WO2002014541A1 - Method of examining infection with helicobacter pylori and diagnostic kit - Google Patents
Method of examining infection with helicobacter pylori and diagnostic kit Download PDFInfo
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- WO2002014541A1 WO2002014541A1 PCT/JP2001/006894 JP0106894W WO0214541A1 WO 2002014541 A1 WO2002014541 A1 WO 2002014541A1 JP 0106894 W JP0106894 W JP 0106894W WO 0214541 A1 WO0214541 A1 WO 0214541A1
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- helicobacter pylori
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56922—Campylobacter
Definitions
- the present invention relates to a test method and a diagnostic kit that can detect the presence or absence of infection with Helicobacter pylori with high sensitivity using feces as a sample.
- Helicobacter pylori is a bacterium found in human gastric mucosa.
- the prevalence of Helicobacter pylori is closely related to the social and economic status, with developing countries tending to have a higher infection rate and developed countries having a lower infection rate.
- the infection rate of Japanese people is remarkably high in developed countries, and it is said that 80 ⁇ ⁇ / 0 people are infected if they are over 40 years old.
- it has been revealed that Helicobacter pylori can cause various stomach and duodenal diseases such as gastric ulcer, duodenal ulcer, chronic gastritis, and gastric cancer.
- H. pylori can reduce the likelihood of these diseases.Helicobacter.
- duodenal ulcers, malignant lymphoma of the stomach, and stomach after early gastric cancer resection are recognized as indications for eradication.
- Helicobacter pylori eradication therapy As a new treatment for gastric and duodenal diseases, the Japanese Society of Gastroenterology has created clinical trial guidelines in Japan, and has diagnosed the presence and eradication of Helicobacter pylori infection. The judgment method was shown (Journal of the Japanese Society of Gastroenterology, Vol. 96, 199—207, 199).
- the presence diagnosis is performed by culture, microscopy, and urease test of gastric biopsy tissue, which is an invasive test method. It has been shown that a urea breath test, which is a standard test method, is required. In addition, it has been shown that in special cases, such as when the subject is a child, the determination is made using blood anti-Helicobacter pylori antibody test and presence diagnosis in combination.
- these methods for detecting Helicobacter pylori infection have the following problems. In the invasive examination method, the subject suffers a great deal of pain due to insertion of a gastroscope and biopsy. Non-invasive testing significantly improves the pain of the subject, but the urea breath test requires fasting before testing.
- the urea breath test requires devices such as a mass spectrum and an infrared spectrometer, and can be performed only in specific facilities, and has the disadvantage of increasing costs.
- Antibody tests are not suitable for judging the eradication because the antibody titer in the blood remains high for a long time after the eradication. Therefore, there is a demand for a non-invasive test method that can directly and specifically detect Helicobacter pylori infection with high accuracy in place of these tests.
- the present invention has been made in view of the above circumstances, and provides a test method and a diagnostic kit that can determine infection to Helicobacter pylori at low cost and with excellent sensitivity without causing pain to a subject and without requiring a special device.
- the purpose is to provide.
- the present invention relates to a test method for determining infection with Helicobacter pylori by measuring catalase activity of Helicobacter pylori in the digestive tract and Z or feces. It is.
- the present invention is also a diagnostic kit for judging infection with Helicobacter pylori by measuring the activity of helicobacter pylori in the digestive tract and / or feces.
- the present inventor surprisingly found that the helicobacter pylori catalase was contained in the feces of helicobacter pylori-positive persons in a naive state. And found that this would be an indicator for determining Helicobacter pylori infection, and completed the present invention.
- Helicobacter pylori catalase has a molecular weight of 200 kDa and is known to have a tetramer structure composed of four subunits having a molecular weight of 50 kDa (J. Gen. Mi crobiol (199 1), 1 37, 57 — 6 1).
- Helicobacter pylori catalase unlike other catalase, has the characteristic that the base is 1 "raw.
- Catalase occupies a percentage of the total protein in the cells of Helicobacter pylori as determined by the increase in specific activity during the catalase purification process described in J. Gen. Microbiol. (1991), 137, 57-61. Approximately, the weight conversion is at most 0.5%.
- the inventor of the present invention has investigated the ability of fecal helicobacter pylori antigen-positive and negative An attempt was made to confirm the presence or absence of tallase activity. As a result, catabolase activity was detected in the stool-derived basic fractions of all positive persons, whereas catalase activity was not detected in the stool-derived basic fractions of negative persons.
- catalase activity could be used as an indicator to determine infection with Helicobacter pylori.
- the strain of Helicobacter pylori detected by the detection method of the present invention is not particularly limited.
- genotype of Helicobacter pylori detected by the test method of the present invention is not particularly limited.
- it may or may not have VacA or cagA. May have any of the sequences 31 &, Slb, and S2, and may have any of the sequences ml and m2.
- the method of separating Helicobacter pylori catalase from contaminating catalase is not particularly limited.
- catalase of Helicobacter pylori is basic unlike other catalase, ion-exchange resin, Chemical methods such as an ion exchange separation method using an ion exchange filter paper and the like; immunological methods using a polyclonal antibody or monoclonal antibody using helicobacter pylori catalase as an antigen, and the like.
- the polyclonal antibody using helicobacter pylori catalase as an antigen is not particularly limited, and a polyclonal antibody prepared by a known method can be used.
- the antibody is sensitized using Helicobacter pylori catalase as an antigen. It can be obtained from the serum, egg yolk, milk, etc. of an animal.
- Examples of the monoclonal antibodies include those produced from hybridomas such as 21G2 (accession number FERM P-17604), 41A5 (accession number FERM P-17605), and 82B9 (accession number FERM P-17606). Can be mentioned.
- a monoclonal nanore antibody having helicobacter pylori catalase as an antigen is preferable.
- a monoclonal antibody having Helicobacter pylori catalase as an antigen it is possible to accurately capture Helicobacter pylori catalase.
- the method for measuring catalase activity is not particularly limited, and examples thereof include the following methods (1) to (3).
- a method for detecting generated oxygen using hydrogen peroxide as a substrate (hereinafter, also referred to as measurement method (1)).
- Examples of the above measurement method (1) include a method of observing the bubbling of generated oxygen, a method of measuring the degree of coloration of oxygen by a color former, and a method of measuring the amount of oxygen generated by an oxygen electrode. it can. .
- measurement method (2) A method for detecting the consumption of hydrogen peroxide using hydrogen peroxide as a substrate (hereinafter, also referred to as measurement method (2)).
- Examples of the measurement method (2) include a potassium permanganate titration method and a spectrophotometric method in which the consumption of hydrogen peroxide is measured by a change in absorbance of 240 nm.
- measurement method (3) A method utilizing the peroxidase action of catalase (hereinafter, also referred to as measurement method (3)).
- Examples of the above-mentioned measurement method (3) include a method of detecting a color generated by using hydrogen peroxide and a coloring reagent which is a hydrogen donor such as tetramethylbenzidine as a substrate.
- the sample to be subjected to the test method of the present invention is not particularly limited, and includes, for example, stomach contents, gastric lavage fluid, gastrointestinal excrement, etc., in that collection is easy and the burden on the subject is small. Digestive tract excretions such as feces are preferred.
- test method of the present invention may be used to check for the presence of infection before the eradication treatment, or may be used to determine the success or failure after the eradication treatment.
- CM-cellulose or other pretreatment ion examples thereof include an exchange resin, a substrate of catalase such as hydrogen peroxide, and a buffer composed of a buffer such as a phosphate buffer.
- the diagnostic kit having such a configuration, first, feces provided by a subject are suspended in a buffer solution, the suspension is loaded on a pretreatment ion exchange resin, and a substrate is added to the eluate. By confirming the presence or absence of oxygen generation, infection with Helicobacter pylori can be determined.
- the presence of Helicobacter pylori can be detected with extremely high sensitivity and specificity. Further, since the present invention can use gastrointestinal excrement as a sample, it is possible to easily and efficiently determine Helicobacter pylori infection without causing pain to a subject. Further, the present invention is easy to measure and is very useful in medical practice. BEST MODE FOR CARRYING OUT THE INVENTION
- Example 2 Detection of Helicobacter pylori catalase in feces using anti-Helicobacter pylori catalase yolk antibody
- the obtained cells were scraped off with a platinum loop and suspended in PBS.
- Helicobacter pylori cells centrifuged at 3000 rpm for 15 minutes at 4 ° C were suspended in 0.5% formalin and left inactivated for 4 to 1 minute to inactivate. Cells were inactivated, using centrifugation (4 ° C, 3000 r pm s 15 min), washed three times with PBS, and suspended again in PBS.
- the cell suspension was sonicated using an ultrasonic crusher (Model 17250, manufactured by Seiko Denshi Kogyo Co., Ltd.) for 30 minutes under the conditions of output 4 and 50% dtycycle to crush the cells.
- the crushed cells were subjected to ultracentrifugation (4, 30,000 rpm, 30 minutes) to obtain 4 OmL of supernatant (cell-soluble fraction) (protein concentration: 4.9 mg / mL).
- the anti-Helicobacter pylori catalase monoclonal antibody 21G2 was converted to CNB r-activated Sepharose 4B (Amersham Pharmacia Biotech).
- An immobilized column was prepared (10 mL). The column was loaded with 2 OmL of the soluble fraction of Helicobacter pylori cells (flow rate: about 4.5 mL / h). The column was washed with 60 mL of PBS (flow rate of about 30 mL Zh) and eluted with 6 OmL of 0.2 M glycine HC1 buffer (pH 3.0) (flow rate of about 30 nxL / h).
- Helicobacter pyloricatalase was mixed with an equal volume of sample buffer (2% SDS, 5% mercaptoethanol) and boiled for 5 minutes. Using 5 L of this solution, SDS-polyacrylamide gel electrophoresis (4-20% atarylamide) was performed. As the molecular weight markers, phosphorylase II>, yeast serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and ⁇ -lactalbumin were used. After the electrophoresis, the gel was stained using Sylperstin® (Kanto Chemical). Helicobacter pylori catalase was a single 59 kDa protein.
- Helicobacter pyloricatalase was mixed in equal amounts with Freund's complete adjuvant, and then injected into the laying hens (white Leghorn strain) via intramuscular injection. Immunization was performed once every two weeks, for a total of three times. Yolk antibodies were prepared from 6 immunized eggs laid 9 to 11 weeks after the first immunization. After the immunized egg was split and the yolk was removed, an equal volume of purified water was added (total volume: 156 mL). This was mixed with 2 volumes (312 mL) of a 0.15% ⁇ one-strength lugginan aqueous solution, left at room temperature for 30 minutes, and centrifuged at lOOO rpm for 15 minutes.
- Ammonium sulfate was added to the obtained supernatant to a saturation of 40%, and the mixture was stirred, followed by centrifugation to obtain a precipitate.
- the precipitate was dissolved in PBS, dialyzed against the same buffer, and the dialysate was recovered (anti-Helicobacter pylori catalase egg yolk antibody 288 mg protein).
- the reaction 14 between the anti-Helicobacter pylori catalase egg yolk antibody and Helicobacter pylori catalase 14 was confirmed by enzyme immunoassay (ELISA). After diluting Helicopter.pyrrolicatalase to 0.1 mgZmL with PBS, 0.1 mL was added to each well of a 96-well LISA plate.
- the reaction solution was collected from each well, and the dissolved oxygen was measured using a dissolved oxygen meter (manufactured by Kemet DO Model K-7512 CHEMetris).
- a dissolved oxygen meter manufactured by Kemet DO Model K-7512 CHEMetris.
- infection with Helicobacter pyloricatalase could be determined by a method for detecting the enzyme activity after capturing Helicobacter pyloricatalase in the stool sample with a specific antibody.
- Example 3 Detection of Helicobacter pylori catalase in feces using anti-Helicobacter pylori catalase monoclonal antibody
- Example 2 The same procedure as in Example 2 was carried out using stool samples of 4 helicobacter pylori positive and negative persons. As a result, in the stool specimen of the positive person, the dissolved oxygen showed a high value of 8 to 12 ppm or more, whereas in the stool specimen of the negative person, there was no difference from the control (about 1 p ⁇ m).
- gastrointestinal excretion can be used as a sample, and Helicobacter pylori infection can be detected simply and efficiently without causing pain to the subject.
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Abstract
Description
へリコパクター ·ピロリ感染の検査方法及び診断キット 技術分野 Helicobacter pylori infection testing method and diagnostic kit
本発明は、 糞便を検体とし、 感度良くへリコパクター 'ピロリへの感染の有無 を検査することができる検査方法及び診断キットに関する。 背景技術 TECHNICAL FIELD The present invention relates to a test method and a diagnostic kit that can detect the presence or absence of infection with Helicobacter pylori with high sensitivity using feces as a sample. Background art
ヘリコパクター · ピロリ （He l i c o b a c t e r p y l o r i ) はヒ ト の胃粘膜に見られる細菌である。 へリコパクター · ピロリへの感染率は、 社会経 .済状態と密接に関連しており、 発展途上国ほど感染率が高く、 先進国ほど感染率 が低くなる傾向がある。 しかしながら、 日本人の感染率は先進国の中でも際立つ て高く、 40歳以上では 80°/0の人が感染しているとも言われている。 近年、 へ リコパクター'ピロリが胃潰瘍、 十二指腸潰瘍、 慢性胃炎、 更には胃癌等のさま ざまな胃、 十二指腸疾患の原因となりうることが明らかにされてきた。 Helicobacter pylori is a bacterium found in human gastric mucosa. The prevalence of Helicobacter pylori is closely related to the social and economic status, with developing countries tending to have a higher infection rate and developed countries having a lower infection rate. However, the infection rate of Japanese people is remarkably high in developed countries, and it is said that 80 で は / 0 people are infected if they are over 40 years old. In recent years, it has been revealed that Helicobacter pylori can cause various stomach and duodenal diseases such as gastric ulcer, duodenal ulcer, chronic gastritis, and gastric cancer.
へリコパクター . ピロリを除菌することにより、 これらの疾患に罹患する可能 性を低減できることが明らかになったので.、 へリコパクター.ピロリの除菌対象 疾患について国際的な議論がなされ、 現在では胃潰瘍、 十二指腸潰瘍、 胃悪性リ ンパ腫、 早期胃癌切除後胃等が除菌の適応対象疾患として認知されている。 ヘリコパクター · ピロリの除菌療法が胃、 十二指腸疾患の新しい治療法として 認知されるのに伴い、 我が国でも日本消化器病学会により治験ガイドラインが作 成され、 へリコパクター ·ピロリ感染の存在診断と除菌判定法が示された （日本 消化器病学会雑誌、 9 6卷、 1 9 9— 20 7、 1 9 9 9年） 。 上記治験ガイドラ インでは、 存在診断は侵襲的検査法である胃部生検組織の培養、 鏡検、 ゥレアー ゼ試験にて行い、 除菌判定は胃部生検組織の培養と鏡検及び非侵襲的検查法であ る尿素呼気試験を必須とすることが示されている。 また、 被験者が小児である等 の特殊な場合、 血中抗へリコパクター ·ピロリ抗体検査と存在診断とを併用して 判定を行うことが示されている。 し力 し、 これらのヘリコパクター ·ピロリ感染の検查法には以下の問題点があ る。 侵襲的検查法は、 胃内視鏡の挿入及び生検等により被験者が多大な苦痛を強 いられることとなる。 非侵襲的検査法では、 被験者の苦痛は大幅に改善されるが 、 尿素呼気試験では、 検査前の絶食が必要である。 また、 尿素呼気試験は、 マス スぺクトルや赤外分光高度計等の装置が必要であり、 特定の施設でしか実施でき ず、 コストも高くなる欠点がある。 抗体検査は、 除菌後も血中抗体価が長期にわ たり高値であるので、 除菌判定には適さない。 従ってこれらの検査に代わる、 非 侵襲的で且つへリコパクター ·ピロリ感染を直接、 特異的に精度良く検出できる 検査方法が望まれている。 Helicopter. It has been clarified that the elimination of H. pylori can reduce the likelihood of these diseases.Helicobacter. In addition, duodenal ulcers, malignant lymphoma of the stomach, and stomach after early gastric cancer resection are recognized as indications for eradication. With the recognition of Helicobacter pylori eradication therapy as a new treatment for gastric and duodenal diseases, the Japanese Society of Gastroenterology has created clinical trial guidelines in Japan, and has diagnosed the presence and eradication of Helicobacter pylori infection. The judgment method was shown (Journal of the Japanese Society of Gastroenterology, Vol. 96, 199—207, 199). According to the above guidelines, the presence diagnosis is performed by culture, microscopy, and urease test of gastric biopsy tissue, which is an invasive test method. It has been shown that a urea breath test, which is a standard test method, is required. In addition, it has been shown that in special cases, such as when the subject is a child, the determination is made using blood anti-Helicobacter pylori antibody test and presence diagnosis in combination. However, these methods for detecting Helicobacter pylori infection have the following problems. In the invasive examination method, the subject suffers a great deal of pain due to insertion of a gastroscope and biopsy. Non-invasive testing significantly improves the pain of the subject, but the urea breath test requires fasting before testing. In addition, the urea breath test requires devices such as a mass spectrum and an infrared spectrometer, and can be performed only in specific facilities, and has the disadvantage of increasing costs. Antibody tests are not suitable for judging the eradication because the antibody titer in the blood remains high for a long time after the eradication. Therefore, there is a demand for a non-invasive test method that can directly and specifically detect Helicobacter pylori infection with high accuracy in place of these tests.
従来、 消化管感染菌の直接検査法として消化管排泄物、 特に糞便からの感染菌 の選択培地を用いた分離培養が行われてきた。 しかし、 へリコパクター ' ピロリ に関しては数多くの試みにも拘らず、 糞便から分離培養された報告はほとんどな レ、。 その理由として、 ヘリコパクター 'ピロリは i n v i t r oで、 低温、 栄 養欠乏、 酸素欠乏等のように環境条件が悪化すると、 通常のらせん状体から培養 不能な球状体へ形態変化することから、 下部消化管においても分離培養不能な球 状体に変化していることが考えられる。 Conventionally, as a direct test method for infectious bacteria in the gastrointestinal tract, separation culture using a selective medium for infectious bacteria from gastrointestinal excreta, particularly feces, has been performed. However, in spite of many attempts at helicobacter pylori, there have been few reports of isolation and culture from feces. The reason for this is that Helicobacter pylori is in vitro, and when environmental conditions such as low temperature, nutrient deficiency, and oxygen deficiency deteriorate, the morphology changes from a normal spiral to a non-cultureable spheroid, and It is also conceivable that the spheres have changed to spheres that cannot be separated and cultured.
一方、 抗原抗体反応に基づく免疫学的方法による糞便からのヘリコパクター - ピロリの直接検出に関して、 ヘリコパクター .ピロリに対するポリクローナル抗 体を用いたィムノアッセィにより糞便等のお泄物検体中のヘリコパクター ·ピロ リを検出する方法が報告されている （J. C l i n. Mi c r o b i o l . 、 3 3卷、 2162— 2165、 1 9 95年、 特開平 10— 10128号公報） 。 発明の要約 On the other hand, regarding the direct detection of Helicobacter pylori from feces by an immunological method based on an antigen-antibody reaction, helicobacter pylori was detected in feces and other faecal samples by immunoassay using polyclonal antibodies against Helicobacter pylori. (J. Clin. Microbiol., Vol. 33, 2162-2165, 1995, JP-A-10-10128). Summary of the Invention
本発明は、 上記現状に鑑み、 被験者に苦痛を与えず、 特別の装置を必要とせず に、 安価に、 優れた感度でヘリコパクター ·ピロリへの感染を判定することがで きる検査方法及び診断キットを提供することを目的とするものである。 The present invention has been made in view of the above circumstances, and provides a test method and a diagnostic kit that can determine infection to Helicobacter pylori at low cost and with excellent sensitivity without causing pain to a subject and without requiring a special device. The purpose is to provide.
本発明は、 消化管中及ぴ Z又は糞便中のへリコパクター ' ピロリのカタラーゼ 活性を測定することによりヘリコパクター ·ピロリへの感染を判定する検査方法 である。 The present invention relates to a test method for determining infection with Helicobacter pylori by measuring catalase activity of Helicobacter pylori in the digestive tract and Z or feces. It is.
本発明はまた、 消化管中及び/又は糞便中のヘリコパクター .ピロリの力タラ ーゼ活性を測定することによりヘリコパクター ·ピロリへの感染を判定する診断 キットである。 発明の詳細な開示 The present invention is also a diagnostic kit for judging infection with Helicobacter pylori by measuring the activity of helicobacter pylori in the digestive tract and / or feces. Detailed Disclosure of the Invention
以下に本発明を詳述する。 Hereinafter, the present invention will be described in detail.
本発明者は、 上記課題を解決するために鋭意検討を重ねた結果、 驚くべきこと にへリコパクター .ピロリ陽性者の糞便中にはへリコパクター .ピロリのカタラ ーゼが Na t i v eな状態で含まれていることを見出し、 これがへリコパクター •ピロリ感染の判断の指標になることに想到し、 本発明の完成に至った。 As a result of intensive studies to solve the above problems, the present inventor surprisingly found that the helicobacter pylori catalase was contained in the feces of helicobacter pylori-positive persons in a naive state. And found that this would be an indicator for determining Helicobacter pylori infection, and completed the present invention.
ヘリコパクター ·ピロリのカタラーゼは 200 kD aの分子量を有し、 50 k D aの分子量を有するサブュニットが 4個集合した 4量体の構造を有することが 知られている （J. Ge n. Mi c r o b i o l . (199 1) , 1 37, 57 — 6 1) 。 へリコパクター .ピロリのカタラーゼは他のカタラーゼと異なり、 塩 基 1"生であるという特徴を有する。 Helicobacter pylori catalase has a molecular weight of 200 kDa and is known to have a tetramer structure composed of four subunits having a molecular weight of 50 kDa (J. Gen. Mi crobiol (199 1), 1 37, 57 — 6 1). Helicobacter pylori catalase, unlike other catalase, has the characteristic that the base is 1 "raw.
ヘリコパクター ·ピロリの菌体中の総タンパク質のうちカタラーゼの占める割 合は、 J. Ge n. Mi c r o b i o l . (199 1) , 137, 57— 61に 記載されたカタラーゼの精製過程における比活性の上昇度から概算して、 重量換 算でたかだか 0. 5%である。 Catalase occupies a percentage of the total protein in the cells of Helicobacter pylori as determined by the increase in specific activity during the catalase purification process described in J. Gen. Microbiol. (1991), 137, 57-61. Approximately, the weight conversion is at most 0.5%.
糞便中に存在するヘリコパクター · ピロリのカタラーゼがサブュニットごとに 解離したものや消化されたものではなく、 4個のサブユニットを保有する N a t i v eなものであるということは今まで全く知られていない。 通常タンパク質は 消化管中でタンパク質分解酵素により分解されてしまうことに鑑みると、 へリコ パクター 'ピロリのカタラーゼが消化管内で消化されずに、 活性を有する N a t i v eな酵素として排出され、 糞便中に存在していたということは全く予想でき ないことであった。 It has never been known that the catalase of Helicobacter pylori present in feces is not a dissociated or digested one for each subunit, but a Nat ive with four subunits. In view of the fact that proteins are usually degraded in the digestive tract by proteolytic enzymes, the catalase of Helicobacter pylori is not digested in the digestive tract, but is excreted as an active native enzyme, which is present in feces. Its existence was completely unpredictable.
本発明者は、 糞便中へリコパクター .ピロリ抗原陽性及び陰性の被験者の、 力 タラーゼ活性の有無の確認を試みた。 その結果、 全ての陽性者の糞便由来塩基性 画分に力タラーゼ活性を検出することができた一方、 陰性者の糞便由来塩基性画 分には、 カタラーゼ活性は検出されなかった。 The inventor of the present invention has investigated the ability of fecal helicobacter pylori antigen-positive and negative An attempt was made to confirm the presence or absence of tallase activity. As a result, catabolase activity was detected in the stool-derived basic fractions of all positive persons, whereas catalase activity was not detected in the stool-derived basic fractions of negative persons.
この結果より、 カタラーゼ活性を指標としてヘリコパクター .ピロリへの感染 を判定できることが明らかとなった。 These results revealed that catalase activity could be used as an indicator to determine infection with Helicobacter pylori.
本発明の検查方法で検出するヘリコパクター · ピロリの菌株種としては特に限 定されない。 The strain of Helicobacter pylori detected by the detection method of the present invention is not particularly limited.
また、 本発明の検査方法で検出するヘリコパクター ·ピロリの遺伝子型として も特に限定されず、 例えば、 V a c Aや c a g Aを有していても有していなくて もよく、 更に、 &。 が31 &、 S l b、 S 2のいずれの配列を有していても よく、 ml、 m 2のどちらの配列を有していてもよい。 In addition, the genotype of Helicobacter pylori detected by the test method of the present invention is not particularly limited. For example, it may or may not have VacA or cagA. May have any of the sequences 31 &, Slb, and S2, and may have any of the sequences ml and m2.
消化管及び糞便中にはヘリコパクター · ピロリのカタラーゼ以外の他の力タラ ーゼも存在する。 このため、 本発明の検査方法では、 これらの影響を排除するた めに、 検体中のへリコパクター 'ピロリのカタラーゼを夾雑カタラーゼから分離 した後、 カタラーゼ活性を測定することが好ましい。 In addition to the Helicobacter pylori catalase, there are other power tarases in the digestive tract and feces. Therefore, in the test method of the present invention, in order to eliminate these effects, it is preferable to measure the catalase activity after separating catalase from Helicobacter pylori in the sample from contaminating catalase.
夾雑カタラーゼからヘリコパクター .ピロリのカタラーゼを分離する方法とし ては特に限定されず、 例えば、 へリコパクター ·ピロリのカタラーゼは他のカタ ラーゼと異なり塩基性であるということを利用して、 イオン交換樹脂、 イオン交 換ろ紙等を用いるイオン交換分離法等の化学的方法；ヘリコパクター · ピロリの カタラーゼを抗原とするポリクローナル抗体やモノクローナル抗体を用いる免疫 学的方法等を挙げることができる。 The method of separating Helicobacter pylori catalase from contaminating catalase is not particularly limited.For example, utilizing the fact that catalase of Helicobacter pylori is basic unlike other catalase, ion-exchange resin, Chemical methods such as an ion exchange separation method using an ion exchange filter paper and the like; immunological methods using a polyclonal antibody or monoclonal antibody using helicobacter pylori catalase as an antigen, and the like.
上記ヘリコパクター ·ピロリのカタラーゼを抗原とするポリクローナル抗体と しては特に限定されず、 公知の方法により作成されたものを用いることができ、 例えば、 へリコパクター ' ピロリのカタラーゼを抗原とし、 感作された動物の血 清、 卵黄、 乳等から得ることができる。 The polyclonal antibody using helicobacter pylori catalase as an antigen is not particularly limited, and a polyclonal antibody prepared by a known method can be used.For example, the antibody is sensitized using Helicobacter pylori catalase as an antigen. It can be obtained from the serum, egg yolk, milk, etc. of an animal.
上記モノクローナル抗体としては、 例えば、 21G2 (受託番号 FERM P - 17604) 、 41A5 (受託番号 FERM P— 17605) 、 82 B 9 ( 受託番号 FERM P- 17606) 等のハイプリ ドーマから産生されるものを 挙げることができる。 Examples of the monoclonal antibodies include those produced from hybridomas such as 21G2 (accession number FERM P-17604), 41A5 (accession number FERM P-17605), and 82B9 (accession number FERM P-17606). Can be mentioned.
上記免疫学的方法に用いる抗体としては、 へリコパクター ·ピロリのカタラー ゼを抗原とするモノクローナノレ抗体が好ましい。 ヘリコパクター ·ピロリのカタ ラーゼを抗原とするモノクローナル抗体を用いることにより、 ヘリコパクター · ピロリのカタラーゼを精度良く捕捉することができる。 As the antibody to be used in the above immunological method, a monoclonal nanore antibody having helicobacter pylori catalase as an antigen is preferable. By using a monoclonal antibody having Helicobacter pylori catalase as an antigen, it is possible to accurately capture Helicobacter pylori catalase.
本発明において、 カタラーゼ活性を測定する方法としては特に限定されず、 例 えば、 以下の （1 ) 〜 （3 ) の方法等を挙げることができる。 In the present invention, the method for measuring catalase activity is not particularly limited, and examples thereof include the following methods (1) to (3).
( 1 ) 過酸化水素を基質として、 生ずる酸素を検出する方法 （以下、 測定方法 （ 1 ) ともいう） 。 (1) A method for detecting generated oxygen using hydrogen peroxide as a substrate (hereinafter, also referred to as measurement method (1)).
上記測定方法 （1 ) としては、 例えば、 生ずる酸素の発泡を観察する方法、 酸 素の発色剤による呈色度合いを測定する方法、 酸素電極により生ずる酸素の量を 測定する方法等を挙げることができる。 . Examples of the above measurement method (1) include a method of observing the bubbling of generated oxygen, a method of measuring the degree of coloration of oxygen by a color former, and a method of measuring the amount of oxygen generated by an oxygen electrode. it can. .
( 2 ) 過酸化水素を基質として、 過酸化水素の消費を検出する方法 （以下、 測定 方法 （2 ) ともレヽう） 。 (2) A method for detecting the consumption of hydrogen peroxide using hydrogen peroxide as a substrate (hereinafter, also referred to as measurement method (2)).
上記測定方法 （2 ) としては、 例えば、 過マンガン酸カリウム滴定法、 過酸化 水素の消費を 2 4 0 n mの吸光度の変化により測定する分光光度法等を挙げるこ とができる。 Examples of the measurement method (2) include a potassium permanganate titration method and a spectrophotometric method in which the consumption of hydrogen peroxide is measured by a change in absorbance of 240 nm.
( 3 ) カタラーゼのペルォキシダーゼ作用を利用する方法 （以下、 測定方法 （3 ) ともいう） 。 (3) A method utilizing the peroxidase action of catalase (hereinafter, also referred to as measurement method (3)).
上記測定方法 （ 3 ) としては、 例えば、 過酸化水素とテトラメチルベンチジン 等の水素供与体である発色試薬とを基質として、 生ずる呈色を検出する方法等を 挙げることができる。 Examples of the above-mentioned measurement method (3) include a method of detecting a color generated by using hydrogen peroxide and a coloring reagent which is a hydrogen donor such as tetramethylbenzidine as a substrate.
本発明の検查方法に供する検体としては特に限定されず、 例えば、 胃内容物、 胃洗浄液、 消化管排泄物等を挙げることができるが、 採取が容易で被験者への負 担が少ない点で糞便等の消化管排泄物が好ましい。 The sample to be subjected to the test method of the present invention is not particularly limited, and includes, for example, stomach contents, gastric lavage fluid, gastrointestinal excrement, etc., in that collection is easy and the burden on the subject is small. Digestive tract excretions such as feces are preferred.
本発明の検査方法は、 除菌治療前に感染の有無を調べるために用いてもよく、 除菌治療後にその成否の判定を行うために用いられてもよい。 The test method of the present invention may be used to check for the presence of infection before the eradication treatment, or may be used to determine the success or failure after the eradication treatment.
本発明の検査方法を実施するためには、 本発明の診断キットを用いることがで さる。 In order to carry out the test method of the present invention, it is possible to use the diagnostic kit of the present invention. Monkey
本発明の診断キットの構成としては、 消化管中及び/又は糞便中のヘリコパク ター ·ピロリのカタラーゼ活性を測定することができれば特に限定されず、 例え ば、 CM—セルロース等の前処理用のイオン交換樹脂、 過酸化水素等のカタラー ゼの基質、 リン酸バッファ一等の緩衝液から構成されるもの等を挙げることがで さる。 The configuration of the diagnostic kit of the present invention is not particularly limited as long as the catalase activity of Helicobacter pylori in the digestive tract and / or feces can be measured. For example, CM-cellulose or other pretreatment ion Examples thereof include an exchange resin, a substrate of catalase such as hydrogen peroxide, and a buffer composed of a buffer such as a phosphate buffer.
このような構成を有する診断キットによれば、 まず、 被験者から提供された糞 便を緩衝液に懸濁し、 これを前処理用イオン交換樹脂に負荷し、 その溶出液に基 質を添加し、 酸素の発生の有無を確認することにより、 へリコパクター ·ピロリ への感染を判定することができる。 According to the diagnostic kit having such a configuration, first, feces provided by a subject are suspended in a buffer solution, the suspension is loaded on a pretreatment ion exchange resin, and a substrate is added to the eluate. By confirming the presence or absence of oxygen generation, infection with Helicobacter pylori can be determined.
本発明によれば、 極めて高感度かつ特異的に、 ヘリコパクター .ピロリの存在 を検出することができる。 また、 本発明は、 消化管排泄物を検体とすることがで きるので、 被験者に苦痛を与えることなく、 簡便かつ効率よくへリコパクター - ピロリ感染を判定することができる。 更に、 本発明は、 測定が簡便であり、 医療 現場で非常に有用である。 発明を実施するための最良の形態 According to the present invention, the presence of Helicobacter pylori can be detected with extremely high sensitivity and specificity. Further, since the present invention can use gastrointestinal excrement as a sample, it is possible to easily and efficiently determine Helicobacter pylori infection without causing pain to a subject. Further, the present invention is easy to measure and is very useful in medical practice. BEST MODE FOR CARRYING OUT THE INVENTION
以下に実施例を掲げて本発明を更に詳しく説明するが、 本発明はこれら実施例 のみに限定されるも.のではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
[実施例 1] 糞便中塩基性カタラーゼの検出 [Example 1] Detection of basic catalase in feces
糞便中へリコパクター ·ピロリ抗原陽性及ぴ陰性のポランティア （各 4名） の 糞便約 10 gを 3倍量のリン酸緩衝生理食塩液 (PBS) に懸濁し、 遠心分離 ( 7000 r pmN 30分） して得られた上清を更に超遠心分離 （30000 r p m、 30分） して上清を得た。 これらを蒸留水にて透析後、 更に Ι ΟπχΜリン酸 緩衝液 (pH7. 0) に対して透析した。 透析内液を 10 mMリン酸緩衝液 ( p H7. 0) にて平衡化した陽イオン交換樹脂 CM〜S e p h a d e X C— 50 のカラム （l cmX l cm) に負荷した。 同緩衝液 5 m Lで洗浄後、 PB S 3m Lにて溶出し、 塩基性物質を含有する画分 （塩基性物質画分） を得た。 溶出画分 lmLに 30%H2O2 0. 1 m Lを添加し、 気泡の発生を観察することにより 、 カタラーゼ活性の有無を確認した。 その結果、 全ての陽性者の糞便由来塩基性 画分にカタラーゼ活性を検出することができた。 一方、 陰性者の糞便由来塩基性 画分には、 活性は検出されなかった。 Suspend about 10 g of feces of Helicobacter pylori antigen positive and negative polenters (four each) in 3 times volume of phosphate buffered saline (PBS) and centrifuge (7000 rpm N 30 min.) ) Was further subjected to ultracentrifugation (30000 rpm, 30 minutes) to obtain a supernatant. These were dialyzed against distilled water, and further dialyzed against ΟππχΜphosphate buffer (pH 7.0). The dialysis solution was applied to a column (1 cm × 1 cm) of a cation exchange resin CM to Sephade XC-50 equilibrated with 10 mM phosphate buffer (pH 7.0). After washing with 5 mL of the same buffer, 3 mL of PBS Elution with L gave a fraction containing a basic substance (basic substance fraction). 0.1 mL of 30% H 2 O 2 was added to 1 mL of the eluted fraction, and the presence or absence of catalase activity was confirmed by observing the generation of bubbles. As a result, catalase activity could be detected in the fecal basic fraction of all positive persons. On the other hand, no activity was detected in the basic fraction derived from feces of a negative person.
この結果より、 カタラーゼ活性を指標としてへリコパクター ' ピロリへの感染 を判定できることがわかつた。 From these results, it was found that infection with Helicobacter pylori can be determined using catalase activity as an index.
[実施例 2] 抗へリコパクター · ピロリカタラーゼ卵黄抗体を用いた糞便中ヘリ コパクター ' ピロリカタラーゼの検出 [Example 2] Detection of Helicobacter pylori catalase in feces using anti-Helicobacter pylori catalase yolk antibody
(1) へリコパクター 'ピロリカタラ一ゼの精製 (1) Purification of Helicobacter pylori catalase
5%ゥマ脱繊血を添加したブレインハートインヒユージョン寒天培地 （ディフ コネ土製） 330枚.にへリコパクター ' ピロリ （ATCC43504 108CF U/mL) を 25 枚にて塗布し、 37°C、 微好気条件下にて 4日間培養し た。 Apply Helicopter 'pylori (ATCC43504 10 8 CF U / mL) to 330 Brain Heart Infusion Agar Medium (Diff Kone Soil) supplemented with 5% ゥ decentratured blood at 25 ° C and 37 ° C The cells were cultured under microaerobic conditions for 4 days.
得られた菌体を白金耳にてかきとり、 PBSに懸濁した。 4°C、 3000 r p mで 1 5分間遠心分離したヘリコパクター ·ピロリ菌体を 0. 5 %ホルマリンに 懸濁し、 4 、 1晚放置して不活化した。 不活化した菌体は、 遠心分離 （4°C、 3000 r pms 15分間） を用い、 P B Sにて 3回洗浄した後、 再度 P B Sに 懸濁した。 菌体懸濁液を超音波破砕機 （セイコー電子工業社製、 Mo d e 1 72 50) を用いて、 o u t p u t 4、 50% d t y c y c l eの条件にて 30分間超音波処理して菌体を破碎した。 この菌体破砕物を超遠心分離 （4で、 30000 r pm、 30分間） し、 上清 （菌体可溶性画分） 4 OmLを得た （タ ンパク質濃度 4. 9mg/mL) 。 The obtained cells were scraped off with a platinum loop and suspended in PBS. Helicobacter pylori cells centrifuged at 3000 rpm for 15 minutes at 4 ° C were suspended in 0.5% formalin and left inactivated for 4 to 1 minute to inactivate. Cells were inactivated, using centrifugation (4 ° C, 3000 r pm s 15 min), washed three times with PBS, and suspended again in PBS. The cell suspension was sonicated using an ultrasonic crusher (Model 17250, manufactured by Seiko Denshi Kogyo Co., Ltd.) for 30 minutes under the conditions of output 4 and 50% dtycycle to crush the cells. The crushed cells were subjected to ultracentrifugation (4, 30,000 rpm, 30 minutes) to obtain 4 OmL of supernatant (cell-soluble fraction) (protein concentration: 4.9 mg / mL).
アマシャムフアルマシアバイオテク社ァフィ二ティークロマトグラフィーハン ドプックに記載の方法により、 抗ヘリコパクター ·ピロリカタラーゼモノクロ一 ナル抗体 21 G 2を CNB r—活性化 S e p h a r o s e 4B (アマシャムファ ルマシアバイオテク社製） に固定化したカラムを作製した （10mL) 。 このカラムにへリコパクター · ピロリ菌体可溶性画分 2 OmLを負荷した （流 速約 4. 5mL/h) 。 PBS 60mL (流速約 30mLZh) にてカラムを洗 浄し、 0. 2Mグリシン HC 1緩衝液 （pH3. 0) 6 OmL (流速約 30nxL /h) にて溶出した。 洗浄、 溶出とも 1 OmLずつ分画した。 各画分 5 / Lに 3 0/όΗ2Ο2及び 0. 1%ツイーン 20を含む PBSを 100 L添加し、 泡の発 生を観察することによりカタラーゼ活性を調べた。 その結果、 溶出画分 （画分 9 及び 10) に強いカタラーゼ活性を認めた。 これらの画分を合わせ、 5mLに濃 縮し、 へリコパクター .ピロリカタラーゼを得た。 （タンパク質濃度 0. 8mg /mL) 。 According to the method described in Amersham Pharmacia Biotech Affinity Chromatography Handbook, the anti-Helicobacter pylori catalase monoclonal antibody 21G2 was converted to CNB r-activated Sepharose 4B (Amersham Pharmacia Biotech). An immobilized column was prepared (10 mL). The column was loaded with 2 OmL of the soluble fraction of Helicobacter pylori cells (flow rate: about 4.5 mL / h). The column was washed with 60 mL of PBS (flow rate of about 30 mL Zh) and eluted with 6 OmL of 0.2 M glycine HC1 buffer (pH 3.0) (flow rate of about 30 nxL / h). Washing and elution were fractionated by 1 OmL. And each fraction 5 / L to 3 0 / όΗ added 100 L of PBS containing 2 Omicron 2 and 0.1% Tween 20, was examined catalase activity by observing the occurrence of bubbles. As a result, strong catalase activity was observed in the eluted fractions (fractions 9 and 10). These fractions were combined and concentrated to 5 mL to obtain Helicobacter pylori catalase. (Protein concentration 0.8 mg / mL).
へリコパクター ·ピロリカタラーゼを等量のサンプル緩衝液 （2 %SDS、 5 %メルカプトエタノール） と混合し、 5分間煮沸した。 この溶液 5 Lを用いて SDS—ポリアクリルァミドゲル電気泳動 （4— 20 %アタリルァミド） を行つ た。 分子量マーカーは、 ホスホリラーゼ¾>、 ゥシ血清アルブミン、 卵白アルブミ ン、 炭酸脱水素酵素、 大豆トリプシンインヒビター及ぴ α—ラクトアルブミンを 用いた。 泳動後、 シルパースティン ΚΑΝΤΟΠΙ (関東化学社製） を用いてゲル を染色した。 へリコパクター ·ピロリカタラーゼは 59 kD aの単一なタンパク 質であった。 Helicobacter pyloricatalase was mixed with an equal volume of sample buffer (2% SDS, 5% mercaptoethanol) and boiled for 5 minutes. Using 5 L of this solution, SDS-polyacrylamide gel electrophoresis (4-20% atarylamide) was performed. As the molecular weight markers, phosphorylase II>, yeast serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and α-lactalbumin were used. After the electrophoresis, the gel was stained using Sylperstin® (Kanto Chemical). Helicobacter pylori catalase was a single 59 kDa protein.
(2) 抗へリコパクター ' ピロリカタラーゼ卵黄抗体の作製 (2) Preparation of Anti-Helicopter '' Pyloricatalase Egg Yolk Antibody
ヘリコパクター ·ピロリカタラーゼをフロイント ·コンプリートアジュパンド と等量混和した後、 産卵鶏 （白色レグホーン系） に lmL筋肉注射した。 免疫は 2週間に 1回、 合計 3回行った。 初回免疫から 9〜11週目に産卵した 6個の免 疫卵より卵黄抗体を調製した。 免疫卵を割卵し、 卵黄を取り出した後、 等量の精 製水を加えた （全量 156m L) 。 これに 2倍量 （312mL) の 0. 15 % λ 一力ラギーナン水溶液を混合し、 室温で 30分間放置した後、 l O O O O r pm 、 15分間遠心分離した。 得られた上清に硫酸アンモニゥムを 40%飽和になる ように加えて撹拌した後、 遠心分離により沈渣を得た。 この沈渣を P B Sに溶解 し、 同緩衝液に対して透析を行い、 透析内液を回収した （抗ヘリコパクタ一 · ピ ロリカタラーゼ卵黄抗体 288mg タンパク質） 。 抗ヘリコパクター · ピロリカタラーゼ卵黄抗体とヘリコパクター ·ピロリカタ ラーゼとの反応 14は、 酵素免疫測定 （EL I SA) 法にて確認した。 へリコパク ター .ピロリカタラーゼを PB Sにて 0. lmgZmLに希釈した後、 96穴£ L I S Aプレートの各穴に 0. lmL加え、 4。C、 1晚放置することにより固相 化した。 PBSにて洗浄した後、 0. 1%スキムミルク一PB Sを各穴に 0. 2 5mL力 13え、 4 で 1時間放置してマスキングを行った。 へリコパク 'ター 'ピロ リカタラーゼを固相化した EL I S Aプレートの各穴に 0. 1%スキムミルク一 PB Sにて適宜希釈した抗ヘリコパクター ·ピロリカタラーゼ卵黄抗体 0. lm Lを加え、 室温にて 1時間放置した。 0. 05%ツイーン 20— PB S (洗浄液 ) にて 5回洗浄した後、 ペルォキシダーゼ標識抗 I gY (ポストンエキスプレス 社製、 1 : 10, 000) 0. lmLを加え、 室温にて 1時間放置した。 洗浄液 にて 5回洗浄後、 基質液 （テトラメチルベンチジン +過酸化水素、 B i o FX社 製） 0. lmLを各穴に添カ卩し、 室温で 10分間反応させた。 反応後、 各穴に 1 N硫酸を 50 Lずつ添加し酵素反応を停止し、 吸光度 （450n m— 630 η m) を測定した。 対照には、 免疫する前に産んだ卵より同様の方法にて調製した 卵黄抗体を用いた。 その結果、 抗へリコパクター ' ピロリカタラーゼ卵黄抗体は 、 へリコパクター 'ピロリカタラーゼと強く反応した。 結果を表 1に示した。 Helicobacter pyloricatalase was mixed in equal amounts with Freund's complete adjuvant, and then injected into the laying hens (white Leghorn strain) via intramuscular injection. Immunization was performed once every two weeks, for a total of three times. Yolk antibodies were prepared from 6 immunized eggs laid 9 to 11 weeks after the first immunization. After the immunized egg was split and the yolk was removed, an equal volume of purified water was added (total volume: 156 mL). This was mixed with 2 volumes (312 mL) of a 0.15% λ one-strength lugginan aqueous solution, left at room temperature for 30 minutes, and centrifuged at lOOO rpm for 15 minutes. Ammonium sulfate was added to the obtained supernatant to a saturation of 40%, and the mixture was stirred, followed by centrifugation to obtain a precipitate. The precipitate was dissolved in PBS, dialyzed against the same buffer, and the dialysate was recovered (anti-Helicobacter pylori catalase egg yolk antibody 288 mg protein). The reaction 14 between the anti-Helicobacter pylori catalase egg yolk antibody and Helicobacter pylori catalase 14 was confirmed by enzyme immunoassay (ELISA). After diluting Helicopter.pyrrolicatalase to 0.1 mgZmL with PBS, 0.1 mL was added to each well of a 96-well LISA plate. C, solidified by leaving 1 晚. After washing with PBS, 0.1% skim milk-PBS was applied to each well with 0.25 mL 13 and left at 4 for 1 hour to perform masking. To each well of the ELISA plate immobilized with Helicopaque 'Ter' Pyrolicatalase, add 0.1 μL of anti-Helicobacter pylori catalase egg yolk antibody appropriately diluted with 0.1% skim milk-PBS, and at room temperature. Left for 1 hour. After washing 5 times with 0.05% Tween 20-PBS (washing solution), 0.1 μL of peroxidase-labeled anti-IgY (manufactured by Poston Express, 1: 10,000) was added and left at room temperature for 1 hour. . After washing with a washing solution 5 times, 0.1 mL of a substrate solution (tetramethylbenzidine + hydrogen peroxide, manufactured by BioFX) was added to each well, and reacted at room temperature for 10 minutes. After the reaction, 50 L of 1 N sulfuric acid was added to each well to stop the enzyme reaction, and the absorbance (450 nm-630 ηm) was measured. As a control, an egg yolk antibody prepared by the same method from eggs laid before immunization was used. As a result, the anti-Helicobacter pylori catalase yolk antibody strongly reacted with the Helicobacter pylori catalase. The results are shown in Table 1.
E L I S Aの吸光度 （4 50 n m— 63 0 n m) Absorbance of ELISA (450 nm-630 nm)
(3) 抗へリコパクター ' ピロリカタラーゼ卵黄抗体固相化プレートの作製 抗ヘリコパクター . ピロリカタラーゼ卵黄抗体を P B Sにて 20 μ g/mLと なるように希釈した後、 9 6穴 EL I S Aプレートの各穴に 0. 2mL加え、 4 °C、 1晚放置した後、 PB Sにて洗浄した。 洗浄後、 0. 1%スキムミルク一 P B Sを各穴に 0. 2 5mL加え、 4°Cで 1時間放置してマスキングを行った。 (4) 抗へリコパクター.ピロリカタラーゼ卵黄抗体固相化プレートを用いた粪 便中へリコパクター .ピロリカタラーゼの捕捉及びその酵素活性の検出 · へリコパクター .ピロリ陽性者及び陰性者各 4名の糞便検体を使用した。 糞便 検体約 3 gを 1 2mLの PB Sに懸濁し、 4°C、 3 000 r p m、 1 5分間遠心 分離して上清 （糞便抽出液） を得た。 糞便抽出液 （対照 PB S) を抗へリコパク ター .ピロリカタラーゼ卵黄抗体固相化プレートの各穴に 0. 2mLずつ 24穴 に分注し、 室温にて 1時間放置した後、 洗浄液にて 5回洗浄した。 予めオートク レイプ処理にて脱気した P B Sを用いて調製した 5 mM 11202を0. 2mL /穴分注して、 室温にて 1時間反応した。 反応後、 各穴から反応液を集め、 溶存 酸素測定計 （ケメット DO計型式 K— 75 1 2 CHEMe t r i e s社製） に て溶存酸素を測定した。 その結果、 陽性者糞便検体では、 溶存酸素は 8~1 2 p pm以上と高値を示したのに対し、 陰性者糞便検体では、 対照と差がなかった （ 約 1 p p m) 。 (3) Preparation of Anti-Helicopter '' Pylori Catalase Egg Yolk Antibody Immobilized Plate After diluting the anti-Helicobacter pylori catalase yolk antibody to 20 μg / mL with PBS, add 0.2 mL to each well of a 96-well ELISA plate, leave at 4 ° C for 1 晚Washed with S. After washing, 0.25 mL of 0.1% skim milk-PBS was added to each well, and left at 4 ° C. for 1 hour to perform masking. (4) Anti-helicopter. Helicobacter pylori catalase using egg yolk antibody-immobilized plate. Capture of fecal helicopter. Pylori catalase and detection of its enzymatic activity. Helicobacter. It was used. Feces Approximately 3 g of the specimen was suspended in 12 mL of PBS and centrifuged at 4 ° C and 3000 rpm for 15 minutes to obtain a supernatant (fecal extract). Dispense 0.2 mL of the fecal extract (control PBS) into each well of the anti-helicopter.pyrrolicatalase egg yolk antibody-immobilized plate into 24 wells, leave it at room temperature for 1 hour, Washed twice. The 5 mM 11 2 0 2 was prepared with PBS was degassed in advance Otoku rape processing aliquoted 0. 2 mL / well min was reacted at room temperature for 1 hour. After the reaction, the reaction solution was collected from each well, and the dissolved oxygen was measured using a dissolved oxygen meter (manufactured by Kemet DO Model K-7512 CHEMetris). As a result, in the stool samples of positive subjects, the dissolved oxygen showed a high value of 8 to 12 ppm or more, whereas in the stool samples of negative subjects, there was no difference from the control (about 1 ppm).
この結果から、 糞便検体中のへリコパクター'ピロリカタラーゼを特異抗体に よって捕捉した後、 酵素活性を検出する方法により、 へリコパクター ·ピロリカ タラーゼへの感染を判定できることがわかった。 From these results, it was found that infection with Helicobacter pyloricatalase could be determined by a method for detecting the enzyme activity after capturing Helicobacter pyloricatalase in the stool sample with a specific antibody.
[実施例 3] 抗へリコパクター 'ピロリカタラーゼモノクローナル抗体を用いた 糞便中ヘリコパクター ·ピロリカタラーゼの検出 [Example 3] Detection of Helicobacter pylori catalase in feces using anti-Helicobacter pylori catalase monoclonal antibody
(1) 抗へリコパクター ' ピロリカタラーゼモノクローナル抗体固相化プレート の作製 (1) Preparation of anti-helicopter '' pyloricatalase monoclonal antibody-immobilized plate
抗ヘリコパクタ一 .ピロリカタラーゼモノクローナル抗体を PBSにて 5 IX g ノ mLとなるように希釈した後、 96穴 EL I S Aプレートの各穴に 0. 2mL 加え、 4 C、 1晚放置した後、 PB Sにて洗浄した。 洗浄後、 0. 1%スキムミ ルクー PBSを各穴に 0. 25mL加え、 4 °Cで 1時間放置してマスキングを行 つに。 After diluting the anti-Helicobacter pyloricatalase monoclonal antibody to 5 IX g / mL with PBS, add 0.2 mL to each well of a 96-well ELISA plate, leave for 4 C and 1 晚Was washed. After washing, 0.1% skim Add 0.25 mL of Lucu PBS to each well and leave at 4 ° C for 1 hour for masking.
(2) 抗ヘリコパクター ·ピロリカタラーゼモノクローナル抗体固相化プレート を用いた糞便中ヘリコパクター ·ピロリカタラーゼの捕捉及ぴその酵素活性の検 出 (2) Capture of Helicobacter pyloricatalase in feces using anti-Helicobacter pylori catalase monoclonal antibody-immobilized plate and detection of its enzyme activity
へリコパクター . ピロリ陽性者及び陰性者各 4名の糞便検体を用いて、 実施例 2と同様の方法で行った。 その結果、 陽性者糞便検体では、 溶存酸素は 8〜12 p pm以上と高値を示したのに対し、 陰性者糞便検体では、 対照と差がなかった (約 1 p ρ m) 。 産業上の利用可能性 The same procedure as in Example 2 was carried out using stool samples of 4 helicobacter pylori positive and negative persons. As a result, in the stool specimen of the positive person, the dissolved oxygen showed a high value of 8 to 12 ppm or more, whereas in the stool specimen of the negative person, there was no difference from the control (about 1 p ρm). Industrial applicability
本発明は、 上述の構成よりなるので、 消化管排泄物を検体とすることができ、 被験者に苦痛を与えることなく、 簡便かつ効率よくへリコパクター ·ピロリ感染 を検出することができる。 Since the present invention has the above-mentioned configuration, gastrointestinal excretion can be used as a sample, and Helicobacter pylori infection can be detected simply and efficiently without causing pain to the subject.
Claims
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| JP2002519666A JP5095065B2 (en) | 2000-08-11 | 2001-08-10 | Test method and diagnostic kit for Helicobacter pylori infection |
| AU2001277749A AU2001277749A1 (en) | 2000-08-11 | 2001-08-10 | Method of examining infection with helicobacter pylori and diagnostic kit |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08233823A (en) * | 1995-02-28 | 1996-09-13 | Dai Ichi Seiyaku Co Ltd | Detection of urease derived from helicobactor pylori |
| EP0806667A1 (en) * | 1996-05-09 | 1997-11-12 | Meridian Diagnostics, Inc. | Immunoassay for h. pylori in fecal specimens |
| US5702911A (en) * | 1996-08-15 | 1997-12-30 | Chek-Med Systems, Inc. | Diagnostic test composition |
| WO1998006853A1 (en) * | 1996-08-15 | 1998-02-19 | Csl Limited | Treatment and prevention of helicobacter infection |
| WO2001027613A2 (en) * | 1999-10-12 | 2001-04-19 | Connex Gesellschaft Zur Optimierung Von Forschung Und Entwicklung Mbh | Improved method for the detection of acid resistant microorganisms in a stool |
| WO2001029198A1 (en) * | 1999-10-15 | 2001-04-26 | Csl Limited | Polypeptide fragments comprising c-terminal portion of helicobacter catalase |
-
2001
- 2001-08-10 WO PCT/JP2001/006894 patent/WO2002014541A1/en not_active Ceased
- 2001-08-10 AU AU2001277749A patent/AU2001277749A1/en not_active Abandoned
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08233823A (en) * | 1995-02-28 | 1996-09-13 | Dai Ichi Seiyaku Co Ltd | Detection of urease derived from helicobactor pylori |
| EP0806667A1 (en) * | 1996-05-09 | 1997-11-12 | Meridian Diagnostics, Inc. | Immunoassay for h. pylori in fecal specimens |
| US5702911A (en) * | 1996-08-15 | 1997-12-30 | Chek-Med Systems, Inc. | Diagnostic test composition |
| WO1998006853A1 (en) * | 1996-08-15 | 1998-02-19 | Csl Limited | Treatment and prevention of helicobacter infection |
| WO2001027613A2 (en) * | 1999-10-12 | 2001-04-19 | Connex Gesellschaft Zur Optimierung Von Forschung Und Entwicklung Mbh | Improved method for the detection of acid resistant microorganisms in a stool |
| WO2001029198A1 (en) * | 1999-10-15 | 2001-04-26 | Csl Limited | Polypeptide fragments comprising c-terminal portion of helicobacter catalase |
Non-Patent Citations (2)
| Title |
|---|
| DATABASE BIOSIS [online] accession no. Dialog Database accession no. 200100014362 * |
| HOKKAIDO JOURNAL OF MEDICAL SCIENCE, vol. 75, no. 5, September 2000 (2000-09-01), pages 287 - 298 * |
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