JP2747643B2 - Colorectal cancer screening method and kit therefor - Google Patents

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 large bowel polyps and colorectal cancer, and more particularly to a method for reliably detecting large bowel cancer and large bowel polyps by measuring lactoferrin and myeloperoxidase in feces.

[0002]

2. Description of the Related Art In various gastrointestinal diseases, chronic bleeding to the gastrointestinal tract often occurs due to inflammation of the gastrointestinal mucosa and tumor formation. In addition, protein leaks into the intestinal tract due to abnormal permeability of capillaries, increased lymphatic pressure, and the like, often presenting a condition such as so-called protein leakage gastroenteropathy.

[0003] Therefore, attempts have been made to measure hemoglobin and various proteins in feces, and in particular, various fecal occult blood reactions have been developed for the purpose of colorectal cancer screening.
For this occult blood reaction, not only a chemical method such as the guaiac method, but also an immunological fecal occult blood reaction capable of specifically detecting human hemoglobin has been developed.

[0004]

However, since the guaiac method utilizes the peroxidase activity of heme in hemoglobin, it reacts not only with human hemoglobin in feces but also with hemoglobin in animal meat or fish meat. There is a problem. That is, there is a drawback that non-specific and false-positive results require dietary restriction, and sensitivity cannot be increased.

On the other hand, the immunological fecal occult blood reaction does not require dietary restriction due to its high specificity and has high sensitivity, but on the other hand, the stability of hemoglobin in feces is poor, resulting in false negative results. There is a problem. In addition, there is also a problem that any of the occult blood reactions causes false positives due to contamination of blood other than the digestive tract such as anal bleeding and physiological bleeding. The present invention has been made in view of these problems, and has as its object to provide a method for reliably detecting colorectal cancer and the like by a new test method for occult blood reaction.

[0006]

Means for Solving the Problems In order to achieve the above object, according to the present invention, colorectal cancer is screened by measuring intragranular components of leukocytes in feces. Intragranular components include lactoferrin, granulocyte elastase, myeloperoxidase, lysozyme, cathepsin G, and the like. In particular, lactoferrin and myeloperoxidase are preferably measured. In order to detect lactoferrin, myeloperoxidase, etc., enzyme immunoassay (ELISA) and radioimmunoassay (RIA)
Method), luminescent enzyme immunoassay, latex agglutination reaction, and other commonly used immunological measurement methods can be used.

[0007] In order to detect colorectal cancer and the like accurately and efficiently, it is considered advantageous to measure a substance produced locally at a lesion. Multinuclear leukocytes, which infiltrate the site of infection first,
Focusing on the release components of neutrophils, neutrophils are
It engulfs and sterilizes invading microorganisms. This action is one of the biological defense mechanisms together with the immune response and is important. The neutrophils sense the concentration of various chemotactic factors and gather at the site of microbial infection or lesion, and release various substances to kill the captured microorganisms. The substances released by neutrophils are various hydrolases and low-molecular-weight basic proteins contained in neutrophil granules, and active enzymes produced and released by activated neutrophils.

[0008] Among the neutrophil released components, the neutrophils separated and collected from the blood of a healthy person are used for phagocytosis of latex particles and PMA (Ph
Orbol myristate acetate) The release state of intragranular components upon stimulation was examined for lactoferrin, granulocyte elastase, myeloperoxidase, lysozyme, and cathepsin G. It was found to be significantly more. In addition, when examining what intragranular components are excreted in faeces and which pathology specifically changes, lactoferrin and myeloperoxidase, especially in the control group and disease group, A clear difference was observed.

By the way, the following facts are known about the action of lactoferrin at the site of infection. Neutrophils that recognize chemotactic factors migrate from the bloodstream to the site of infection and aggregate. Neutrophils that reach the site of infection release extracellular lactoferrin extracellularly due to high concentrations of chemotactic factors and bacterial phagocytosis. This lactoferrin causes macrophages to release neutrophil migration inhibitory factors. High concentrations of lactoferrin also act directly on the surrounding neutrophils, preventing them from migrating and stopping them at the site of infection. From the above facts, it can be seen that lactoferrin is suitable as a measurement target.

It is also known that in cancer, granulocytes are mobilized by cytokines such as tumor necrosis factor and interferon and aggregate at tumor sites to act as antitumors. It is thought that this may be one of the factors that increase the medium lactoferrin level.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in more detail with reference to embodiments. [1] Test of effectiveness of the present invention (A) Clinical samples A total of 17 cases in which no abnormalities were observed by upper gastrointestinal endoscope and colonoscopy or enema X-ray examination and 18 healthy subjects were 3 in total.
Five cases were set as a control group. As lower gastrointestinal diseases, a. Colon polyp 16 cases b. Colorectal cancer 13 cases (early cancer 1 case, advanced cancer 12 cases) c. Ulcerative colitis 28 cases (38 specimens in active phase, 20 specimens in non-active phase) d. A total of 75 cases (139 samples) were studied, including 18 cases of Crohn's disease (36 samples during the active period and 16 samples during the non-active period).

(B) Collection of feces Instructed to directly defecate in a vinyl chloride container having a diameter of 15 cm and a depth of 12 cm, and collect the entire amount of defecation,
The stool was homogenized by sufficiently stirring with a hand mixer attached to the lid of the container. Immediately after defecation, they were immediately stored in a refrigerator at 4 ° C.

(C) Preparation of stool sample 20 mg of stool was measured and collected, and a TBS buffer solution (0.1 mol / l Tr
2 ml of is-HCl buffer, pH 8.0, containing 0.1 mol / l NaCl).

(D) Measurement of lactoferrin by ELISA [Immobilization of antibody on microplate] 0.1 M containing 5 µg / ml of anti-human lactoferrin antibody (DAKOPATTS, Denmark) in each well of a microplate (SUMILON, Japan). T
Dispense 100 μl of the ris buffer solution at room temperature overnight at 4 ° C. to allow physical adsorption to solidify on the surface. [Preparation of Enzyme-Labeled Antibody] Separately, an alkaline phosphatase (Beehringer-Mannheim, FRG) is enzymatically labeled with an anti-human lactoferrin antibody by the periodate method.

[Measurement of lactoferrin in feces]
0 μl of Tris buffer (0.1 mol / l pH 8.0) containing 1% BSA (Beehringer-Mannheim, FRG) was dispensed, then 50 μl of a stool sample was added and mixed, and the mixture was mixed at 37 ° C. for 1 hour. Let react. Next, it is washed three times with deionized water containing 0.05% of Tween20. Thereafter, an alkaline phosphatase-labeled anti-human lactoferrin antibody solution (Tris buffer containing 1% BSA) is added to each well in an amount of 100 μl, mixed and reacted at 37 ° C. for 1 hour, and washed three times as before.

Further, 100 μl of substrate buffer for the Kind-King method
Is added to each well and reacted at 37 ° C. for 30 minutes. Here, the substrate buffer is Disodium Phenyl-phosphate (WAKO Junya
ku Japan) 0.215g and 4-aminoantipyrine (WAKO Junyaku
Japan) (0.09 g) in carbonate buffer (0.05 mol / l pH 10.1)
5) Dissolved in 100 ml. Next, 100 μl of a coloring solution is added to each well to cause coloration. Here, the color liquid is 20
2.6 g boric acid (WAKO Junyaku Jap) in 0 ml deionized water
ans) and 0.38 g of Potassium ferricyani
de (WAKO Junyaku Japan) is dissolved.

Finally, the color of each well was measured using a colorimeter for microplate (manufactured by Sanko Junyaku Japan) at 510/68.
Colorimetry is performed with light at a wavelength of 0 nm, and the lactoferrin concentration in feces is calculated from a calibration curve. The results are as shown in the lactoferrin concentration shown in FIG.

[Calibration Curve] The calibration curve is as shown in FIG. 6, and the calibration curve is 1 μg / ml of human lactoferrin.
(Manufactured by Sigma, USA) as a standard solution, which was determined by the same operation as for lactoferrin measurement. The measurement range was 10 ng / ml to 1000 ng / ml, and the reproducibility was good.

(E) Measurement of myeloperoxidase [Immobilization of antibody on microplate] Each well of a microplate (SUMILON, Japan) contains 5 µg / ml of an anti-human myeloperoxidase antibody (DAKOPATTS, Denmark). Dispense 100 μl of 1 M Tris buffer for 4 nights.
Leave at ℃ for physical adsorption to solidify on the surface. [Preparation of Enzyme-Labeled Antibody] Separately, alkaline phosphatase (Behringer-Mannheim, FRG) is enzymatically labeled with an anti-human myeloperoxidase antibody by the periodate method.

[Measurement of fecal myeloperoxidase]
100 μl of 1% BSA (Beehringer-Mannheim, FR)
G) containing Tris buffer (0.1 mol / l pH 8.0), then adding 50 μl of a stool sample and mixing.
Incubate at 37 ° C for 1 hour. Next, Tween20 0.05%
Wash three times with deionized water. Thereafter, 100 μl of an alkaline phosphatase-labeled anti-human myeloperoxidase antibody solution (Tris buffer containing 1% BSA) was added to each well.
After each addition and mixing, the mixture is reacted at 37 ° C. for 1 hour and washed three times as before.

Further, 100 μl of substrate buffer for the Kind-King method
Is added to each well and reacted at 37 ° C. for 30 minutes. Next, 100 μl of a coloring solution is added to each well to cause coloration. Finally, the color of each well is measured using a colorimeter for microplate (Sank
o Junyaku Japan) and colorimetrically measure 510/680 nm wavelength light, and calculate the myeloperoxidase in feces from the calibration curve. The results are as shown in the myeloperoxidase concentration shown in FIG.

(F) Measurement of myeloperoxidase activity [Immobilization of antibody] The means for immobilizing an antibody on a microplate is the same as in the above (E) for the measurement of myeloperoxidase. [Measurement of fecal myeloperoxidase activity] 10 per well
0 μl of Tris buffer containing 1% BSA (0.1 mol / l pH
8.0), then add 50 μl of stool sample,
After mixing, react at 37 ° C. for 1 hour.

Next, the substrate is washed three times with deionized water containing 0.05% of Tween20. Thereafter, 50 μl of a TMB peroxidase substrate is added, 50 μl of a hydrogen peroxide reagent is further added, and the mixture is left for 15 minutes. After that, a 2.7 g / dl NaF solution is added to stop color development. Next, the color of each well was measured using a colorimeter for microplate with light having a wavelength of 680/510 nm, and the myeloperoxidase activity in feces was derived from a calibration curve. As a result, almost the same results as those in FIG. 2 were obtained.

(G) Measurement of Granulocyte Elastase Amount of granulocyte elastase was measured in the same manner. The results are as shown in the granulocyte elastase concentration shown in FIG. (H) Measurement of Hemoglobin Amount For the same sample, the amount of hemoglobin was measured using a conventional immunological fecal occult blood reaction and compared. The results are as shown in the hemoglobin concentration shown in FIG.

[2] The results of the measurement are summarized as follows. (A) Lactoferrin amount (FIG. 1). The lactoferrin concentration in the feces of the control group (healthy persons and those who did not recognize abnormalities) was 0.75 ± 0.83 μg / g stool (mean ± standard deviation σ: the same applies hereinafter), and the upper limit of normality (the judgment value was the average The value ± 2σ) was 2.4 μg / g stool. In the colon polyps, the amount of lactoferrin was 6.1 ± 9.1 μg / g stool, and 7 out of 16 cases (43.8%) had stool of 2.4 μg / g or more. In colon cancer, the amount of lactoferrin is 89.7 ± 82.4 μg / g
All of the 13 cases (100%) had stool with a stool of 2.4 μg / g or more.

In the case of inflammatory bowel disease and ulcerative colitis, the amount of lactoferrin in the active phase was 307.4 ± 233.9 μg / g stool, and all 38 cases (100%) had a stool of 2.4 μg / g or more. Lactoferrin amount is 63.3 ± 144.6 μg even during active period
/ G stool and 15 out of 20 cases (75.0%) were 2.4 μg / g
It was more than a stool. In the case of Crohn's disease, the amount of lactoferrin in the active phase was 191.7 ± 231.1 μg / g stool, and all of the 36 cases (100%) had stool of 2.4 μg / g or more. / G flight and 1
Fifteen out of the six cases (93.8%) had stool of 2.4 μg / g or more.

(B) Amount of myeloperoxidase (FIG. 2). The concentration of myeloperoxidase in the feces of the 35 control group was
2.4 ± 0.9 μg / g stool, normal upper limit is 4.2 μg / g
It was a stool. The colorectal polyps had 5.9 ± 7.8 μg / g stool, and 7 out of 16 cases (43.8%) had more than 4.2 μg / g stool. In colorectal cancer, stool was 72.3 ± 68.2 μg / g, and all 13 cases (100%) had stool of 4.2 μg / g or more.

In the case of ulcerative colitis, the active phase is 206.2 ±
196.6 μg / g stool and 36 out of 38 cases (94.7%) showed 4.
More than 2 μg / g stool, 37.8 ± 89.6 μg in inactive period
/ G stool and 17 out of 20 cases (85.0%) were 4.2 μg / g
It was more than a stool. 246.9 for Crohn's disease during active phase
± 206.1 μg / g stool and all 36 cases (100%) showed 4.
More than 2 μg / g stool, 59.2 ± 66.2μg even in inactive period
/ G stool and 11 out of 16 cases (68.8%) were 2.4 μg / g
It was more than a stool.

(C) Amount of granulocyte elastase (FIG. 3) In the control group, the concentration of faecal granulocyte elastase was 0.7 ±
The dose was 0.4 μg / g stool, and the upper normal limit was 1.5 μg / g stool. 1.2 ± 1.1 μg / g stool in colorectal polyps, 5.2 ± 4.1 μg / g stool in colorectal cancer, 52.2 ± 41.3 μg / g stool in active phase for ulcerative colitis, 12.8 ± 9.6 in inactive phase
μg / g stool, 35.6 ± 32.3 in active phase in case of Crohn's disease
μg / g stool and 13.4 ± 10.3 μg / g stool in the inactive period.

(D) Hemoglobin amount (FIG. 4). In the control group, the concentration of hemoglobin in feces was 1.9 ± 2.0 μ
g / g stool, and the upper limit of normal is 5.9 μg / g stool. 33.1 ± 68.8 μg / g stool in colorectal polyps, 116 in colorectal cancer
2.8 ± 1705.2 μg / g stool, in the case of ulcerative colitis, 2989.5 ± 3435.5 μg / g stool in the active phase, 117.5 ± 481.
8 μg / g stool, 175 ± 36 in active phase for Crohn's disease
3.3 μg / g stool and inactive 39.2 ± 120.9 μg / g stool.

[3] Disease Positive Rate of Various Markers FIG. 5 shows the comparison of the disease detection (positive) rates of various diseases with various markers based on the above results.
According to this comparison table, it is found that the positive rate of healthy subjects is lower when lactoferrin / myeloperoxidase / granulocyte elastase is used as a marker than in the conventional fecal occult blood method (the number of false positives is small). The lactoferrin method and myeloperoxidase method have a detection rate of 100% in colorectal cancer.
And even colon polyps outperform the occult blood test.

[4] Other studies on the efficacy of the present invention (A) Examination of antibody specificity Using a sample containing milk-derived lactoferrin (bovine lactoferrin), an anti-human lactoferrin antibody and an enzyme-labeled anti-human lactoferrin antibody were used. As a result, no reaction was shown (see FIG. 6). That is, according to the present invention, it is clear that human lactoferrin can be specifically detected and is not affected by dairy products in the diet.

(B) Stability in stool Human lactoferrin and human hemoglobin were added to stool juice prepared from stool of a healthy subject (whole blood was added to a solution in which 1 g of stool was dissolved in 3 ml of deionized water). Hemoglobin amount is 1mg / m
l, add human lactoferrin and add 1 μg /
ml) at 37 ° C. for 30 minutes, 1 hour, 2 hours, and 4 hours at a constant temperature.
The amount of lactoferrin was measured by the method A, and the amount of hemoglobin was measured by the conventional immunological fecal occult blood test.

The result was as shown in FIG. From this result, the amount of hemoglobin was reduced to 75% after 30 minutes of sample storage, and only 20% remained after 4 hours. On the other hand, lactoferrin showed almost no decrease in activity even after 4 hours, and lactoferrin was reduced to hemoglobin. It turns out to be suitable as a marker for comparison. FIG.
Fig. 9 shows another experimental result for examining the stability of leukocyte granule components in feces.

(C) Influence of Blood Incorporation into Feces To examine the effect of blood invasion due to bleeding into feces, first, the lactoferrin concentration in the blood of a healthy individual was measured. Then, the lactoferrin concentration in whole blood of 10 healthy subjects was 6.
The concentration was 88 ± 2.35 μg / ml, and the concentration of lactoferrin in plasma was 86.4 ± 28.5 ng / ml. This whole blood sample was added to 1 g of stool of a healthy subject, mixed, and the lactoferrin concentration was measured. When the total blood was 250 μl or less, the lactoferrin concentration in the stool was 2.4 μg / g stool or less. It was confirmed that blood contamination of the lactoferrin had no effect on lactoferrin measurement.

(D) Influence of lactoferrin in saliva Since lactoferrin is also contained in saliva in an amount of about 5 to 10 μg / ml, there is a concern that the lactoferrin may remain in the feces. Concentration is 0.75 ± 0.83
Since only a small amount of μg / g stool was detected, it is considered that most of the stool was digested and decomposed, and it is considered that there is almost no effect on the method of the present invention.

[0037]

As described above, if intragranular components of leukocytes in feces, particularly lactoferrin and myeloperoxidase, are measured, colorectal cancer and the like can be reliably screened. In other words, colorectal cancer and the like can be reliably screened without being affected by dairy products in the diet. In addition, lactoferrin and myeloperoxidase have excellent stability in feces, and furthermore, blood contaminants of the order of occult blood have no effect.

[Brief description of the drawings]

FIG. 1 is a graph showing lactoferrin concentration in feces for each disease group.

FIG. 2 is a graph showing the concentration of myeloperoxidase in feces for each disease group.

FIG. 3 is a graph showing the concentration of granulocyte elastase in feces for each disease group.

FIG. 4 is a graph showing the concentration of hemoglobin in feces for each disease group.

FIG. 5 is a table showing disease positive rates of various markers.

FIG. 6 is a graph showing the relationship between the absorbance measured by a colorimeter and the lactoferrin concentration.

FIG. 7 is a graph showing temporal changes of lactoferrin and hemoglobin in feces.

FIG. 8 is a graph showing the temporal change of leukocyte granule components in feces.

Claims (5)

(57) [Claims] 1. A method for screening for colorectal cancer, wherein intragranular components of leukocytes in feces are measured. 2. The method for screening for colorectal cancer according to claim 1, wherein lactoferrin or myeloperoxidase in feces is measured. 3. The method for screening colorectal cancer according to claim 1 or 2, wherein an immunological assay such as an enzyme immunoassay or a latex agglutination is used. 4. A method for diagnosing colorectal cancer comprising at least an anti-human lactoferrin antibody, an enzyme-labeled anti-human lactoferrin antibody and a reagent for measuring the enzyme, and measuring the lactoferrin concentration in feces by an immunological assay. A colorectal cancer screening kit, comprising: 5. A method for measuring colorectal cancer comprising at least an anti-human myeloperoxidase antibody, an enzyme-labeled anti-human myeloperoxidase antibody and a reagent for measuring the enzyme, wherein the concentration of fecal myeloperoxidase is measured by an immunological assay. A colorectal cancer screening kit, characterized in that it is diagnosed.