JP2014048074A - Artificial stool composition having good addition recovery ratio - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an artificial stool composition which allows hemoglobin or transferrin to be measured to fully diffuse and dissolve into a stool dilution from a matrix without being trapped.SOLUTION: By adding gelatin, collagen, degradation products thereof, or the like to an artificial stool as an absorption inhibitor for powder, such as rice powder, used as a base material of an artificial stool, added hemoglobin or transferrin can be fully eluted into a dilution, which leads to more accurate measurements.

Description

  The present invention relates to a composition of artificial artificial stool used for surveys and the like in the field of colorectal cancer screening, and a method for producing the same.

Fecal hemoglobin and fecal transferrin are important screening tests for colorectal cancer. However, this important medical examination item has not been standardized yet, and it is almost unknown what the measurement value of each facility is. Surveillance (hereinafter referred to as “survey”) is not actually conducted. One reason for this was that the stability of hemoglobin and transferrin in actual stool samples was extremely poor, and it was virtually impossible to distribute and store actual stool samples for surveys. Therefore, for stability, instead of using a fecal sample, a solution containing rice flour, wheat flour, potato starch, corn flour, soy protein, etc. as the main component, hemoglobin or transferrin and its stabilizer is used. An application has been filed for a technique to solve the problem by adding artificial stool and using stable stool for distribution and storage.
However, stabilization of hemoglobin or transferrin with artificial stool does not enable standardization by itself. A reagent for measuring actual hemoglobin or transferrin and a stool collection device for collecting stool are also a major factor in standardization. FIG. 1 shows a method of using a stool collector that collects stool currently in use. The collected stool enters a dilution container in which a diluent is enclosed, and is dispersed therein, and hemoglobin or transferrin is dissolved out of the stool.
Specifically, it is collected with a stool collection instrument, inserted into a dilution container containing a diluent attached to the stool collection instrument, and pseudo stool is dispersed, dissolved, and diluted, and hemoglobin or transferrin as a measurement item is added. In the case of conventional stool, a part of hemoglobin or transferrin is captured in the matrix of insoluble stool material powder and is not recovered in a 100% diluted solution, so that hemoglobin or transferrin can be measured accurately. I didn't mean. Furthermore, since the stool collection apparatus of each measuring reagent manufacturer was different, and the dilutions were also different, the amounts of hemoglobin or transferrin supplemented in the matrix were also different. That is, hemoglobin or transferrin to be measured was reduced by matrix supplementation, and 100% could not be measured accurately. In this case, the survey conducted to contain a certain amount of hemoglobin or transferrin in simulated stool and to unify the measured values to that amount is incomplete in such simulated stool, which is an important colorectal cancer screening item stool It interfered with the standardization of medium hemoglobin or transferrin measurements.

Conventionally, many attempts have been made to improve the stability of hemoglobin or transferrin due to its instability. No attempt has been made to incorporate 100% into the reagent system to be measured, that is, to make the addition recovery rate 100%. Specifically, we have not studied pseudo stool composition or diluting solution from the aspect of adding hemoglobin or transferrin to the powder matrix and dispersing and dissolving it all in the diluting solution. There are no known examples of what you have.
In such a situation, it was impossible to collect survey data including the entire company with different toilet collection devices.

Japanese Patent Laid-Open No. 11-242027 JP 2003-185654 A Japanese Patent Application No. 2012-185962

Therapeutic Res. vol. 15 suppl. 2 18-23; 1994

There has been a demand for a pseudo stool composition in which hemoglobin or transferrin is dispersed and dissolved in a 100% diluent from a pseudo stool matrix.

As a result of various studies, the inventor unexpectedly used bovine serum albumin, which is often used in enzyme immunoassays as an adsorption inhibitor, rather, adsorbs hemoglobin or transferrin on a pseudo-stool material and retains it in the matrix to increase the recovery rate. While it has been found that it has a detrimental effect, it was surprisingly found that gelatin, collagen, or a degradation product thereof maintained the effect of eluting the recovery rate in a 100% diluted solution, thus completing the present invention.

Gelatin solutions are known to form gels or flowable sols depending on the temperature, and are usually used to prevent adsorption in enzyme immunoassays because of their difficulty in handling. I did not come. In addition, the adsorption partner in the case of enzyme immunoassay is a plastic such as styrene, but in the case of artificial stool, it is a natural product mainly composed of cellulose such as rice flour. Has never been done.

For this purpose, the inventor considered a method of calculating the recovery rate by the following method as an index of the adsorption prevention effect. That is, a solution containing a known amount of hemoglobin or transferrin together with an additive for which a recovery rate is to be obtained (a hemoglobin or transferrin concentration is A) in a fixed amount (1 g) of powder that is pseudo fecal material in a test tube A suitable amount (BmL) is added to make a pseudo-stool-like paste. After standing for 1 hour, add a fixed amount (2 mL) of purified water to the test tube to elute hemoglobin or transferrin in the solution, and measure the hemoglobin or transferrin in the supernatant after centrifugation (concentration is C). Then, the recovery rate is calculated. In addition, it must be considered that the powder of the artificial stool material itself adsorbs and binds only a part of moisture in the solution, which becomes fixed moisture that cannot be re-eluted. Since this immobilized water content (referred to as D) is excluded from the solution in which hemoglobin or transferrin is dissolved, the calculation is roughly calculated by the following equation.

By conducting surveys or performing quality control using simulated stool supplemented with a certain amount of hemoglobin or transferrin prepared by the method of the present invention and the patent previously filed by the inventor (Japanese Patent Application No. 2012-185962) This makes it possible to standardize commercially available stool hemoglobin or transferrin measuring instruments and measuring reagents.

FIG. 1 illustrates a process of collecting simulated stool using one of commercially available stool collectors. A is a stool collection rod equipped in the stool collection device. B is a dilution container holding a diluent. C shows the artificial stool scraped by the entrance when the stool collection rod is inserted into the entrance of the dilution container. D is a sample collection nozzle of an analyzer such as hemoglobin. 1 is the state which removed the stool collection rod of the toilet stool. 2 shows a state in which simulated stool is collected using a stool collection stick. 3 shows a state in which a stool collection rod is inserted from the entrance of the dilution container into the inside of the dilution container. The narrowness of the entrance rubs the stool, and a certain amount of stool is dispersed and dissolved in the diluent. Show. The fake stool that remains after rubbing remains on the outside of the container. 4 shows a state in which the artificial stool is dissolved in the diluent in the dilution container. 5 shows a state where the sample collection nozzle of the analyzer is injected from behind with the container upside down.

As gelatin and collagen to be added, those mainly derived from animal tissues are suitable, but degradation products thereof can also be used. For example, it is made from cow bone, cow skin, pig bone, pig skin, fish scale, fish skin, etc., and commercially available edible gelatin may be used. In addition, those obtained by decomposing them into low molecules with an enzyme or the like can also be suitably used. As the concentration to contain these, an effect is seen at 0.01 g / L to 50 g / L, but preferably 0.1 to 10 g / L, and in view of the viscosity of the dissolved solution and the temperature at which the gel is formed. It is good to use. In general, collagen that has been decomposed into low molecules dissolves better in water.

As a conventional method for producing artificial stool, a method of adding hemoglobin or transferrin solution to powdered artificial stool material such as rice flour to obtain physical properties of stool is performed, but in the present invention, it is added. Add gelatin etc. to a solution in which hemoglobin or transferrin is dissolved to make a solution once and mix it with the powder material, and add and mix a solution in which gelatin or collagen is dissolved in a powder such as rice flour, homogenize, Thereafter, the powder is separated by filtration, and further dried. The powder is pretreated in advance, and finally mixed with a solution in which hemoglobin or transferrin is dissolved, either of which is possible. Further, the artificial stool may be prepared by adding the gelatin or collagen of the present invention or a degradation product thereof to a solution in which a third substance such as detectable glycerol shown in (Japanese Patent Application No. 2012-185962) is added. Is possible. In addition, saccharide added as a substance that stabilizes hemoglobin or transferrin, sodium azide, EDTA-Fe, N-acetylcysteine having an SH group, or the like may be added.

A treatment solution was prepared by dissolving 500 mg of cowhide-derived gelatin (manufactured by Wako Pure Chemical Industries, Ltd.), 500 mg of sucrose, and 100 mg of sodium azide in 100 mL of 50 mM MOPS buffer (pH 7.5). Next, 90 mL of the above processing solution was added to 30 g of commercially available flour (made by Tohirada Co., Ltd.), once stirred and homogenized with a spatula, allowed to stand at room temperature for 1 hour, filtered through a glass filter, and directly in a vacuum dryer. And dried under reduced pressure for 1 day.
On the other hand, instead of cowhide-derived gelatin, two types of control treatment solutions of bovine serum albumin 500 mg and cowhide-derived gelatin were prepared and processed in the same manner as above to produce a total of three types of processed top powder. did.

A solution (referred to as Solution A) in which human hemoglobin A0 (manufactured by Sigma-Aldrich) was dissolved at 0.2 mg / mL in 10 mM phosphate buffer (pH 7.5) containing 5 g / L sodium chloride was prepared. did. In each test tube, 1 g of each of the three types of treated top powder of Example 1 was taken, and 1.1 mL of Solution A was added thereto. When stirred well with a spatula, three types of paste-like stool were prepared. Simulated stool 1: No addition, Simulated stool 2: Cow skin derived gelatin added, Simulated stool 3: Bovine serum albumin added.
After standing at 5 ° C. for 1 hour, 2 mL of purified water was added to each test tube and stirred, then left at 5 ° C. for an additional hour with occasional stirring with a test tube mixer, and finally at 3000 rpm for 5 minutes in a centrifuge. The supernatant was obtained by centrifugation.
The hemoglobin concentration in the supernatant was measured by the following measuring reagent and procedure.
The measurement reagent composition of hemoglobin is as shown in Table 1.

The hemoglobin measurement procedure was performed by adding 3 mL of the measurement reagent to 50 μL of the supernatant and stirring, followed by heating at 37 ° C. for 15 minutes, and then measuring the absorbance at 666 nm. For the calculation of the hemoglobin concentration, a solution prepared by dissolving human hemoglobin A0 of Example 1 in 100 μg / mL as a standard solution of hemoglobin was prepared and compared in the same manner. In addition, B in the calculation formula was 1.1 mL, D of the upper powder was separately measured and was 0.43 mL, and the A measurement value was measured by diluting solution A three times, and the result was tripled.
The recovery rate was calculated by Equation 1 and the results are shown in Table 2.
What was added to the artificial stool gave better results than conventional albumin or no additive.

A part of the solution A of Example 2 was prepared by adding gelatin derived from bovine bone (manufactured by Wako Pure Chemical Industries, Ltd.) so as to be 5 mg / mL. 1 g of untreated top powder was taken in two test tubes, and 1.1 mL each of the A and A ′ solutions was added thereto. Two types of artificial stool were prepared by stirring well with a spatula to make it uniform.
After standing at 5 ° C. for 1 hour, 2 mL of purified water was added to each test tube and stirred, then left at 5 ° C. for an additional hour with occasional stirring with a test tube mixer, and finally at 3000 rpm for 5 minutes in a centrifuge. The supernatant was obtained by centrifugation. By operating in the same manner as in Example 2, each recovery rate was calculated. The results are shown in Table 2.
In addition, B corresponding to the calculation formula 1 in the case of Example 3 is 1.1 mL, D is separately measured, 0.43 mL, and A measurement values are obtained by diluting the solutions A and A ′ solutions three times, and the result. Was tripled.
As in the case of Example 2, a recovery rate of 98% or more was obtained when gelatin was directly used in a solution containing hemoglobin. On the other hand, when gelatin was not used, the recovery rate was about 90%.

An experiment similar to that in Example 2 was performed using Nippi Collagen 100 (manufactured by Nippi Collagen Cosmetics Co., Ltd.), which is a degradation product of gelatin, instead of the gelatin of Example 2, and almost the same results were obtained.

When a solution obtained by adding 10% glycerol (manufactured by Wako Pure Chemical Industries, Ltd.) to the solution A of Example 2 was used in place of the solution A, an experiment was performed in the same manner as in Example 3, and almost the same result was obtained. . In addition, the amount of artificial stool collected from this simulated stool could be estimated by measuring the glycerol concentration.

Since the present invention completes the situation in which hemoglobin and transferrin are not supplemented inside the artificial fecal material, the added hemoglobin and transferrin as expected can be recovered regardless of the company's stool collection system. A nationwide survey of medium hemoglobin and transferrin items will be possible and standardization will be possible.

The symbols used in FIG. 1 are as follows.
A A stool collection rod B Dilution container C A fake stool that has been scraped off and remains in the mouth D The specimen nozzle 1 of the hemoglobin measuring device is in a state in which the stool collection rod is removed.
2 shows a sample of artificial stool collected using a stool collection stick.
3 shows a stool collection rod inserted into the dilution container from the entrance of the dilution container.
4 shows the simulated stool dissolved in the diluent in the dilution container.
5 shows that the sample collection nozzle of the analyzer was injected from behind with the container upside down.

Claims (6)

A pseudo stool characterized by containing at least one selected from gelatin, collagen, and a degradation product of collagen, which is hemoglobin or transferrin prepared by infiltrating a powder with an aqueous solution. A simulated stool characterized by adding one or more selected from gelatin, collagen, and a degradation product of collagen when producing hemoglobin and / or simulated stool for measurement of transferrin prepared by infiltrating a powder with an aqueous solution Manufacturing method. The artificial stool in which the degradation product of gelatin, collagen or collagen of claim 1 is a degradation product of gelatin, collagen or collagen derived from animal tissue. A method for performing quality control of fecal hemoglobin or transferrin items using the artificial stool of claim 1 and claim 3. The artificial stool according to claim 3, wherein the animal-derived gelatin, collagen, or collagen degradation product of claim 3 is derived from cow bone or cow skin, pig bone, pig skin, fish scale, or fish bone. A method for performing quality control of fecal hemoglobin or transferrin items using the artificial stool of claim 5.

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