JP2003028861A - Human hemoglobin detecting device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inexpensive human hemoglobin detecting device provided with both easiness in operation and determination and stability in the results of determination so as to measure occult blood in dejections even at home and capable of removing discomfort for operators and detecting human Hb by easy operations in a short time. SOLUTION: The device for detecting human hemoglobin in a sample comprises both a porous matrix with a section (1) and a porous matrix with a reaction section (2). The section (1) is labelled with coloring particles, and impregnated with a first antibody which is specifically combined with the human hemoglobin in the sample. A second antibody which is specifically combined with the human hemoglobin in the sample is fixed to the reaction section (2). The porous matrix with the section (1) and the porous matrix with the reaction section (2) are each formed of a different matrix, and the distance between the section (1) and the reaction section (2) is >=0.5 cm and <=4 cm or less in the human hemoglobin detecting device.

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention 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 an apparatus for detecting fecal occult blood, which is important for diagnosis and treatment of diseases causing neoplastic mechanisms of the gastrointestinal tract. [0002] Until now, as a method for detecting occult blood in feces, a non-specific detection method utilizing the peroxidase-like action of Hb has been used. However, this method also requires a strict dietary restriction before the test because of a positive reaction due to hemoglobin and other substances in meat, and also has a problem in accuracy. In recent years, immunoassays for human Hb using anti-human Hb antibodies (Barrows, American Journal of Clinical Pathology (Am. J. Cli.
athol.) 69 342-346, 1978), and detection methods for fecal occult blood that do not require dietary restriction have been developed one after another. JP-A-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, since the operation procedure is many and complicated, and the measurement requires a long time, there is a drawback that the skill of the user is required and the use scene is limited. JP-A-59-125064 describes a method of detecting Hb by immunological latex agglutination using an anti-Hb antibody-sensitized latex. This method has an advantage that the operation is simple. But,
If the reaction time is not strictly observed, there is a problem in accuracy, and the judgment is made by confirming the presence or absence of latex agglutination with the naked eye. In addition, since the stool suspension must be reacted on the slide plate, it has the disadvantage of giving the user discomfort such as odor and uncleanness. In this connection, 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 measured quantitatively, the confusing of the judgment has been eliminated, and the automation has improved the operability and the discomfort of the operator. However, the machine itself becomes very expensive and is suitable for processing multiple samples for mass screening, but is not suitable for measuring a small number of samples at home and in a clinic. [0007] JP-A-59-182667 and JP-A-61-228351 disclose a method in which human Hb is adsorbed and washed by directly contacting a stool with sticks or saji, and then subjected to a specific reaction. Has been described. Further, Japanese Patent Application Laid-Open No. 60-173471 describes a method of applying stool to a filter paper on which an anti-Hb antibody is immobilized and washing the filter paper. However, these methods all require operations such as washing and coloring, and have the disadvantage of being complicated. [0008] JP-A-2-141665 discloses an anti-H
It describes a method for detecting a monoclonal antibody by a change in color tone due to aggregation of sensitized colloidal gold particles. This method is excellent in stability of results, easiness of determination, and simplicity of operation, but has a drawback that the reaction time is as long as tens of minutes. SUMMARY OF THE INVENTION The present invention combines the easiness of operation, the determination, and the stability of the determination result so that occult blood in feces can be measured even at home, and furthermore, discomfort to workers. It is an object of the present invention to provide an inexpensive apparatus which can easily detect human Hb in a short time except for the operation. Means for Solving the Problems The present inventors have made intensive studies in view of the above object, and as a result, have found that in human Hb detection,
A sandwich immunoassay in which an antibody that specifically binds to human Hb in the sample is incorporated into a chromatographic medium;
It has been found that the objective can be achieved by an apparatus that performs an immunochromatography method that combines chromatography for separation of reacted and unreacted components under constant conditions that are not hindered by an agglutination reaction between human Hb and an antibody. The present invention has been completed. [0011] The present invention provides a method comprising the steps of:
A porous matrix having a site (1) impregnated with a first antibody that specifically binds to human hemoglobin in a sample, and a second matrix that specifically binds to human hemoglobin in the sample.
A device for detecting human hemoglobin in a sample having a porous matrix having a reaction site (2) on which an antibody is immobilized, comprising the porous matrix having the site (1) and the reaction site (2) The present invention relates to a human hemoglobin detection device, wherein a porous matrix is formed of another porous matrix, and a distance between the site (1) and the reaction site (2) is 0.5 cm or more and 4 cm or less. Next, the device of the present invention will be described. The device of the present invention is a device comprising a chromatographic medium (3) having a site (1) impregnated with a first antibody and a reaction site (2) immobilized with a second antibody. As the first antibody of the present invention, a monoclonal antibody against human Hb is used. As the second antibody, if it is an antibody that can specifically bind to human Hb,
There is no particular limitation. [0015] The first antibody is labeled with a colored particle. 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 preferably, a metal colloid such as a gold colloid or a colored latex is 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 also be used. The labeling of the first antibody with such particles can be performed by adsorbing the antibody on the surface of the colored particles. For example, when colloidal gold is used as the colored particles, the antibody and the colloidal gold are mixed immediately, and incubated for 1 to 2 minutes, and then polyethylene glycol or bovine serum albumin is added thereto to prevent nonspecific aggregation. Subsequently, centrifugation is performed at a high speed to remove the supernatant by suction. The precipitate obtained is resuspended in a buffer containing polyethylene glycol or bovine serum albumin, etc., and the remaining unlabeled antibody is removed by centrifugation to obtain the antibody labeled with the colored particles. (Geoghegan et al., Journal of Immunological Methods (J Immunol Method)
s) 34, 11-21, 1980, etc.). The first antibody labeled with the colored particles may be impregnated and dried on a part of the chromatographic medium, or may be impregnated and dried on a porous matrix of fibers separate from the chromatographic medium. , The matrix may be contacted with a chromatographic medium. As such a matrix, a nonwoven fabric or felt is preferably used. [0018] The second antibody is immobilized at a certain site on the chromatographic medium. The method of immobilization varies depending on the type of the chromatographic medium, but may be a conventional filter paper, glass filter,
In the case of a nylon membrane or the like, the antibody is first bound to latex particles. As the latex particles to be used, particles having a particle size larger than the size of the filter (retained particle size) or particles having a smaller particle size are aggregated and used. The solid phase latex in which the second antibody and latex particles are bound is prepared and prepared by a usual method. For example, the antibody and the latex emulsion are incubated at room temperature for about 2 hours, and then a buffer containing bovine serum albumin and the like is added, followed by centrifugation. The obtained precipitate is washed by carrying out suspension and centrifugation operations 1 to 3 times with the same buffer to obtain an antibody bound to latex particles, that is, a solid phase latex. The antibody bound to the latex particles is immobilized by spotting or direct jet printing while suspended in the buffer. On the other hand, in the case of a nitrocellulose membrane or a certain activated membrane, the antibody solution is immobilized by spotting or directly jet-printing the antibody solution as it is. The site where the second antibody is immobilized is 0.5 cm or more and 4 cm or less, and preferably 0.5 cm or more, on the chromatographic medium in the downstream direction from the site where the first antibody is impregnated, for the following reason. It must be at a distance of 2 cm or less. That is, since human Hb has a special structure of being a tetramer composed of two subunits of α and β, two identical antigenic determinants are present per hemoglobin.
One by one. For this reason, 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 cannot be expanded. However, when the distance between the labeled antibody and the reaction site is 4 cm or less, particularly 2 cm or less, the conjugate of human Hb / labeled antibody is rapidly developed before the development 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 time required for the antigen-antibody reaction is sufficient. Because it cannot be obtained, sufficient sensitivity cannot be obtained. 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 stool suspension sample. However, since a sample is not adsorbed and a uniform and high developing speed is obtained, a glass filter is used. Is particularly preferred. The porous matrix of the chromatographic medium is used after being shaped into a strip. The device of the present invention may have a sample pad (4) for temporarily absorbing and filtering the stool suspension sample when applying the sample to the device. As such a sample pad, a porous matrix of fibers, preferably a nonwoven fabric or a felt, having a size sufficient to hold a sample amount necessary for chromatographic development is used. The sample pad partially overlaps the first antibody-impregnated site on the downstream side of the site and is located on the chromatographic medium. Since the stool suspension is filtered by the sample pad, it is not necessary to perform an operation for removing insoluble matters such as fibers contained in the stool in advance. The device of the present invention may have a water absorbing pad (6) for holding the developed sample. This water-absorbing pad is made of a highly water-absorbing porous matrix. The water-absorbing pad is large enough to hold all of the developed sample, and overlaps the downstream end of the chromatographic medium. The device according to the invention may have a housing (5). The housing is made of a non-water immersible moldable material, such as polyvinyl chloride, polystyrene, polyethylene, carp or polypropylene, covers the entire device consisting of the sample pad, the chromatographic medium and the water absorbing pad, and has a sample application site and It has a shape in which a window is provided in the second antibody immobilization site. By providing this housing, it is possible to prevent the odor and the stool suspension from leaking. Human H in stool suspension using the device of the present invention
The detection of b is performed as follows. That is, the test sample is brought into contact with the side of the site impregnated with the first antibody (1), and human Hb is reacted with the labeled first antibody;
The formed human Hb / first antibody / colored particle complex is further moved on the chromatographic medium (3), reacted with the immobilized second antibody (2), and supplemented, thereby obtaining The presence or absence of human Hb is expressed as a colored 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 stool is not particularly limited. For example, phosphate-buffered saline containing 0.1% by weight of bovine serum albumin is used. Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Examples (Assay of human Hb by immunochromatography) (a) Preparation of solid phase latex A commercially available latex emulsion (N-450, manufactured by Sekisui Chemical Co., Ltd.) was adjusted to phosphoric acid so that the solid concentration became 0.6% by weight. It was diluted with buffered saline (hereinafter referred to as "PBS"). 1 ml of the mixture 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 concentration 0.1
Wt% bovine serum albumin (hereinafter referred to as "BSA")
The solid phase latex was prepared by washing three times by centrifugation at 8000 G for 10 minutes using PBS containing, and resuspending to a final volume of 2 ml. (B) Preparation of colloidal gold particles The preparation of colloidal gold particles was carried out according to the method of G. Frens (Nature Physics).
ICALSCIENCE) 241 Jan.1 1973).
That is, 200 ml of an aqueous solution of gold chloride having a concentration of 0.01% by weight was boiled, and 2 ml of an aqueous solution of sodium citrate having a concentration of 1% by weight was added thereto. By heating and boiling until the color of the solution changed from pale yellow to purple or red, a gold colloid dispersion having an average particle diameter of 0.04 μm was prepared. (C) Preparation of Colloidal Gold Particle-Labeled Antibody
The pH was adjusted to 6.7 using M potassium carbonate solution. To this was added a monoclonal antibody against human Hb at a rate of 5 μg per ml of colloidal gold dispersion. 1-2
After a minute, 0.1 ml of BSA having a concentration of 10% by weight is added to the 10 ml,
The supernatant was removed by centrifugation at 10,200 G for 1 hour,
A three times with PBS containing 0.1% by weight of A
After washing by centrifugation at 200 G for 1 hour, a first antibody labeled with colloidal gold particles was prepared. This is the P
The suspension was redispersed in 10 ml of BS, and 0.1 ml of the solution was impregnated with a 5 × 10 mm Bemliese nonwoven fabric (trade name, manufactured by Asahi Kasei Corporation) and freeze-dried. (D) Preparation of a chromatographic medium A commercially available glass filter (GC-
From 50), a 10 × 100 mm membrane strip was cut, placed on a glass plate, and fixed with an adhesive tape for easy handling. 0.2, 0.5, 1. from the end of the strip
At the position of 0, 2.0, 3.0, 4.0 or 5.0 cm, using an Acura Jetter liquid ejector (trade name, manufactured by Nordson) and an XY table aero stage (trade name, manufactured by THK), perpendicular to the developing direction, That is, 1 μl of the solid latex was jet-printed to a length of 10 mm in parallel with the short side of the strip. (E) Assembly of the Apparatus The chromatographic strip was placed on a 10 × 120 mm adhesive sheet with the same width of 10 mm, and the ends farthest from the portion where the latex was jet-printed were aligned.
10 x 40mm filter paper (Whatman, 17C
hr) and taped. On the other end of the chromatographic strip, the non-woven fabric containing the gold colloid-labeled antibody was placed so as to overlap by 2.5 mm. Further, a 10 × 20 mm Bemliese nonwoven fabric was placed as a sample pad so as to overlap the above-mentioned nonwoven fabric containing the gold colloid-labeled antibody by 2.5 mm. (F) Preparation of a sample containing human Hb Human Hb (manufactured by Sigma) was diluted with PBS containing BSA at a concentration of 0.1% by weight so that the concentration of human Hb was reduced.
Samples containing human Hb at 0.1, 1.0, 10 and 100 μg / ml were prepared. (G) Assay 200 μl of the human Hb-containing sample or 200 μl of PBS containing BSA at a concentration of 0.1% by weight as a blank was dropped on the sample pad of the device obtained in (e) above, and the sample was developed. I let it. After a lapse of 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 observed ±, when a line was observed +, and when a clear line was observed ++. . Table 1 shows the results. [Table 1] Table 1 shows the following. That is,
When the distance between the labeled antibody-impregnated site and the reaction site is 5.0 cm and the Hb concentration in the sample is 10 μg / ml or more, the line at the reaction site becomes ambiguous and the Hb concentration becomes 100 μg / ml.
Above g / ml, negative results are obtained. On the other hand, when the distance between the labeled antibody-impregnated site and the reaction site is 0.2 cm 2, when the Hb concentration in the sample is 1 μg / ml, the line at the reaction site becomes slightly ambiguous. On the other hand, when the distance between the labeled antibody-impregnated site and the reaction site is 0.5 cm or more and 4.0 cm or less, a stable determination result can be obtained in a wide concentration range in the sample. According to the present invention, there is provided a simple Hb detecting apparatus which can easily obtain a stable determination result in a short time even at home and eliminates discomfort of an operator.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a plan view of the device of the present invention. FIG. 2 is a plan view of the device of the present invention provided with a sample pad and a water absorbing pad. FIG. 3 is a side view of the apparatus of FIG. 2; FIG. 4 is a plan view of the device of the present invention with a housing. FIG. 5 is a side sectional view of the apparatus of FIG. 4; [Description of Signs] 1 Labeled antibody impregnated site 2 Reaction site 3 Chromatographic medium 4 Sample pad 5 Housing 6 Water absorption pad 7 Adhesive sheet

   ────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Takeshi Doe             3810 405 Ninamachi, Nara City F term (reference) 2G045 AA22 BA11 BB03 CB04 DA51                       FA11 FA18 FB03 FB15

Claims (1)

Claims: 1. A porous matrix labeled with colored particles and having a site (1) impregnated with a first antibody that specifically binds to human hemoglobin in a sample; And a porous matrix having a reaction site (2) to which a second antibody that specifically binds to human hemoglobin in the sample has a reaction site (2). A human, wherein the porous matrix and the porous matrix having the reaction site (2) are different porous matrices, and the distance between the site (1) and the reaction site (2) is 0.5 cm or more and 4 cm or less. Hemoglobin detector.