JPH0569466B2 - - Google Patents

Description

Detailed Description of the Invention A. Object of the Invention (a) Field of Industrial Application This invention relates to a method for detecting hemoglobin in feces.

(b) Prior Art As a screening method for early detection of abnormalities in the digestive system, such as cancer and ulcers, it is common to detect hemoglobin, a typical protein in blood mixed in feces. Conventionally, hemoglobin in feces has been detected by a chemical fecal occult blood reaction method such as the benzidine method or by an immunological method.

In general, chemical reaction methods have the disadvantage of being very sensitive and producing many false positives.Although the chemical reaction method detects the peroxidase activity of hemoglobin in the blood, peroxidase activity is also present in meat, vegetables, etc. It is necessary to restrict food intake.

It also detects hemoglobin that has been broken down by digestive enzymes, so it detects bleeding in the entire digestive system, making it impossible to obtain information on the site of bleeding, making it difficult to prove bleeding from the lower part of the digestive system, such as in cases of colon cancer. It has the drawback that it cannot be used as a stand-alone method.

In contrast, recently Barrow (Am.J.Cli.Pathol.69:
342, March 1978) have developed an immunological measurement method based on fecal occult blood reaction using an antibody-antigen reaction between human hemoglobin and anti-human hemoglobin. According to this immunological fecal occult blood measurement method, hemoglobin loses most of its antigenicity when subjected to the action of digestive enzymes in the digestive system. It has the characteristic of being able to detect only bleeding from the lower part of the system. This method includes, for example, a method in which feces is applied to a filter paper and mixed with carrier particles sensitized with human hemoglobin antibodies, as described in JP-A-61-137064, and the state of aggregation of the carrier particles in the mixture is determined. As described in Japanese Patent Application No. 60-069598, feces is brought into contact with a hydrophobic resin to adsorb hemoglobin, an antigen-antibody reaction is generated using an enzyme-labeled antibody reagent, and the feces is immersed in a substrate solution to be detected by coloration. Methods and the like are publicly known.

(c) Problems to be solved by the invention In general, hemoglobin has a weak titer against antibodies, so the sensitivity of immunoassays is low and disadvantageous, and the action of digestive enzymes and proteolytic enzymes produced by intestinal bacteria is disadvantageous. It is disadvantageous for immunological detection because it easily undergoes structural changes due to In particular, when detecting hemoglobin in feces using an immunological reaction method, (a) hemoglobin binds to mucus components secreted from the gastrointestinal tract and cell wall components of intestinal bacteria, reducing immunological reactivity (b) ) Hemoglobin in feces is easily decomposed by intestinal bacteria, and as it decomposes and decreases over time, there is a problem that detection becomes unstable.

B. Structure of the Invention (a) Means for Solving the Problems The present invention involves adding or allowing glycosidase-type cell wall lytic enzymes (hereinafter referred to as bacteriolytic enzymes) to act on collected feces to determine the amount of hemoglobin in the feces by an immunological method. This method eliminates the drawbacks of conventional methods.

The use of glycosidase-type bacteriolytic enzymes, such as lysozyme, as preservatives for foods, medicines, etc. is known, and when mixed into feces, it has the effect of preventing the decomposition of hemoglobin by intestinal bacteria over a long period of time. (Disadvantage (b) above) As a result of various experiments, the present inventor found that glycosidase-type bacteriolytic enzymes have an antiseptic effect (preventing decomposition by intestinal bacteria), as well as mucus components in feces and cell wall components of intestinal bacteria. The present invention was made based on the discovery that it also has the effect of restoring immunological reactivity by releasing hemoglobin binding in a short period of time.

(b) Action As mentioned above, hemoglobin in feces is bound to mucus components in feces and bacterial cell wall components, and is degraded by bacteria over time and decreases. For example, when hemoglobin is detected using an immunological method with the addition of lysozyme, the hemoglobin is released from mucus and cell wall components in a short period of time and can be completely detected, and accurate detection results can be obtained even if the sample is stored for a long time. can get.

In this case, the lytic enzyme may be any glycosidase-type lytic enzyme, but lysozyme is generally commercially available and easy to use.

The addition may be done by simply soaking the collected feces in a solution of the lytic enzyme or by dissolving the feces. Alternatively, various methods may be used, such as a method in which a fecal sample is inserted into an agar gel container containing a lytic enzyme and absorbed, or a method in which fecal matter is smeared onto a filter paper containing a lytic enzyme.

Further, as a detection method, a common immunoassay method may be used.

For example, a plastic stool sample with a grooved tip is coated by soaking it in Tris buffer containing anti-human hemoglobin antibody overnight, and this sample is inserted several times into different locations in the feces.
Wipe off excess feces with toilet paper and collect a certain amount of feces in the groove. On the other hand, add commercially available agarose (agar) to a concentration of 2% in a phosphate buffer solution containing sodium chloride, dissolve it by heating, and lower the temperature to about 60°C. Add 1 lytic enzyme (lysozyme) to the agar solution.
Add 3 mg of NaN ₃ per ml to a final concentration of 0.1%, dispense into an agar gel container, and cool to gel to create agar containing the lytic enzyme. The stool collection tube containing the feces is inserted into the agar gel and left for a certain period of time. Then, the hemoglobin in the feces diffuses into the agar gel, and at this time, the hemoglobin bound to mucus and cell wall components is released by the action of the lytic enzyme and binds to the anti-human hemoglobin antibody coated on the stool sample. It is completely captured on the surface of the feces sample. In this state, the lytic enzyme
The preservative effect of NaN ₃ also prevents hemoglobin from being degraded by intestinal bacteria. This stool sample is taken out, washed, and immersed in an alkaline phosphatase-labeled antibody prepared using a known method (for example, Yoshitake et al., Immunology Experimental Methods XI, pp. 3497-3519, 1982) to perform an antigen-antibody reaction. let Next, after washing the stool sample, alkaline phosphatase was added to Kind-
Measure using the King method. In other words, place a stool sample into a small test tube containing phenylphosphate substrate solution and incubate at 37℃ for 30 minutes.
After incubating for a minute, a coloring solution is added to stop the reaction, and the degree of coloring can be measured. In this way, the hemoglobin in the feces is completely captured by the antibodies of the feces sample, making it possible to detect hemoglobin with high accuracy.

Alternatively, the solution in which the lytic enzyme (lysozyme) and NaN ₃ are dissolved may be used as a sample by applying a thin layer of feces on a thick impregnated filter paper with a spatula, or as a sample by dissolving feces collected with a thin stick in the solution. Of course it's a good thing.

(c) Examples Example 1 Using the ELISA method (Enzyme-Linked
Measured by Immunosorbent Assay).

Collect a small amount of feces with a thin stick and add NaN ₃ per ml to saline phosphate buffer (PH7.2, 0.1M).
The cells were immersed in a small bottle containing 1 ml of a buffer containing 1 mg of lysozyme and 4 mg of lysozyme as a lytic enzyme per 1 ml of the buffer.

Add anti-human hemoglobin antibody (commercially available from DAKO) to each well of the ELISA microplate at the final concentration.
Tris buffer (PH8.4,
0.05M) was dispensed in 100μ portions and left at 4-8°C for 12 hours for coating.

After washing the antibody-coated plate with deionized water, dispense 100 μl of saline phosphate buffer containing 1% bovine albumin into each well. Inject 50μ of the fecal juice (fecal juice) mentioned above into this, and heat it for 60 minutes at 37°C.
Allow antigen-antibody reaction to occur for 1 minute.

After washing well, dispense 100μ of alkaline phosphatase-labeled antibody reagent into each well and incubate at 37°C.
After allowing antigen-antibody reaction for 60 minutes and washing with deionized water, add substrate solution for alkaline phosphatase measurement.
Dispense 100 μl into each well and react at 37°C for 30 minutes, then immediately add 100 μl of coloring reagent to stop the reaction. The colored liquid in each well was side-illuminated using a microplate photometer.

When the results were plotted, the degree of coloration of the sample without additives was plotted on the horizontal axis and the coloration of the lysozyme-added sample was plotted on the vertical axis, and the result was as shown in FIG.

The measured value of the sample to which lysozyme was added was larger than the measured value without the addition (above the 45 degree line), indicating that hemoglobin was completely measured. Normally, in the above-mentioned detection, the absorbance is determined as a limit of 100 to 200, but it was found that a considerable number of samples were negative without the additive, but became positive with the addition, increasing the accuracy of detection. .

Example 2 Whole blood was added at a final concentration of 10,000 times to approximately 25% stool juice prepared by mixing the stool juices of 30 healthy subjects, and the temporal change in the amount of hemoglobin was investigated. The result is second
It was as shown in the picture.

This indicates that the progress of decomposition of hemoglobin by intestinal bacteria is inhibited in stool juice to which lysozyme has been added.

C. Effects of the Invention As explained in detail above, the present invention has advantages over the conventional method for immunologically detecting hemoglobin in feces.
By simply adding a lytic enzyme to the test subject's feces, hemoglobin bound to mucus components and intestinal bacterial wall components can be separated and accurately detected. This is a very effective method for detecting hemoglobin in feces, which allows accurate detection even if the sample is stored for a long time.

[Brief explanation of drawings]

Figure 1 is a graph comparing the hemoglobin measurement results when lytic enzyme was added to the same stool and when it was not added. Figure 2 shows the results of comparing the temporal changes in the amount of hemoglobin in the stool. It is a graph.

Claims (1)

[Scope of Claims] 1. A method for detecting hemoglobin in feces, which comprises adding or allowing a glycosidase-type lytic enzyme to act on collected feces and detecting hemoglobin by an immunoassay method. Detection method 2 Method for detecting hemoglobin in feces according to claim 1, characterized in that lysozyme is used as a glycosidase-type bacteriolytic enzyme. The method according to claim 1 or 2, wherein the sample is inserted into a gel containing a lytic enzyme, the sample is preserved for a certain period of time, and then hemoglobin is detected by immunoassay. How to detect hemoglobin.