JPS63315950A - Method for testing occult blood of feces - Google Patents

Abstract

PURPOSE:To exactly detect the occult blood of feces with high sensitivity by determining the human albumin contained in excrements for inspection by an enzyme immune adsorption measurement method. CONSTITUTION:A diluted anti-human albumin antibody soln. is put into the pores of a polystyrene microplate to sensitize the plate, then the plate is cleaned. Filter paper coated with the excrements for inspection is put together with a physiological salt soln. into the pores sensitized by the anti-albumin antibody soln. to effect the reaction. A diethanol amine liquid contg. nitrophenyl phosphate as a substrate is put into the same to effect reaction. Since the color forming condition varies according to the content of the human albumin of the filter paper, the color forming condition is measured by microplate photometry. The content of the human albumin in the exrements for inspection is determined by comparing the result of the measurement with the result of the case in which a standard albumin soln. is used.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel fecal occult blood test method in which human albumin in feces is measured with high sensitivity by enzyme immunosorbent assay in order to detect colorectal cancer at an early stage.

1st part 4 Generally for detecting human gastrointestinal bleeding.

A fecal test must be performed to detect specific blood-derived substances in the feces. Hemoglobin, which is most abundantly contained in blood, is used as the above-mentioned specific substance. The conventional fecal occult blood test is a test that utilizes the pseudoperoxidase activity of hemoglobin. Generally, a test method using guaiac is performed. Since hemoglobin is the most abundant protein in the blood, it has the advantage of being suitable for detection.1 Normally, a fecal occult blood test refers to the detection of hemoglobin.

However,
In the conventional fecal occult blood test that detects gastrointestinal bleeding using the pseudoperoxidase activity of hemoglobin, peroxidase and catalase contained in meat, fish, fruits, and raw vegetables also react sensitively. It is not specific for hemoglobin alone, has the problem of many false positives, and requires strict dietary restrictions before testing. Furthermore, since conventional fecal occult blood tests are affected by various substances, it is necessary to interrupt other treatments. Furthermore, in order to obtain a positive fecal occult blood test rate of 80 to 90%, it is necessary to have blood loss of 201 ffIIQ or more per day, and the sensitivity is low.

Therefore, in order to overcome the drawbacks of the conventional fecal occult blood test method, the present invention proposes a new enzyme-linked immunosorbent assay method that can specifically measure human albumin, which is not normally present in feces, with high sensitivity. The purpose of this study is to provide a method for testing fecal occult blood.

In order to achieve the above-mentioned object, the present invention involves placing an anti-human albumin antibody solution diluted with a diluent into a predetermined number of holes formed in a microplate, leaving it to stand for a predetermined period of time, and performing the above-mentioned number of holes. After washing the hole, insert a filter paper coated with test feces together with 0.2 mQ of phosphate buffered saline to allow antigen-antibody reaction at room temperature for a predetermined time, and then close the hole. After washing, add alkaline forsterase labeling-anti-human albumin antibody solution into the holes and allow to react. After washing each hole, add enzyme substrate and allow to react, and determine the color value using an absorbance tester. The content of human albumin in the test feces was compared with the measured value when an absorbance test similar to the above was conducted using a standard sample prepared in advance to contain a predetermined amount of human albumin. Provided is a fecal occult blood test method characterized by calculating fecal occult blood.

According to the present invention having the above-mentioned configuration, since the amount of absorption into the filter paper differs depending on the content of human albumin contained in the test stool, the coloring value also differs. Therefore, the content of human albumin is calculated from the measurement results of the absorbance test.

An embodiment of the fecal occult blood test method by calculating the content of human albumin in feces according to the present invention will be described below.

First, 0.2 mQ of an anti-human albumin antibody solution diluted with bicarbonate buffer (pH 9, 6) is poured into a predetermined number of holes formed in a polystyrene microplate, and the plate is sensitized at 4°C overnight, followed by washing. Next, a filter paper coated with feces for testing together with 0.2 mQ of phosphate-buffered saline was placed into the hole sensitized with the anti-human albumin antibody solution, and the filter paper was placed at room temperature for 4 hours.
Allow time to react. Separately, 0.2 ml of a predetermined amount of human albumin diluted with phosphate buffered physiological saline is placed in the hole as a standard. After washing, a jetanolamine solution containing nitrophenyl phosphate is added as a substrate and the reaction is continued. Then, since each filter paper exhibits a different coloring state depending on the content of human albumin, the coloring state is measured using microplate photometry. Therefore, the human albumin content in the test feces can be quantitatively determined by comparison with the results of the absorbance test of the hole containing the albumin standard source Bet 13 solution.

The detailed method of the above liquid absorption measurement method will be further explained. First, 3,125-800n was prepared using human albumin.
Five standard solutions with a concentration range of g/rnQ were prepared.

Furthermore, in order to examine species specificity based on this method, monkey, cow,
Blood from pigs, sheep, chickens, and tuna was diluted with phosphate-buffered saline to determine the albumin concentration of each species.
A solution of g/mQ to 1 mg/mQ was prepared, and the albumin reactivity of each animal was measured by the standard enzyme immunosorbent d111 method in the same manner as described above.

Mark r da! The first relationship between the albumin concentration of the solution and the adsorption rate is
As shown in the figure. That is, the albumin concentration is 3.125°1205
．． There are five levels of 50, 200, and 800, and the adsorption rates for solutions of each concentration are plotted. The graph shown in FIG. 1 is the standard curve for this method.

In order to examine species specificity based on the original characteristics, monkeys, cows, pigs,
As a result of adsorption reactions using sheep, chicken, and tuna blood, we found that when the albumin concentration was 1 mg/m Q, 2.
A 7% reactivity was observed, but no reactivity was observed with other blood.

Next, to prepare feces for standard 1 (1!), a dilution series was made using blood with a hemoglobin concentration of 15 gdl and an albumin concentration of 4 g/di, and 80 μm was added to 2 g of healthy human feces with a negative hemocalto test. The amount of blood added was 4°1.0.25.O per 100g of feces.
There were 5 levels of 0,0625 mQ. FIG. 2 is a graph showing the relationship between the blood concentration mixed in the standard feces prepared in this manner and the measured albumin concentration. As shown in the figure, it is recognized that there is a specific correlation between the concentration of blood mixed in feces and the measured albumin level. It is possible to know the albumin concentration in the test feces by comparing it with the curve 19 shown in FIG. 1 above.

Next, Table 1 shows the results of a comparison between the enzyme immunosorbent assay method according to the present invention and the conventional hemocaltox method (positive).

That is, it is clear that the albumin detection value to which the present invention is applied has higher detection sensitivity than the hemoglobin detection result using Hemocalto II rb.

Clarifying effect As explained in detail above, the enzyme immunoadsorption of human albumin in feces according to the present invention? According to the standard method, an anti-human albumin antibody solution diluted with a diluent is poured into a number of holes formed in a microplate, and a filter paper coated with test feces is placed in the holes, and the mixture is heated at room temperature. The absorbance test was conducted in the same manner as above using a standard sample prepared in advance containing a predetermined amount of human albumin by allowing an adsorption reaction for a predetermined time and measuring the color development value in each hole using an absorbance tester. Since the content of human albumin in the test stool is calculated by comparing it with the actual 211'l constant value, the following effects are brought about. That is, human albumin in feces can be detected quantitatively and with high sensitivity in order to detect bleeding in the digestive system in early detection of colon cancer and the like. In addition, unlike when detecting human hemoglobin, it does not react with peroxidase or tacalase contained in meat, fish, etc., or with various drugs, so the specificity of human albumin can be utilized, and strict dietary restrictions and drugs are not required during testing. It has the advantage of not requiring interruption of use. In addition, in the study of species specificity based on this method, a reaction was observed only when the albumin concentration in monkeys was 1 mg/mQ; There is no clinical problem because there is no possibility of its existence.

Furthermore, according to the present invention, the detection rate of colon cancer can be improved without increasing false positives, and it is also effective for screening not only for the above-mentioned colon cancer but also for tin protein-losing gastroenteropathy.

[Brief explanation of the drawing]

Figure 1 is a graph showing the relationship between the albumin concentration of the standard solution used in the present invention and the adsorption rate, and Figure 2 is a graph showing the relationship between the blood concentration mixed in feces and the measured albumin concentration. .

Claims (1)

[Claims] An anti-human albumin antibody solution diluted with a diluent is poured into a predetermined number of holes formed in a microplate and left for a predetermined period of time to adhere to the wall of each hole. Insert the coated filter paper and allow the antigen-antibody reaction to occur at room temperature for a predetermined time. Next, after washing each hole, add an alkaline foster enzyme-labeled anti-human albumin antibody solution to each hole and allow the reaction to occur. Measurement when performing the same absorbance test as above using a standard sample prepared in advance containing a predetermined amount of human albumin by adding a substrate and reacting and measuring the color value with an absorbance tester. A fecal occult blood test method, characterized in that the content of human albumin in the test feces is calculated by comparing the human albumin content with the fecal feces.