JPH05203643A - Method for detecting occult blood in dejection - Google Patents

Abstract

PURPOSE:To detect occult blood in dejection in order to perform screening of micro bleeding due to the cancer and ulcer of the digestive system. CONSTITUTION:Of three components hemoglobin, IgG, and C two kinds are employed as occult blood markers and are measured and detected by immunological method. The components of the two kinds of markers are detected by simultaneous use of the antibody of each of the components. The antibody is deposited on the surface of a solid and the marker components in a dejection sample are captured and a corresponding enzyme-labeled antibody is reacted therewith and a substrate solution and a coloring liquid are added thereto to detect coloring. Also the antibody is sensitized with latex particles and a suspension of the particles and the dejection sample are admixed on a agglutination reaction slide plate and are detected in an agglutinated state.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting microhemorrhage in the digestive tract for the purpose of screening cancer, ulcer, etc. of the digestive system.

[0002]

2. Description of the Related Art Conventionally, hemoglobin, which is a typical protein of blood, has been detected by a chemical or immunological method to detect bleeding from the digestive tract.

[0003]

However, the detection of hemoglobin alone has the drawbacks that the chemical method requires dietary restriction prior to the test and that false positives are more frequent. However, there is a problem that the immunological method is not suitable due to a decrease in the immunological reactivity due to the above-mentioned, which causes poor reactivity at a low concentration and a drawback that hemoglobin in the sample decreases with time due to intestinal cells.

On the other hand, the present inventors have discovered that transferrin, which is a protein contained in blood, can be used as a marker for detecting intestinal microbleeding, and a method for detecting it by an immunological assay method. Invention of
The application was filed as 62-80741 (JP-A-63-246668).
However, there was a problem that transferrin alone lacked reliability. On the other hand, when hemoglobin was stabilized with a glycosidase-type lytic enzyme and both hemoglobin and transferrin were simultaneously detected by an immunological method, it was found that even better results were obtained, and Japanese Patent Application No. No. (JP-A-63-246669)
Filed as an invention. However, it is not preferable to limit the combination of hemoglobin and transferrin as a component (marker) for detection because the detection means is limited.

[0005]

Means for Solving the Problems After the invention of the method for simultaneously measuring hemoglobin and transferrin, the present inventor conducted a search on many blood components, and as a result, found that not only transferrin but also immunoglobulin IgG in blood components. , And the C3 component, which is complement (C), serve as a marker for occult blood reaction.

That is, according to the present invention, hemoglobin and immunoglobulin IgG, or hemoglobin and C in a fecal sample.
A method for detecting occult blood in feces, which comprises simultaneously detecting three components or a combination of any two components among immunoglobulin IgG and C3 components by an immunological measurement method, which solves the above problems. Is.

In that case, when hemoglobin is used as a marker, glycosidase-type lytic enzyme, especially lysozyme, is added to a stool sample to prevent hemoglobin in the stool sample from being decomposed by bacteria and to be stably detected.

There are the following methods for implementing the present invention. 1. Antibody-antibody sandwich method Two types of anti-human hemoglobin antibody, anti-human IgG antibody or anti-human C3 component antibody are insolubilized on the surface of a solid such as plastic. A stool sample is brought into contact with this to capture components corresponding to these in the sample. After washing, the used solution prepared by mixing equal amounts of the enzyme-labeled antibodies against two kinds of components corresponding to the above-mentioned captured components among hemoglobin, IgG or C3 components is reacted with the components in the captured sample, and the substrate solution and the color are developed. After adding the liquid and reacting, the color development is measured with a colorimeter.

2. Latex agglutination reaction method Latex particles having a small particle size are suspended, and a liquid containing equal amounts of anti-human hemoglobin antibody and anti-human IgG antibody is added thereto to sensitize the latex particles. The obtained latex particles are washed and suspended in Tris buffer (hereinafter referred to as TBS). A stool sample is dropped on the slide plate for agglutination reaction, and the above-mentioned sensitized latex reagent is dropped on it and shaken to mix to determine the agglutination state.

A combination of anti-human hemoglobin antibody and anti-human C3 component antibody or anti-human I in the same manner as described above
Sensitizing the latex with a combination of gG antibody and anti-human C3 component antibody can detect the presence of those components.

[0011]

【Example】

1. 50 μl of feces juice mixed with 1 ml of deionized water containing 15 mg of feces of the patient to be examined or hemochaser (trade name of occult blood test kit for feces sold by Shionogi Pharmaceutical Co., Ltd. (according to Japanese Patent Application No. 62-80741)) One drop of the container is used as the sample.

On the other hand, 5 μg / ml of anti-human hemoglobin antibody (manufactured by Special Immune Research Institute, Monoclonal antibody) and 5 μg / ml (0.1 mol of anti-human IgG antibody (manufactured by Dako))
A solution containing both Tris buffer and pH 8.4) was dispensed in 100 μl aliquots to each well of a microplate and left overnight at 4 ° C to immobilize (insolubilize) the surface. 1% BSA (0.1 mol Tris buffer, pH
8.0) in 100 μl and add the above stool sample to 37 ℃,
The reaction is allowed to proceed for 1 hour to capture hemoglobin and IgG in the sample.

After washing, an equal amount of both the alkaline phosphatase-labeled anti-human hemoglobin antibody and the alkaline phosphatase-labeled anti-human IgG antibody used solution (1% BSA: 0.1 mol Tris buffer diluted to 1000 times with pH 8.0) was used. Dispense 100 μl of the mixed solution into each well and incubate at 37 ° C for 1 hour. After washing, 100 μl of phenyl phosphate substrate solution is dispensed into each well and reacted at 37 ° C. for 30 minutes, and 100 μl of color developing solution is added to each well to develop color.

This color development was measured at two wavelengths of 510 nm and 630 nm using a colorimeter (Sanko Junyaku Co., Ltd. auto reader). As a result, the presence of one or both of hemoglobin and IgG, which are blood components in the stool sample, can be simultaneously detected, that is, occult blood in the stool sample can be detected.

2. By using the anti-human C3 antibody instead of the anti-human IgG antibody in the same manner as in Example 1, it is possible to detect the presence of one or both of hemoglobin and the C3 component, and thus to detect occult blood in a stool sample as well. You can

3. By using the anti-human IgG antibody and the anti-human C3 antibody in the same manner as in Example 1 without using the anti-human hemoglobin antibody, it is possible to simultaneously detect the presence of one or both of the IgG and C3 components, and thus, to detect the presence of stool in the stool sample. Occult blood can be detected.

4. As a sample, 50 ml of feces juice mixed with 1 ml of deionized water containing 15 mg of the feces of the patient to be examined or one drop of a hemochaser container is used. 1 mg of latex particles having a particle size of 0.1 to 1.0 μm is suspended in TBS so as to be 1%. To 4 volumes of this suspension, 1 volume of TBS containing 1 mg of anti-human hemoglobin antibody and 1 volume of anti-human IgG antibody per ml was added, and the cells were sensitized at room temperature for 6 hours.

The sensitized latex obtained is washed twice with TBS and suspended in TBS containing 0.1% bovine serum albumin at a ratio of 0.5%. To preserve this solution, a preservative such as sodium azide is added. On the slide plate for agglutination reaction, drop 50 μl of the sample solution, add 50 μl of the above-mentioned sensitized latex reagent on it, shake the slide plate up, down, left and right to mix well, and after 3 minutes, check for the presence of aggregation. To judge. The state of aggregation is judged according to the criteria shown in FIG. Hemoglobin and IgG are determined by the positive judgment at this time.
The presence of either one or both of
Therefore, occult blood in the stool sample could be detected.

5. The same method as in Example 4 is used to detect the presence of occult blood in a stool sample by using a combination of an anti-human hemoglobin antibody and an anti-human C3 component antibody or a combination of an anti-human IgG antibody and an anti-human C3 component antibody. You can

6. For many stool samples, detection is performed from the stool sample for each combination of hemoglobin and IgG, hemoglobin and C3 component, IgG and C3 component, using the detection method by absorption using the antibody-antibody sandwich method of the present invention. It was On the other hand, the same stool sample was detected by a combination of hemoglobin and transferrin by the same detection method by absorption and compared. In addition, the measurement of the absorbance
Performed for a wavelength of 510 nm.

The results are shown in FIGS. 2, 3 and 4. In the figure, n is the number of samples and r is the value of the correlation function. In all cases, r> 0.8, and both have the same accuracy. It can be seen that occult blood in a stool sample can be effectively detected in the same manner as 80472 (JP-A-63-246669).

[0022]

INDUSTRIAL APPLICABILITY As described in detail above, the method of the present invention can detect occult blood in the stool of a subject as a screening method for early detection of cancer, ulcer, etc. of the digestive system, Unlike conventional methods, the reagent used is not limited to anti-human hemoglobin antibody and anti-human transferrin antibody, but can be detected with the same degree of accuracy, and it is possible to improve accuracy by diversifying reagents. Is.

[Brief description of drawings]

FIG. 1 is a diagram showing a judgment standard when the latex method of the present invention (Example 4) is used.

FIG. 2 is a graph showing a comparison of the absorbance of many stool samples when using the combination of hemoglobin and IgG which is the detection method of the present invention and when using the combination of hemoglobin and transferrin of the conventional method.

FIG. 3 is a graph showing a comparison of absorbance when a combination of hemoglobin and a C3 component is used and when a combination of a conventional method of hemoglobin and transferrin is used.

FIG. 4 is a graph showing a comparison of absorbance when a combination of IgG and C3 component is used and when a conventional combination of hemoglobin and transferrin is used.

[Explanation of symbols] 1 latex agglomerate n number of samples r correlation coefficient

Claims (6)

[Claims] 1. A method for detecting occult blood in feces, which comprises simultaneously measuring hemoglobin and immunoglobulin IgG in a stool sample. 2. A method for detecting occult blood in feces, which comprises simultaneously measuring hemoglobin in a stool sample and C3 component in blood. 3. The method for detecting occult blood in feces according to claim 1 or 2, wherein the glycosidase-type lytic enzyme is added to the stool sample and then the measurement is performed. 4. A method for detecting occult blood in feces, which comprises simultaneously measuring immunoglobulin IgG in a stool sample and C3 component in blood. 5. A stool sample is brought into contact with a solid whose surface is insolubilized with any two kinds of anti-human hemoglobin antibody, anti-human IgG antibody and anti-human C3 component antibody, and hemoglobin, IgG,
Any two kinds of C3 components are captured, and a working solution in which equal amounts of the enzyme-labeled antibodies corresponding to the two kinds of insolubilized above are mixed is allowed to act, and a substrate solution and a coloring solution are added,
The method for detecting occult blood in feces according to claim 1, wherein the color development is detected by measuring the absorbance with a colorimeter. 6. A solution in which latex particles are sensitized with a solution containing an equal amount of any two kinds of anti-human hemoglobin antibody, anti-human IgG antibody and anti-human C3 component antibody, and a stool sample and the latex particles are suspended. The method for detecting occult blood in feces according to any one of claims 1 to 4, characterized in that the agglutination degree is dropped onto a slide plate for agglutination reaction to determine and detect the degree of agglutination.