KR980009467A - Antibody diagnostic composition for Helicobacter pylori - Google Patents

Abstract

The present invention relates to a diagnostic reagent for infection of Helicobacter pylori (H. pylori), which comprises a protein (A545) consisting of 545 amino acids of N-terminal of a cytotoxin associated gene A (CagA) of H. pylori, (Ub-A545) linked to ubiquitin can be used for the diagnosis of H. pylori-related diseases.

Description

Antibody diagnostic composition for Helicobacter pylori

The present invention relates to an antibody diagnostic reagent for Helicobacter pylori (H. pylori), and more particularly to a cytotoxin associated gene A (hereinafter, referred to as " H. pylori " (Hereinafter referred to as "Ub-A545") in which a protein (hereinafter referred to as "A545") consisting of 545 amino acids from the N-terminus of a CagA protein is linked to Ubiquitin The present invention relates to a composition for diagnosing antibodies against H. pylori that diagnoses whether H. pylori is infected in the stomach and duodenum by measuring an antibody against H. pylori present in human serum.

H. pylori is a microaerophilic gram-negative spiral bacterium initially isolated from a biopsy of a duodenal ulcer patient (Warren and Marshall, Lancet, 1, 1273-1275 (1983)), originally referred to as Campylobacter pilolidis , H. pylori (H. pylori), and H. pylori (H. pylori) were newly identified as Helicobacter pylori (Goodwin et al., Int. J. Syst. Bacterial, 39, 397-405 In the gastric mucosa of the gastrointestinal tract, once it starts to live, it becomes parasitic throughout its lifetime and is found in the gastric mucosa of patients with various stomach and duodenal diseases. According to recent reports, H. pylori is found in gastritis, duodenum (Alper J., et al., Science, 260, 159-160 (1993)) have also been reported to cause gastric cancer (Parsonnet, et al., New Eng. J. Med., 325, 1127-113 4 (1991); The Eurogast Study Group, Lancet, 341, 1359-1362 (1993)).

In 1994, the consensus conference organized by the National Institute of Health (USA) defined stomach and duodenal ulcer as a disease caused by H. pylori and eradication of H. pylori by using antibiotics instead of existing gastric acid therapy (NIH, NIH concensus development conference, Helicobacter pylori in peptic ulcer disease (1994)). It has been reported that various antibiotic therapies currently being practiced have an effect of 90% or more on eradication of H. pylori and have an extremely low recurrence rate, which is a problem in the treatment of gastric and duodenal ulcers Company, 1359, 22-23).

The most basic method for eradicating H. pylori is the accurate diagnosis. Currently, there is a method of infecting the organism in the body and a method of not infiltrating the organ. Examples of invasive methods for inserting a device into the body include a method of histologically identifying the presence of bacteria by biopsy of the stomach tissue after endoscopic examination, a method of detecting the activity of urease enzyme in the tissue after biopsy (Korean Patent Publication No. 94-6322 And a method of culturing bacteria after biopsy. In a non-invasive method, there is a method of measuring the activity of urease enzyme indirectly by taking urea which is labeled with ¹³ C or ¹⁴ C. However, these methods are time-consuming, difficult to perform many tests at the same time, and have a disadvantage that it is difficult to perform repeated tests after treatment.

Because of the above-mentioned problems, a method of diagnosing H. pylori infection using serum is preferred. This is because of the presence of antibodies formed in the serum of a person infected with H. pylori using a specific antigen of H. pylori infection . However, most of the currently used antigens are cell extracts of H. pylori, or partially purified mixed proteins, which can be useful in advanced countries with low infection rates. However, in developed or developing countries with infection rates of up to 90% It is almost impossible to isolate the infected person using the sample.

H. pylori can be divided into 'H. pylori', which can express toxins and related proteins, and 'H. pylori,' which does not express these proteins. In patients with duodenal ulcer, only the first type of H. pylori is detected (Telford, JL et al., Trends in Biotechnology, 12, 420-426 (1994)). Therefore, in countries where H. pylori infection is prevalent like in Korea, it is more desirable to develop a diagnostic method for screening only H. pylori-infected persons who are highly likely to develop gastric and duodenal ulcers among H. pylori infected persons .

Therefore, the present inventors have continued to develop a diagnostic reagent capable of diagnosing the infection of the first type H. pylori. As a result, it has been found that a part of CagA (A545) among the proteins expressed in the first type H. pylori A serologic study to determine the presence of H. pylori infection when diagnosing antibodies to H. pylori using recombinant Ub-A545 fusion protein (Korean Patent Application No. 95-5096) linked to ubiquitin as a specific antigen So that the present invention has been completed.

It is an object of the present invention to provide a composition for diagnosing antibodies against H. pylori that can be used for diagnosing H. pylori infection which is mainly found in gastric and duodenal ulcer patients.

According to the above object, in the present invention, a fusion protein comprising a 545 amino acid N-terminal amino acid (A545) of Helicobacter pylori (cytotoxin associated gene A (CagA) (Ub-A545). ≪ / RTI >

1 shows the amino acid sequence of a fusion protein (UB-A545) obtained by fusing a portion (A545) of a toxin-related protein (CagA) of Helicobacter pylori with ubiquitin.

Figure 2 shows the results of SDS-PAGE of the unconjugated protein (UB-A545) obtained by fusing a portion (A545) of the toxin-related protein (CagA) of H. pylori with ubiquitin.

Figure 3 shows the results of a combination of Ub-A545, a fusion protein of ubiquitin with a portion of A645 (A545) of H. pylori toxin-related protein (CagA) The results of western blotting are shown.

Figure 4 shows the detection of antibodies to H. pylori in Korean sera by using a diagnostic composition containing the uni-protein (UB-A545), which is a fusion of ubiquitin with a part of the toxin-related protein (CagA) of H. pylori The results are shown.

1 shows the amino acid sequence of a fusion protein (UB-A545) obtained by fusing a portion (A545) of a toxin-related protein (CagA) of H. pylori with ubiquitin.

In the present invention, the Ub-A545 fusion protein as described above is adsorbed to each well of a 96-well ELISA plate as a solid support, and the H. pylori antibody in the sample serum is reacted in the following steps Black diagnosis.

First, sample serum is diluted in each well using a sample dilution, and reacted at the appropriate time and temperature. In this case, when an antibody against H. pylori is present in the serum of the sample, an antigen-antibody complex is formed, but when no antibody against H. pylori is present, an antigen-antibody complex will not be formed.

Second, anti-human IgG-HRP, which is labeled with horseradish peroxidase (HRP), is added to each well to specifically bind to the antibody region of the antigen-antibody complex formed in the above step . In this case, when the antigen-antibody complex is not formed in the above step, the antigen-antibody-anti-human IgG-HRP complex is not formed, so that the HRP conjugated secondary antibody as the chromogenic enzyme is removed in the next washing step.

Third, a coloring solution composed of O-phenylenediamine hydrochloride (OPD) and hydrogen peroxide is added. At this time, when the antigen-antibody-anti-human IgG-HRP complex is formed, a color reaction occurs between peroxidase and -O-phenylenediamine hydrochloride and appears orange. Then, diluted sulfuric acid was added to terminate the chromogenic reaction, and the chromaticity of the final color reaction was measured with a Microwell reader to determine the presence of H. pylori antibody in the sample serum.

In diagnosing H. pylori antibodies in a sample according to the above-described known three-step method, a solid support for antigen attachment can be used. As the solid support, a polystyrene 96-well ELISA plate, a polystyrene bead or a nitrocellulose strip Is preferably used.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1

Western blotting for confirmation of immunological specificity of antigen

First, Escherichia coli cells expressing Ub-A545 (Korean Patent Application No. 95-5096) were induced with IPTG (isopropyl- beta -D-thiogalactopyranoside), samples were taken with time, and 10% sodium dodecylsulfate (SDS) - < / RTI > polyacrylamide gels. FIG. 2 shows the results of SDS-PAGE of a fusion protein (UB-A545) obtained by fusing a portion (A545) of a toxin-related protein (CagA) of H. pylori with ubiquitin.

The column at the leftmost position represents a standard molecular weight labeled protein, and the 0th column shows a sample just before induction of expression by adding IPTG. In columns 1, 3, 5 and 7, 1, 3, 5, and 7 hours, respectively. Proteins separated on the gel were purified on a nitrocellulose filter (Bio-Rad Lab., Pore size 0.22 占 퐉, according to Tobin's method (Towbin, et al., Proc. Natl. Acad. Sci. USA 76, 4750 CA, USA). The filter was shaken in PBS (pH 7.0, 10 mM phosphate, 0.15M sodium chloride) containing 1% bovine serum albumin for 1 hour at room temperature to block the adsorption site of the extra protein. Serum and normal serum of the patients with duodenal ulcer confirmed by H. pylori infection were diluted with PBS (pH 7.0) containing 1% bovine serum albumin and reacted at room temperature for 2 hours. The paper was sufficiently washed with 10 mM PBS buffer (pH 7.0) containing 0.05% Tween 20 to nonspecifically remove the antibody attached to the nitrocellulose filter, and then the secondary antibody labeled with horseradish peroxidase (anti-human -IgG-HRP) and reacted at room temperature for 1 hour. After sufficiently washing with 10 mM PBS buffer (pH 7.0) containing 0.05% Tween 20, a horseradish peroxidase substrate solution (4-chloro-1-naphthol solution) To confirm the immunological specificity of the Ub-A545 protein.

Figure 3 is a graph showing the effect of a fusion protein (UB-A545) obtained by fusing a portion (A545) of the toxin-related protein (CagA) of H. pylori with ubiquitin on the serum of H. pylori infected persons (a) Respectively.

Example 2

Diagnostic reagent manufacturing and diagnostic methods

Step 1

Using the Ub-A545 fusion protein as an antigen, the solid support was coated in the following manner. That is, the Ub-A545 fusion protein was diluted with a 0.1 M NaHCO ₃ buffer solution (pH 9.6) to a concentration of 0.1 to 10 μg / ml, and 100 μl of a 96-well ELISA plate (96-well ELISA plate, Dynatech, Immulon I) And then reacted at room temperature for 16 hours or more to adsorb the antigen. The contents of each well were then removed with an inhaler and washed with phosphate buffered saline (PBS, pH 7.4) containing 0.05% (v / v) Tween 20 and resuspended in 0.1% (w / v) gelatin Lt; / RTI > buffer (pH 7.5). After each well was washed again with the washing solution, residual water in the well was removed with a moisture absorber to dry the plate on which the antigen was adsorbed.

Step 2

Using the H. pylori antigen-adsorbed microwell plate and solutions prepared in step 1 above, the H. pylori antibody can be diagnosed as follows. First, a serum sample was added to each well to prepare a sample dilution (buffer solution containing 1% (v / v) bovine serum: 10 mM Tris, pH 7.5, 1M NaCl, 0.2% Triton X-100, 0.1 mM EDTA, 0.02% Diluted 1: 10 to 1: 100, and added at 100 each at 37 째 C for 1 hour to prepare an antigen-antibody complex.

Each well was washed 5 times with PBS (pH 7.4) containing 0.05% (v / v) Tween-20 and incubated with anti-human IgG-HRP labeled with horseradish peroxidase (10% (v / v) bovine serum-containing buffer solution: 10 mM Tris pH 7.5, 0.5 M NaCl, 0.02% thimerosal) at 37 ° C for 1 hour. The cells were washed five times with PBS (pH 7.4) containing 0.05% (v / v) Tween 20, and 100 μl of O-phenylenediamine hydrochloride (OPD) solution was added thereto. The cells were developed at room temperature for 30 minutes, The reaction was stopped by adding each. At this time, the absorbance at 492 nm is measured for the degree of color development.

Example 3

Identification of H. pylori antibodies from Korean sera

Two clinical serologic samples (P1, P2, P3 and P4) and clinically normal H. pylori negative serum samples (clinically diagnosed as duodenal ulcer and H. pylori positive in Seoul National University Hospital) N1 and N2), and H. pylori antibody was assayed according to the diagnostic method of (Step 2) of Example 2, and the results are shown in the following table.

In addition, H. pylori antibodies were tested using serum samples from 23 children with hepatitis C and 78 samples from normal adults. Figure 4 shows the detection of antibodies to H. pylori in Korean sera by using a diagnostic composition containing the uni-protein (UB-A545), which is a fusion of ubiquitin with a part of the toxin-related protein (CagA) of H. pylori The results are shown. Herein, FIG. 4A shows the case of a child and FIG. 4B shows a case of a general adult. More than 60% to 70% of Korean general adults are infected with H. pylori, and more than 80% Respectively.

By using the composition for the diagnosis of H. pylori according to the present invention, it is possible to reduce the mortality due to gastric cancer if the gastric and duodenal ulcers are treated as needed while continuing diagnosis from the time of childhood. It is expected to contribute greatly to the public health.

Claims (3)

A fusion protein (Ub-A545) in which ubiquitin is linked to a protein (A545) composed of N-terminal 545 amino acids of a cytotoxin associated gene A (CagA) of Helicobacter pylori (H. pylori) Antibody diagnostic kits for H. pylori. A solid support with ub-A545 antigen attached to a ubiquitin-linked protein (A545) consisting of N-terminal 545 amino acids of the cytotoxin associated gene A (CagA) of Helicobacter pylori Wherein the antibody is an antibody to H. pylori. The diagnostic kit according to claim 2, wherein the solid support is a polystyrene 96-well ELISA plate, a polystyrene bead or a nitrocellulose strip. ※ Note: It is disclosed by the contents of the first application.