WO2002066631A1 - Antibodies recognizing helicobacter pylori and method of detecting helicobacter pylori - Google Patents

Abstract

Antibodies recognizing Helicobacter pylori and a method of detecting Helicobacter pylori are provided. A monoclonal antibody recognizing the core oligosaccharide moiety of Helicobacter pylori LPS; a monoclonal antibody recognizing an LPS antigen moiety carried by some S-type Helicobacter pylori strains which is the antigen moiety remaining after removing the Lewis antigen from the Helicobacter pylori S-type LPS O-antigen polysaccharide moiety; a monoclonal antibody recognizing an LPS antigen moiety carried commonly by S-type Helicobacter pylori strains which is the antigen moiety remaining after removing the Lewis antigen from the Helicobacter pylori S-type LPS O-antigen polysaccharide moiety; a monoclonal antibody recognizing low antigenic LPS of Helicobacter pylori strains; and a method of detecting Helicobacter pylori by using at least one of these antibodies.

Description

 TECHNICAL FIELD An antibody recognizing Helicobacter pylori and a method for detecting Helicobacter pylori

 The present invention relates to a monoclonal antibody recognizing the core oligosaccharide portion of LPS (lipopolysaccharide) of Helicobacter pylori, a hybridoma producing the antibody, and an LPS antigen (Helicopter) commonly owned by strains of S-type Helicobacter pylori. · H. pylori species-specific LPS), that is, helicobacter · Helicobacter · S-type LPS antigen of H. pylori, which is the antigen portion excluding the Lewis antigen, and is the LPS antigen that S-type helicobacter and H. pylori have in common Monoclonal antibody (anti-Helicobacter pylori species-specific monoclonal antibody) that specifically recognizes the part, and the hybridoma that produces the antibody, Lewis of the 〇-antigen polysaccharide part of the S-type LPS of Helicobacter pylori The LPS antigen portion of the S-type Helicobacter pylori strain, which is the antigen portion excluding the antigen, is recognized. A monoclonal antibody that recognizes the antibody, a hybridoma that produces the antibody, and a monoclonal antibody that recognizes the low antigenic LPS of low antigenic Helicobacter pylori / H. Pylori (low antigenic epitopes present in LPS) and the antibody that produces the antibody About hybridoma. Furthermore, the present invention relates to a reagent and a kit for detecting Helicobacter pylori containing one or several of these antibodies, a method for detecting Helicobacter 1 'pylori using one or several of these antibodies, and a kit thereof. . These reagents, kits and methods are effectively used for diagnosis of gastric diseases and the like.

Re-scenery technique fe

 Recent studies have revealed that Helicobacter pylori is a major cause of gastric ulcer, duodenal ulcer and chronic gastritis. Attention has also been focused on the relationship between Helicobacter pylori and gastric cancer.

Many species of the genus Helicobacter are known, and it is confirmed whether the bacteria isolated from the stomach biopsy are Helicobacter pylori (differentiated). H. pylori needs to be specifically detected.

 To this end, in addition to morphological observation with a microscope, it is necessary to carry out a number of tests, such as confirmation that enzymes such as urease, ribonuclease, and oxidase are positive, nalidixic acid resistance, and cephalotin sensitivity. Since a large amount of time and labor is required to perform these tests, it has been desired to develop a reagent that can easily detect Helicobacter pylori.

 It is also known that many Helicobacter pylori-infected patients have antibodies against LPS, which is a surface antigen of Helicobacter pylori, that is, that LPS contains Helicobacter pylori and a major epitope of H. pylori. Had been.

However, antibodies against Lewis antigen portion of LPS have been obtained as antibodies against L. pylori, but these were specific to each type of Lewis antigen. Therefore, multiple antibodies to each type of Lewis antigen (Le ^x , Le ^y , Le ^a , Le ^b , Le ^d ) were required to detect Helicobacter pylori widely regardless of strain.

 Therefore, development of a method for reliably detecting Helicobacter pylori with a smaller number of antibodies regardless of the strain of Helicobacter pylori and development of an antibody used in the method have been desired.

 In addition, recently, in relation to Helicobacter pylori lipopolysaccharide (LPS) and patient antibodies, Helicobacter pylori is a strain with a polysaccharide that raises the antibody titer to humans (highly antigenic Helicobacter pylori strain). , Which are classified into strains with non-elevated polysaccharides (low antigenic Helicobacter-1 and H. pylori), which have high antigenic LPS and low antigenic LPS, respectively.Low antigenic Helicobacter is a strain derived from cancer patients. (Shin-ichi Yokota et al .: Antigenicity of Helicobacter pylori lipopolysaccharide in humans, Endotoxin research 1-Basic and clinical-, Motoharu Kondo et al., Pp. 113-121, Nanade N. Edition, Tokyo, 1998).

Further, a reagent utilizing the same has been disclosed as a patent (publication number: JP-A-10-218900). According to the report, LPS, a strain of Helicobacter pylori from gastric cancer patients, is missing, but is present in helicobacter strains from patients with chronic gastritis, gastric ulcer, or duodenal ulcer. It tests malignancy using antibodies that recognize determinants. In other words, helicopters that do not react with this serum are said to be highly malignant. However, this method requires the preparation of LPS from the isolated Helicobacter pylori strain by hot phenol extraction or proteinase K treatment in the presence of sodium lauryl sulfate (SDS). In addition, the prepared LPS must be subjected to polyacrylamide electrophoresis or immobilized on a microplate for use in enzyme-linked immunosorbent assay (EIA). Furthermore, the antibody is reacted with these prepared LPS, and if negative, the malignancy is determined to be high. These tests require a lot of time and effort and emphasize negative rather than positive. Based on these facts, on the other hand, an antibody that recognizes an antigen specific to a low antigenic strain, that is, an antibody that recognizes a low antigenic epitope specifically present in LPS (low antigenic LPS) of Helicobacter pylori is used. The development of a simple reagent has been desired.

Disclosure of the invention

The present invention provides a monoclonal antibody for specifically and simply detecting Helicobacter pylori, a hybridoma that produces the antibody, a Helicobacter pylori, and a specific, simple, An object of the present invention is to provide a kit for detecting Helicobacter pylori specifically, simply and in a short time. Specifically, a monoclonal antibody recognizing the core oligosaccharide portion of LPS of Helicobacter pylori, a hybridoma producing the antibody, and an LPS antigen (Helicobacter p. H. pylori species-specific LPS), that is, the antigenic portion of the O antigen polysaccharide portion of the S-type LPS of Helicobacter pylori except for the Lewis antigen, and the LPS antigen portion that S-type Helicobacter pylori has in common Monoclonal antibody (specific monoclonal antibody specific for Helicobacter pylori) and the hybridoma producing the antibody, the Helicobacter pylori, the Lewis antigen in the 0-antigen polysaccharide portion of the S-type LPS of Helicobacter pylori Monoclonal that recognizes the LPS antigen portion of the S-type Helicobacter pylori strain, excluding the antigen portion The present invention relates to a null antibody, a hybridoma producing the antibody, a monoclonal antibody recognizing Helicobacter 1 'pylori low antigenic LPS, and a hybridoma producing the antibody. Furthermore, the present invention relates to a reagent for detecting Helicobacter pylori containing one or more of these antibodies, and one or more of these antibodies. It is intended to provide a method for detecting Helicobacter pylori using a species and a kit for detecting the same.

 The present inventors proceeded with the study of the serological classification of Helicobacter pylori (Japanese Patent Publication No. 2000-262291), and discovered that spleen cells of mice immunized with Helicobacter pylori 220-B as an antigen were A number of hybridomas were produced by fusion with a myeloma cell line (myeloma). A large number of monoclonal antibodies reactive with Helicobacter pylori were screened using the endotoxin (LPS) extracted from Helicobacter pylori as an antigen to produce monoclonal antibodies reactive widely with Helicobacter pylori strains. Was obtained. As shown in Fig. 1, Helicobacter pylori contains a normal strain called Smooth (S) with 〇_antigen polysaccharide and a rough (0-antigen polysaccharide) (R type) It was known that there was a rough type (R type) strain having LPS. Most Helicobacter pylori strains are smooth, and the 0-antigen polysaccharide is composed of an antigenic portion that is common to the Lewis blood group antigen that reacts with the terminal anti-Lewis antibody and a repeating portion. Since most Helicobacter pylori strains are smooth, it can be said that the smooth Helicobacter pylori is a normal Helicobacter pylori. Therefore, when a population of normal Helicobacter pylori is targeted, the antigenic portion of the O-antigen polysaccharide portion of the S-type LPS of Helicobacter pylori of the present invention excluding the Lewis antigen, Helicobacter pylori has a monoclonal antibody that specifically recognizes the LPS antigen portion that is common to Helicobacter pylori. Helicobacter pylori is a monoclonal antibody that recognizes the LPS antigen that is common to H. pylori strains. It is a monoclonal antibody that recognizes the LPS antigen portion of the Helicobacter pylori strain, which is the portion of the O-antigen polysaccharide portion on the LPS antigen of the H. pylori strain except for the Lewis antigen.

The present inventors have found that the antibody titer against Lewis antigen in human anti-Helicobacter pylori serum is not high, and the antibody in human serum recognizes a different epitope from the Lewis antigen of 0-antigen polysaccharide. It was confirmed. Therefore, when monoclonal antibodies against this epitope were prepared, a monoclonal antibody recognizing the core oligosaccharide portion of the LPS of Helicobacter pylori was found on the S-type LPS of Helicobacter pylori. Monoclonal antibody that recognizes the LPS antigen portion of the O-antigen polysaccharide portion of S. cerevisiae, excluding the Lewis antigen portion, which is common to smooth Helicobacter pylori strains, and S. of Helicobacter pylori. Monoclonal antibodies that recognize the LPS antigen portion of the S-type Helicobacter pylori strain, which is the antigen portion of the 〇-antigen polysaccharide portion of the LPS-type antigen except for the Lewis antigen, and monoclonal antibodies thereof Was obtained. As a result of the study of the reactivity of the monoclonal antibody, the monoclonal antibody recognizing the core oligosaccharide portion of the LPS and the antigen portion of the O-antigen polysaccharide portion of the S-type LPS of Helicobacter 1 'pylori except for the Lewis antigen were detected. It has been found that almost all of the Helicobacter pylori strains can be detected by using in combination with a monoclonal antibody recognizing the LPS antigen portion shared by the S-type Helicobacter pylori strains. In addition to the combination of antibodies, the antigenic portion of the 多 -antigen polysaccharide portion of Helicobacter pylori S-type LPS excluding the Lewis antigen, and the LPS antigen portion of some S-type Helicobacter pylori strains All Helicobacter pylori strains can be detected by using a combination of monoclonal antibodies that recognize And completed the invention.

 In addition, the present inventors have proposed a Helicobacter pylori strain derived from a gastric cancer patient, which is a low antigenic Helicobacter pylori strain having a low antigenic LPS that does not react with the sera of patients with chronic gastritis, gastric ulcer and duodenum. The mouse is immunized using the antigen as the antigen, and mouse spleen cells are fused with mouse myeloma cells (myeloma), and the LPS of L. pylori, ie, low antigenic LPS, is converted from the obtained hybridoma to low antigenicity. We succeeded in obtaining a monoclonal antibody that specifically recognizes it, and completed the present invention. The present inventors have found that monoclonal antibodies recognizing low antigenic LPS are abundant in the O-antigen polysaccharide portion of LPS of Helicobacter pylori from gastric cancer patients, but from chronic gastritis patients, gastric ulcer patients and duodenal ulcer patients Of Helicobacter pylori was found to recognize the low-antigenic epitope of LPS, which is not present in LPS.

 The present inventors have further developed a reagent, kit and method which can detect all Helicobacter pylori by combining the above-mentioned monoclonal antibodies, and further can detect Helicobacter pylori from gastric cancer patients. I found it.

That is, the present invention is as follows. (1) Monoclonal antibody that recognizes the core oligosaccharide of LPS of Helicobacter pylori

 (2) Monoclonal antibody (1) that recognizes surface LPS of 26695, CA9, CA6, and CA4 strains among 6 Helicobacter pylori strains, 26695, CA9, CA6, CA4, CA2, and GU2.

 (3) Hypridoma strain HPCA / D42-3 (Accession No. FERM BP-7894, deposited on August 15, 2001 (Hara Deposit), National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary Center (Tsukuba, Ibaraki, Japan) (1) or (2), which is deposited at 1-chome No. 1, 1 Chuo No. 6) (a request for transfer from the original deposit to a deposit based on the Budapest Treaty on February 14, 2002) Antibodies,

(4) the monoclonal antibody according to any one of (1) to (3), which is Fab, Fab ', F (ab') ₂ or an Fv fragment;

 (5) Hypridoma strain HPCA / D42-3 (Accession number FERM BP-7894),

 (6) a monoclonal antibody that recognizes the LPS antigen portion of the O-antigen polysaccharide portion of the S-type LPS of Helicobacter pylori except for the Lewis antigen, .

 (7) Monoclonal antibody (6) that recognizes surface LPS of CA6, CA4, CA2, and GU2 strains among 26695, CA9, CA6, CA4, CA2, and GU2 out of 6 Helicobacter pylori strains

 (8) Hypri-Dorma strain HP220 / B25-3 (Accession No. FERM BP-7893, deposited on August 15, 2001 (Hara Deposit), Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (AIST)

(Deposit received at Tsukuba East 1-chome 1-Chome No. 6 Ibaraki, Japan) (Request for transfer of deposit from the original deposit to the deposit based on the Budapest Treaty on February 14, 2002)) 6) or the monoclonal antibody of (7),

(9) The monoclonal antibody according to any one of (6) to (8), which is a Fab, Fab ', F (ab') ₂ or Fv fragment.

 (10) eighty bridoma strain HP220 / B25-3 (Accession number FERM BP-7893), eighty bridoma,

(11) Helicobacter pylori S-type LPS 0-antigen polysaccharide portion of the LPS antigen, excluding the Lewis antigen, recognizes the LPS antigen portion shared by the S-type helicobacter 'H. pylori strain Monoclonal antibodies, (12) Recognize surface LPS of 6 strains of Helicobacter pylori, 26695, CA9, CA6, CA4, CA2 and GU2, of all 26695, CA9, CA6, CA4, CA2 and GU2 (11) the monoclonal antibody,

 (13) Hypridoma strain HP220 / 2D10-2 (Accession No. FERM BP-7891; deposited on February 19, 2001 (Hara Deposit), National Institute of Advanced Industrial Science and Technology (AIST), Patent Organism Depositary Center 1 (Tsukuba, Ibaraki, Japan) (1 1) or (1 2) which was deposited at Ichihigashi 1-1-chome 1 Chuo No. 6) (a request for transfer of the original deposit to a deposit based on the Budapest Treaty on February 14, 2002) Monoclonal antibodies,

(14) the monoclonal antibody of any of (11) to (13), which is a Fab, Fab ', F (ab') ₂ or Fv fragment;

 (15) A hybridoma which is a hybridoma strain HP220 / 2D10-2 (Accession number FERM BP-7891),

 (16) Monoclonal antibody that recognizes low antigenic LPS of Helicobacter pylori strain,

 (17) Recognize low antigenic LPS, which is abundant in Helicobacter pylori strains derived from gastric cancer patients and does not react with serum of patients with chronic gastritis, gastric ulcer and duodenal ulcer,

(16) the monoclonal antibody,

 (18) Of the six strains of Helicobacter pylori, 26695, CA9, CA6, CA4, CA2 and GU2, recognize the surface LPS of CA6, CA4 and CA2 strains. (16) or (17) monoclonal Antibodies,

 (19) Hypridoma strain HP217 / B15-4 (Accession number: FEM BP-7892, deposited on January 19, 2001 (Hara Deposit), National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center-(Tsukuba, Ibaraki, Japan) Deposited at Ichihigashi 1-1-chome 1 Chuo No. 6) (reception of transfer request from the original deposit to the deposit based on the Budapest Treaty on February 14, 2002) produced (16)-(18) Any of the monoclonal antibodies,

(20) the monoclonal antibody of any one of (16) to (19), which is a Fab, Fab ', F (ab') ₂ or Fv fragment;

 (2 1) A hybridoma which is a hybridoma strain HP217 / B15-4 (Accession number FERM BP-7892),

(22) The monoclonal antibody of any of (1) to (4) and (1 1) to (1 4) A method for detecting LPS of Helicobacter pylori strain using any one of the monoclonal antibodies,

 (23) The method of (22) for detecting LPS of Helicobacter pylori strain using the monoclonal antibody of any one of (6) to (9),

 (24) The method of (23) for detecting LPS of Helicobacter pylori strain using the monoclonal antibody of any one of (16) to (20),

 (25) Using the monoclonal antibody of any one of (16) to (20), it is abundant in Helicopaque Yuichi pylori strains derived from gastric cancer patients and does not react with the serum of patients with chronic gastritis, gastric ulcer or duodenal ulcer A method for detecting low antigenic LPS,

 (26) A method for detecting Helicobacter pylori using the monoclonal antibody of any one of (1) to (4) and the monoclonal antibody of any one of (11) to (14),

 (27) The method for detecting Helicobacter pylori according to (26) using the monoclonal antibody according to any one of (6) to (9),

 (28) The method for detecting Helicobacter pylori according to (27), further comprising using the monoclonal antibody according to any one of (16) to (20),

 (29) Using the monoclonal antibody of any one of (16) to (20), a low level that is abundant in Helicobacter pylori strains derived from gastric cancer patients and does not react with the serum of patients with chronic gastritis, gastric ulcer, or duodenal ulcer A method for detecting low antigenic Helicobacter pylori, which comprises detecting antigenic LPS,

 (30) Any one of the monoclonal antibodies (1) to (4), any one of the monoclonal antibodies (6) to (9), any one of the monoclonal antibodies (11) to (14), and (1) 6) a kit for detecting Helicobacter pylori comprising at least one monoclonal antibody selected from the group consisting of the monoclonal antibodies of any of (20) to (20);

 (31) the kit for detecting Helicobacter pylori according to (30), comprising the monoclonal antibody of any one of (1) to (4) and the monoclonal antibody of any of (11) to (14); and

(32) A kit for detecting low antigenic Helicobacter pylori, comprising the monoclonal antibody according to any one of (16) to (20). Hereinafter, the present invention will be described in detail.

Production of monoclonal antibodies

 The monoclonal antibody of the present invention can be prepared by a known method such as the method of Kohler and Milstein (Kohler, G. and Milstein, C., Nature, 256, 495-497, 1975).

 Helicobacter pylori strain inactivated by formalin treatment or the like can be used as an immunogen. The immunogen can be prepared by suspending the inactivated strain in a buffer such as PBS at a concentration of 0.5 to: I. 5 mg / mL, preferably 1 mg / mL. The immunogen is desirably administered with an appropriate adjuvant such as commercially available complete Freund's adjuvant, incomplete Freund's adjuvant, BCG, aluminum hydroxide gel, pertussis vaccine, and the like. In addition, mice, rats, guinea pigs, and the like can be used as animals to be immunized, but mice are generally used. In the case of a mouse, a mouse 3 to 10 weeks old, preferably 4 weeks old is used. The prepared immunogen can be administered via any route such as subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal of animals. The interval of immunization is not particularly limited, but it is preferable to immunize 2 to 5 times, for example, every 1 to 2 weeks. The dose per dose is not limited. For example, the immunogen prepared as described above may be mixed with an appropriate adjuvant and administered in an amount of several tens to several hundreds of X1. Three to ten days after the final immunization, antibody-producing cells are collected from the animal to be immunized. Examples of antibody-producing cells include spleen cells, lymph node cells, thymocytes, and peripheral blood cells, and spleen cells are generally used. The spleen, lymph nodes, thymus, peripheral blood, etc. are removed or collected from the immunized animal, and these tissues are disrupted. The crushed material is suspended in a buffer such as PBS or a medium such as DMEM, RPMI-1640, or E-RDF, and then filtered using a stainless steel mesh of 200 to 250 / xm and centrifuged. Target antibody-producing cells can be prepared.

The antibody-producing cells thus prepared are fused with myeloma cells. As myeloma cells, cell lines available to those skilled in the art can be used. Myeloma cells are generally obtained from animals of the same species as the animal to be immunized, but xenogeneic cells may be possible. The cell line used has drug resistance and cannot survive in a selective medium such as hypoxanthine / aminopterin / thymidine medium (HAT medium) in an unfused state, but survives only in a state fused to antibody-producing cells. Those that have the property that can be New Generally, an 8-azaguanine resistant strain is used. This cell line lacks hypoxanthin-guanine phospholiposyltransferase (HGPRT-) and cannot grow on a HAT medium. As myeloma cells, Sp2 / 0—Agl4 [ATCC CRL-1581; Nature, 276, 271 (1978)], P3X63Ag8 [ATCC TIB-9; Nature, 265, 495-497 (1978)], P3X63 Ag8U. 1 (P3U1) [ATCC CRL-1580; Current Topics in Microbiology and Immunology, 81, 1-7 (1978)], P3X63Ag8.65 [ATCC TIB-18; European

J. Immunology, 6, 511-519 (1976)], and mouse myeloma cell lines such as P2 / NSI / 1—Ag4-1 (ATCC CRL-1581; Nature, 276, 269-270 (1978)). .

Cell fusion, MEM, MEM, RPMI_1640, the 10 ⁷ to 10 ⁸ cells / ml of myeloma cells and antibody-producing cells in animal cell culture media, such as E-RDF, mixing ratio 1: 1 to 1: 10 For example, the cells can be efficiently contacted with each other at a ratio of 1: 5 in the presence of a fusion promoting agent at 30 to 37 for 1 to 3 minutes. As the fusion promoter, polyethylene glycol or polyvinyl alcohol having an average molecular weight of 1000 to 600 can be used. Cell fusion can also be performed using a fusion virus such as Sendai virus. Furthermore, cell fusion can also be performed by a method using electrical stimulation such as electroporation. A commercially available cell fusion device using electroboration can be used.

Hybridomas can be selected from the cells after the cell fusion treatment by a method utilizing the selective growth of cells in a selective medium such as a HAT medium. For example, a fusion suspension of cells HAT supplement (Gibco BRL) and interleukin - 6 (lu ni t / mL ) so as to be 10 ³ to 10 ⁷ cells / mL in Iscove's medium (IMDM) supplemented with dilutions Then, seed cells in a 96-well cell culture microplate at a cell density of 10 ² to 10 ⁶ cells / well, add a selection medium such as HAT medium to each well, and then replace the cells while replacing the selection medium appropriately. Culture and select for hybridomas.

 When 8-azaguanine resistant strains are used as myeloma cells and HAT medium is used as the selection medium, unfused myeloma cells die about 7 to 10 days after culture, and antibody-producing cells, which are normal cells, are in vitro. Do not survive long, and die about 7-10 days after culture. As a result, cells that grow from around 6 to 10 days of culture can be obtained as hybridomas.

For the culture supernatant of the proliferating cells, use Helicobacter '' H. pylori on LPS antigen. The presence or absence of production of an antibody against the core oligosaccharide portion of the above is determined, and a hybridoma producing the desired antibody is screened. Screening of the hybridoma can be performed by a usual method. For example, a portion of the culture supernatant contained in the wells where the hybridomas grew is collected and used to determine whether the target antibody is contained by enzyme-linked immunosorbent assay (EIA, ELISA) or radioimmunoassay (RIA). Can be tested. For example, a 96-well microtiter plate was used for adsorbing LPS prepared from Helicobacter pylori strain and LPS co-oligosaccharide prepared from other Helicobacter pylori strains used as immunogens on a plate. Evening Add the culture supernatant containing the monoclonal antibody to the plate and react with the antigen. Next, the bound specific antibody is reacted with an enzyme-labeled anti-mouse immunoglobulin antibody, an enzyme substrate is added to each well to develop color, and whether the monoclonal antibody in the culture supernatant reacts with immobilized LPS is determined. Is tested. At this time, i3-D-galactosidase, peroxidase, alkaline phosphatase, glucose oxidase and the like are used as enzymes. The color development can be measured using a signal reader corresponding to the Atsushi system, for example, a microphone port plate reader. By screening the culture supernatant containing a monoclonal antibody that reacts and binds to the LPS core oligosaccharide, a hybridoma that produces a monoclonal antibody that recognizes the core oligosaccharide portion of the LPS of Helicobacter pylori is screened. be able to.

 Also, without purifying the LPS core oligosaccharide, a hybridoma producing a monoclonal antibody recognizing LPS was selected, and the reactivity of the culture supernatant with LPS was confirmed to recognize the core oligosaccharide portion of LPS. The monoclonal antibody-producing hybridomas can be selected. The reactivity with LPS was determined by separating LPS obtained by treating Helicobacter pylori cells with hot phenol or proteinase K by polyacrylamide gel electrophoresis, reacting it with a monoclonal antibody, and labeling the secondary antibody. The color can be confirmed by immunoblot method (Western plot method) or the like.

In this way, a hybridoma producing the desired monoclonal antibody is cloned from the selected hybridomas in the well. Cloning of hybridomas can be performed by the limiting dilution method, soft agar method, fibrin gel method, fluorescence-excited cell sorter method, and the like. A hybridoma producing a monoclonal antibody that recognizes the glycose moiety can be obtained.

Produced Helicobacter Pylori Monoclonal antibody recognizing the core oligosaccharide of LPS (anti-Helicobacter Pylori LPS core oligosaccharide monoclonal antibody) Produced from hybridomas using standard cell culture method, ascites formation method, etc. Helicobacter pylori LPS core oligosaccharide monoclonal antibody can be collected. For cell culture, IMDM, RPMI-1640, MEM, E-RDF, or 10-20% lipoprotein / H. Pylori LPS-core oligosaccharide-producing hybridomas containing spores or fetal serum are included in the piles. in a medium for animal cell culture such as serum-free medium under conventional culture conditions (e.g., 37 ° C, 5% C0 2 concentration) that in cultured 2-14 days, to obtain the antibody from the supernatant that culture Youe it can. In the ascites formation method, a mineral oil such as pristane (2,6,10,14-tetramethylpentyldecane) is administered to the abdominal cavity of an animal of the same species as the animal derived from myeloma cells in advance, and then 1 × 10 ⁷ to 1 X 10 ⁹ cells, preferably rather is a 5 X 10 ⁷ ~1 X 10 ⁸ Rikopakuta pylori LPS core oligosaccharide monoclonal antibody producing High Priestess dormer cell anti to the administered intraperitoneally, mass proliferating High Priestess dormer Let it. After 1 to 4 weeks, preferably 2 to 3 weeks, ascites or blood can be collected to obtain the antibody.

 If antibody purification is required, ammonium sulfate precipitation, ion exchange chromatography using an anion exchanger such as DEAE cellulose, molecular sieving chromatography by molecular weight and structure, hydroxyapatite It can be purified by appropriately selecting a known method such as matography or a combination thereof.

Thus, the monoclonal antibody of the present invention can be obtained. The monoclonal antibody may be a complete antibody or a fragment having an antigen-binding site such as Fab, Fab ', F (ab') ₂ , or Fv.

 Further, as a specific monoclonal antibody against the LPS core oligosaccharide of Helicobacter pylori, for example, one produced by the hybridoma strain HPCA / M2-3 can be used.

Also, using the LPS of several types of S-type Helicobacter pylori strains, the 0-antigen polysaccharide portion of the S-type LPS antigen of Helicobacter pylori excluding the Lewis antigen Can be obtained. At this time, if a substance that reacts with LPS of S. helicobacter pylori strain widely is selected, it is the part of the 0-antigen polysaccharide part on the S-type LPS antigen of Helicobacter pylori that excludes the Lewis antigen. Thus, it is possible to obtain an antibody that recognizes the LPS antigen portion commonly possessed by S-type Helicobacter pylori strains. Furthermore, if one that reacts with LPS of some S-type Helicobacter pylori strains is selected, the portion of the 0-antigen polysaccharide portion on the S-type LPS antigen of Helicobacter pylori excluding the Lewis antigen is considered. Thus, it is possible to obtain an antibody that recognizes the LPS antigen portion of some S-type Helicobacter pylori strains. Here, “an antibody recognizing the LPS antigen portion possessed by L-pylori and H. pylori to a part of the S-type” refers to a plurality of S-type L-reactors isolated from gastric disease patients such as gastric cancer, chronic gastritis, gastric ulcer, and duodenal ulcer. · Of H. pylori, for example, an antibody that reacts with and recognizes 80% or less of a helicopter, and preferably an antibody that reacts with and recognizes 70% or less of a helicopter. Specifically, when reacting with 7 out of 14 S-type Helicobacter pylori strains, it falls under "an antibody recognizing the LPS antigen portion of some S-type Helicobacter pylori". As the former antibody, the hybridoma strain HP220 / 2D10-2 can also be used. Alternatively, the hybridoma HP220 / B-25-3 can be used as the latter antibody. In the same manner as in obtaining monoclonal antibodies recognizing the core oligosaccharide part of Helicobacter pylori LPS, the antigenic part of the O-antigen polysaccharide part of Helicobacter pylori S-type LPS excluding the Lewis antigen was used. A monoclonal antibody recognizing the LPS antigen portion commonly possessed by S-type Helicobacter pylori strains or a portion of the O-antigen polysaccharide portion on the S-type LPS antigen of Helicobacter pylori excluding the Lewis antigen However, in obtaining a monoclonal antibody that recognizes the LPS antigen portion of some S-type Helicobacter pylori strains, it is necessary to purify the antigen portion of the O-antigen polysaccharide portion of the S-type LPS excluding the Louis antigen. SDS-PAGE and immunoblot analysis using S-type LPS and Lewis antigens among monoclonal antibodies that recognize S-type LPS (Western plot Analysis), it is possible to select a monoclonal antibody that recognizes the antigen portion of the 0-antigen polysaccharide portion of the S-type LPS excluding the Lewis antigen.

Furthermore, a monoclonal antibody that recognizes LPS of Helicobacter pylori with low antigenicity can be obtained as follows. About the culture supernatant of the proliferating cells, The presence or absence of the production of an antibody that recognizes LPS of Helicobacter pylori with low antigenicity is examined, and screening for eight hybridomas that produce the desired antibody is performed. Hybridoma screening can be performed by a conventional method. For example, a portion of the culture supernatant contained in the wells where the hybridomas grew is collected and used to determine whether the antibody of interest is contained by enzyme immunoassay (EIA, ELISA) or radioimmunoassay (RIA). Can be tested. For example, LPS prepared from low antigenic Helicobacter pylori strains from multiple gastric cancer patients in a 96-well microtiter plate and high antigenic Helicobacter pylori from multiple chronic gastritis patients, gastric ulcer patients or duodenal ulcer patients The culture supernatant containing the monoclonal antibody is added to a 96-well microphone-mouth titer plate to which LPS prepared from the strain is adsorbed, and reacted with the antigen. Next, the enzyme-labeled anti-mouse immunoglobulin antibody is reacted with the bound specific antibody, an enzyme substrate is added to each well to develop color, and it is assayed whether the monoclonal antibody in the culture supernatant reacts with solid LPS. I do. At this time, j3-D-galactosidase, peroxidase, alkaline phosphatase, glucose oxidase, etc. are used as enzymes. The color development can be measured using a signal reader corresponding to the Atsushi system, for example, a microphone plate reader. By selecting a culture supernatant containing a monoclonal antibody that reacts with LPS derived from a low antigenic Helicobacter pylori strain, an antibody that recognizes and binds to the LPS antigen common to the low antigenic Helicobacter pylori strain is produced. Hybridomas can be screened. In this way, hybridomas producing the desired monoclonal antibody are cloned from the hybridomas in the thus selected ゥ, エ ル. Hybridomas can be cloned by limiting dilution, soft agar, fibrin gel, fluorescence-excited cell sorter, etc., and finally obtain hybridomas that produce the desired monoclonal antibody. .

 A monoclonal antibody that recognizes the LPS of Lactobacillus pylori from the obtained low-antigenic Helicobacter pylori LPS using a standard cell culture method, ascites formation method, etc. Can be collected.

In the cell culture method, low-antigenic Helicobacter pylori LPS-recognizing monoclonal antibodies producing 10-20% In an animal cell culture medium such as IM RPM, RPMI-1640, MEM, E-RDF or serum-free medium, contain ₂ to 14 at normal culture conditions (eg, 37 ° (5% C02 concentration)). Antibodies can be obtained from the culture supernatant, and in the ascites formation method, pristane (2, 6, 10, 14-) is intraperitoneally injected into an animal of the same species as the animal derived from myeloma cells. administered tetramethyl pen evening decane) mineral oils, such as, then 1 X 10 ⁷ ~1 X 10 ⁹ cells, preferably recognizes ^{5 X 10 7 ~1 X 10 8} LPS low antigenicity Helicobacter one pylori cells The monoclonal antibody-producing hybridoma is administered intraperitoneally to proliferate the hybridoma in a large amount.After 1 to 4 weeks, preferably 2 to 3 weeks, ascites or blood is collected to obtain the antibody.

 If antibody purification is required, ammonium sulfate precipitation, ion exchange chromatography using an anion exchanger such as DEAE cellulose, molecular sieving chromatography sieving based on molecular weight and structure, hydroxyapatite chromatography It can be purified by appropriately selecting a known method such as, or a combination thereof.

In this manner, a specific monoclonal antibody against LPS of the low antigenic Helicobacter pylori of the present invention can be obtained. The monoclonal antibody may be a complete antibody or a fragment having an antigen binding site such as Fab, Fab ', F (ab') ₂ , Fv, or the like.

 As the monoclonal antibody, for example, one produced by the hybridoma strain HP217 / B15-4 can be used.

The reactivity of the monoclonal antibody of the present invention with LPS was determined by separating the LPS obtained by treating Helicobacter pylori cells with hot phenol or proteinase K by polyacrylamide gel electrophoresis and reacting the monoclonal antibody with the target. Color can be confirmed by immunoblotting or the like, which looks at the color developed by the secondary antibody. To confirm whether Helicobacter pylori isolated from a patient's stomach biopsy has low antigenicity, LPS prepared from the test Helicobacter pylori strain was solid-phased on a microtiter plate, and gastric ulcer or duodenal ulcer was detected. It can be performed by the EIA method in which the serum of the patient is reacted. In other words, a Helicobacter pylori strain having LPS in which stomach ulcer or duodenal ulcer patient's serum does not react is regarded as a low antigenic Helicobacter pylori. Helicobacter pylori detection

 Using the Helicobacter pylori LPS core oligosaccharide-specific monoclonal antibody obtained in this way, helicobacter pylori strains having R-type LPS and a part of Helicobacter pylori strains having S-type LPS were used. It is possible to detect.

 It also recognizes the part of the 0-antigen polysaccharide moiety on the S-type LPS antigen of Helicobacter pylori, excluding the Louis antigen, and the LPS antigen part that is common to S-type Helicobacter pylori strains. It is possible to detect Helicobacter pylori strains with most S-type LPS using anti-HRP. Furthermore, an antibody that recognizes the LPS antigen portion of the S-type Helicobacter pylori strain, which is a portion of the 0-antigen polysaccharide portion on the S-type LPS antigen of Helicobacter pylori except for the Lewis antigen Can be used to detect Helicopaque Yuichi pylori strains having some S-type LPS. The risk of gastric disease of a subject can also be evaluated by detecting whether or not Helicobacter pylori is present in the stomach of the subject using the monoclonal antibody of the present invention. When Helicobacter pylori is detected in the stomach of a subject by the monoclonal antibody of the present invention, helicobacter pylori is removed by washing the subject's stomach or administering antibiotics, etc., and the risk of stomach disease is increased. Can be reduced or eliminated. The monoclonal antibody of the present invention can also be used to determine the effect of removing Helicobacter pylori.

It is also possible to detect Helicobacter pylori derived from gastric cancer patients by using the monoclonal antibody that recognizes LPS of Helicobacter pylori obtained as described above. Further, by detecting whether or not the low antigenic Helicobacter 1 'pylori LPS of the present invention is present in the subject's stomach by a monoclonal antibody recognizing LPS of Helicobacter pylori, the subject is exposed to gastric cancer. You can also assess the danger of When low antigenic Helicobacter pylori is detected in the stomach of a subject by the monoclonal antibody of the present invention, the low antigenic Helicobacter pylori is generally removed by washing the subject's stomach or administering an antibiotic. However, the risk of stomach cancer can be reduced or eliminated. The monoclonal antibody of the present invention can also be used to determine the effect of removing low antigenic Helicobacter pylori. The anti-Helicobacter pylori LPS core oligosaccharide monoclonal antibody and helicopter Combination of a monoclonal antibody that recognizes the LPS antigen portion of the S-type Helicobacter pylori strain that is the antigen portion of the O-antigen polysaccharide portion of the S-type LPS excluding the Lewis antigen This makes it possible to detect almost all Helicobacter pylori strains. Here, "detecting almost all" means that it is possible to detect preferably 95% or more of a plurality of Helicobacter pylori isolated from patients with gastric diseases such as gastric cancer, chronic gastritis, gastric ulcer, and duodenal ulcer. More preferably, it means that 98% or more can be detected. For example, if 179 of L. pylori could be detected in 181 separations, it corresponds to “detect almost all”. In addition, these two types of monoclonal antibodies were further added to the above-mentioned 0-antigen polysaccharide portion of the S-type LPS antigen of Helicobacter pylori, excluding the Lewis antigen, and some S-type Helicobacter pylori. By using a combination of monoclonal antibodies that recognize the LPS antigen portion of the strain, it becomes possible to detect all Helicobacter pylori strains. Here, "to detect all" means that preferably 99% or more of Helicobacter pylori isolated from gastric disease patients can be detected, and more preferably 100% of Helicobacter pylori can be detected. What you can do. For example, this is the case where all 181 of L. pylori can be detected in 181 separations. However, Helicobacter pylori does not have a normal LPS antigen structure and has the above-mentioned anti-Helicobacter pylori LPS core oligosaccharide monoclonal antibody and the O-antigen polysaccharide portion of S-type LPS of Helicobacter pylori. Monoclonal antibodies recognizing the LPS antigen portion of the S-type Helicobacter pylori strain, which is the antigen portion excluding the Lewis antigen, and 0-antigen polysaccharide portion on the S-type LPS antigen of Helicobacter pylori. Among them, there are very few strains that exclude the Lewis antigen and cannot recognize any of the monoclonal antibodies that recognize the LPS antigen portion of some S-type Helicobacter pylori strains. Abnormal strains should be excluded from the detection rate calculation.

The above-mentioned anti-Helicobacter pylori LPS core oligosaccharide monoclonal antibody, the antigenic portion of the O-antigen polysaccharide portion of Helicobacter pylori S-type LPS excluding the Lewis antigen, which is common to S-type Helicobacter pylori strains Helicobacter pylori, a monoclonal antibody recognizing the LPS antigen portion of the O-antigen polysaccharide portion of the S-type LPS antigen, excluding the Lewis antigen, and a portion of the S-type Helicobacter Detection of all Helicobacter pylori by using all monoclonal antibodies recognizing the LPS antigen portion of the H. pylori strain and the low antigenic helicopter and LPS of H. pylori In addition, low antigenic Helicobacter pylori and H. pylori strains derived from gastric cancer patients can be detected.

 Helicobacter pylori can be detected by immunoblotting, enzyme immunoassay (EIA, ELISA), radioimmunoassay (RIA), fluorescent antibody, agglutination, immunochromatography, etc. Can be carried out by a method known in the art. In this case, as the sample, for example, bacteria separated and cultured from a gastric biopsy of a patient with a gastric disease such as a gastric ulcer can be used, but it is not limited thereto.

 For example, in the method utilizing the agglutination reaction, a monoclonal antibody recognizing the LPS core oligosaccharide of Helicobacter pylori and an antigen part excluding the Lewis antigen in the O-antigen polysaccharide part of the S-type LPS of Helicobacter pylori are used. Helicobacter pylori can be detected using a carrier sensitized with a monoclonal antibody recognizing the LPS antigen portion shared by S-type Helicobacter pylori strains. Furthermore, in order to increase the detection rate, the O-antigen polysaccharide portion on the S-type LPS antigen of Helicobacter pylori is a portion of the O-antigen polysaccharide portion excluding the Lewis antigen, and the LPS contained in some S-type Helicobacter pylori strains A monoclonal antibody that recognizes the antigen portion may be sensitized. Furthermore, in order to detect a low antigenic Helicobacter pylori strain derived from a gastric cancer patient-derived strain, a monoclonal antibody recognizing LPS of the low antigenic Helicobacter pylori may be sensitized.

As a carrier for sensitizing these monoclonal antibodies, any carrier may be used as long as it is insoluble, does not cause nonspecific reaction, and is stable. For example, latex particles, bentonite, collodion, kaolin, fixed sheep erythrocytes and the like can be used, but it is preferable to use latex particles. As the latex particles, for example, polystyrene latex particles, styrene-butadiene copolymer latex particles, polyvinyl toluene latex particles and the like can be used, but it is preferable to use polystyrene latex particles. When latex particles are used, the antibody can be easily sensitized to the carrier without any special treatment, and the aggregation image generated by the reaction between the target strain and the carrier becomes clear. It is further advantageous in that the reactivity with respect to it can be easily and accurately determined. At this time, two to four of the above-mentioned four types of antibodies may be bound to the carrier simultaneously, or these antibodies may be separately bound to the carrier, and the four types of carriers to which each antibody is bound may be bound. It is also possible to use a mixture of 2 to 4 types. Furthermore, a carrier to which each antibody is separately bound may be used separately for the detection of Helicobacter pylori by each antibody. The low-antigenic Helicobacter pylori monoclonal antibody that recognizes LPS can detect low-antigenic Helicobacter pylori strains derived from gastric cancer patient strains separately from the other three species. It is preferable to bind to a carrier separately from the three types.

The method for sensitizing the carrier with the monoclonal antibody is not particularly limited. For example, the monoclonal antibody may be physically adsorbed to a carrier or chemically bound. More specifically, for example, after mixing a monoclonal antibody and a carrier, the antibody can be sensitized to the carrier by heating and shaking at 30 to 37 ° C. for 2 hours. The amount of the antibody sensitizing the carrier can be appropriately set according to the particle size of the carrier used. After the antibody is sensitized to the carrier, it is preferable to block the unsensitized portion on the carrier surface with serum albumin, human serum albumin, heron serum albumin, ovalbumin and the like. The carrier sensitized with the monoclonal antibody is preferably retained as a medium dispersion until the reaction with the target strain belonging to Helicobacter pylori. At this time, as the medium, for example, a phosphate buffer, a glycine buffer, or the like can be used. The content of the carrier sensitized to the monoclonal antibody can be usually 0.2 to 0.5% by weight based on the medium dispersion, but is preferably 0.25 to 0.3% by weight. preferable. If necessary, pepsin albumin, gelatin, gum arabic and the like may be added to the medium. The monoclonal antibody-sensitized carrier prepared in this way is reacted with the target strain, and the reactivity of the target strain with the monoclonal antibody is determined based on the presence or absence of agglutination or the degree thereof, whereby Helicobacter pylori can be detected. At this time, the Helicobacter pylori strain may be previously treated with an appropriate extraction reagent, for example, an enzyme or acetic acid and sodium nitrite. In the enzyme immunoassay (EIA), the antigenic portion of the 0-antigen polysaccharide portion of Helicobacter pylori LPS-specific monoclonal antibody and the 0-antigen polysaccharide portion of Helicobacter pylori S-type LPS was excluded from the Lewis antigen. Helicobacter pylori Monoclonal antibodies recognizing the LPS antigen portion that the strains have in common are immobilized on a carrier such as a plate, resin beads, or magnetized beads by physical adsorption or chemical bonding. Furthermore, in order to increase the detection rate, the O-antigen polysaccharide moiety on the S-type LPS antigen of Helicobacter pylori is a part of the O-antigen polysaccharide excluding the Lewis antigen, and some S-type lycopac Yuichi pylori strains A monoclonal antibody recognizing the LPS antigen portion may be immobilized. Furthermore, in order to detect a low antigenic Helicobacter pylori strain derived from a gastric cancer patient, a monoclonal antibody recognizing LPS of low antigenic Helicobacter pylori may be immobilized.

 At this time, 2 to 4 types of these antibodies may be mixed and immobilized on a carrier, and a carrier on which 2 to 4 types of antibodies may be immobilized may be prepared and used for detection of Helicobacter pylori. Alternatively, a carrier in which each antibody is immobilized may be prepared, and the reactivity with each antibody may be measured separately. Low antigenic Helicobacter '' Monoclonal antibodies that recognize H. pylori LPS can detect low antigenic Helicobacter 1-H. Pylori strains derived from gastric cancer patients separately from the other three species. It is preferable to bind to a carrier separately from the carrier.

The amount of solid phase immobilization is not particularly limited, but is preferably several ng to several tens / zg per well when the carrier is a microtiter plate. The immobilization can be performed by dissolving the antibody to be immobilized in an appropriate buffer and bringing the antibody into contact with a carrier. For example, when using a microtiter well, the antibody solution can be dispensed into a well of a microtiter plate and immobilized by placing it for a certain period of time. After the substrate is immobilized, blocking is preferably performed using a blocking solution containing serum albumin, human serum albumin, egret serum albumin, ovalbumin, etc. to prevent non-specific binding in Atsusei. . Next, the sample is reacted with the solid-phased carrier, washed, and then reacted with a labeled monoclonal antibody that recognizes Helicobacter pylori. Labeling can be performed using enzymes such as j3_D-galactosidase, peroxidase, alkaline phosphatase and glucose oxidase. For example, in enzyme-linked immunosorbent assay (ELISA), a substrate is immobilized on a microtiter plate having a large number of wells (for example, 96 wells), and an antigen-antibody reaction is performed in the well. Measurement becomes possible. In addition, the amount of antibody and sample cells to be used can be extremely reduced. In addition, a fully automatic EIA measurement device Any automatic measuring equipment can be used.

 Another object of the present invention is to provide a kit capable of detecting almost all of Helicobacter pylori strains. The kit comprises at least a monoclonal antibody recognizing the LPS core oligosaccharide of Helicobacter pylori and a Helicobacter pylori strain. Includes a monoclonal antibody that recognizes the LPS antigen portion of the S-type Helicobacter pylori strain, which is the antigen portion of the S-type LPS of H. pylori except for the Lewis antigen among the ◦-antigen polysaccharide portions. Another object of the present invention is to provide a kit which enables the detection of all Helicobacter pylori strains. The kit further comprises a 0-antigen polysaccharide moiety on the S-type LPS antigen of Helicobacter pylori. And a monoclonal antibody recognizing the LPS antigen portion of some S-type Helicobacter pylori strains except for the Lewis antigen. Further, the present invention also aims to provide a kit which enables detection of low antigenic Helicobacter pylori strains derived from gastric cancer patients, and the kit further comprises a low antigenic Helicobacter pylori strain. Includes monoclonal antibodies that recognize LPS. When the kit is based on the EIA method, the kit may contain a carrier for immobilizing the antibody, or the antibody may be bound to the carrier in advance. When the kit is based on an agglutination method using a carrier such as latex, the kit may contain a carrier to which the antibody is adsorbed. Further, the kit may appropriately contain a blocking solution, a reaction solution, a reaction stop solution, a reagent for treating a sample, and the like.

As described above, the monoclonal antibody recognizing the core oligosaccharide of Helicobacter pylori LPS of the present invention and the antigen part of the 0-antigen polysaccharide part of the S-type LPS of Helicobacter 1 ′ pylori except for the Lewis antigen, By using a monoclonal antibody that recognizes the LPS antigen portion of S. helicobacter and H. pylori strains in common, almost all Helicobacter pylori and H. pylori can be detected. -All the Helicobacter pylori using a monoclonal antibody that recognizes the LPS antigen part of the S-type Helicobacter pylori strain except for the Lewis antigen in the antigen polysaccharide part. From a gastric cancer patient using a monoclonal antibody that recognizes LPS of low antigenic Helicobacter pylori Since low antigenic Helicobacter pylori can be detected, bacteria isolated from gastric biopsy can be quickly distinguished from Helicobacter pylori, and Helicobacter pylori can be detected. Investigate chemical properties Time and effort can be greatly reduced.

 The monoclonal antibody that recognizes the core oligosaccharide of LPS of Helicobacter pylori and the antigenic portion of the 0-antigen polysaccharide portion of the S-type LPS of Helicobacter pylori except for the Lewis antigen, which is an S-type helicobacter · Almost all helicobacters are detected using a monoclonal antibody that recognizes the LPS antigen portion shared by H. pylori strains · H. pylori is detected, or even helicopters · O-antigen polysaccharide on S-type LPS antigen of H. pylori Whether all Helicobacter pylori is detected using a monoclonal antibody that recognizes the LPS antigen part of the S-type Helicobacter pylori strain except for the Lewis antigen, Alternatively, whether to detect low antigenic Helicobacter pylori strains derived from gastric cancer patients may be determined by the target detection rate, diagnosis or The selection can be made according to the disease to be sought, and the cost and time required for preparation can be reduced by reducing the number of antibodies used. it can.

Further, Helicobacter pylori can be classified based on the reactivity pattern with the four kinds of monoclonal antibodies of the present invention, and the classification can be associated with various gastric diseases. Furthermore, in addition to the four types of monoclonal antibodies of the present invention, anti-Lewis ^{(Le x, Le y, Le} a, Le b, Le d) further Rikobaku evening one 'to the H. subclassified into the pattern of reactivity with the antibody Can be.

BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing a putative structure of Helicobacter pylori LPS.

 Figure 2 shows the antigenic portion of the O-antigen polysaccharide portion of the S-type LPS of Helicobacter pylori except for the Helicobacter pylori LPS core oligosaccharide monoclonal antibody (MoAb 1). Monoclonal antibody (MoAb I 1) recognizing the LPS antigen portion shared by S-type Helicobacter and H. pylori strains, excluding Lewis antigens from the O-antigen polysaccharide portion on the S-type LPS antigen of Helicobacter pylori And a monoclonal antibody (MoAb III) that recognizes the LPS antigen portion of some S-type Helicobacter pylori strains and L of low antigenic Helicobacter pylori.

Using a monoclonal antibody (MoAB IV) that recognizes PS, helicopter- FIG. 4 is a diagram showing the results of Western blot using H. pylori LPS.

BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these embodiments. Example 1 Preparation of monoclonal antibody against Helicobacter pylori

Preparation of immunizing antigen

 Incubate Helicobacter Pylori 220-B strain on blood agar medium under microaerobic conditions for 3-4 days at 37 ° C. The grown cells are suspended in phosphate buffered saline (PBS) and inactivated by formalin treatment. The inactivated cells are adjusted to a concentration of 0.5 to 1.5 mg / mL, preferably 1 mg / mL with PBS, and used as an immunizing antigen.

Preparation of monoclonal antibody

A 5-week-old female BALB / c mouse was immunized with the above immunizing antigen IOOL together with complete Freund's adjuvant, and two weeks later, 50 L of the same antigen was boosted with Freund's incomplete adjuvant. Two weeks later, 25 L of the antigen was injected intravenously, and three days later, spleen cells were removed. The extracted spleen cells were fused with mouse myeloma cells (P3X63) by the method of Keller et al. (Kohler et al., Na. Ture, vol. 256, p495-497 (1975)), and placed in a carbon dioxide incubator. The cells were cultured at 37 ° C. Using the obtained culture supernatant of the hybridoma as a specimen, screening of the hybridoma was performed by ELISA using Helicobacter pylori strain S-type LPS (217, 220, CA4 strain) as an antigen. Hybridomas in the culture supernatant that reacted with all S-type LPS were selected. The hybridomas screened were plated at 0.5 cells / well in a 96-well plate by limiting dilution, and only hybridomas that grew as a single colony in the well were cloned, and this limiting dilution was repeated twice. Cloning was performed. Based on the above, the monoclonal antibody-producing cell line against the core oligosaccharide of LPS of Helicobacter pylori and the antigenic portion of the O-antigen polysaccharide portion of the S-type LPS of Helicobacter pylori excluding the Louis antigen, Monoclonal antibody-producing cell lines and helicobacter that recognize the LPS antigen portion that are commonly found in Helicobacter pylori strains Monoclonal antibody-producing cell line that recognizes the LPS antigen portion of the S-type Helicobacter pylori strain, excluding the Lewis antigen, from the O-antigen polysaccharide portion of the S-type LPS antigen of T-pylori I got The obtained cell line was intraperitoneally administered to pristane-treated BALB / c mice, and about 2 weeks later, antibody-containing ascites was collected. IgG was purified from the ascites fluid by ammonium sulfate precipitation.

Example 2 Preparation of Monoclonal Antibody Against Low Antigenic Helicobacter Y. pylori Preparation of Immunization Antigen

 Incubate the low antigenic Helicobacter pylori CA2 strain on a blood agar medium under microaerobic conditions for 3-4 days at 37 ° C. The grown cells are suspended in phosphate buffered saline (PBS) and inactivated by formalin treatment. The inactivated cells are adjusted to a concentration of 0.5 to 1.5 mg / mL, preferably 1 mg / mL with PBS, and used as an immunizing antigen.

Preparation of monoclonal antibody

 A 5-week-old female BALB / c mouse was immunized with the above-mentioned immunizing antigen IOOL together with complete Freund's adjuvant, and 2 weeks later, the same antigen 50zL was boosted with Freund's incomplete adjuvant. Two weeks later, the antigen was injected intravenously at 25 / zL, and three days later, spleen cells were removed. The isolated spleen cells were fused with mouse myeloma cells (P3X63) by the method of Keller et al. (Kohler et al., Nature, vol. 256, p495-497 (1975)), and were placed in a CO2 incubator at 37 ° C. And cultured. Hybridomas were screened by the ELIS A method using LPS of three low antigenic Helicobacter pylori strains and three high antigenic Helicobacter pylori strains as antigens. That is, a hybridoma that responded to LPS of low antigenic Helicobacter pylori and did not react to LPS of high antigenic Helicobacter pylori was selected. The screened hybridomas were plated at 0.5 cells / well in a 96-well plate by limiting dilution, and only hybridomas that grew as a single colony in the well were cloned, and this limiting dilution was repeated twice. Thus, cloning was performed.

As described above, several types of monoclonal antibody-producing cell lines against low antigenic Helicobacter pylori LPS were obtained. The obtained cells were intraperitoneally administered to a pristane-treated BALB / c mouse, and about 2 weeks later, antibody-containing ascites was collected. Ammonium sulfate from the ascites fluid obtained The IgG was purified by a salting-out method and used as a monoclonal antibody. Example 3 Reactivity of monoclonal antibody with LPS

 Helicobacter pylori LPS was obtained in two ways. The first is hot phenol water extraction, which yields purified LPS. 1 g of the cultured cells of Helicobacter pylori treated with 1% phenol was suspended in 80 mL of distilled water, and mixed with an equal volume of 90% phenol water. The mixture was stirred at 68 ° C for 15 minutes, then returned to room temperature, and then centrifuged (2500 rpm, 20 minutes) to remove the aqueous layer (supernatant). Add 80niL distilled water to the remaining phenol layer, and stir again at 68 ° C for 15 minutes. After cooling to room temperature, the mixture was centrifuged (2500 rpm, 20 minutes), the aqueous layer was taken out, and dialyzed together with the previous aqueous layer (for 3 days with exchange of distilled water twice a day).

 After dialysis, the dialysis solution was freeze-dried, dissolved in a small amount of distilled water, and LPS was precipitated by ultracentrifugation (100,000 xg, 3 hours). The precipitate was washed twice by ultracentrifugation (100,000 × g, 3 hours) and finally freeze-dried to obtain purified LPS.

 Second, LPS from proteinase K treatment is not purified, but can be used for immunoreactions such as immunoblotting. Dissolve the dried cell solution (2mg) in lmL of Lysing buf fer (20% glycerol, 10% 2-mercaptoethanol, 6% SDS, 10% SDS-upper Tris buf fer used for PAGE), 100 ° C, 10 minutes Heated. After cooling to room temperature, 200 zL (2.5 mg / mL) of proteinase K was added, and the mixture was reacted at 37 ° C for 14 hours and further for 65 and 2 hours. The obtained sample was used for SDS-PAGE and the like.

 Reactivity of LPS with monoclonal antibodies was determined by SDS-PAGE and immunoblotting. As a sample for electrophoresis, about 0.8 g of purified LPS and about 100 g of proteinase K-treated LPS were added to 1 μl in terms of the initial cells, and 12.5% gel concentration of SDS-PAGE was added. Perform in the usual way. After electrophoresis, transfer the gel to a PVDF membrane filter. Monoclonal antibody (500-fold dilution) as primary antibody, anti-mouse Ig antibody as secondary antibody

(Diluted 500 times) and developed with diaminobenzene. The result is shown in figure 2. As shown in FIG. 2, the anti-helicopaylose-pylori LPS core oligosaccharide monoclonal antibody (antibody I) of the present invention reacts with a low molecular weight core oligosaccharide to form 0_ antigen polysaccharide of S-type LPS of Helicobacter pylori. A part that recognizes the LPS antigen part that is common to S-type Helicobacter pylori strains except for the Lewis antigen. The monoclonal antibody (antibody II) reacted with S-type LPS in a ladder form. Furthermore, a monoclonal antibody that recognizes the LPS antigen portion of the S-type Helicobacter pylori strain, which is the portion of the O-antigen polysaccharide portion of the S-type LPS antigen of Helicobacter pylori except for the Lewis antigen. (Antibody III) also reacted with S-type LPS in a ladder-like manner, but the reactivity pattern for each strain was different from that of Antibody II. Furthermore, a monoclonal antibody (antibody IV) that recognizes LPS of low antigenic Helicobacter pylori also reacted with LPS in a ladder-like manner, but the reactivity pattern for each strain was different from that of other monoclonal antibodies. Example 4 Confirmation of Low Antigenic Helicobacter Pylori

 Identification of low antigenic Helicobacter pylori was performed by hot phenol aqueous extraction method, immunoblot using proteinase K-treated LPS, or detection method by ELISA.

 The test bacteria, Helicobacter pylori strain Helicobacter pylori, were cultured on blood agar medium at 37 ° C for 4 days under microaerobic conditions. The grown cells were suspended in physiological saline, and an equal amount of hot phenol was added to extract LPS. Next, the aqueous layer was collected by centrifugation and dialyzed against running water to obtain an LPS standard. This was diluted 50-fold with 50 mM sodium carbonate buffer (pH 9.6), dispensed into a microplate, and left overnight at 4 ° C to solidify. Then, blocking was performed with bovine serum albumin (BSA) to prepare an EIA plate. The stomach ulcer patient serum was diluted 200-fold with 0.05 Tween 20 phosphate buffered saline (PBST), and 100 l of the solution was added dropwise to each well and reacted at room temperature for 1 hour. The reaction solution was removed by suction, and 200 L of PBST was added to each well, followed by washing three times. After washing, peroxidase-labeled goat anti-human IgG antibody diluted 50,000-fold with PBST was added to each well, and left at room temperature for 1 hour. Thereafter, 200 L of PBST was added to each well, and the wells were washed four times. After washing, 100 L of a substrate solution (3, 3 ', 5, 5'-tetramethylbenzidine, hydrogen peroxide) was added, and the mixture was allowed to stand at room temperature for 30 minutes.

Next, the reaction was stopped by adding IO L of a reaction stop solution (0.6N sulfuric acid), and the absorbance was measured with an auto-reader (wavelength 450 nm / 630 nm). Example 5 Identification of Helicobacter pylori by latex slide agglutination method ①Preparation of monoclonal antibody sensitized latex

 Spherical particles made of polystyrene having a diameter of 0.3 m (latex: commercially available) were used as a support. The monoclonal antibody and latex particles obtained in Examples 1 and 2 were each dissolved in phosphate buffered saline (pH 7.0), and the antibody was supported on a support so that the weight ratio of the antibody to latex particles was 1:10. Immobilized. This was washed with phosphate-buffered saline and blocked with bovine serum albumin to obtain antibody-sensitized latex.

 ② Sample processing method

 Collect all the test bacteria cultured on one plate of blood agar medium and suspend them in physiological saline 250 '. To this, Extraction reagent 1 (acetic acid) and Extraction reagent 2 (sodium nitrite) were added at 250 / zL, respectively, and left for 10 minutes. Thereafter, 250 L of Extraction Solution 3 (buffer) was added and neutralized to obtain a sample.

 ③Slide agglutination reaction

 The sample obtained by the method of (1) was dropped into the circle of the paper slide plate, and 50 L of the antibody-sensitized latex prepared in (2) was added thereto and mixed. After stirring for 1 minute, the presence or absence of aggregation was visually observed, and those with aggregation were regarded as positive, and those without were determined as negative. Example 6 Monoclonal Antibody Recognizing LPS of Helicobacter pylori with Low Antigenicity (Antibody IV) Specificity of Sensitized Latex Reagent

 Helicobacter pylori strains isolated from various gastric disease patients were tested for low antigenicity or high antigenicity by the method described in Example 4 and then tested as in Example 5. The results are shown in Table 1. All strains belonging to low antigenic Helicobacter pylori showed positive with this sensitized latex. On the other hand, highly antigenic Helicobacter pylori did not react with this sensitized latex.

Table 1 Specificity of sensitized latex reagent

Antigenic test strains Positive strains Low antigenic Helicobacter pylori 5 5 High antigenic Helicobacter pylori 162 0 Example 7 LPS antigen (Helicobacter pylori strain-specific LPS) commonly possessed by strains of S-type Helicobacter pylori, that is, Lewis antigens out of the 0 antigen polysaccharide portion of S-type LPS of Helicobacter pylori Specificity of a monoclonal antibody (antibody II) sensitized latex reagent that specifically recognizes the LPS antigen portion that is common to S-type Helicobacter pylori, excluding the antigen portion

 The tests were performed as in Example 5 using Helicobacter pylori strains isolated from patients with various stomach diseases, strains other than Helicobacter pylori belonging to the genus Helicobacter, and Campylobacter.

 The results are shown in Table 2. All strains whose biochemical properties belonged to Helicobacter pylori were positive with this sensitized latex. On the other hand, strains classified as Helicobacter other than Helicobacter pylori and strains classified as Campylobacter were all negative. Table 2 Reactivity between sensitized latex and various bacteria

実施例 8 4種類の抗体 （抗体 I、 I I、 I I Iおよび I V) 感作ラテックス試 薬を用いたヘリコパクター · ピロリ菌株 181株の反応性パターン

Example 8 Reactivity Patterns of 181 Helicobacter pylori Strains Using Four Antibodies (Antibodies I, II, III and IV) Sensitized Latex Reagent

 The test was carried out as in Example 5 using 181 Helicobacter pylori strains isolated from various gastric disease patients and antibodies I, II and III. .

Table 3 shows the results. By using antibody II, almost all 179 strains out of 181 strains can be detected, and by using antibodies I, II and III, all 181 strains can be detected. Table 3 Reaction pattern of Helicobacter pylori isolates by sensitized latex (using monoclonal antibodies I, II and III)

 181

I: Anti-Helicopter '' H. pylori LPS Core Oligosaccharide Monoclonal Antibody

 II: Monoclonal antibody that recognizes the LPS antigen portion of the Helicobacter pylori S-type LPS O-antigen polysaccharide portion excluding the Lewis antigen from the O-antigen polysaccharide portion, which is shared by the S-type Helicobacter pylori strain

 III: Helicobacter pylori S-type LPS antigen on S-type LPS antigen, excluding the Louis antigen, recognizes the LPS antigen portion of some S-type Helicobacter pylori strains Monoclonal Monoclonal Antibody Further, the test was similarly performed using 181 strains using Antibody IV.

 Table 4 shows the results. There were 17 out of 181 helicobacter strains detected by antibodies that recognize LPS of low antigenic Helicobacter pylori. Table 4 Reaction pattern of Helicobacter and H. pylori isolates by sensitized latex (when monoclonal antibodies I, II, III and IV are used) Reaction pattern with sensitized antibodies Number of positive strains

 Antibodies I, II, II I and IV 5

Antibodies I, II and IV 3 Antibodies I, II and III 21

 Antibodies I I, I I I and IV 3

 Antibodies I and I I 42

 Antibodies I I I and IV 1

 Antibodies II and IV 5

 Antibodies I I and I I I 28

 Antibodies I and I I I 0

 Antibodies I and I V 0

 Antibody I 3

 Antibody I I I 1

 Antibody II 69

 Antibody I V 0

 Total 1 8 1

 Example IV Monoclonal Antibody Recognizing LPS of Low Antigenic Helicobacter Yuichi Example 9 Detection Using Four Antibodies (Antibodies I, II, II and IV) and 14 Helicobacter pylori Strains

According to the method described in Example 3, LPS was purified from 14 strains of Helicobacter yuuichi 'H. pylori (Japanese Bacteriological Journal, 56 (2): 421-433, 2001, etc.), and 5 types of commercially available anti-Lewis Hara antibody (Le ^x: Seikagaku Corporation, Le ^a; Chemicon, ^{Inc.; Le y, Le b, Le} d: Signet Lab oratories Ltd.) and three Rikopakuta pylori LPS monoclonal antibodies to anti (anti Hp monoclonal Antibody) was confirmed by the immunoblot described in Example 3.

Table 5 shows the results. From the results of the electrophoresis, among the 14 Helicobacter pylori strains, 10 strains of CG, 8 strains of DU and 01 strains had the R-type LPS without the O-antigen polysaccharide in FIG. Eleven strains were found to have S-type LPS with 0-antigen polysaccharide. The helicobacter pylori LPS core oligosaccharide monoclonal antibody (antibody I) of the present invention reacted with two of the three R-type LPS strains and reacted with nine of the eleven S-type LPS strains. In addition, O-on the S-type LPS antigen of Helicobacter pylori A monoclonal antibody (antibody III) that recognizes the LPS antigen portion of the S-type Helicobacter pylori strain, which is a portion of the antigenic polysaccharide portion excluding the Lewis antigen, is an S-type LPS of 11 strains. Reacted with 7 strains. Helicobacter pylori Monochrome recognizing the LPS antigen portion of the 0-antigen polysaccharide portion of the S-type LPS of H. pylori, excluding the Lewis antigen portion, which is shared by the S-type helicobacter and H. pylori strains One null antibody (Antibody II) did not react with the rough strains, but reacted with all smooth strains. A monoclonal antibody (antibody IV) that recognizes LPS of low antigenic Helicobacter pylori reacted with 4 out of 14 strains.

 In addition, there was a Helicobacter pylori strain that did not react with any of the anti-Helicobacter pylori LPS monoclonal antibodies and anti-Lewis antibodies, such as the Ol strain.

Table 5 Reactivity of Hp-LPS with anti-Lewis antibody and anti-Hp monoclonal antibody

Hp-LPS silver stain Anti-Lewis antibody Anti-Hp monoclonal antibody

Le Le ^y Le ^a Le ^b Le ^d III III IV

GU2 S + Sat + + + Sat

DU1 S Sat ++ + one + ++ +

CG10 R +

Luo R + Shi

 CA1 S ++

 CA2 s ++++++++

CA4 s + + + + + +

CA5 s ++++++

CA6 s + + + + +

CA8 s Sat + + + +

 CA9 s one + + +

 0 1 R Sat

 Sydney s ++ +

 26695 s + + + +

 ++: Strong positive, +: Positive, Chi and I: Negative

I: Pile helicopter '' H. pylori LPS core oligosaccharide monoclonal antibody II: Recognition of the LPS antigen portion of the O-antigen polysaccharide portion of Helicobacter pylori S-type LPS, excluding the Lewis antigen portion, which is common to the S-type Helicobacter pylori strains Monoclonal antibodies

 III: Recognition of the LPS antigen portion of the S-type Helicobacter pylori strain, which is the portion of the 0-antigen polysaccharide portion on the S-type LPS antigen of Helicobacter pylori except for the Lewis antigen. Monoclonal antibodies

 IV: Monoclonal antibody that recognizes LPS of low antigenic helicopter

 *: LPS purified from Helicobacter pylori strains

 **: Analysis of electrophoresis pattern by silver staining S: Smooth type

 : Rough industrial applicability

As shown in the Examples, the anti-Helicobacter pylori LPS core oligosaccharide monoclonal antibody of the present invention recognizes a portion of the O-antigen polysaccharide portion of S-type LPS of Helicobacter pylori excluding the Lewis antigen portion. Helicopter '' H. pylori strains that are not recognized by monoclonal antibodies can be recognized. Anti-Helicobacter '' H. pylori LPS Core Oligosaccharide Monoclonal Antibody and Helicobacter pylori S-type LPS on O-Antigen Polysaccharide Excluding Lewis Antigen The detection of Helicobacter pylori using a monoclonal antibody that recognizes the LPS antigen portion of S-type Helicobacter pylori strains in common allows almost all Helicobacter pylori strains to be detected. It becomes possible to detect. Furthermore, it recognizes the LPS antigen portion of the S-type Helicobacter pylori strain, which is a portion of the 0-antigen polysaccharide portion on the S-type LPS antigen of Helicobacter pylori of the present invention except for the Lewis antigen, and which is part of the S-type Helicobacter pylori strain. The detection of Helicobacter pylori using a monoclonal antibody to be detected makes it possible to detect all Helicobacter pylori strains. Furthermore, the use of the antibody that recognizes LPS of Helicobacter pylori of the present invention can detect Helicobacter pylori derived from gastric cancer patients, and can diagnose the possibility of gastric cancer. Therefore, the monoclonal antibody of the present invention can be effectively used widely for diagnosis of gastric diseases and the like. All publications cited herein are incorporated by reference in their entirety. It will be readily apparent to those skilled in the art that various modifications and alterations of the present invention are possible without departing from the technical concept and the scope of the invention described in the appended claims. The present invention is intended to cover such modifications and variations.

Claims

The scope of the claims  1. A monoclonal antibody that recognizes the core oligosaccharide of LPS of Helicobacter pylori.  2. The monoclonal antibody according to claim 1, which recognizes surface LPS of 26695, CA9, CA6 and CA4 strains among 2.6 Helicobacter pylori strains, 26695, CA9, CA6, CA4, CA2 and GU2.  3. The monoclonal antibody according to claim 1 or 2, which is produced by the Hypri-Doma strain HPCA / D42-3 (Accession number: FERM BP-7894). 4. The monoclonal antibody according to any one of claims 1 to 3, which is a Fab, Fab ', F (ab') ₂ or Fv fragment.  5. A hybridoma, which is a hybridoma strain HPCA / D42-3 (accession number FERM BP-7894).  6. A monoclonal antibody that recognizes the LPS antigen portion of the Helicobacter pylori S-type LPS 0-antigen polysaccharide portion of the S-type LPS excluding the Lewis antigen, which is part of the S-type Helicobacter pylori strain.  7. The monoclonal antibody according to claim 6, which recognizes the surface LPS of CA6, CA4, CA2 and GU2 strains among 7.6 Helicobacter pylori strains, 26695, CA9, CA6, CA4, CA2 and GU2.  8. The monoclonal antibody according to claim 6 or 7, which is produced by the hybridoma strain HP220 / B25_3 (Accession number: FERM BP-7893). 9. The monoclonal antibody according to any one of claims 6 to 8, which is a Fab, Fab ', F (ab') ₂ or Fv fragment.  10. A hybridopoma strain, which is a hybridoma strain # 20 / # 25-3 (accession number FERM BP-7893).  1 1. Monoclonal that recognizes the LPS antigen portion of the Helicobacter pylori S-type LPS O-antigen polysaccharide portion excluding the Lewis antigen from the O-antigen polysaccharide portion, which is shared by the S-type Helicobacter pylori strain antibody. 12.6 Of the 26 Helicobacter pylori strains, 26695, CA9, CA6, CA4, CA2, and GU2, the surface LPS of all 26695, CA9, CA6, CA4, CA2, and GU2 strains was identified. 12. The monoclonal antibody according to claim 11, which is known.  13. The monoclonal antibody according to claim 11 or 12, which is produced by the hybridoma strain HP220 / 2D10-2 (Accession No. FE-Registration BP-7891). 14. The monoclonal antibody according to any one of claims 11 to 13, which is a Fab, Fab ', F (ab') ₂ or Fv fragment.  1 5. A hybridoma that is a hybridoma strain HP220 / 2D10-2 (Accession number FERM BP-7891).  1 6. Monoclonal antibody that recognizes low antigenic LPS of Helicobacter pylori strain.  17. The monoclonal antibody according to claim 16, which recognizes low antigenic LPS that is abundantly present in Helicobacter pylori strains derived from gastric cancer patients and does not react with serum of patients with chronic gastritis, gastric ulcer, and duodenal ulcer.  18. The monoclonal antibody according to claim 16 or 17, which recognizes the surface LPS of CA6, CA4 and CA2 strains out of 18.6 strains of Helicobacter pylori strains, 26695, CA9, CA6, CA4, CA2 and GU2.  19. The monoclonal antibody according to any one of claims 16 to 18, which is produced by the hybridoma strain HP217 / B15_4 (Accession No. FERM BP-7892). 20. The monoclonal antibody according to any one of claims 16 to 19, which is a Fab, Fab ', F (ab') _2, or Fv fragment.  2 1. Hypridoma strain, which is Hypridoma strain 1 ^ 217 / 815-4 (accession number? £ 丽 BP-7892).  22. A method for detecting LPS of Helicobacter pylori strain using the monoclonal antibody according to any one of claims 1 to 4 and the monoclonal antibody according to any one of claims 11 to 14 .  23. The method for detecting LPS of Helicobacter pylori strain according to claim 22, further comprising using the monoclonal antibody according to any one of claims 6 to 9. 24. The method for detecting LPS of Helicobacter pylori strain according to claim 23, further comprising using the monoclonal antibody according to any one of claims 16 to 20. 25. The monoclonal antibody according to any one of claims 16 to 20, which is abundant in Helicobacter pylori strains derived from gastric cancer patients, and is used for chronic gastritis and gastric ulcer disease. Or low-antigenic LPS that does not react with the serum of patients with duodenal ulcer. 26. A method for detecting Helicobacter pylori using the monoclonal antibody according to any one of claims 1 to 4 and the monoclonal antibody according to any one of claims 11 to 14.  27. The method for detecting Helicobacter pylori according to claim 26, further comprising using the monoclonal antibody according to any one of claims 6 to 9. 28. The method for detecting Helicobacter pylori according to claim 27, further comprising using the monoclonal antibody according to any one of claims 16 to 20.  29. The monoclonal antibody according to any one of claims 16 to 20, wherein the serum is present in patients with chronic gastritis, gastric ulcer or duodenal ulcer, which is abundant in Helicobacter pylori strains derived from gastric cancer patients. A method for detecting low antigenic Helicobacter pylori, which comprises detecting low antigenic LPS that does not react with E. coli.  30. The monoclonal antibody according to any one of claims 1 to 4, the monoclonal antibody according to any one of claims 6 to 9, and the monoclonal antibody according to any one of claims 11 to 14. A kit for detecting Helicobacter pylori comprising at least one monoclonal antibody selected from the group consisting of the monoclonal antibody according to claim 1 and the monoclonal antibody according to any one of claims 16 to 20.  31. Detecting Helicobacter pylori according to claim 30, which comprises the monoclonal antibody according to any one of claims 1 to 4 and the monoclonal antibody according to any one of claims 11 to 14 Kit for.  32. A kit for detecting low antigenic Helicobacter pylori, comprising the monoclonal antibody according to any one of claims 16 to 20.