JP3393855B2 - Monoclonal antibodies, hybridomas, immunoassays and diagnostic kits - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a Helicobacter
A monoclonal antibody whose antigen is Helicobacter pylori catalase,
The present invention relates to a hybridoma producing the monoclonal antibody, an immunological assay method, and a diagnostic kit.

[0002]

2. Description of the Related Art Helicobacter pylori
bacterium pylori) is a bacterium found in human gastric mucosa. The infection rate to Helicobacter pylori is
It is closely related to socio-economic conditions, and developing countries tend to have higher infection rates and developed countries tend to have lower infection rates.
However, the infection rate of Japanese people is outstandingly high in developed countries, and it is said that 80% of people over the age of 40 are infected. In recent years, it has been revealed that Helicobacter pylori can cause various gastric and duodenal diseases such as gastric ulcer, duodenal ulcer, chronic gastritis, and gastric cancer.

[0003] Since it was revealed that the susceptibility to these diseases can be reduced by eradicating Helicobacter pylori, the diseases to be eradicated by Helicobacter pylori have been internationally discussed, and now, gastric ulcer is present. , Duodenal ulcer, gastric malignant lymphoma, stomach after resection of early gastric cancer, etc. are recognized as diseases targeted for eradication.

Helicobacter pylori eradication therapy is for the stomach,
Following the recognition as a new treatment method for duodenal disease, the Japanese Society of Gastroenterology also prepared clinical trial guidelines in Japan, and the diagnosis of Helicobacter pylori infection and the eradication judgment method were presented (Japan Gastroenterological Society. Magazine, 96
Vol., 199-207, 1999). In the above guidelines for clinical trials, the presence diagnosis is performed by the invasive test method of culturing gastric biopsy tissue, microscopic examination, urease test, and the eradication judgment is performed by culturing the gastric biopsy tissue, microscopic examination and non-invasive test. It has been shown that the method urea breath test is mandatory. In addition, in a special case where the subject is a child, it is shown that the determination is performed by using the blood anti-Helicobacter pylori antibody test and the presence diagnosis in combination.

However, these inspection methods for Helicobacter pylori infection have the following problems. In the invasive examination method, a subject suffers great pain due to insertion of a gastroscope, biopsy, and the like. Non-invasive tests significantly reduce subject distress, while the urea breath test requires fasting prior to testing. Further, the urea breath test requires a device such as a mass spectrum or an infrared spectrophotometer, and can be carried out only in a specific facility, and there is a drawback that the cost becomes high. The antibody test is not suitable for eradication because the antibody value in the blood remains high for a long time even after eradication. Therefore, non-invasive and Helicobacter alternatives to these tests
A test method capable of directly and specifically detecting H. pylori infection with high accuracy is desired.

[0006] Conventionally, as a direct test method for gastrointestinal infectious bacteria, separation culture using gastrointestinal excrement, in particular, a selective medium for infectious bacteria from feces has been carried out. However, in spite of many attempts on Helicobacter pylori, there have been few reports of separate culture from feces. The reason for this is that Helicobacter pylori transforms from normal helices into non-culturable spheroids in vitro when environmental conditions such as low temperature, nutrient deficiency, and oxygen deficiency deteriorate in vitro. It is conceivable that even in the case of the above, it has been changed to a spherical body that cannot be separated and cultured.

On the other hand, regarding the direct detection of Helicobacter pylori from feces by an immunological method based on the antigen-antibody reaction, the detection of Helicobacter pylori in excrement samples such as feces was detected by an immunoassay using a polyclonal antibody against Helicobacter pylori. Has been reported (J. Clin. Microbiol., 33.
Vol. 2162-2165, 1995, JP-A-10-1
No. 0128 (Patent No. 3043999)).

However, since polyclonal antibodies generally have cross-reactivity and are inferior in specificity, and the antibody titer and specificity vary from lot to lot of antiserum, the production of diagnostic agents using polyclonal antibodies Has a problem that quality control is essentially difficult. Actually, Patent No. 3043
With regard to the fecal Helicobacter pylori antigen detection kit "HpSA" using a polyclonal antibody manufactured by Meridian, which is the patentee of No. 999, the appearance of false negatives and low specificity pose problems ( Medi
cal Tribune, 4-5, June 3, 1999 issue; Am. J. Gastroenterol. , 94
Vol. 1830-1833, 1999).

On the other hand, in Japanese Patent Laid-Open No. 10-10128, Helicobacter pylori causes mutations in the strain. Therefore, a monoclonal antibody capable of reacting with only a single antigen is unsuitable for detecting Helicobacter pylori. On the contrary, it is described that the polyclonal antibody is more suitable for the detection of Helicobacter pylori because it can correspond to various antigens and epitopes.

[0010]

SUMMARY OF THE INVENTION In view of the above situation, the present invention is capable of diagnosing Helicobacter pylori infection at low cost without causing pain to the subject and requiring no special device.
In addition, by using a single antibody, there is no cross-reactivity and excellent specificity, there is no lot-to-lot variation, and quality control is easy. Furthermore, even when a single monoclonal antibody is used, excellent sensitivity is achieved. It is an object of the present invention to provide a diagnostic method having the above.

[0011]

The present invention is a monoclonal antibody which uses Helicobacter pylori catalase as an antigen. The present invention is also a hybridoma that produces a monoclonal antibody against Helicobacter pylori catalase as an antigen. An immunological assay method using at least one of the monoclonal antibodies of the present invention is also one of the present invention. A diagnostic kit containing at least one of the monoclonal antibodies of the present invention is also part of the present invention. In this specification, catalase means SDS.
It does not contain a protein corresponding to a subunit that has been denatured and dissociated by a denaturing agent such as, and whose three-dimensional structure has been unfolded. The present invention is described in detail below.

The monoclonal antibody of the present invention uses Helicobacter pylori catalase as an antigen.
The monoclonal antibody of the present invention can be produced by the hybridoma of the present invention. For example, the monoclonal antibody of the present invention can be obtained by culturing the hybridoma of the present invention.
However, the method for producing the monoclonal antibody of the present invention is not particularly limited, and even if it is obtained by genetic engineering, as long as it can specifically bind to the catalase of Helicobacter pylori, It is included in the range.

The hybridoma of the present invention produces a monoclonal antibody using Helicobacter pylori catalase as an antigen, and is a fusion of splenocytes or lymph node cells of an animal immunized with Helicobacter pylori and myeloma cells. It is obtained by doing.

The hybridoma of the present invention can be produced by a known cell fusion method. That is, a non-human animal is immunized with Helicobacter pylori as an immunogen, and its spleen cells or lymph node cells are fused with myeloma cells to prepare hybridomas, from which Helicobacter
The hybridoma of the present invention can be obtained by selecting a hybridoma that produces a monoclonal antibody that recognizes H. pylori.

The above-mentioned immunogen is not particularly limited as long as it contains Helicobacter pylori catalase. For example, a culture obtained by culturing a Helicobacter pylori strain in an appropriate medium, or a helical form. Examples thereof include bacterial cells, spherical bacterial cells, crushed products of these bacterial cells, lysates, extracts, and fractionated products thereof. The cells may be dead cells or live cells.

The catalase is not particularly limited,
For example, it may be inactivated, partially broken in three-dimensional structure, partially deleted, etc., but preferably has 4 subunits, and more preferably is native. In addition, in this specification, Nati
The ve enzyme refers to an enzyme that retains a unique structure taken under almost physiological conditions, has all subunits, and is active.

Among the above immunogens, a crushed product of spherical bacterial cells is preferable. Helicobacter pylori is in vi
In tro, when environmental conditions such as low temperature, nutrient deficiency, and oxygen deficiency deteriorate, the morphology of normal helices changes to non-culturable spheroids. It is considered to be changing. Also,
The cells of Helicobacter pylori are considered to have been disrupted in the lower digestive tract.

Helicobacter used as the immunogen
The strain species of Helicobacter pylori is not particularly limited, and examples thereof include standard strains ATCC43504 strain and NCTC11638.
Etc., and other strains collected from infected persons may be used. The genotype of Helicobacter pylori used as the immunogen is not particularly limited, and examples thereof include va
may or may not have cA and cagA,
Further, vacA may have any sequence of S1a, S1b and S2, and may have any sequence of m1 and m2.

The immunized animal used for producing the hybridoma of the present invention is not particularly limited, and examples thereof include goat, sheep, guinea pig, mouse, rat, rabbit, etc., among which mouse is preferable.

As a method for immunizing the immunized animal,
Known methods can be used, for example, when immunizing a mouse, 1 to 100 μg, preferably 50 to 1 at a time.
Emulsify 00 μg of the immunizing antigen with an equal volume (0.1 mL) of physiological saline and Freund's complete adjuvant or RIBI adjuvant system, and subcutaneously or intraperitoneally in the back or abdomen of the immunized animal every 2-3 weeks. And a method of inoculating 3 to 6 times.

In the present invention, after immunizing the above-mentioned immunized animal, an individual with a high antibody titer is selected, and 3 to 5 days after the final immunization, the spleen or lymph node is removed, and a known cell fusion method is used.
The antibody-producing cells contained in these tissues can be fused with myeloma cells in the presence of the fusion promoter.

The fusion promoter is not particularly limited,
For example, polyethylene glycol (hereinafter referred to as PEG), Sendai virus and the like can be mentioned.
Preferably PEG is used.

The myeloma cells are not particularly limited,
For example, P3U1, NS-1, P3x63. Ag 8.6
Examples thereof include mouse-derived cells such as 53; and rat-derived cells such as AG1 and AG2.

The above cell fusion method is not particularly limited,
For example, spleen cells and myeloma cells are mixed at a ratio of 1: 1 to 10: 1, and P having a molecular weight of 1,000 to 6,000 is added to the mixture.
Examples include a method of adding EG at a concentration of 10 to 80%, and incubating at 20 to 37 ° C, preferably 30 to 37 ° C for 3 to 10 minutes.

In the present invention, selection of hybridomas producing a monoclonal antibody that recognizes Helicobacter pylori is carried out by culturing in a selective medium such as HAT medium in which only the hybridoma can grow, and fixing the antibody activity in the hybridoma culture supernatant. -Phase enzyme-linked immunosorbent assay (ELISA)
It can be performed by measuring using a method such as. Furthermore, in the present invention, the establishment of a hybridoma that produces a monoclonal antibody that recognizes Helicobacter pylori, for example, for a hybridoma that produces a monoclonal antibody that recognizes Helicobacter pylori,
It can be performed by repeating cloning by a method such as limiting dilution.

The hybridoma of the present invention includes, for example, 21G2 (accession number FERM BP-7336),
41A5 (accession number FERM BP-7337) and 82B9 (accession number FERM BP-7338) can be mentioned. These hybridomas were found on October 14, 1999 by the Institute of Biotechnology, Ministry of International Trade and Industry, Institute of Biotechnology (1-3, Higashi, Tsukuba, Ibaraki, Japan).
No.) deposited (original deposit)
Microdevelopment microorganism contribution No. P-17604 deposited on October 23
(21G2), P-17605 (41A5), and P
-17606 (82B9), respectively.

The method for preparing a large amount of the monoclonal antibody of the present invention is not particularly limited, and examples thereof include a method in which a hybridoma is transplanted into the abdominal cavity of a mouse to which pristane has been administered in advance, and which is obtained from the collected ascites fluid. . The monoclonal antibody of the present invention in ascites can be purified by a known method using a protein A or protein G column or the like.

The monoclonal antibody of the present invention uses catalase as an antigen. The catalase is not particularly limited as long as it retains an epitope, and may be, for example, inactivated, partially sterically damaged, partially deficient, or the like. Those having 4 subunits are preferred, Native
More preferred. The catalase includes those having mutations and the like.

The strain species of Helicobacter pylori producing the above catalase is not particularly limited. The genotype of Helicobacter pylori that produces the above catalase is not particularly limited, and examples thereof include vacA and ca.
It may or may not have gA.
cA may have any sequence of S1a, S1b and S2, and may have any sequence of m1 and m2.

The form of Helicobacter pylori which produces the above-mentioned catalase is not particularly limited, and examples thereof include a helical Helicobacter pylori and a spherical Helicobacter pylori.

The class / subclass of the monoclonal antibody of the present invention is not particularly limited, and IgG ₁ , Ig
G ₂ , IgG ₃ , IgG ₄ , IgM, IgE, Ig
It may be any of A ₁ , IgA ₂ , and IgD, and the L chain is not particularly limited, and may be a λ chain or a κ chain.

Further, the monoclonal antibody of the present invention is not particularly limited as long as it can specifically bind to Helicobacter pylori catalase, and for example, F (a) which is a degradation product of the monoclonal antibody of the present invention.
b ′) ₂ , Fab ′, Fab, or a chimeric antibody.

The above-mentioned 21G2 (accession number FERM B
P-7336), 41A5 (accession number FERM BP-
7337) and 82B9 (accession number FERM BP
-7338) produced by the hybridoma (hereinafter, referred to as monoclonal antibody 21G2,
Monoclonal antibody 41A5, monoclonal antibody 82
(Also called B9) is the Helicobacter pylori Nati
It recognizes ve catalase.

The present inventors have confirmed that the monoclonal antibody 21G
2, the monoclonal antibody 41A5 and the monoclonal antibody 82B9 were used to carry out antibody-immobilized affinity chromatography on the lysate of Helicobacter pylori, which revealed that the 270 kDa contained in the lysate of Helicobacter pylori. A protein having a molecular weight of was detected. When this protein was subjected to SDS-polyacrylamide gel electrophoresis, a single band having a molecular weight of 59 kDa was detected. In addition, when the N-terminal amino acid sequence of this protein was examined, it coincided with the amino acid sequence of Helicobacter pylori catalase. Helicobacter pylori catalase has a molecular weight of 200 kDa and is known to have a tetrameric structure in which four subunits having a molecular weight of 50 kDa are assembled (J. Gen. Microb.
iol. (1991), 137, 57-61).

Therefore, from the above results, the proteins detected by the monoclonal antibody 21G2, the monoclonal antibody 41A5, and the monoclonal antibody 82B9 were identified as Helicobacter pylori catalase having four subunits, and each of the above monoclonals was identified. It was revealed that the antibody recognizes a catalase having four subunits.

The activity of catalase detected by the above-mentioned antibody-immobilized affinity chromatography was measured and found to be active.
It was also found to be a live enzyme. In addition, the monoclonal antibody 21G2, the monoclonal antibody 41A5,
Neither of the monoclonal antibodies 82B9 reacted with the dissociated subunit of catalase.

The proportion of catalase in the total protein in the cells of Helicobacter pylori is as described in J. Gen.
Microbiol. (1991), 137, 57-6.
Estimated from the degree of increase in specific activity in the purification process of catalase described in 1 above, it is at most 0.5% in terms of weight. In view of this point, the monoclonal antibody 21G2,
It was surprising that the monoclonal antibody 41A5 and the monoclonal antibody 82B9 recognized catalase having four subunits, which was an unexpected matter.

Catalase is extremely well conserved among different species, and Helicobacter pylori catalase is known to have high homology with other bacterial catalase (J. Bacteriol. (1996), 178).
(23), 6960-6967). However, monoclonal antibody 21G2, monoclonal antibody 41A
As shown in Example 3, 5 and the monoclonal antibody 82B9 did not react with catalase of bacteria other than Helicobacter pylori at all.

It is known that there are various mutant strains of Helicobacter pylori (Molecu.
lar Microbiology (1996), 2
0,833-842), surprisingly, monoclonal antibody 21G2, monoclonal antibody 41A5, and
As shown in Example 3, the monoclonal antibody 82B9 reacted well with any Helicobacter pylori.

Considering that the monoclonal antibody of the present invention reacts well with various mutant strains of Helicobacter pylori, the epitope recognized by the monoclonal antibody of the present invention is extremely well conserved among the catalase mutants. It seems to be a part that is being done.

As described in detail below, the monoclonal antibody of the present invention can determine the presence or absence of infection with Helicobacter pylori with extremely high accuracy using feces as a sample.

Monoclonal antibody 21G2, monoclonal antibody 41A5, and monoclonal antibody 82B9
When the immunological measurement was carried out using stool as a sample, each of the above monoclonal antibodies reacted only with the stool of a subject infected with Helicobacter pylori. This revealed that the feces of a subject infected with Helicobacter pylori contained a Helicobacter pylori catalase having four subunits. It has not been known at all until now that Helicobacter pylori catalase present in feces does not have dissociated subunits, but possesses four subunits. Is surprising in view of the fact that it is degraded by proteolytic enzymes in the digestive tract.

Further, using the feces of a Helicobacter pylori-infected person as a sample, a column on which the monoclonal antibody 21G2 was immobilized was prepared, affinity chromatography was carried out, and ELISA and catalase activity measurement were carried out on the eluate fraction. Catalase activity and antigenicity were in agreement.

From the above, it was found that the faeces of Helicobacter pylori-infected persons contained native catalase having four subunits and also having catalase activity. Catalase of Helicobacter pylori is active without being digested in the digestive tract
It was completely unpredictable that it was excreted as an e-enzyme and was present in the feces.

Japanese Unexamined Patent Publication No. 9-322772 and Inf
ect. Immun. (1997), 65, 4668-
4674 is Helicobacter pylori catalase S
There is disclosed a monoclonal antibody that reacts with a protein corresponding to the unfolded subunit obtained by denaturation / dissociation by DS-PAGE. However, whether or not these monoclonal antibodies react with native, three-dimensionally-retaining and active catalase, and whether or not Helicobacter pylori can be detected in excrement samples such as feces. Unknown.

On the other hand, the monoclonal antibody of the present invention is capable of recognizing the presence or absence of infection with Helicobacter pylori by using the digestive tract excrement such as feces, which is easy to collect, as a sample. .

The use of the monoclonal antibody of the present invention is not particularly limited, but it can be used for the determination of infection with Helicobacter pylori in the immunoassay.

The immunoassay method of the present invention is carried out using at least one of the monoclonal antibodies of the present invention, and can be carried out using only one kind.

In general, as an antibody used in an immunoassay, a monoclonal antibody having a high specificity and a low background is superior to a polyclonal antibody having a low specificity and a high background. It is said to be preferable.

However, it is known that there are various mutant strains of Helicobacter pylori, and therefore it is considered that it is extremely difficult to detect the infection and the presence thereof using a single monoclonal antibody. It was being done. As described in JP-A-10-10128, a polyclonal antibody capable of responding to various antigens is more suitable for detecting infection of bacteria having various antigens and the presence of the bacteria. In addition, a method for detecting Helicobacter pylori using a plurality of monoclonal antibodies in combination has been disclosed (WO00 / 26671).
However, a method for detecting Helicobacter pylori using a single monoclonal antibody has not been developed so far.

On the other hand, surprisingly, the monoclonal antibody of the present invention was able to detect Helicobacter pylori with extremely excellent accuracy by using only one kind of antibody.

The monoclonal antibodies described in WO00 / 26671 include urease, heat shock protein, alkaline hydroperoxidase-reductase, 20 kDa protein (3-dehydrokinase type 2), 16.9 kDa protein (neutrophil activity. Protein) and 33.8 kDa protein (fructose-bisphosphatase-aldolase) as antigens, and catalase is not mentioned at all. Further, the detection sensitivity of the measuring method described in WO00 / 26671 was not sufficient.

Examples of the immunological assay of the present invention include enzyme immunoassay (EIA), solid-phase enzyme immunoassay (ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), Western blotting, immunochromatography and the like can be mentioned. The various immunological measurement methods described above can measure the target antigen or antibody using an antigen or antibody labeled with a labeling agent by a competitive method or a sandwich method. Among the above various immunological assay methods, the ELISA method and the immunochromatographic method are preferable.

The above-mentioned competitive method is based on, for example, a quantitative competitive reaction of binding of Helicobacter pylori catalase in a sample with a known amount of labeled Helicobacter pylori catalase to the monoclonal antibody of the present invention. Is. In the competitive method exemplified above, a certain amount of antibody retained on a carrier is added to a sample solution containing Helicobacter pylori catalase, and further a certain amount of Helicobacter pylori catalase labeled with a labeling agent is added. Then, the activity of the labeling agent retained on the carrier or the labeling agent not retained on the carrier is measured. At this time, it is preferable that the antibody and the labeled antigen are added almost simultaneously.

The above sandwich method is, for example, to sandwich Helicobacter pylori catalase in a sample with the immobilized monoclonal antibody of the present invention and the monoclonal antibody of the present invention labeled with a labeling agent. , A substrate for a labeling agent such as an enzyme is added, and color is developed to detect catalase of Helicobacter pylori in a sample.

Examples of the labeling agent include radioisotopes such as ¹²⁵ I (hereinafter referred to as RI), enzymes, enzyme substrates, luminescent substances, fluorescent substances, biotin, coloring substances and the like. The maleimide method [J. Biochem. (1976), 7
9, 233], activated biotin method [J. Am. Che
m. Soc. (1978), 100, 3585], the hydrophobic binding method and the like are used.

Examples of the enzyme include peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase and the like.
As the substrate used at this time, one suitable for the selected enzyme may be selected, and examples thereof include ABTS and luminol-H ₂ O.
_2, o-phenylenediamine _-H 2 _{O 2} (for peroxidase), p-nitrophenyl phosphate, methylumbelliferyl phosphate, 3- (2'-spiroadamantane) -4-methoxy-4- (3 "- phosphoryloxy ) Phenyl-1,2-dioxetane (for alkaline phosphatase), p-nitrophenyl-β-D-galactose, methylumbelliferyl-β-D-galactose (for β-galactosidase) and the like can be mentioned.

The above-mentioned measurement can be carried out by reacting at 4 to 40 ° C. for 1 minute to 18 hours, and measuring the produced coloration, fluorescence amount, luminescence amount or coloring amount. In addition, 4 to 40 ° C.
A so-called rate method may be employed in which heating is performed within the range.

Radiolabeling to the above-mentioned antigen or antibody can be easily carried out by using a commercially available Bolton Hunter reagent. For example, by adding a Bolton-Hunter reagent to an antigen or antibody solution dissolved in a 0.1 M sodium hydrogencarbonate aqueous solution and after 1 to 2 hours, the unreacted Bolton-Hunter reagent is removed using a G-25 desalting column or the like. It can be carried out. In addition, radiolabeling with ¹²⁵ I can be easily performed by employing the chloramine T method, the iodozine method, or the like.

Examples of the light emitting substance include isoluminol and acridine ester, and examples of the fluorescent substance include fluorescein and rhodamine. At this time, the labeling method can be easily performed by employing the activated ester method or the isocyanate method (“Enzyme-linked immunosorbent assay”, Medical Shoin, 1987). Examples of the coloring substance include colored latex particles and gold colloid.

To carry out the above-mentioned competitive method as the immunological assay method of the present invention, for example, an unknown amount of Helicobacter
A sample containing Helicobacter pylori catalase is added to the solid phase to which the monoclonal antibody of the present invention is bound by a known means physically or chemically and reacted. At the same time, a certain amount of Helicobacter pylori catalase labeled with a labeling agent is added and reacted.

In order to carry out the above-mentioned sandwich method as the immunological assay method of the present invention, for example, a sample containing an unknown amount of Helicobacter pylori catalase is physically or chemically tested by a known method. The reaction is performed by adding to the solid phase bound with the monoclonal antibody of. Then, the monoclonal antibody of the present invention labeled with a labeling agent is added and reacted.

Then, in any method, the solid phase is thoroughly washed, if necessary, and the activity of the labeling agent bound to the solid phase is measured. When the labeling agent is RI, it is measured using a well counter or a liquid scintillation counter. If the labeling agent is an enzyme,
The substrate is added and left to stand, and the enzyme activity is measured by a colorimetric method or a fluorescence method. Even if the labeling agent is a fluorescent substance, a luminescent substance, or a coloring substance, measurement is performed according to known methods.

Since the immunological assay method of the present invention uses the monoclonal antibody of the present invention, it can recognize an epitope specifically present in Helicobacter pylori catalase, and crosses other substances. It has an extremely excellent feature that it is possible to carry out a measurement with extremely high specificity without being erroneously detected due to a reaction.

The diagnostic kit of the present invention can be used to carry out the immunological assay method of the present invention. The diagnostic kit of the present invention contains at least one kind of the monoclonal antibody of the present invention, and may contain only one kind of monoclonal antibody. The monoclonal antibody of the present invention used in the diagnostic kit of the present invention is not particularly limited as long as it recognizes Helicobacter pylori catalase, and F (ab ′) ₂ , which is a degradation product of the monoclonal antibody of the present invention, It may be Fab ′, Fab or the like.

The diagnostic kit of the present invention comprises a Helicobacter
The presence of Helicobacter pylori or Helicobacter pylori catalase can be detected immunologically, and the infection to Helicobacter pylori is determined. In the diagnostic kit of the present invention, the monoclonal antibody of the present invention may be immobilized in advance, or the monoclonal antibody of the present invention may be labeled with the above labeling agent in advance.

The solid phase used in the diagnostic kit of the present invention is not particularly limited, and examples thereof include polymers such as polystyrene, glass beads, magnetic particles, microplates, filter paper for immunochromatography, and insoluble carriers such as glass filters. it can.

The diagnostic kit of the present invention may further contain other components. The above-mentioned other components are not particularly limited, and examples thereof include an enzyme used for labeling, a substrate thereof, a radioisotope, a luminescent substance, a fluorescent substance, a coloring substance, a buffer solution, a plate, and the like. The ones listed can be used.

The form of the diagnostic kit of the present invention is not particularly limited, but for quick and simple diagnosis, it is preferable that the diagnostic kit of the present invention is an integrated diagnostic kit in which the constituent components are integrated. . The integrated diagnostic kit is not particularly limited, and examples thereof include a cassette type and a cartridge type.

Examples of the above cassette type embodiment include:
Using the immunochromatography method, the membrane is housed in the reaction cassette, the monoclonal antibody of the present invention is immobilized on one end (downstream side) of the membrane, and the other end (upstream side) of the membrane is immobilized. ) Is loaded with a developing solution, a pad to which the substrate of the labeling agent is added is arranged on the downstream side in the vicinity thereof, and a book labeled with the labeling agent is provided in the middle part of the membrane. Examples include a mode in which a pad to which the monoclonal antibody of the invention is added is arranged.

When the diagnostic kit having the cassette type embodiment exemplified above is used, the sample is added onto the pad to which the monoclonal antibody of the present invention labeled with the above-mentioned labeling agent is added, and the Helicobacter After forming a conjugate of Helicobacter pylori catalase and the monoclonal antibody of the present invention labeled with the labeling agent, when the developing solution is developed, the formed conjugate is immobilized with the monoclonal antibody of the present invention. A complex of Helicobacter pylori catalase, the monoclonal antibody of the present invention labeled with the above-mentioned labeling agent, and the immobilized monoclonal antibody of the present invention is formed therein. Next, the labeling agent reacts with the substrate to develop color. By detecting this color development or the like, it is possible to determine the infection with Helicobacter pylori.

In the case of adopting a mode in which the sample is diluted with a sufficient amount of developing solution and then dropped on the membrane, the developing solution need not be mounted on the membrane in advance.
Further, when using a colored substance such as colored latex particles as the labeling agent, no substrate is required, and whether or not the complex is formed at the position where the monoclonal antibody of the present invention is immobilized is colored. It is judged by the coloring of the substance.

As the above-mentioned cartridge type embodiment, for example, when the reaction is the above-mentioned competitive method, it is a cartridge having a plurality of wells, (a) a well containing the monoclonal antibody of the present invention, (b) Liquid reagent containing Helicobacter pylori etc. labeled with the above labeling agent (eg:
Examples include a cartridge in which a well containing a buffer solution) and (c) a well containing a liquid reagent (eg, a buffer solution) containing a substrate of the above labeling agent are integrally formed. Is a cartridge having a plurality of wells in the case of sandwich method,
(A) a well containing an insoluble carrier in which the monoclonal antibody of the present invention is immobilized, (b) a liquid reagent containing the monoclonal antibody of the present invention labeled with the above labeling agent (eg:
Examples thereof include a cartridge in which a well containing a buffer solution) and (c) a well containing a liquid reagent (eg, a buffer solution) containing a substrate for the labeling agent are integrally formed.

When the cartridge-type diagnostic kit of the present invention exemplified above is used, the reaction and the measurement can be carried out in the same manner as when carrying out the competitive method or the sandwich method. The diagnostic kit of the present invention may carry out any of the above-mentioned competitive method and the above-mentioned sandwich method, but preferably carries out the above-mentioned sandwich method. The sandwich method has the advantages of high sensitivity, short reaction time, and excellent accuracy.
When the immunochromatography method is used as the sandwich method, it is possible to prepare a kit in which the test operation is simple and the determination of the result can be easily performed visually.
In addition, when the above sandwich method is used as an ELISA, an enzyme reaction product is produced depending on the amount of the substance to be measured, so that a system that allows visual confirmation of color development etc. when Helicobacter pylori infection is positive Is easy to set.

The sample used in the diagnostic kit of the present invention is not particularly limited, and examples thereof include gastric contents, gastric lavage fluid, gastrointestinal excretion, etc., but it is easy to collect and the burden on the subject is small. Therefore, gastrointestinal excrement such as feces is preferable. The diagnostic kit of the present invention may be used for examining the presence or absence of infection before eradication treatment, and may be used for determining the success or failure of eradication treatment.

Since the diagnostic kit of the present invention uses a monoclonal antibody, there is no difference between lots and the detection sensitivity is high,
No false negative or false positive problems occur. According to the present invention, by using a monoclonal antibody whose antigen is Helicobacter pylori catalase, it is possible to eliminate the influence of other substances in the sample, so that the presence of Helicobacter pylori can be detected with extremely high sensitivity and specificity. can do. In addition, the establishment of a monoclonal antibody-producing hybridoma against Helicobacter pylori catalase makes it possible to semi-permanently produce the same monoclonal antibody. Since the diagnostic kit using the monoclonal antibody of the present invention can use gastrointestinal excretion as a sample, Helicobacter pylori infection can be detected easily and efficiently without causing pain to the subject. In addition, the diagnostic kit using the monoclonal antibody of the present invention exhibits extremely excellent accuracy even when only one type of monoclonal antibody is used, is stable because there is no difference between lots, and is therefore unique. The Helicobacter pylori infection can be detected accurately and accurately. In addition, the above-mentioned diagnostic kit is easy to measure and is very useful in the medical field.

[0077]

EXAMPLES The present invention will be described in more detail below with reference to Examples and Test Examples, but the present invention is not limited to these.

Example 1 [Preparation of Monoclonal Antibody] (1) Preparation of Helicobacter pylori Spherical Cell Suspension (Immunogen) Brain Heart Infusion Agar Medium (manufactured by Difco) supplemented with 5% horse defibrinated blood Helicobacter pylori (ATCC 43504) was streaked on the top. After culturing this plate in a microaerobic environment at 37 ° C for 3 to 4 days, the plate was further incubated in an anaerobic environment at 37 ° C for 7 days to be spherical. The obtained colonies were scraped off with a platinum loop or the like, and suspended in phosphate buffered saline (PBS). At 4 ° C
The Helicobacter pylori cells, which had been centrifuged at 10,000 × g for 10 minutes, were suspended in 0.5% formalin and left at 4 ° C. for 4 days to be inactivated. Then, the operation of suspending in PBS and centrifuging at 10,000 × g for 10 minutes at 4 ° C. was repeated 3 times to wash the above-mentioned bacterial cells and then suspend in PBS.

(2) Preparation of Helicobacter pylori globular microbial cell lysate (immunogen) The microbial cell suspension obtained in (1) was sonicated (Seiko Denshi Kogyo, Model 7250). O
The cells were disrupted by ultrasonication for 10 minutes under the conditions of output 3 and 50% duty cycle.

(3) Immunity and cell fusion (1), immunogen prepared in (2) (Helicobacter.
A suspension of Helicobacter pylori or a disrupted product of Helicobacter pylori) and Freund's complete adjuvant (manufactured by Calbiochem) were mixed in an equivalent amount to obtain an oil emulsion. 0.2 mL of this was subcutaneously administered to the back of BALB / cA mice (CLEA Japan, 6 weeks old, female). Booster immunization was performed on the 7th and 14th days after the first immunization, and 0.2 mL of the above immunogen was intraperitoneally administered 3 days before cell fusion. Mouse spleen cells on the third day after the final immunization were extracted, and myeloma cells (P3x63.Ag8.653 strain, RC
B0146, RIKEN GENE BANK) was mixed at a ratio of 10: 1 and fused with 50% polyethylene glycol 4000, and then hybridomas were selectively cultured in HAT medium (manufactured by Gibco).

(4) Selection of hybridoma 12 days after cell fusion, the antibody activity in the culture supernatant was determined by ELIS.
Measured by A. 200 μL of the culture supernatant of fused cells was added to each well of a 96-well ELISA plate (manufactured by Coaster) on which 10 μg / mL of immunogen was immobilized, and the mixture was incubated at 37 ° C.
After reacting for a period of time, it was washed with PBS containing 0.05% Tween 20 (washing solution) and labeled with peroxidase-anti-mouse IgG.
200 μL (manufactured by Cappel, 1: 20,000) was added. After reacting at 37 ° C. for 1 hour, it was washed with the above washing solution. After washing, a substrate solution (0.1 M o-phenylenediamine and 0.012% hydrogen peroxide solution) was added to each well in an amount of 200 μL and reacted at room temperature for 15 minutes. After the reaction, 50 μL of 3.5 N sulfuric acid was added to each well to stop the enzyme reaction,
The absorbance value at 492 nm was measured. Absorbance value is 0.1
Hybridoma clones producing antibodies reactive with the above immunogens, which showed 5 or more, were selected. Each clone was cloned twice by the limiting dilution method. As a result of transplanting the hybridoma after cloning into BALB / cA mice, 32 clones of hybridoma produced a monoclonal antibody that could be collected as ascites fluid.

Example 2 [Selection of monoclonal antibody specifically recognizing Helicobacter pylori in digestive tract excretion by sandwich ELISA] (1) Preparation of monoclonal antibody-immobilized plate 1 mL of ascitic fluid of 32 clones each with PBS Dilute 2 times,
2 mL of saturated ammonium sulfate was added dropwise and the mixture was left at 4 ° C. for 4 hours. After that, centrifugation was performed at 3000 rpm for 20 minutes, and the precipitate was suspended in 2 mL of PBS and dialyzed. These monoclonal antibodies were subjected to 96-well ELI by the following method.
It was immobilized on an SA plate. That is, after diluting each monoclonal antibody to 5 μg / mL, 0.2 mL was added to each well of a 96-well ELISA plate and left overnight at 4 ° C.
It was washed with PBS. After washing, 1% skim milk-PBS
0.25 mL was added to each hole, and masking was performed by leaving at 4 ° C. for 1 hour. After masking, it was washed with the above washing solution.

(2) Preparation of Biotin-Labeled Monoclonal Antibody 3 mg of the monoclonal antibody of 32 clones prepared in (1) was mixed with 10 mg of biotinyl-N-hydroxysuccinimide ester (Zymed) and 0.1 M hydrogen carbonate was added. The reaction was carried out in sodium (pH 8) at room temperature with stirring for 3 hours. Reaction solution at 4 ° C against 5 L of PBS
It was dialyzed overnight with to obtain a biotin-labeled monoclonal antibody.

(3) Helicobacter in digestive tract excrement
Selection of Monoclonal Antibody that Recognizes Helicobacter Pylori 250 mg of fecal sample of 1 person each of which was determined to be Helicobacter pylori positive and negative by urea breath test
After suspending in 0.5 mL of 1% skim milk-PBS, 30
The supernatant obtained by centrifugation at 00 rpm for 10 minutes was collected and used as a fecal extract. 0.2 mL of the fecal extract was added to each well of each monoclonal antibody-immobilized plate prepared in (1). After standing at 37 ° C. for 1 hour, the plate was washed 5 times with the above washing solution, and 0.2 mL of each biotin-labeled monoclonal antibody prepared in (2) was added. After standing at 37 ° C. for 1 hour, the plate was washed with the above washing solution, and 0.2 mL of peroxidase-labeled avidin (manufactured by Zymed) was added. 1 at 37 ° C
After leaving for a while, the substrate solution (0.1M
0.2 mL each of o-phenylenediamine and 0.012% hydrogen peroxide solution) was added to each hole, and the mixture was reacted at room temperature for 10 minutes. After the reaction, 50 μL of 3.5 N sulfuric acid was added to each well to stop the enzyme reaction, and the absorbance value at 492 nm was measured. The results are shown in Table 1.

[0085]

[Table 1]

As shown in Table 1, the sandwich ELISA in which three monoclonal antibodies 21G2, 41A5 and 82B9 were used alone or in combination was found to show high reactivity to fecal samples of Helicobacter pylori infected persons. . The hybridoma producing each monoclonal antibody was hybridoma 21G2 (FERM BP-
7336), hybridoma 41A5 (FERM BP
-7337) and hybridoma 82B9 (FER
MBP-7338) has been deposited with the Institute of Biotechnology, Industrial Technology Institute of Industrial Technology. The immunoglobulin subclass of each monoclonal antibody was examined using an immunoglobulin typing kit mouse (manufactured by Wako Pure Chemical Industries, Ltd.). As a result, 3 clones were IgG ₁ and the L chain was κ type.

Example 3 [Comparison of Reactivity with Strains and Reactivity with Other Bacteria] (1) Preparation of Helicobacter pylori Cell Suspension Brain Heart Infusion Agar Medium supplemented with 5% horse defibrinated blood Helicobacter pylori (ATCC43
504; Tokai University Hospital Clinical Isolate No. 504; 130 and
No. 112; Hyogo College of Medicine clinical isolate No. 526,
No. 4484, No. 5017, No. 5025, N
o. 5049, No. 5142, No. 5287, N
o. 5308, No. 5314, and No. 533
0) was streaked. This plate at 37 ° C for 3-4
Helical fungus colony obtained by microaerobic culture for a day,
Alternatively, a spherical fungus colony obtained by leaving it at 37 ° C in an anaerobic environment for 7 days is scraped off with a platinum loop or the like,
Suspended in. The cells that had been centrifuged at 10,000 × g for 10 minutes at 4 ° C. were suspended in 0.5% formalin and left at 4 ° C. for 4 days to be inactivated. Then, suspend in PBS,
The operation of centrifuging at 10,000 xg for 10 minutes at 4 ° C is repeated 3 times, and after washing the cells, the cells are suspended again in PBS to suspend Helicobacter pylori spiral cell suspension and spherical cell suspension. A liquid was obtained.

(2) Preparation of bacterial suspension of human intestinal bacterium and Campylobacter jejuni Two typical bacterial species of intestinal bacterium Bacteroides vulgatus and Escherichia coli separated from human feces and spiral form Campylobacter jejuni, which is a bacterium, was cultured by the following method, and a microbial cell suspension of each bacterium was prepared from the obtained colonies by the same method as (1). That is, Bacteroides vulgartus (IFO 14291) was anaerobically cultured at 37 ° C. for 2 days on a BL agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) supplemented with 5% horse defibrinated blood. Escherichia coli (ATCC
25922) was aerobically cultured on a brain heart infusion agar medium at 37 ° C. for 1 day. Campylobacter jejuni (80068 Tokai University Hospital Clinical Isolate) was microaerobically cultured on Brain Heart Infusion agar medium supplemented with 5% horse defibrinated blood at 37 ° C. for 2 days.

(3) Preparation of cell suspension of other Helicobacter genus Helicobacter felis (ATCC49179) and Helicobacter hepaticas (ATCC51448) on a Brain Heart Infusion agar medium supplemented with 5% horse defibrinated blood ( Culturing was carried out in the same manner as in 1), and a spiral bacterial cell suspension was obtained in the same manner as in (1) from the obtained colony of spiral fungi.

(4) Preparation of disrupted cell bodies Each cell suspension obtained in (1), (2) and (3) was
Ultrasonic crusher (Seiko Denshi Kogyo, Model 7
250), output 3, 50% duty
The cells were sonicated under cycle conditions for 10 minutes to disrupt the cells.

(5) Sandwich ELISA Each cell crushed product obtained in (4) (protein concentration 10 μm
g / mL) 0.2 mL of sandwich ELISA (monoclonal antibody 41A5 immobilized plate: biotin-labeled monoclonal antibody 82B9, monoclonal antibody 21G2 immobilized plate: biotin-labeled monoclonal antibody 41A5, respectively) by the method of Example 2 (3). Monoclonal antibody 21 G2 immobilized plate: biotin-labeled monoclonal antibody 82B9, monoclonal antibody 21
G2 immobilized plate: biotin-labeled monoclonal antibody 21G2 and HpSA manufactured by Meridian) were used. The results are shown in Table 2.

[0092]

[Table 2]

As shown in Table 2, monoclonal antibody 2
It was found that 1G2, 41A5, and 82B9 show high reactivity with respect to the helicoidal cells and the spherical cells of each strain of Helicobacter pylori. On the other hand, it did not react at all against Bacteroides burgatas, Escherichia coli, Campylobacter jejuni, Helicobacter felis, and Helicobacter hepaticas.
On the other hand, Meridian HpSA showed reactivity with Helicobacter felis and Helicobacter hepaticas.

Example 4 [Detection of Helicobacter pylori in Fecal Samples by Sandwich ELISA (1)] As a result of the urea breath test, 250 mg of fecal samples provided by each of three positive and negative Helicobacter pylori samples were analyzed. The suspension was suspended in 0.5 mL of 1% skim milk-PBS and tested in the same manner as in Example 2 (3). Further, the same stool sample was tested according to the operating method using Meridian HpSA ELISA, and the absorbance value at 450 nm was measured. Table 3 shows the respective absorbance values and the urea breath test results.

[0095]

[Table 3]

The fecal specimens provided by the subjects (No. 4, 5, 6) having a positive result of the urea breath test show remarkably high absorbance values as compared with the negative specimens (No. 1, 2, 3). I understood it.

Example 5 [Detection of Helicobacter pylori in fecal sample by sandwich ELISA (2)] (1) Preparation of monoclonal antibody Hybridoma 21G2 was transplanted to BALB / cA mice and 5 ml of ascites collected was doubled with PBS. The mixture was diluted, 10 mL of saturated ammonium sulfate was added dropwise, and the mixture was left at 4 ° C. for 4 hours. Then, centrifuge at 3000 rpm for 20 minutes,
The precipitate was dissolved in 10 mL of PBS and then dialyzed against PBS.

(2) Preparation of monoclonal antibody-immobilized plate The monoclonal antibody 21G2 solution prepared in (1) was immobilized on a 96-well ELISA plate by the following method. After diluting with PBS to an antibody concentration of 5 μg / mL,
Add 0.2 mL to each well of a 6-well ELISA plate, 4
After being left overnight at ℃, washed with PBS.

(3) Preparation of Peroxidase-Labeled Monoclonal Antibody A peroxidase-labeled monoclonal antibody was prepared using the maleimide method (Eiji Ishikawa, Biochemistry Experimental Method 27, Enzyme Labeling Method, p. 51, 1991, Academic Publishing Center). 5 mg of the monoclonal antibody prepared in (1) and S
-Acetylmercaptosuccinica
nhydride (manufactured by Aldrich Co.) 0.6 mg is mixed, and 0.1 M phosphate buffer (pH 6.5) 0.5 mL
The reaction was carried out at 30 ° C. for 30 minutes. 0.1M in the reaction solution
EDTA 20 μL, 0.1 M Tris-HCl buffer (pH
7.0) 0.1 mL, 1M hydroxylamine (pH
7.0) Add 0.1 mL, leave at 30 ° C. for 5 minutes,
The supernatant was obtained by centrifugation at 10,000 rpm for 5 minutes. The supernatant was subjected to ultrafiltration with Centricon 30 (manufactured by amicon) to remove the reagent, and then 5 mM EDTA-0.1M
It was replaced with 1 mL of a phosphate buffer (pH 6.5) to obtain a thiol group-introduced monoclonal antibody.

5 mg of peroxidase (horseradish, Toyobo Co.) and N-succinimidyl-4- (N
-Maleimidomethyl) -cycle
xane-1-carboxylate- (ICN B
1 mg (manufactured by iomedicals) is mixed, and 0.1M
In 0.5 mL of phosphate buffer (pH 7.0) at 30 ° C.,
The reaction was carried out for 1 hour. The reaction solution was centrifuged at 10,000 rpm for 5 minutes to obtain a supernatant. The supernatant was subjected to ultrafiltration with Centricon 30 to remove the reagent, and then replaced with 1 mL of 0.1 M phosphate buffer (pH 7.0) to obtain a maleimide group-introduced peroxidase.

The thiol group-introduced monoclonal antibody and the maleimide group-introduced peroxidase were mixed at a molar ratio of 1: 5 and reacted at room temperature for 30 minutes. The reaction solution is Seph
gel filtration column of acryl-S300HR column (diameter 26 x length 870 mm, 0.1 M phosphate buffer (pH
6.5)), and the fractions containing the peroxidase-labeled monoclonal antibody were collected.

(4) Detection of Helicobacter pylori in Fecal Samples by Sandwich ELISA 250 mg of fecal samples from each of 10 persons who were determined to be Helicobacter pylori-positive and negative Helicobacter pylori in the urea breath test were treated with 0.1% skim milk-PBS. After suspending in 5 mL,
The supernatant obtained by centrifugation at 3000 rpm for 10 minutes was collected and used as a fecal extract. 50 μL of fecal extract and 50 μL of peroxidase-labeled monoclonal antibody prepared in (3)
Was added to each well of the monoclonal antibody-immobilized plate. After leaving it at 25 ° C. for 1 hour, it was washed 5 times with a washing solution (0.05% Tween 20-PBS), and then a substrate solution (tetramethylbenzidine + hydrogen peroxide, manufactured by BioFX) 0.1.
mL was added to each well and reacted at room temperature for 10 minutes. After the reaction, 50 μL of 1N sulfuric acid was added to each well to stop the enzyme reaction, and the absorbance value (450 nm-630 nm) was measured.
Further, the same fecal sample was tested using HpSA, and the absorbance value (450 nm-630 nm) was measured. The results are shown in Table 4.
It was shown to.

[0103]

[Table 4]

As shown in Table 4, monoclonal antibody 2
The sandwich ELISA using 1G2 alone showed high reactivity to fecal samples of Helicobacter pylori-infected persons, and was completely in agreement with the determination result by the urea breath test. On the other hand, with HpSA, 2 samples (sample numbers 16 and 19)
Was a mismatch (false positive).

Example 6 [Detection of Helicobacter pylori in feces by immunochromatography method] (1) Preparation of antibody-immobilized support Linearly immobilized antibody of anti-Helicobacter pylori monoclonal antibody and anti-rabbit IgG antibody To prepare a solid-phased support, a nitrocellulose sheet (manufactured by Whatman) was cut into 5 mm × 20 mm, and 10 was cut from the lower end thereof.
solution of monoclonal antibody 21G2 at 15 mm position, 15
A solution of an anti-rabbit IgG goat polyclonal antibody (manufactured by Cappel) is placed at the position of mm in Biojet Q300.
0 (manufactured by Biodot) was used for coating. After drying at room temperature for 2 hours, 1% skim milk (manufactured by Difco) -0.
Masking was carried out by immersing in 1% Tween 20-PBS for 10 minutes. Then, it was sufficiently dried.

(2) Preparation of colored latex particle label a. Anti-Helicobacter pylori antibody labeled with red latex particles Red latex particle dispersion (PL-Latex, 10
%, 450 nm, Polymer Laborator
1.2 mL of PBS was added to 300 μL of 1)
Centrifugation was performed at 3000 rpm for 5 minutes. To the precipitate, 1 mL of the monoclonal antibody 21G2 solution (5 mg / mL) was added, mixed well, and reacted at room temperature for 1 hour. 13,000r to remove unreacted monoclonal antibody
pm, centrifuge for 5 minutes, and deposit with 1.5 mL of PBS.
The cells were suspended in and centrifuged again. 1% skim milk 1
mL was added, and masking was performed by reacting at room temperature for 1 hour. Then, centrifugation was performed at 13000 rpm for 5 minutes, and the precipitate was suspended in 1.5 mL of PBS containing 1% skim milk-0.01% sodium azide.

B. Rabbit IgG labeled with blue latex particles Blue latex particle dispersion (PL-Latex, 10
%, 450 nm, Polymer Laborator
ies) and rabbit IgG (0.5 mg / mL, C
A blue latex particle-labeled rabbit IgG was prepared by the same procedure as above using an Appel company).

C. Colored Latex Particle Labeled Product An equal amount of the above-mentioned two types of colored latex particle labeled antibodies is mixed, and Benmize (registered trademark) non-woven fabric (manufactured by Asahi Kasei Corporation) 5 m
10 μL was impregnated into m × 5 mm and dried by ventilation.

(3) Preparation of immunochromatographic test strip A colored latex label was placed up to 2.5 mm from the lower end of the antibody-immobilized support. Further, a carrier for dipping a test liquid (3MM Chr, manufactured by Whatman) was placed on the colored latex labeled material up to a position 2.5 mm from the lower end. Also,
A water-absorbent carrier (3MM Chr, manufactured by Whatman) was stacked up to a position 2 mm from the upper end of the antibody-immobilized support, and finally, a transparent tape was attached and fixed on the top to give an immunochromatographic test piece.

(4) Detection of Helicobacter pylori in Fecal Samples Feces of 6 persons (3 persons each for positive and negative) described in Example 4 were used for the test. Collect 0.1g of each stool, 0.1% B
Suspended in 1 mL of SA-0.05% Tween 20-PBS. Impurities were removed by centrifugation at 3000 rpm for 1 minute, and 50 μL of the supernatant was added dropwise to the carrier for immersing the test solution in the immunochromatographic test piece prepared in (3). 10
After a minute, the presence or absence of a red line appearing at the immobilized site of the anti-Helicobacter pylori monoclonal antibody was determined. As a result, the red line could not be confirmed in all the negative samples, and the red line could be confirmed in all the positive samples. The test was successful because the blue line was confirmed in all the samples.

Example 7 [Detection of Helicobacter pylori in Feces by Latex Aggregation Method] (1) Preparation of Latex Particle-Labeled Anti-Helicobacter pylori Antibody White latex particle dispersion (PL-Latex, 10
%, 440 nm, Polymer Laborator
0.4 ml of PBS was added to 0.1 ml of ies), and 1
Centrifugation was performed at 3000 rpm for 5 minutes. PB in the sediment
S0.5mL and monoclonal antibody 21G2 solution (1
(mg / mL) 0.5 mL, mixed well, at room temperature,
The reaction was carried out overnight. In order to remove unreacted monoclonal antibody, centrifugation was performed at 13000 rpm for 10 minutes, the precipitate was suspended in 1 mL of PBS, and centrifugation was performed again. Masking was performed by adding 1 mL of 1% skim milk and reacting at room temperature for 1 hour. After that, 13000 rp
Centrifuge for 5 minutes and remove the sediment with 1% skim milk-
It was suspended in 1 mL of PBS containing 0.01% sodium azide.

(2) Detection of Helicobacter pylori in Fecal Samples The feces of 6 persons (3 persons each for positive and negative) described in Example 4 were used for the test. Collect 0.1g of each stool, 0.1% B
Suspended in 1 mL of PBS containing SA-0.05% Tween 20. The mixture was centrifuged at 3000 rpm for 1 minute to remove impurities and obtain a supernatant. 50 μL of this stool suspension supernatant and 50 μL of latex particle-labeled anti-Helicobacter pylori antibody prepared in (1) were dropped on a latex agglutinator,
It mixed using the slide rotor (Eiken Chemical Co., Ltd.).
After 5 minutes, the presence or absence of aggregation was visually determined. As a result, it was not possible to confirm aggregation in all cases in the negative sample, and it was possible to confirm aggregation in all cases in the positive sample.

Example 8 [Molecular Weight Measurement of Fecal Antigen by Gel Filtration Method] Helicobacter pylori positive (No. 4 and No. 5 in Table 3)
PBS100 ice-cooled 20g of stool sample provided by
Suspended in mL. The residue was removed by centrifugation at 10,000 xg for 10 minutes, and further centrifuged at 90,000 xg for 30 minutes to obtain a supernatant. 1.5 mL of this supernatant is added to Se
gel filtration column (1.5 × 140 cm, 0.1) packed with phacryl-S300HR (manufactured by Pharmacia).
M phosphate buffer, pH 6.5) and fractionated every 1.5 mL. Molecular weight markers include thyroglobulin,
Ferritin, catalase, bovine serum albumin and cytochrome c were used. Sandwich ELISA described in Example 2 (3) (monoclonal antibody 41A5 immobilized plate, biotin-labeled monoclonal antibody 82B9,
Also, the antigen of each fraction was detected with a monoclonal antibody 21G2 immobilized plate and a biotin-labeled monoclonal antibody 41A5). As a result, the molecular weight of the Helicobacter pylori-specific antigen in the feces was found to be 270 kDa.

Example 9 [Identification of antigen] (1) Purification of antigen Monoclonal antibody 82B9 was treated with CNBr-activated Sep by the method described in Amersham Pharmacia Biotech Affinity Chromatography Handbook.
A column immobilized on harose 4B (manufactured by Amersham Pharmacia Biotech) was prepared (10 mL). After sonicating the Helicobacter pylori (ATCC 43504) cells, 5 mL of the supernatant (protein concentration 4 mg / mL) obtained by ultracentrifugation was loaded on this column. After standing at room temperature for 2 hours, PBS 120 mL (flow rate about 2 mL
/ Min) to wash the column with 0.2 M glycine HCl
Elution was performed with 130 mL of buffer solution (pH 3.0). Washing,
The elution was fractionated by 10 mL, and the absorbance at 280 nm and the antigenicity were measured. The antigenicity was measured by sandwich ELISA (immobilized 21G2, labeled 82B) described in Example 2.
9). As a result, as shown in Fig. 1, the antigenicity was confirmed only in the eluted fractions (fraction Nos. 14 to 17).

(2) Purity of purified antigen and 50 μL of molecular weight elution fraction (No. 14) were mixed with an equal volume of sample buffer (2% SDS-5% mercaptoethanol) and boiled at 100 ° C. for 5 minutes. . Using 20 μL of this solution, SDS-polyacrylamide gel electrophoresis (4-2
0% acrylamide gel). The molecular weight markers used were phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and α-lactalbumin. After the electrophoresis, the gel was stained with Silver Stain KANTO III (manufactured by Kanto Chemical Co., Inc.). As a result, the purified antigen showed a single band and its molecular weight was 59 kDa. Further, 1 mL of the elution fraction (No. 14) was subjected to gel filtration of Sephacryl-S300HR by the same method as described in Example 6. About the obtained fractions, sandwich EL
As a result of examining the antigenicity by ISA, it was found that the molecular weight of this purified antigen was 270 kDa, which was the same as the fecal antigen.

(3) Determination of the amino-terminal amino acid sequence of the purified antigen Using 300 μL of the elution fraction (No. 14), HP G1
005A Protein Sequencing S
The amino acid sequence on the amino-terminal side of the purified antigen was determined by a system (manufactured by Hewlett-Packard Japan). The sequence of 8 residues from the amino terminus is Met-Val-Asn.
It was -Lys-Asp-Val-Lys-Gln.
This sequence is from the amino terminus of Helicobacter pylori catalase (J. Bacteriol.
96), 178, 6960-6967).

(4) Measurement of Catalase Activity and UV-Visible Absorption Spectrum of Purified Antigen Since the amino terminal amino acid sequence of the purified antigen coincided with that of catalase, the catalase activity of each fraction was measured. PBS containing 11 mM hydrogen peroxide was used as the reaction solution. After diluting each fraction 100-fold with PBS, 50 μL thereof was added to 2 mL of the reaction solution to start the reaction. The reaction was performed at room temperature. The absorbance at 240 nm was measured over time, and the decrease in absorbance per minute was determined. PBS for blank
Was used. As a result, as shown in FIG. 1, catalase activity was detected in the antigen fraction.

To confirm the heme contained in catalase, the elution fraction No. The UV-visible absorption spectrum of 14 was measured. As a result, absorption maxima were observed at 407 and 277 nm. This value is the literature value of Helicobacter pylori catalase (405 and 280 nm, J. Gen. Mic.
robiol. (1991), 137, 57-61).

(5) Storage stability of purified antigen A solution obtained by diluting the eluted fraction (No. 14) with PBS containing 0.1% BSA at a concentration of 1.4 μg / mL was added to 25
It was stored at ℃ for 1 week. The antigenicity of the purified antigen after storage was measured by the sandwich ELISA method described in Example 5, and the catalase activity was measured by the method described in (4). As a result of comparison with the purified antigen cryopreserved at −80 ° C., the antigenicity hardly changed upon storage at 25 ° C. However, the catalase activity was significantly inactivated, and the residual activity rate was 5% or less.

Example 10 [Reactivity between Anti-Helicobacter pylori Monoclonal Antibody and Fecal Antigen Subunit] Anti-Helicobacter
In order to measure the reactivity between the Helicobacter pylori monoclonal antibody and the subunit of the fecal antigen, 50 μL of the Helicobacter pylori-positive fecal suspension supernatant described in Example 8 was used to describe in Example 9 (2). SDS-polyacrylamide gel electrophoresis was performed. As a control, 50 μL of the purified antigen of Example 9 (eluted fraction No. 14) was also electrophoresed in the same manner. After the electrophoresis, the protein was transferred to a nitrocellulose membrane by Western blotting. After immersing the nitrocellulose membrane in 1% skim milk for 1 hour to mask it, the monoclonal antibody 21G2 or 82B9 and 1 were used.
Reacted for hours. The nitrocellulose membrane was washed 5 times with 0.05% Tween 20-PBS and then reacted with a peroxidase-labeled anti-mouse IgG antibody. The nitrocellulose membrane was washed 5 times with 0.05% Tween 20-PBS, and a substrate solution (hydrogen peroxide + 3,3′-diaminobenzidine) was added and incubated for 10 minutes, but fecal antigen and purified antigen were electrophoresed. No color development due to antigenicity was observed in any of the lanes using any of the monoclonal antibodies. In order to clarify the reason why the subunit molecule was not found to be antigenic, it was examined by dot blotting whether the antigenicity was changed by dissociation into subunits. 5 μL of each of the specimens before and after the dissociation treatment with 1% SDS was dropped on a nitrocellulose membrane and air-dried. As a result of investigating the antigenicity of the protein adsorbed on the nitrocellulose membrane by the same method as above, it was found that both fecal antigen and purified antigen had 1% S
It was found that the antigenicity was significantly reduced after the dissociation treatment with DS. Therefore, it was suggested that the monoclonal antibody of the present invention does not recognize the subunit of catalase, that is, the epitope on the primary structure, but recognizes a more native higher-order structure as the epitope.

Example 11 [Purification of Fecal Antigen] (1) Molecular Weight of Fecal Antigen Fecal Helicobacter pylori antigen showed the highest value. Positive feces (Sample No. 8 in Table 4) provided 165 g of feces.
Suspended in 4 volumes of PBS and centrifuged (7000 rp
Ultracentrifugation (30 minutes)
(000 rpm, 30 minutes) to obtain a supernatant. 680m of supernatant
Ammonium sulfate (165 g) was added to L so as to be 40% saturated, and the mixture was stirred and the resulting precipitate was centrifuged (7,000 rpm, 30 minutes).
Removed by. Ammonium sulfate (214 g) was added to 750 mL of the supernatant so as to be 80% saturated, and the mixture was stirred, and the generated precipitate was collected by centrifugation (7,000 rpm, 30 minutes). The precipitate was suspended in PBS and dialyzed against 10 L of distilled water. Gel filtration using a Sephacryl-S300HR column was performed in the same manner as in Example 6 by using 2 mL of the dialyzed inner solution (82 mL). The obtained fraction was examined for antigenicity by a sandwich ELISA in the same manner as in Example 5. As a result, the molecular weight of the antigen in the present feces was 270 kDa, and the feces of the two positives described in Example 8 were confirmed. It was the same as the molecular weight of the antigen and the purified antigen described in Example 9.

(2) Purification of Fecal Antigen 40 mL of the dialysis inner solution of (1) was added with 10 mM phosphate buffer (p
C-Sephadex C-5 cation exchange resin equilibrated with the same buffer after diluting to 100 mL with H7.0).
0 (Amersham Pharmacia Biotech) column (1 × 2.5 cm) was loaded. After washing with 10 mL of the same buffer, it was eluted with 10 mL of PBS. 320% of the fractions of the tower, the wash, and the elution with PBS containing 0.1% skim milk.
Sandwich ELIS as described in Example 5 after doubling dilution
Antigenicity was measured by A. Antigenicity was found in the eluted fraction.

Monoclonal antibody 21G2 was treated with CNBr- by the method described in Amersham Pharmacia Biotech Affinity Chromatography Handbook.
A column immobilized on activated Sepharose 4B (manufactured by Amersham Pharmacia Biotech) was prepared (2
× 3 cm). 6 mL of the above-mentioned elution fraction was loaded onto a monoclonal antibody-immobilized column and left standing at room temperature for 2 hours. PB
After washing with S50 mL, it was eluted with 0.2 M glycine HCl buffer (pH 3.0) and fractionated into 10 mL each. After diluting each fraction 10-fold with PBS containing 0.1% skim milk, the antigenicity was measured by the sandwich ELISA described in Example 5. Further, the catalase activity was measured by the method described in Example 9. As a result, as shown in Fig. 2, the antigenicity was observed in the elution fractions (fraction Nos. 7 to 8) immediately after the start of elution indicated by the arrow, and the catalase activity was also in agreement with the antigenicity. From the above, 4 in the feces
Which has 3 subunits and also has catalase activity
It was found that a live antigen was included.

[0124]

INDUSTRIAL APPLICABILITY Since the present invention has the above-mentioned constitution, it can provide a monoclonal antibody capable of recognizing an epitope specifically present in Helicobacter pylori catalase, and further, the monoclonal antibody of the present invention. Can be used to recognize Helicobacter pylori very specifically. In addition, since a monoclonal antibody-producing hybridoma that recognizes Helicobacter pylori catalase was successfully established, the same monoclonal antibody can be semipermanently produced. The diagnostic kit using the monoclonal antibody of the present invention can use gastrointestinal excretion as a sample, and can detect Helicobacter pylori infection simply and efficiently without causing pain to the subject. Moreover, the diagnostic kit using the monoclonal antibody of the present invention exhibits extremely excellent accuracy even when only one type of monoclonal antibody is used, is stable with no difference between lots, and is always specific. In addition, Helicobacter pylori infection can be detected accurately.

[Brief description of drawings]

FIG. 1 is a diagram showing an elution pattern of affinity chromatography in Example 9.

FIG. 2 is a diagram showing an elution pattern of affinity chromatography in Example 11.

Continuation of front page (51) Int.Cl. ⁷ identification code FI C12R 1:91) Microbial accession number FERM BP-7337 Microbial accession number FERM BP-7338 Preliminary examination subject application (56) References Flat 10-10128 (JP, A) Infection and Immunity, 65 (11), p. 4668-4674, 1997 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C07K 16/00-16/46 BIOSIS / MEDLINE / WPID S (STN)

Claims (2)

(57) [Claims] 1. 21G2 (FERM BP-733
6), 41A5 (FERMBP-7337), or 8
A hybridoma characterized in that it is 2B9 (FERM BP-7338). 2. A monoclonal antibody produced by the hybridoma according to claim 1 .