, the line at the reaction site becomes a little vague when the Hb concentration in the sample is 1 μg / ml. On the other hand, when the distance between the labeled antibody-impregnated portion and the reaction portion is 0.5 cm or more and 4.0 cm or less, stable determination results can be obtained in a wide concentration range in the sample.

[0040]

As described above, the present invention provides a simple Hb detecting device which can easily obtain a stable judgment result in a short time even at home and which eliminates the discomfort of an operator.

[Brief description of drawings]

FIG. 1 is a plan view of the device of the present invention.

FIG. 2 is a plan view of an apparatus of the present invention including a sample pad and a water absorption pad.

FIG. 3 is a side view of the device of FIG.

FIG. 4 is a plan view of the device of the present invention with a housing.

5 is a side cross-sectional view of the device of FIG.

[Explanation of symbols]

1 Labeled antibody impregnation site 2 reaction sites 3 Chromatographic media 4 sample pad 5 housing 6 water absorption pad 7 Adhesive sheet

Claims (5)

(57) [Claims] 1. A porous matrix having a site (1) impregnated with a first antibody that specifically binds to human hemoglobin in a sample, labeled with colored particles, and specific to human hemoglobin in a sample. Having a reactive site (2) to which a second antibody that binds to the site is immobilized, the site (1)
An apparatus for detecting human hemoglobin in a sample, which comprises a chromatographic medium (3), which is in contact with, but is different from the porous matrix having the site (1), which is a porous matrix containing no protein denaturing agent. The distance between the site (1) and the reaction site (2) is 0.5 cm.
A device having a size of 4 cm or more. 2. A device according to claim 1, wherein the porous matrix having the sites (1) comprises a non-woven pad. 3. An apparatus according to claim 1, wherein the chromatographic medium (3) comprises a glass filter. 4. The device according to claim 1, 2 or 3, comprising a sample pad (4). 5. A housing having a housing (5),
The device according to 2, 3, or 4.