JPH06507494A - Method for detecting and treating diseases caused by bacterial allergens - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for detecting and treating diseases caused by bacterial allergens field of invention The present invention relates to the field of allergens, particularly the identification of bacterial allergens, as well as the diagnosis and diagnosis of diseases. The etiology of many idiopathic relapsing diseases is unknown and their use in therapy.

Some of these diseases are believed to be related to infection by microorganisms. but However, the causal relationship between microorganisms and diseases is often not confirmed. chronic gastritis and This category also includes twin diseases of inflammatory ulcers.

Chronic gastritis and peptic ulcer disease are extremely important diseases. 5-10% of people will live forever Chronic gastritis or gastroduodenal ulcer develops. Ulcers are a major cause of morbidity It has become.

The annual incidence of symptomatic peptic ulcer disease in the United States is approximately 18% per 1,000 adults. (i.e. 4,500.000 people).

Approximately 350,000 new patients are diagnosed with peptic ulcer disease each year.

Diagnosis of the above diseases is generally performed by gastroduodenoscopy; This is a costly method that damages healthy tissue. Treatment is oral medication, dietary system Including limited medical and surgical procedures. It is rarely definitive and such chronic conditions can be reversed. It often occurs repeatedly.

reported that these microorganisms were physically associated with the foci of chronic gastritis. A great deal of research is being conducted to clarify the causal relationships between diseases. H,pylo ri-induced local pH changes, toxin release (Huperts et al. l, (1988), Eur J, C11n Microbiol Infec tDis 7:576], and harmful enzymes (destructive enzymes). early speculations regarding the differences between various bacterial strains (Eatonet et al. , (1989), no firm conclusion regarding Infect Immu has been obtained. . Furthermore, the recognition that a significant number of clinically healthy patients harbor the microorganisms , the search for rational explanations of causal relationships has become even more complex.

Related technology Ce5ka et al., (1972), Radi.

immunosorbent As5ay of Allergens [J, Allergy and C11n.

Immunol, soil 1:1] to detect IgE against specific allergens. Discloses the Radioallergosorbent (RAST) method. ing.

Na1ebuff et al, (1979), The 5 study of IgE in the Diagnosis of Allergic Dis orders in an Otolarygology Practice (Otolaryngol Head Neck Lambert et al, , (1978), Diffuse Varioliform Gastriti s (Digestion Upper Uni 159), the focus of chronic gastritis is IgE positive. They report experiments showing that it contains cells, which they believe are responsible for the disease's allergy. - Interpreted as indicating the source.

Andre et al., (1983), Evidence for Ana phylactic Reacttons in Peptic Ulcer and Varioliform Ga5tritis (Annals of Allergy 5↓:325) shows that various types of The average number and class distribution of IgE-containing cells in patients with gastritis/ulcers were investigated. and their results suggest that mucosal anaphylaxis may be the cause of gastritis. This is interpreted as confirmation of this theory.

Calenoff et al., (1983), Bacteria-3pec ific IgE in Patents with Na5al Po1y posis (Arch Otolaryngol 109:372) is a chronic nasal To detect IgE specific for bacterial antigens in patients exhibiting luminal polybosis describes the use of a modified RAST method.

Warren (1983), Unident i f 1edCurved Bacilli on GastricEpithelium in Acti ve Chr.

nic Ga5tritis (Lancet, 1273) is slightly curved and S-shaped Bacilli were found in 135 gastritis biopsy specimens. It is reported that the Bacillus species are most often associated with inflammation and acute and is almost always present in chronic gastritis.

Peterson (1991), Helicobacter pylori and Peptic UlcerDi 5ease [New English d　JoMed.

374:1043) is H. pylori (formerly Campylobacte Research literature on the relationship between R. pylori) and gastritis/ulcer It is.

Description of the invention The presence of IgE against the specific subfraccidin protein allergen of L↓ It was found that there was a high positive correlation between the two. These results are valid for the above bacteria. The harmful immune response caused by pathological reactions, especially hypersensitivity mediated by specific IgE. This indicates that it is the cause of the symptomatic reaction.

In the modified RAST method, purified protein allergens are bound to a solid support. . Sera to be tested before reacting with Hopylori protein allergens was treated to remove IgA and IgG. This “Scrubbing” ng)” step was essential for the detection of allergen-specific IgE.

The disease can be detected with relatively little damage to healthy tissue. Additionally, purified protein alleles Genes can also be used to treat the disease by immunotherapy.

Accordingly, one aspect of the invention provides that IgE immunologically binds to bacterial allergens. A method for measuring IgE, wherein serum presumed to contain IgE is bound to a solid support. The bacterial extract was then washed, reacted with labeled anti-1gE, and transferred to a solid support. consists of detecting labeled anti-1gE bound to a solid support. a composition capable of removing IgA and IgG from serum prior to reaction with bacterial extract; It has been improved to react with serum, and the amount of the composition used is A method that is sufficient to remove IgA and IgG that interfere with binding to allergens It is.

Another aspect of the invention is a method of preparing purified protein allergens from bacteria. (a) Treat bacteria containing protein allergens with acetone to remove lipid components. (b) The acetone-treated bacteria are treated with buffers, salts, metal chelators, and proteins. (c) protein-containing (d) a fraction with a molecular weight of about 1.000 or more is separated from complex carbohydrates and nucleic acids; Collect the above protein-containing composition, and inject the proteins in the compositions (e) and (d) into This method consists of isolation by on-exchange chromatography.

Yet another aspect of the invention provides for the treatment of diseases resulting from allergic reactions to bacterial infections. immunotherapy for treating an individual with a protein substantially derived from bacteria. The method consists of introducing a composition consisting of an allergen, and the conditions for said introduction are such that the allergen is A method that is sufficient to alleviate the symptoms of allergic reactions to pylori - A method for determining whether a reaction occurs, the method comprising: (a) against H, pylori allergen; (b) provide serum from an individual estimated to contain IgE, and (b) substantially contain H, py lori protein allergen, and (C) administer the serum of (a) to ( Immunological binding occurs between the composition of b) and the IgE and the allergen thereto. (d) the composition of IgE in the serum of (a) and (b), if any; Detects IgE-allergen complexes formed between protein allergens in foods. This is a method that consists of putting out.

Yet another aspect of the present invention is that substantially all protein allergens of H. pylori It is a composition consisting of

Yet another embodiment of the present invention provides for the H8 pylori protein to be bound to a solid support. It is a quality allergen.

Yet another aspect of the invention provides one or more epitopes capable of H. pylori-induced gastritis. comprising introducing a composition comprising a polypeptide comprising said polypeptide; is sufficient to alleviate the allergic reaction to H. pylori in the individual. The composition further comprises a suitable excipient.

Yet another aspect of the invention provides a H. pylori epitope, enclosed in a suitable container. polypeptide containing at least one epitope that can be immunologically identified using the polypeptide, and the IgE, if present, in the biological sample. methods for detecting immunological conjugates containing structural analogs of diagnostic epitopes. a composition in which the structural analog binds to an IgE baratope.

Yet another aspect of the present invention is directed to a polypeptide allergen of H. pylori. A composition comprising purified polyclonal antibodies.

Yet another aspect of the present invention is directed to a polypeptide allergen of H. pylori. Compositions Containing Monoclonal Antibodies Using Protein A in the Detection of gE It is a graph which shows the effect of scrubbing a cleansing liquid.

Figure 2A shows the H, pylori protein allergen in healthy individuals (control). Figure 2 is a graph showing serum 1gE levels of IgE for subfractions.

Figure 2B shows the subfraction of H. pylori protein allergen in gastritis patients. 1 is a graph showing serum IgE levels in response to action.

Figure 3 shows all used, control and gastritis patients isolated from the HPLCDEAE column. Figure 2 is a graph plotting the net total 1gE immunoreactivity of the derived serum.

Figure 4 shows the HPLCDEAE column flank 59.64.66.68.7. Net total IgE immunity of sera from controls and gastritis patients using proteins 2 and 74. It is a graph plotting epidemiological reactivity.

Embodiment of the invention The present invention provides that individuals suffering from chronic gastritis or gastroduodenal ulcers It has serum IgE specific for protein allergens, which indicates that this microorganism Starting from the knowledge that hypersensitivity to substances may be the cause of such diseases are doing.

The protective mucus is not very harmful to the host or its protection against stomach acid. It receives nutrients from the liquid layer. Due to the inflammatory process observed in chronic gastritis , individuals become prone to general allergies, and furthermore, they are exposed to H, MHCII antigenic framework required to provide pylori allergenic proteins They will have a lot of work. Quantitative and/or secretion of protective mucus by Sakazuki cells Or a qualitative deterioration probably occurs, making the underlying mucosa more vulnerable. Furthermore, allergic reactions In response, local histamine production tends to increase and is absorbed into the gastric venous plexus, Increases stomach acid production. These two phenomena combine to increase the pain of the pre-initial lesion. However, due to a certain allergic reaction to H. pylori, the lesion enlarges and becomes chronic. Sexualization is brought about.

Based on the above new findings, H, p)'fort-induced allergic reactions in individuals Immunoassays can be designed to detect. In one embodiment, the The immunoassay uses purified protein allergen, which is At least one purified protein in the soil can be carried out and is relatively damaging to healthy tissue. A composition containing a wood allergen and/or a protein allele of H. pylori Immunotherapy using allergoids of Gen.

In the practice of this invention, unless otherwise specified, conventional protein purification methods, Those within the skill of the art are used, such as biology, molecular biology, immunology, etc.

The term "allergen" as used herein refers to Ig Refers to antigens that cause allergic sensitization by E antibodies.

The term “allergod” refers to the IgE class, but not the IgE class. chemically modified that produces gG antibodies, thereby reducing allergic symptoms. Refers to allergens.

The term "individual" as used herein refers to vertebrates, especially generally refers to members of mammalian species, including, but not limited to, livestock, sport animals, and humans. Including primates.

The term "allergy" as used herein generally refers to hypersensitivity. refers to a degenerative state of the immune response that gives rise to

As used herein, the expression "immunologically identifiable using/by" means Indicates the presence of epitopes and polypeptides that are also present in the indicated polypeptide. vinegar. Immunological identification may be determined by antibody binding and/or competition for binding. Such methods are well known to those skilled in the art and are described below.

The term "epitope" as used herein refers to an antigenic determinant of a polypeptide. . An epitope consists of three amino acids that form a conformation unique to the epitope. May contain acids. Generally one epitope consists of five or more such amino acids, More typically it consists of 8-10 or more such amino acids. amino acid cornho Methods for determining the mation are well known in the art and include, for example, X-ray crystallography and and two-dimensional nuclear magnetic resonance.

A polypeptide is known when it is "immunologically reactive" with an antibody; When binding to an antibody through antibody recognition of a specific epitope contained within the Immunological reactivity is determined by antibody binding, especially the rate of antibody binding. and/or a known polypeptide containing an epitope for the antibody as a competitor. This can be determined by the binding conflicts used as . consists of a polypeptide Methods for determining immunological reactivity with antibodies are well known in the art. “ An “immunoreactive” polypeptide is also an “immunogenic” polypeptide. The term "sexual polypeptide" refers to cellular and/or humoral, whether in the presence or absence of adjuvant Refers to a polypeptide that elicits an immune response.

The term "antibody" as used herein refers to a antibody containing at least one antibody binding site. Refers to a group of lipeptides or polypeptides. “Antibody combining site” or “binding domain” The antibody molecule's variable region folds to complement the characteristics of the antigen's epitope. It has a typical internal surface topography and charge distribution, which enables immunological reactions with antigens. It is formed by creating a three-dimensional connective space that can be used. The antibody binding site is the antigen-binding Also from the H chain ring (VH) and L end region (VL) that form a hypervariable loop that contributes to can be formed. “Balatopes are epitopes, which are the simplest form of antigenic determinants. It is the antigen binding site for The term “antibody” refers to, for example, vertebrate antibodies, Hybrid antibodies, chimeric antibodies, modified antibodies, -valent antibodies, Fab proteins, and single Contains domain antibodies.

The term "polypeptide" refers to a polymer of amino acids and refers to a product of a specific length. It's not. Therefore, peptides, oligopeptides and proteins are polypeptides. included in the definition of The term also refers to post-expression modifications of polypeptides, such as glycosylation. It does not refer to dylation, acetylation, phosphorylation, etc., and these are excluded. In the above definition Included are, for example, natural or non-natural amino acids (e.g. non-natural amino acids) polypeptides containing one or more analogs of amino acids (including Other modifications well known in the art can be mentioned. “Poly The term "peptide" does not refer to the method of manufacturing the molecule, but rather to the natural molecule, and molecules produced by chemical or recombinant synthesis.

The term "biological sample" as used herein refers to a tissue or sample isolated from an individual. Refers to the use of body fluids, including but not limited to plasma, serum, spinal fluid, lymph fluid, etc. , external sections of the skin, respiratory tract, intestines and genitourinary tract, tears, saliva, milk, blood cells, tumors , organs, and even in vitro cell culture component samples.

The term "coupled" herein refers to a covalent bond or Bonding through strong non-covalent interactions (e.g. hydrophobic interactions, hydrogen bonds, etc.) refers to Covalent bonds include, for example, esters, ethers, phosphoesters, amides, It can be a peptide, an imide, a carbon-sulfur bond, a carbon-phosphorous bond, etc.

The term "support" refers to any material to which the desired polypeptide is attached. refers to any solid or semi-solid surface.

Suitable supports include glass, plastic, metal, polymer gels, etc. It can also take the form of beads, wells, dip rods, membranes, etc.

The term "label" as used herein refers to a detectable (preferably quantifiable) label. A polynucleotide or polypeptide used to give a signal. Refers to any atom or group (moi ety) that can be bonded to

The term "therapy" herein refers to prophylaxis and/or therapy.

The term “immunogenic” means that one or more functions of the immune system are enhanced, resulting in an “immunogenic substance”. Refers to substances used to stimulate the body's immune system to counteract.

In one embodiment of the invention, an individual's allergy to Hlpylori - Sensitivity is determined by detecting IgE specific to H, I) Vlori allergen. will be determined.

Any method known in the art for detecting allergen-specific 1gE can be used. can be done.

For example, in one method allergens (this term refers to allergen polypeptides) one or more epitopes that are immunologically identifiable using epitopes of of the polypeptide is attached to a solid support. Specific to allergens derived from test materials A biological sample presumed to contain specific IgE is reacted with an allergen-support conjugate. and detect IgE that has immunologically reacted with the allergen in the conjugate. This kind of a An example of a test is the RAST method.

Generally, in the RAST method, allergen extracts are added to cellulose particles or disks. Bind to filter paper. Allergen binding of patient serum or standard serum containing IgE antibodies react with immunosorbent. After thorough washing, the labeled anti-IgE was immunosorbed. React with Rubent. After further washing, the label on the separated solvent is measured. This is then a measurement of the amount of specific serum IgE antibodies against the allergen.

In a preferred embodiment, the RAST method interferes with the binding of IgE to the allergen. Increase its sensitivity by removing IgG and/or IgA antibodies obtained It will be improved as follows.

This is especially true when measuring serum 1gE specific to the H. pylori allergen. is important. Reactants capable of removing IgG and/or IgA are known in the art. For example, protein G1 anti-human IgG and anti-human IgA, and One example is Ting A. Conveniently, these reactants are It can be immobilized on a solid support including loin. The amount of reactants used is Although it removes interfering IgG and IgA, it does not remove the IgE that should be detected. Make sure the amount is sufficient. The desired amount is determined by methods well known to those skilled in the art.

Incubating serum with protein A removes interfering IgG and Methods for removing IgA antibodies and/or IgA antibodies are described in the Examples below. Generally used The amount of protein A that the blocking antibody uses will compete with IgE with the same specificity The amount should be sufficient to prevent

Improved RAST methods may also include the use of purified protein allergens. protein Methods for purifying are well known in the art and include fractional extraction, salting out, ion exchange, etc. Chromatography on polymeric resins, affinity chromatography, centrifugation Examples include separation. For example, Methods in Enzymol ogy for a variety of methods for pur See ifying proteins. H. pylori protein allergen An example of a purification method to separate proteins from carbohydrates, lipids and nucleic acids is provided in the Examples. D.E. Further isolation of protein allergens by HPLC chromatography in AE IgE obtained from individuals with chronic gastritis and/or gastroduodenal ulcers by separating H, a sub-fraction of pylori-derived protein allergens that bind to Identified. Such allergenic cancer-specific IgE is not present in normal control individuals. It was qualitatively absent. Allergens derived from the above fractions are specific for immunoassays. It is useful for

Conveniently, immunological identification using an epitope of H. pylori allergen A diagnostic kit may include a polypeptide containing one or more possible epitopes. can. A diagnostic kit consists of a polypeptide contained in a suitable container; Detect immunological complexes, if any, formed with IgE in biological samples. including means for In some cases, immobilizing the polypeptide on a solid support You can also do it. In addition, the kit includes other suitable packaged reagents as required for the particular diagnostic protocol, For example, standard reagents, buffers, and instructions for performing the test using kit components. It may also include.

In another embodiment of the invention, HoI) with a propensity for gastric disease induced by HoI) Individuals suffering from or suspected of having the microorganism are identified as having an allergy to the microorganism. Treat with substances that reduce response. Treatments may include, for example, purified protein allergens. epitope on the H9 pylori protein allergen. recombinant polypeptides that are immunologically identifiable using allergens and immunologically cross-reactive properties. This can be done using lipeptides or anti-idiotype antibodies.

DEAE fraction 59.64.66.6 whose preparation method is described in Example 1 One or more allergens included in 8.72 and 74 may be particularly suitable.

Treatments may further include, for example, using allergoids of the H. pylori protein allergen. You can also do it by Methods for preparing allergoids from antigens are well known in the art. It is well known. Typically, mild formalin or glutaraldehyde of the antigen The treatment removes allergens without affecting antigenicity (IgG “blocking antibody formation”). (I'gE formation).

Treatment may include e.g. protein allergen antigens that bind to the corresponding IgE paratope. It can also be carried out using a composition comprising at least one structural analog of the pitope. Ru. Structural analogs bind to baratopes in a manner that mimics the immunological binding of epitopes. It is an organic molecule that can have an appropriate charge distribution and hydrophobicity/hydrophilicity.

When the aim is to alleviate allergic reactions by immunotherapy in the form of hypersensitivity reduction, The treated individual receives continuous injections of compositions containing one or more relevant allergens.

Treatment begins at doses low enough to avoid any local or systemic reactions. until reaching the highest dose that the patient can tolerate without excessive local or systemic reactions. Administer by injection in increasing doses once or twice a week. then less Continue maintenance dosing infrequently, usually every 2 to 6 weeks depending on individual response. Continue. However, the actual dosage and treatment resin will depend on the individual being treated and determined by the person in charge of the treatment.

In another embodiment of the invention, the immunoreactive polypeptide (including the allergen) Structural analogs of the domain or epitope are prepared into vaccines. More than one type of vaccine immunogenic polypeptides. If recombinant, such ports in a variety of host cells (e.g. bacteria, yeast, insects or mammalian cells). or isolated from bacterial preparations.

Vaccines containing structural analogs of immunogenic polypeptides or epitopes as active ingredients The preparation of tin is well known to those skilled in the art. Typically such vaccines are available as solutions or suspensions. Prepared as a cloudy injectable solution but must be dissolved or suspended in a liquid before injection. It can also be prepared in solid form suitable for use.

Preparations can be emulsified and proteins can be encapsulated in ribosomes. can. The immunogenic active ingredient is pharmaceutically acceptable and compatible with the active ingredient. It is often mixed with excipients. Suitable excipients include, for example, water, physiological Saline, dextrose, glycerol, ethanol, etc., and combinations thereof can be mentioned. Additionally, if desired, the vaccine may contain e.g. wetting agents or Micro-adjuvants such as emulsifiers, pH buffers and/or to enhance vaccine efficacy. The adjuvant may also be included. Examples of adjuvants that may be effective include , but not limited to aluminum hydroxide, N-acetyl-muramyl-thi-tre Oniru D-isoglutamine (thr-MDP), N-acetyl-norm Ramiru L-alanyl D-isoglutamine (CGP 11637.nor-M DP), N-acetylmuramyl-L-alanyl-D-isogluta mini-roux-alanine-2-(1°,2'-dipalmitoyl-5n-glycero- 3-Hydroxyphosphoryloxy)-ethylamine (CGP 19835A, M TP-PE), and RIBI (which is called TP-PE), and RIBI (which is 3 Seed components, monophosphoryl ribide A1 trehalose dimycolate and cell wall scale Luton (MPL 10 TDM + CWS) 2% squalene/Tween 80 emal (included in the Japanese version) can be mentioned. The effectiveness of adjuvants is produced by administering the above polypeptide in a vaccine containing H, pyl.

L↓ Measuring the amount of antibodies to an immunogenic polypeptide containing an immunoreactive sequence It can be determined by

Vaccines are conveniently administered parenterally, for example by subcutaneous or intramuscular injection. is good. Other formulations suitable for other forms of administration include suppositories and, in some cases, oral Mention may be made of formulations. In suppositories, for example, polyalkylene glycol or conventional binders and carriers such as triglycerides. child Suppositories such as the mixture containing 0.5% to 10% of active ingredient, preferably 1% to 2%. It can be molded from a compound. Oral preparations include, for example, pharmaceutical mannitol, Cutose, starch, magnesium stearate, sodium saccharin, cellulose Contains customary excipients such as carbonate and magnesium carbonate. Such compositions may be It takes the form of suspension, tablet, gunpowder, capsule, sustained release preparation or powder, and contains 10% to 9 5%, preferably 25% to 70% active ingredient.

Proteins can be prepared into vaccines in neutral or salt form. pharmaceutically Acceptable salts include acid addition salts (formed with free amino groups of the peptide). This can be an inorganic acid such as hydrochloric acid or phosphoric acid, or acetic acid. , formed using organic acids such as oxalic acid, tartaric acid, and maleic acid. free cal Salts formed with boxyl groups are, for example, sodium, potassium, ammonium, carbon inorganic bases such as lucium or ferric hydroxide, and isopropylamine, Trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. It can also be derived from organic bases.

Vaccines are administered in a manner compatible with the dosage formulation and with prophylactically and/or therapeutically effective administered in amounts. The amount to be administered is usually about 5 μg to about 250 μg per dose. g of the antigen to be administered, the subject to be treated, the ability of the subject's immune system to synthesize antibodies, and according to the degree of protection desired. The exact amount of active ingredient required to be administered is at the discretion of the practitioner. and can be specific to each subject.

The vaccine can be given in a single dose or preferably in multiple doses. . For multiple doses, the initial immunization is administered 1 to 10 times, followed by the immunization. Additional doses and time intervals necessary to maintain and/or enhance the epidemic response, e.g. Administer at a different dose every 1 to 4 months and re-administer after a few months if necessary. can be done. The dosing resin is determined at least in part by individual requirements. It is left to the discretion of the implementer.

In another embodiment of the invention, epitopes of the H. pylori allergen are used to A polypeptide containing one or more immunologically identifiable epitopes is used as an immunizing agent. to the H. pylori epitope using methods well known to those skilled in the art. Produce antibodies. The antibodies produced are purified polyclonal antibodies, heavy chain antibodies, mono It can be a clonal antibody, antibody fragment, etc. These antibodies are, for example, Purify the polypeptide in question by affinity chromatography. It can be used to In particular, immunotherapy using epitopes of the ori allergen. Polypeptides containing epitopes that are epidemiologically identifiable can be purified.

Furthermore, antibodies against H. pylori epitopes can be used to produce anti-idiotype antibodies. It can be used for construction. Such anti-idiotypic antibodies are Contains areas that mimic taupe. Anti-idiotypes can be determined using methods well known in the art. H. pylori epitope is usually used as an immunizing agent. using an antibody against the protein.

Anti-idiotypic antibodies can be used for immunotherapy in individuals susceptible to H. pylori allergens. H9py containing epitopes that immunologically cross-react with anti-idiotype antibodies and anti-idiotype antibodies. It may be useful for purifying and/or detecting antibodies against lori antigens.

Using the immunogenic polypeptide produced as described above, polyclonal antibodies and Antibodies, including monoclonal antibodies, can be produced. polyclonal antibody If desired, the mammal of your choice (e.g. mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide bearing the H. pylori epitope. . Serum from immunized animals is collected and processed according to known methods. H,pylor Serum containing polyclonal antibodies against the i epitope will generate antibodies against other antigens. If present, polyclonal analysis is performed by immunoaffinity chromatography. Antibodies can be purified. How to produce and process polyclonal antisera Well known in the art, for example Mayer and Walker (19 87) IMMUNOCHEMICAL METHODS IN CELL A ND MOLECULAR BIOLOGY (Academic Press, London). Alternatively, individuals infected with H. pylori in advance Polyclonal antibodies can be isolated from the body and purified by the methods described above. .

Monoclonal antibodies against H. pylori epitopes are readily available to those skilled in the art. produced. A general method for producing monoclonal antibodies using hybridomas is well known. Immortalized antibody-producing cell lines can also be created by cell fusion. Yes, it is possible to directly generate 8923 spheres using oncogenic DNA. Transformation or Epstein-Barr virus transfection It can also be produced by other methods. For example, M, 5chreier et al, (1980) HYBRIDOMA TECHNIQUES PR INCIPLESAND PRACTICE, 5E COND EDITION (Springe r-Ve r 1 ag, N, Y,); Hamme rling et al, (1981) MONOCLONAL ANTIBO DY AND T-CELLHYBRIDOMAS; Kennet et al ,.

(1980) MONOCLONAL ANTIBODY.

Further U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427. No. 783; No. 4,444.887; No. 4.466.917; No. 4.472. 500:4,491,632; and 4,493,890. H1pyl Some monoclonal antibodies raised against orf epitopes have various properties. , for example, to screen for isotype, epitope affinity, etc. Antibodies against H. pylori epitopes are monoclonal and polyclonal. All of these are particularly useful for diagnosis, and the neutralizing ones are useful for passive immunotherapy. be. Monoclonal antibodies are used specifically to produce anti-idiotype antibodies can do.

Anti-idiotype antibodies are used to detect the “internal image” (internal image) of the antigen of the infectious agent against which protection is desired. It is an immunoglobulin responsible for t e rna 1 image). For example, N 15onoff, A.

et al., (1981), Cl1n, Immunol.

Immunopathol, 21:397-406; and Dreesman e. tal, (1985), J. Infect, Disease 151 Near 6 See 1. Methods for producing anti-idiotypic antibodies are well known in the art and ag et al., (1985), J, 1mm Example Examples of the present invention are given below for illustrative purposes, but these examples do not fall within the scope of the present invention. It is not intended to limit. In light of the disclosure of the invention, various embodiments may be made within the scope of the claims. It will be clear to those skilled in the art.

Example I Isolation of H. pylori protein allergen and its allergen 4; ATCC, Bet hesda, MD, USA) to substantially the Sm1bert method (Smibert , Ann, Rev. Microbiol, 1978 32:67). did. In particular, aspectical from the American Type y and suspended in 1 ml of sterile Difco Brucella broth. , three separate B by inoculation loop (tnoculat tng 1loop) rucella agar plates (anaerobic, San Jose, CA). P rate in a micro-aerobic atmosphere of 85%N7.10%CO6 and 5%02. and incubated at 35°C for 5 days. After incubation, remove the plate. When I took it out and examined it, I found small grayish-white colonies. Gram staining microscope Examination showed large U-shaped and looped Gram-negative rods (5μ). This ± into a new rucella plate and incubate the plate at 35°C for 3-5 days. Microorganisms were incubated under aerobic conditions. After 3 days, growth of H. pylori colonies was more popular. Such colonies were cultured in broth seeds (br.

As an inoculum for th5eed culture I used it.

Several 10ml screw cap test tubes containing 5% horse serum (GIBCOBRL) Add 5 ml of sterile Brucella broth and the swab collected from the plate. I moved with Ronnie. All test tubes were placed in a micro-aerobic atmosphere at 35°C for 3-5 days. Incubated. If severe turbidity is observed in the test tube after this period, The purity of the culture fluid was investigated by microscopic examination of Gram-stained slides.

Transfer the broth seed culture to sterile Difco Brucella containing 5% horse serum. Used as inoculum for 1 liter of broth. 3 liters of inoculated culture solution Incubate for 3-5 days at 35°C in a micro-aerobic atmosphere in a flask. It was propagated by If moderate turbidity is observed, the purity of the culture solution is determined as described above. I checked. A typical yield of 2.0 g of unwashed cells is obtained from 1 liter of culture medium. Ta.

Live microorganisms obtained from 1 liter of culture to isolate protein allergens Materials were pelleted by centrifugation at 3.00 Orpm for 15 minutes at 4°C.

These are attenuated, defatted together by suspension, and water Stirred in cold acetone for 15 minutes. Attenuated bacteria are centrifuged in the same way and pelleted again. did. The pellet was washed with 50mM sodium phosphate, pH 7.3, 150mM Na C1゜5mM EDTA, 5mM EGTA, 100μg/ml PMSF and 1 00 μg/ml resuspended in 20 ml of cold buffer containing benzamidine I let it happen. 10ml or 150-210μ acid-washed glass beads (Si gma.

St., Louis, MO, USA) and the suspension was transferred to a 40°C tube with a standard tip. Using a Branson 5onifier II ultrasonic cell disruption device, It was set to No. 7 and subjected to ultrasonic treatment. While sonicating the suspension for 15 min, methane Cooled in a cool water bath. The resulting mixture was then centrifuged as described above, and the supernatant was drained. saved.

density gradient centrifugation The supernatant was transferred to a Beckman SW 40Tio-tar (Beckman, Pa1 Centrifuge at 100,000 g for 1 hour at 4°C in Ta. 0.456 g/ml of RbC1 (Aldrich Ch. chemical Co., Milwaukee, Wis.

USA) was added. The solution was placed in a Beckman 70Ti rotor (first 2 65,000 rpm for 4 hours and 48.00 Orpm for the next 24 hours) at 4°C. Centrifuged for 48 hours. Stage the contents of each density gradient tube into an equal volume starting from the bottom of the tube. Collected into 10 fractions. Residual complexes present in supernatant before density gradient format The pellet in the tube, consisting mostly of carbohydrates and nucleic acids, was discarded.

ion exchange chromatography Transfer each density gradient fraction to dialysis tubing with a cutoff average molecular weight of 1°000. 20mM sodium phosphate buffer, pH 7,0, dialyzed at 4°C. spectroscopy Protein content per fraction at wavelength 280 nm by photometer The amount was estimated. 90% of the detected proteins were found in fractions 2-6. , these fractions were pooled. The pooled fractions were then divided into Bl o-8il DEAE analysis anion exchange HPLC column (BioRad, Ric hmond, CA, USA) so that the end of the concentration gradient is 100% buffered. A 30 minute linear concentration gradient for solution B was performed. Equilibration buffer (buffer A) buffer The solution (buffer B) was 20mM sodium phosphate containing 1.0M NaCl, pH 7. ,0. The eluted fractions were collected and each protein was determined as described above. Quantified.

The void fraction containing polymers and cationic molecules was collected using Blo-5 il SP cation exchange column (BioRad) and positive for DEAE. The column was operated under precise concentration gradient conditions. Protein of the resulting elution fraction was quantified.

Covalent attachment of H. pylori proteins to disk filters in a substantially Ce5ka manner. (Ceska et al.

J, Allergy and C11n, Immun, 49: 1 (197 2)], a CnBr-activated disk-shaped filter paper was prepared. To explain in detail, 5c hleicherand 5chuell 589 punched from red ribbon filter paper Disc filter paper (diameter 6 mm) was cut using the following. The discs were swollen with water for 30 minutes.

Add CNBr solution (5% aqueous solution) and incubate for 3 minutes in a 19°C water bath using a stirrer. Mixed.

Add NaOH (LM) dropwise to maintain the pH within the range of 10.0-10°5. Ta. The suspension was immediately added to an approximately 10-fold excess of cold N a HCOs solution (5 mM, 4°C). After thorough mixing, the solution was decanted. NaHCO3 Washing was repeated 11 times with the solution. Then, two filter paper disks of 500 ml each Wash twice with increasing concentrations of acetone of 5%, 50% and 75%, and then wash twice with increasing concentrations of acetone. Washed 4 times with 00ml acetone (reagent grade, 4°C). Then they are Place on filter paper under hood ventilation for 3 hours to dry, then dry in a plastic bag. It was packaged in a pouch and stored at -20°C until use.

Take a sufficient amount from each eluted sample collected during the ion exchange experiment and add 50mM carbonate. 3 ml of solution containing 300 μg of protein diluted with sodium buffer, pH 9.6 I got l. Add 30 CNBr-activated disk filters to each and add various proteins. The mixture was heated at 4°C with gentle stirring to covalently bond the I left it for 48 hours. Wash the disk filter paper to which the protein has adhered, and add Ce5ka ( buH,p)’f using ethanolamine as described by (supra) Improved RAST method for detecting IgE specific for ort allergens H. pylori allergen-specific IgE was assayed using a modified RAST method. Said. Some of the methods are substantially similar to those described by Nalebuff et al. etc.

, Otolarygol Head Neck Surg89: 271 ( 1981)). In detail, 100μl Serum aliquots were partially incubated with appropriate allergen discs and 0. 50mM phosphate buffered saline (PBS) containing 1% Tween20, pH 7.3 Washed three times using Next, a 1!5-labeled antibody specific for the De-2 antigenic determinant Another portion was incubated with IgE. After cleaning, replace the allergen disc with a new one. Place it in a new test tube and place it in a gamma counter for a preselected time by adjusting the time. I counted. Time adjustment is 25 units of WHO standard standardized ERI ST anti-I Only the time required for 25,000 counts of IgE to bind to the gE disk It turned out to be a test. This time was used to count all subsequent trials.

The background in each individual patient was scrubbed for protein A. Serum (see below) was tested on four blank discs to determine the background of the individual. Determined by calculating the median value representing the this background level A value of 2 times or more was considered positive. Determine individual background levels for each patient Directly detect total serum IgE (not just bacterial allergen-specific). This will increase the precision of the assay as it will take into account the corresponding variability. Ru.

As shown in Figure 1, in order to detect H, py lo r i I gE, Incubate with discs containing H. pylori protein allergen. Before scrubbing the serum sample to remove most IgG and IgA antibodies It is essential that

Scrubbing was performed using recombinant protein A/Sepharose (Zymed, S, San Francisco, CA.

tJsA). Explain in more detail Then, add 2 ml of serum per 1 ml of Protein A/Sepharose with stirring. Incubated for hours. The suspension was then centrifuged at 1500 rpm for 15 minutes to remove blood. The supernatant was collected.

The results in Figure 1 indicate that one patient had gastritis and H. pylori colonization on paper. Take two aliquots of the same serum from a patient and scrub one aliquot. It was obtained by carrying out the processing. Scruvin from the same amount of serum As mentioned above, H, p y 1 o r-i The remaining work of the RAST method using discs containing protein allergens was performed. . In the scrubbed sample (white square) and non-scrubbed sample (black circle) in the figure. Compare the detected autologous IgE levels. As you can see from the graph, scrubbing The sample was a sample eluted from the DEAE column with a peak at fraction number 66. Enables IgE to bind to Y. yorori protein allergens. Non-school No binding was detected in the rubbed samples. The same results can be obtained even if the assay is repeated. Similar results were obtained by H. pylori-specific 1g-specific 1-view E test of patient serum. 10 patients with persistent gastritis/Gl ulcer who were found to be positive for the disease by endoscopy. Two patients in whom no lesions were found and 12 control patients who were apparently asymptomatic. , tested using a modified RAST method involving scrubbing, as described in Example 2. did.

More than 10 disease-positive patients had measurable amounts of Lpylori-specific Ig in their serum. It had an E. Two patients with normal endoscopy were IgE negative, and 12 patients were asymptomatic. Six of the control patients were IgE positive for some of the HP I, C eluted proteins. showed that. As shown in Figure 2, each 1gE-positive patient had different HPLC fractions. It seems that the protein reacts differently to the protein.

Individual chromatography for positive patients among “asymptomatic” and “gastritis” patients. The prevalence of IgE-positive reactivity to the -fraction was investigated. disease patient group Some H. pylori proteins responded more clearly than “asymptomatic” patients. There was a quality fraction. This more obvious reactivity is due to DEAE fraction 5 9.64.66.68.72 and 74.

Figure 3 shows all available H,pylor isolated from the HPLCDEAE column. Net total Ig of serum from controls and gastritis patients using i protein fraction. E is a diagram plotting immunological reactivity. Figure 4 shows fractions 59.62. Control with proteins in 65.70.64.68.71.73 and 74 and Figure 2 is a plot of the net total 1gE immunological reactivity of serum from gastritis patients.

Industrial advantages (including, but not limited to, purified allergens, recombinant or synthetic pVlori proteins) Polypeptides containing one or more epitopes that are immunologically identifiable using epitopes of pylori is useful in the diagnosis of H. pylori-induced gastritis and in the treatment of such diseases. It can also be useful for medical treatment. Such polypeptides are epitopes of H. pylori. It is useful for producing purified polyclonal and monoclonal antibodies against.

Furthermore, these antibodies can be used in immunological studies using epitopes of the H. pylori protein. It is useful for the purification of polypeptides that contain one or more epitopes that can be identified visually.

Monoclonal antibodies are particularly useful for producing anti-idiotypic antibodies; It may also be useful in the production of vaccines for i-induced diseases.

methods, especially the modified RAST method, are useful for diagnosing H. pylori-induced gastric diseases. Ru.

test tube number Figure 2A DEAE test tube number Figure 2B DEAE test tube number Figure 3 1 dirl: Seal no πl r 劫蜆台) Yuki Suki r Tsuji Namazuji 1Q1 small improvement RAST The method may also include the use of purified protein allergens. Those who purify proteins Methods are well known in the art, e.g. fractional extraction, salting out, on ion exchange resins. Chromatography, affinity chromatography, centrifugation, etc. can be done. For example, Methods in Enzymology fo r a variety of methods for purifying See proteins. H. pylori protein allergen to carbohydrates Examples of purification methods for separation from lipids and nucleic acids are provided in the Examples. in DEAE Further isolation of protein allergens by HPLC chromatography H, py, which binds to IgE in individuals with chronic gastritis and/or gastroduodenal ulcers. A subfraction of protein allergens from L. lori were identified.

Such allergen-specific IgE is virtually absent in normal control individuals. there were. Allergens derived from the above fractions are particularly useful for immunoassays. .

Conveniently, immunological identification using an epitope of H. pylori allergen A diagnostic kit may include a polypeptide containing one or more possible epitopes. can. A diagnostic kit consists of a polypeptide contained in a suitable container; Detect immunological complexes, if any, formed with IgE in biological samples. including means for In some cases, immobilizing the polypeptide on a solid support You can also do it. In addition, the kit includes other suitable packaged reagents as required for the particular diagnostic protocol, For example, standard reagents, buffers, and instructions for performing the test using kit components. It may also include.

ion exchange chromatography Transfer each density gradient fraction to dialysis tubing with a cutoff average molecular weight of 1°000. 20mM sodium phosphate buffer, pH 7,0, dialyzed at 4°C. spectroscopy Protein content per fraction at wavelength 280 nm by photometer The amount was estimated. 90% of the detected proteins were found in fractions 2-6. , these fractions were pooled. The pooled fractions were then divided into Bl o-5il DEAE analysis anion exchange HPLC column (BioRad, Ric hmond, CA, USA) so that the end of the concentration gradient is 100% buffer B. A 30 minute linear concentration gradient was performed. Equilibration buffer (buffer A) was 20mM sodium chloride, pH 7.0. The salt-containing buffer (buffer B) was 1.0M 20mM sodium phosphate containing NaCl, pH 7.0. Elution fraction samples were collected and each protein was quantified as described above. Polymer and Kachi The through-through (void) fraction containing onic molecules is transferred to the Blo-5il SP unit. on-exchange column (BioRad) to generate precise concentration gradient conditions for DEAE. Column operation was performed under the subject. Proteins in the resulting elution fractions were quantified.

Covalent attachment of H. pylori proteins to disk filters in a substantially Ce5ka manner. (Ceska et al.

J, Allergy and C11n, Immun, 4 papers were produced.

As shown in Figure 2, each 1gE-positive patient showed various HPLC-fractionated proteins. They seem to respond differently to quality.

Individual chromatography for positive patients among “asymptomatic” and “gastritis” patients. The prevalence of IgE-positive reactivity to the -fraction was investigated. disease patient group Some H,pylor± proteins responded more clearly than “asymptomatic” patients. There was a quality fraction. This more obvious reactivity is due to DEAE fraction 5 9.64.66.68.72 and 74.

Figure 3 shows all available H-pylor isolated from HPLCDEAE column. Total net 1 g of serum from controls and gastritis patients using i protein fraction. E is a diagram plotting immunological reactivity. Figure 4 shows fractions 59.64. Serum from control and gastritis patients with proteins in 66.68.72 and 74 FIG. 3 is a plot of the net total IgE immunoreactivity of .

The scope of the claims 1. A method for measuring IgE that immunologically binds to bacterial allergens, the method comprising: The serum estimated to contain 1gE was bound to a solid support. gen extract, washed, reacted with labeled anti-IgE, and bound to the solid support. the bacteria bound to said solid support. IgA and IgG are removed from the serum before reacting with the sex allergen extract. The composition has been improved to react with the serum, and the amount of the composition to be used is reduced. , removes IgA and IgG that prevent IgE from binding to bacterial allergens. said method, in an amount sufficient to

2. Claim 1, wherein the bacterial allergen extract is a purified protein allergen. Method described.

3. The method of claim 1, wherein the composition comprises protein A.

4. The method of claim 2, wherein the composition comprises protein A.

5. A method for preparing purified protein allergen from bacteria, comprising: (a) Treat bacteria containing protein allergens with acetone to remove lipid components. , (b) The acetone-treated bacteria are treated with a buffer, salt, metal chelating agent, protea, etc. -se inhibitor and benzamidine in a solution containing (c) separating the protein-containing fraction from complex carbohydrates and nucleic acids; (d) collecting a composition containing a protein having a molecular weight of about 1,000 or more; (e) the protein of the composition of (d) above by ion exchange chromatography; isolate A method consisting of things.

6. Converting the ion exchange chromatography to anion exchange high performance liquid chromatography 6. The method according to claim 5, carried out in a HPL C' column.

7. The purified protein allergen is (a) a bacterium containing a protein allergen. was treated with acetone to remove lipid components, (b) The acetone-treated bacteria are treated with a buffer, salt, metal chelating agent, protea, etc. -se inhibitor and benzamidine in a solution containing (e) separating the protein-containing fraction from complex carbohydrates and nucleic acids; (d) collecting a composition containing a protein having a molecular weight of about 1,000 or more; (e) the protein of the composition of (d) above by ion exchange chromatography; isolate Produced by a method of preparing purified protein allergens from bacteria, consisting of 3. The method according to claim 2.

8. The purified protein allergen is (a) a bacterium containing a protein allergen. was treated with acetone to remove lipid components, (b) The acetone-treated bacteria are treated with a buffer, salt, metal chelating agent, protea, etc. -se inhibitor and benzamidine in a solution containing (c) separating the protein-containing fraction from complex carbohydrates and nucleic acids; (d) collecting a composition containing a protein having a molecular weight of about 1,000 or more; (e) Ion exchange on an anion exchange high performance liquid chromatography (HPLC) column isolating the protein of the composition of (d) above by ion exchange chromatography; Produced by a method of preparing purified protein allergens from bacteria, consisting of 3. The method according to claim 2.

9. A composition comprising a protein allergen prepared by the method according to claim 5. thing.

10. A set containing a protein allergen prepared by the method according to claim 6. A product.

11. Immune therapy to treat individuals for diseases resulting from allergic reactions to bacterial infections. epidemiological therapy, wherein said individual is substantially treated with a protein allergen derived from said bacterium. and the conditions for said introduction are such that said allergic reaction The method is sufficient to alleviate the symptoms of.

12. Individual has allergic reaction to Helicobacter pylori (a) H,py I　o　r　i allergen (b) providing serum from an individual estimated to contain IgE to pylori protein allergen; (C) The serum of (a) above is combined with the composition of (b) above, IgE and an allele thereof. react under conditions that allow immunological bonding with Gen. (d) IgE in the serum of (a) above and protein in the composition of (b) above, if any; detecting the IgE-allergen conjugate formed between the wood allergen and (e) Thereby, the individual is allergic to Helicobacter pylori. Determine if there is a response A method consisting of things.

13. The given serum contains Ig that interferes with the formation of IgE-allergen conjugates. removing IgA and IgG from the serum in an amount sufficient to remove G and IgA; 13. The method according to claim 12, wherein the method comprises reacting with the composition obtained.

14. The method of claim 13, wherein the composition comprises protein A.

15. A composition consisting essentially of H. pylori protein allergen.

16. H, protein allergen of pylori bound to solid support.

17. The H. pylori protein allergen is (a) a protein allergen. Bacteria containing Rugen are treated with acetone to remove lipid components, (b) The acetone-treated bacteria are treated with a buffer, salt, metal chelating agent, protea, etc. -se inhibitor and benzamidine in a solution containing (C) separating the protein-containing fraction from complex carbohydrates and nucleic acids; (d) collecting a composition containing a protein having a molecular weight of about 1.000 or more; (e) the protein of the composition of (d) above by ion exchange chromatography; isolate Produced according to the method for preparing purified protein allergens from bacteria, consisting of The composition according to claim 15.

18. The purified protein allergen of H. pylori is (a) Treat bacteria containing protein allergens with acetone to remove lipid components. , (b) The acetone-treated bacteria are treated with a buffer, salt, metal chelating agent, protea, etc. -se inhibitor and benzamidine in a solution containing (C) separating the protein-containing fraction from complex carbohydrates and nucleic acids; (d) collecting a composition containing a protein having a molecular weight of about 1.000 or more; (e) Ion exchange on an anion exchange high performance liquid chromatography (HPLC) column isolating the protein of the composition of (d) above by ion exchange chromatography; Preparation of purified protein allergen from bacteria consisting of 19, H, pVlor A method of treating an individual for i-induced gastritis, wherein the individual is treated with H. pylori contains one or more epitopes that can be immunologically identified using immunogenic epitopes of a composition comprising a polypeptide containing the polypeptide, wherein the polypeptide H, p71 in the above individual, and the composition further contains a suitable excipient. 20. The method of claim 19, comprising said allergen.

21. The composition contains allergoids of H.pytori protein allergen. 21. The method of claim 20, comprising:

22゜Immunology using H. pylori epitope encapsulated in a suitable container. a polypeptide comprising at least one epitope that is physically identifiable; The immunological conjugate formed between the peptide and the IgE, if present, in the biological sample. A diagnostic kit comprising a means for detecting.

23. The protein allergen according to claim 22, wherein the protein allergen is immobilized on a solid support. Diagnostic kit included.

A composition comprising an individual, wherein the structural analog binds to an IgE paratope. thing.

25. The composition according to claim 24, wherein the structural analog is an anti-idiotype antibody. .

26. Purified polyclonal against H. pylori polypeptide allergen A composition comprising an antibody.

27. Monoclonal antibody against H. pylori polypeptide allergen A composition comprising.

Country@! I umpire, -1+-1-h++Continuation of front page (51) Int, C1, 5 identification symbol Internal reference number Cl2P 21108 82 14-48GOIN 33153 Q 8310-2JI

Claims (27)

[Claims] 1. A method for measuring IgE that immunologically binds to a bacterial allergen, the method comprising: A serum estimated to contain 1gE is bound to a solid support, and an extract of the above bacteria is prepared. The labeled anti-IgE bound to the solid support is reacted, washed, and reacted with labeled anti-IgE. detecting IgE and reacting with the bacterial extract bound to said solid support. The serum is reacted with a composition capable of removing IgA and IgG from the serum before The composition has been improved so that the amount of the composition to be used is such that IgE is in an amount sufficient to remove IgA and IgG that would interfere with binding to the How to write. 2. Furthermore, using a purified protein allergen bound to the solid support The method according to claim 1, comprising: 3. 2. The method of claim 1, wherein the composition comprises Protein A. 4. 3. The method of claim 2, wherein the composition comprises Protein A. 5. A method for preparing purified protein allergens from bacteria, the method comprising: (a) Treat bacteria containing protein allergens with acetone to remove lipid components. , (b) The acetone-treated bacteria are treated with a buffer, salt, metal chelating agent, protea, etc. -se inhibitor and benzamidine in a solution containing (c) separating the protein-containing fraction from complex carbohydrates and nucleic acids; (d) collecting a composition containing a protein having a molecular weight of about 1,000 or more; (e) the protein of the composition of (d) above by ion exchange chromatography; isolate A method consisting of things. 6. The ion exchange chromatography is replaced by an anion exchange high performance liquid chromatography. 6. The method according to claim 5, carried out in a HPLC column. 7. The purified protein allergen is prepared by the method according to claim 5. The method described in claim 2. 8. The purified protein allergen is prepared by the method according to claim 6. The method described in claim 2. 9. A composition comprising a protein allergen prepared by the method according to claim 5. thing. 10. A set comprising a protein allergen prepared by the method according to claim 6. A product. 11. Immunology to treat individuals for diseases resulting from allergic reactions to bacterial infections epidemic therapy, wherein the individual is treated with a protein allergen substantially derived from the bacterium. and the conditions for said introduction are such that said allergic reaction is induced. Said method is sufficient to alleviate symptoms. 12. An individual has an allergic reaction to Helicobacter pylori A method for determining whether (a) H. I against pylori allergen (b) providing serum from an individual presumed to contain H. gE; pylor providing a composition comprising an i protein allergen; (c) The serum of above (a) is combined with the composition of above (b), IgE and an allele thereof. react under conditions that allow immunological bonding with Gen. (d) IgE in the serum of (a) above and protein in the composition of (b) above, if any; Is it possible to detect IgE-allergen complexes formed between wood allergens? How to be different. 13. The given serum contains an Ig that interferes with the formation of IgE-allergen conjugates. removing IgA and IgG from the serum in an amount sufficient to remove G and IgA; 13. The method according to claim 12, wherein the method is reacted with the composition obtained. 14. 14. The method of claim 13, wherein the composition comprises Protein A. 15. Substantially H. A composition comprising a protein allergen of pylori. 16. H. bound to a solid support. pylori protein allergen. 17. Said H. pylori protein allergen is the method according to claim 5. 16. A composition according to claim 15, prepared according to. 18. Said H. The purified protein allergen of pylori according to claim 6 16. A composition according to claim 15, prepared according to the method. 19. H. 1. A method of treating an individual for pylor1-induced gastritis, the method comprising: In, H. One that can be immunologically identified using immunogenic epitopes of pyIori The method consists of introducing a composition containing a polypeptide containing the above epitope; The polypeptide is present in the individual when the polypeptide is present in the individual. Allergic reaction to pylor1 the composition further comprising suitable excipients, in an amount sufficient to moderate the Said method. 20. The composition may contain H. Contains purified protein allergen of pylor1 20. The method according to claim 19. 21. The composition may contain H. Allergoids of pylor1 protein allergen 21. The method of claim 20, comprising: 22. H. sealed in a suitable container. Immunology using pylori epitopes a polypeptide comprising at least one epitope that is physically identifiable; The immunological conjugate formed between the peptide and the IgE, if present, in the biological sample. A diagnostic kit comprising a means for detecting. 23. 23. The protein allergen according to claim 22, wherein the protein allergen is immobilized on a solid support. Diagnostic kit included. 24. H. A composition comprising a structural analog of an epitope of P. pylori allergen. A composition in which the structural analog binds to an IgE paratope. 25. 25. The composition of claim 24, wherein the structural analog is an anti-idiotypic antibody. . 26. H. Purified polyclonal against pylor1 polypeptide allergen A composition comprising an antibody. 27. H. Monoclonal antibody against pylor1 polypeptide allergen A composition comprising.