KR20040046427A - Method for detecting Helicobacter pylori in a stool sample - Google Patents

Abstract

PURPOSE: A method for detecting Helicobacter pylori in a stool sample is provided, thereby easily and accurately detecting Helicobacter pylori in the stool sample without collecting the blood sample. CONSTITUTION: The method for detecting Helicobacter pylori in a stool sample comprises the steps of: collecting the stool sample; preparing a first polyclonal anti-H. pylori antibody; contacting the stool sample with the first polyclonal anti-H. pylori antibody; preparing a second polyclonal anti-H. pylori antibody; contacting the first complex of the stool sample and the first polyclonal anti-H. pylori antibody with second polyclonal anti-H. pylori antibody; and detecting the presence of a detecting agent in the second polyclonal anti-H. pylori antibody, wherein one of the first and second polyclonal anti-H. pylori antibodies is bound to a solid carrier, and another is labeled with the detecting agent; the solid carrier is polyethylene, polystyrene, polypropylene or nitrocellulose membrane; and the detecting agent is enzyme marker, fluorescent or illuminating agent, radiation marker and coloring particles.

Description

Method for detecting Helicobacter pylori in a stool sample

The present invention relates to a non-invasive H. pylori detection method capable of detecting Helicobacter pylori from feces.

H. pylori is a Gram-negative bacterium that infects the gastric mucosa and is responsible for most of the peptic ulcer disease (PUD). Until recently, gastric ulcers and other forms of indigestion have been considered to be related to stress levels or eating habits. Recently, however, it has been confirmed that H. pylori is the cause of gastrointestinal diseases, including gastric ulcer and gastric cancer.

H. pylori is the cause of most stomach and duodenal ulcers, as well as peptic ulcer disease (PUD). The correlation between H. pylori and Peptic Ulcer Disease (PUD) was discovered and published in 1984 by Australian physicians Warren and Marshall ( Lancet I : 1311-1344). Currently, H. pylori infection is known to be the most common cause of gastritis and is believed to be the cause of gastric ulcer, duodenal ulcer, gastric adenocarcinoma and primary B-cell lymphoma.

Peptic ulcer disease has been found to be easily treatable. The cause of most peptic ulcer disease is H.pylori infection. However, H. pylori infection is not routinely diagnosed because the method of testing for H. pylori infection is not satisfactory to physicians, especially primary care physicians (e.g., conventional biopsy). Therefore, primary physicians tend to treat symptomatic patients with antisecretory agents.

Physicians need a simple, accurate and inexpensive method of diagnosing H. pylori infection so that they know when to treat the patient and when to inform the gastroenterologist. However, currently available diagnostic methods of H. pylori infection can be divided into invasive and non-invasive methods, which are not completely satisfactory.

Invasive tests require biopsy procedures using endoscopy. Tissue samples taken by the biopsy process are analyzed by culture, histology or ureidase test.

By culturing the biopsy specimens, the most reliable results of H. pylori testing can be obtained, but the success rate in the equipped experiments has been reported to be only 70-80% (Han, SW, et al. J. Clin. Microbiol.Infect.Dis. (1995), 14: 349-352). Histological examination of stained biopsy specimens may provide direct evidence of acute or chronic inflammatory mucosal cells and lesions. However, this requires a joint effort of endoscopists and pathologists (Genta, RM, et al . , Hum. Pathol . (1994), 25: 221-226). The urease activity test can detect an increase in pH by ammonia produced by the urease of H. pylori that breaks down urea into ammonia and carbon dioxide. However, high density bacteria are required, and false-negative can be caused by any factor that reduces the density of bacteria (Cutler, AF, Am. J. Med. (1996), 100: 35S-39S). .

Since the 1990s, many non-invasive tests have been developed to detect the presence of H. pylori . For example, the urea breath test is based on the urease activity of an organism that breaks down ¹³ C or ¹⁴ C labeled urea into non-radioactive ¹³ CO ₂ or radioactive ¹⁴ CO ₂ . The urea breath test is a widely recommended method to confirm eradication of H. pylori after 4 weeks of treatment.

In addition, as a serological test, there was a method of measuring the antibody titer of anti- H.pylori antibody in serum. This includes complement binding, agglutination, enzyme immunoassay (ELISA) and immunoblotting. ELISA method is a method of measuring IgG, IgM, and IgA in serum. In particular, measuring IgG is of high diagnostic value. According to this method, sensitivity is 72-100% and specificity is 8.8-53%, but the sensitivity and specificity are 94.9% and 94.2% in children. Thus, this method is helpful for epidemiological investigations, public screening and pediatric examinations. However, according to this method, there is a problem that blood must be collected and the specificity is low. In addition, no method of detecting H. pylori has been reported in the conventional method using the feces of the patient as a sample.

It is an object of the present invention to provide a method for detecting H. pylori from fecal samples.

The present invention comprises the steps of providing a fecal sample;

Providing an anti- H.pylori first antibody that is a polyclonal antibody purified by an affinity column;

Contacting the fecal sample with the first antibody;

Providing an anti- H.pylori second antibody that is a polyclonal antibody purified by an affinity column;

Contacting the first complex with the second antibody; And

Detecting the presence of H. pylori in the second complex by detecting the presence of a detector, wherein one of the first and second antibodies is bound to a solid carrier and the other is labeled with a detector Provided are methods for detecting H. pylori antibodies in fecal samples.

Such solid carriers include, for example, polyethylene, polystyrene, polypropylene or nitrocellulose membranes.

The detection agent includes at least one selected from the group consisting of, for example, enzyme markers, fluorescent or luminescent agents, radiolabels and chromophores. Such enzyme markers include alkaline phosphatase or horseradish peroxidase. The fluorescent or light emitting agent includes at least one selected from the group consisting of fluororesin, rhodamine, europium, luminol and acridium. The colored particles include one or more selected from the group consisting of gold, silver, blue latex and selenium.

In the present invention, the first antibody may be bound to the solid carrier, and the second antibody may be labeled with gold.

Fecal samples used in the present invention can be prepared as follows. Stool samples should be taken in clean, dry containers that do not contain media, preservatives, animal serum, or other surfactants. The feces should be kept as fresh as possible, and if not tested immediately after collection, store for 3 days in a refrigerator at 2-8 ℃, and store at -20 ℃ if long-term storage is required. Also, avoid repeated freezing and freezing of samples.

In addition, the anti- H.pylori first antibody and the second antibody used in the present invention is preferably a polyclonal antibody purified by an affinity column. The anti- H.pylori antibody used in the present invention can be produced by a conventional manufacturing process of producing by inducing the injection of H.pylori antigen into the animal. For example, rabbits are immunized with 0.5-1.0 mg of H. pylori antigen in complete Freund's adjuvant through the muscle and boosted every 0.5 weeks with 0.5-1.0 mg of the same antigen in incomplete Freund's adjuvant. After the third boost, test blood is drawn for analysis. When the antibody titer reaches the desired level of about 10 ⁶ , production blood is drawn. The antibody can be separated by conventional antibody separation methods such as ion chromatography, salting out and affinity column chromatography. Preferably, the antibody is separated by affinity column chromatography, and the antibody may be eluted with potassium thiocyanate (KSCN) in water.

The antibody thus separated may be conjugated with a detection agent using conventional conjugation methods. For example, the antibody can be conjugated with the enzyme horseradishperoxidase. The antibody-horseradishperoxidase particle conjugate can be prepared using the method of Nakane (Nakane et al., 1978: p215-220, Elsivier / North-Holland Biomedical Press, Amsterdam).

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

1. Reagents Used

The reagents and instruments used in this example were as follows.

Plates with the anti- H.pylori antibody attached to a solid substrate were used. The plates were 96 wells / plate and adsorbed 100 ng of purified rabbit anti- H.pylori antibody in a solution containing carbonate buffer and Tween 20 per plate.

As the second antibody labeled with the detection agent, a conjugate was used. The conjugate was a purified rabbit anti-H.pylori antibody-horseradish peroxidase. In 10 ml of the conjugate solution, an appropriate amount of bovine serum albumin as a protein stabilizer and Proclin 30 as a preservative 0.05v / v% It includes.

The positive control was an extract of inactivated H. pylori strain ATCC 43504. The positive control contains 0.05v / v% of Proclin 30 as a preservative.

As a negative control, 10 mM phosphate buffer (pH 7.2) was used. The negative control contains 0.05v / v% of Proclin 30 as a preservative.

10 mM phosphate buffer (pH 7.2) was used as the sample dilution. This diluent contains 0.05v / v% of Proclin 30 as a preservative.

200 mM phosphate buffer (pH 6.9) was used as the concentrated washing solution. As the substrate solution, 10 ml of tetramethylbenzidine (TMB) and 30% hydrogen peroxide 0.02v / v% in citric acid-phosphate buffer solution were used. 1.6 N sulfuric acid was used as a reaction terminating liquid.

2. Detection of H. pylori in feces

1 ml of the sample dilution was added to the centrifuge tube, and the sample derived from the well with a diarrheal disease of about 5 to 6 mm in diameter was placed in the sample dilution using a sample collecting wooden rod. Do not test with water-containing fecal samples or diarrhea. The lid was closed and vigorously mixed for 20 seconds to mix. Centrifugation was performed at 3,000-4,000 rpm for about 2 minutes. The supernatant was carefully sucked up using a dropper to drop 3 to 5 drops (about 120 μl to 200 μl) into the wells of the antibody adsorption plate. 100 μl of purified rabbit anti- H.pylori antibody-horseradishperoxidase conjugate solution was added. The reaction was carried out at room temperature for 15 minutes to show a reaction line. After washing four times with PBS, 100 μl of tetramethylbenzidine (TMB) substrate was added to each well and developed. Color intensity in each well was measured at OD 450/650 nm using a microwell reader.

As a result, H. pylori was detected in 10 of the positive control and 90 fecal samples.

According to the method of the present invention, H. pylori in fecal samples can be detected with high specificity.

Claims (4)

Providing a fecal sample; Providing an anti- H.pylori first antibody that is a polyclonal antibody; Contacting the fecal sample with the first antibody; Providing an anti- H.pylori second antibody that is a polyclonal antibody; Contacting the first complex with the second antibody; And Detecting the presence of H. pylori in the second complex by detecting the presence of a detector, wherein one of the first and second antibodies is bound to a solid carrier and the other is labeled with a detector A method for detecting an H. pylori antibody in a fecal sample. The method of claim 1 wherein the solid carrier is a polyethylene, polystyrene, polypropylene or nitrocellulose membrane. The method of claim 1, wherein the detection agent comprises at least one selected from the group consisting of an enzyme marker, a fluorescent or luminescent agent, a radiolabel, and a chromophoric particle. The method of claim 1, wherein said first antibody is bound to said solid carrier and said second antibody is labeled with horseradish peroxidase.