HK1136350A - Method for assaying antibodies in body fluids by immune reaction with glycoprotein 2 (gp2) from zymogenic granules of the pancreas for the differential diagnosis of inflammatory intestinal diseases and chronic pancreatitis - Google Patents

Description

Method for detecting antibodies in body fluids by immunoreaction of glycoprotein 2(GP2) from zymogen granules of pancreas for diagnosing inflammatory bowel disease and chronic pancreatitis

Technical Field

The present invention relates to a method for detecting antibodies in body fluids by immunoreaction with GP2, an immunoreactive sequence or an analogue thereof from zymogenic granules of the pancreas (but not including tissue sections).

The method can be used for diagnosing or treating diseases related to the immune response of GP2 and similar substances. More specifically, therefore, the invention is directed to the use of GP2, an immunoreactive sequence or an analogue thereof for the diagnostic or therapeutic control of chronic inflammatory or autoimmune diseases, particularly Crohn's Disease (CD) and Chronic Pancreatitis (CP).

Background

The present invention is based on the discovery that GP2 is an autoimmune antigen in the course of immunization of Inflammatory Bowel Diseases (IBDs), particularly CD and CP, and thus represents an epitope of disease-associated antibodies.

GP2 is a membrane glycoprotein of acinar cells of the pancreas, having an apparent molecular weight of 78 kDa. In addition, GP2 was detected in brush-border cells (brush-border cells) of the intestine and as a component of free, membrane-unbound peptide in lysosomes or pancreatic juice. Since GP2 makes up 30-45% of the total membrane protein, it is the main component of the zymogen granule membrane. GP2 is a component of lipid rafts (lipid rafts) of the granular membrane, together with other trypsins secreted by zymogens (e.g. tubulin (syncolin), lectin ZG16p, synaptophysin 2 and other sulfated matrix proteoglycans), which interact with GP2. These complexes (including also other proteins, such as ZG46p) form a partially membranous substrate (submembranous matrix).

GP2 binds to the zymogenic granulosa membrane of pancreatic acinar cells via phosphatidylinositol anchors and can be removed by phospholipase C of bacillus cereus, for example. Zymogenic granules are reservoirs of digestive enzymes (e.g., amylase) within the pancreatic acinar cells. After neurostimulation or hormonal stimulation of acinar cells, digestive enzymes are secreted into the pancreatic duct. GP2 is not only localized to the zymogen granule membrane, but also bound to its matrix within the lumen of the golgi and pancreatic vacuole. In addition, GP2 was also found to be a component of lysosomes and thus involved in endocytosis.

During stimulation of pancreatic secretions, GP2 is transferred to the apical membrane surface of acinar cells, is cleaved and released into the lumen of the acinus. Given the considerable amount of GP2 in pancreatic juice, another cell pool (cellular pool) was suspected to be present that secreted GP2. In contrast to digestive enzymes which are activated in the intestine by proteolysis, GP2 has been modified within acinar cells by cleavage. It is speculated that the intracellular sequential proteolysis of GP2 has an effect on its function.

Increased GP2 serum levels in acute pancreatitis and chronic pancreatitis led to a discussion of whether GP2 is suitable as a marker for serodiagnostics of this entity. In the rat model, the serum concentration of GP2 has been shown to be related to the severity of inflammatory bowel disease.

For human cell lines, this relationship has not been determined without doubt; the GP2 level detectable by autoantibodies shows great individual variability.

Despite this deficiency, a number of approaches to developing diagnostic methods rely on the production and detection of antibodies against GP2 to determine the severity of inflammatory bowel disease through antibody responses.

CD and Ulcerative Colitis (UC) represent the two most important of inflammatory bowel diseases. They are characterized by a chronic, recurrent inflammatory process that destroys tissue within the digestive system. To date, the etiology and pathogenesis of CD and UC is still unclear.

While UC inflammation occurs primarily in the mucosa and submucosa of the colon and rectum, CD is characterized by granulomatous inflammatory processes that penetrate through the walls of the entire gastrointestinal tract.

Genetic and environmental factors appear to play a key role in the development of IBD. The link between the mutation in the NOD2 gene and CD appearance was considered to be certain in some groups (cohort). Similarly, a clear association was considered with the appearance of CD in the terminal ileum. To date, no relationship between genetic markers and the course of therapy has been established in any therapeutic approach, including anti-tumor necrosis factor (anti-TNF) therapy.

The incidence of CD in europe is about 5.6 per 100000 per year. The prevalence of CD in germany has been determined to be 1/500 to 1/800.

On average, the first symptoms of CD (first symptoms) occur relatively early at age 30. In turn, CD patients are affected during their working life due to corresponding socio-economic effects. In a manner similar to UC, CD patients have an increased incidence of cancer as Crohn's colitis (Crohn's colitis) and the course of the disease continue for years.

Clinical manifestations include abdominal pain, diarrhea, malabsorption, abscesses, fistulas (fistula), gallstone complications, kidney stones and their related complications.

CD patients can develop many extra-intestinal manifestations (3.5% in total) with pancreatitis, which is a relatively rare event in CD patients. However, hyperamyloidosis (hyperamyloasemia) and hyperlipidaemia (hyperlipasemia) without symptoms of acute enteritis can be observed in 8-17% of patients, indicating an increased incidence of asymptomatic pancreatitis (silent pancreatis). Changes in the pancreatic duct and limitations on pancreatic function have been described in some cases. The levels of hyperamylase and hyperlipidaemia are associated with CD activity. Changes in a similar manner to primary sclerosing cholangitis (primary sclerosing cholangitis) patients occurred with concurrent findings in the bile and pancreatic ducts of 4.6% of CD patients. However, chronic pancreatitis in CD patients is often different from chronic pancreatitis in UC patients, where bile duct involvement, weight loss, and pancreatic duct stenosis (pancreatic duct stenosis) are more common. The existence of idiopathic chronic pancreatitis associated with CD has been discussed. In contrast to chronic pancreatitis associated with UC, intestinal symptoms often appear in CD patients prior to the discovery of pancreatic symptoms. Exocrine pancreatic insufficiency (Exocrine pancreatic insufficiency) commonly seen in CD can be simply attributed to significant acinar degeneration associated with a dense inflammatory infiltrate (inflimatory inflilters) in parenchyma (parenchyma).

Although various studies have indicated that there are significant disadvantages due to the limited or no effect achieved by the active substance 5-aminosalicylic acid, the use of 5-aminosalicylic acid for administration therapy is recommended. However, from the data obtained, it appears to be effective in reducing the severity of the morbidity to moderate use in patients when the treatment is changed in time without efficacy. In cases where no serious morbidity of the complications occurs, it should be considered to administer an equal amount of prednisolone (prednisolone). If the onset is frequent (2 times per year) azathioprine or 6-mercaptopurine can be added for administration.

In germany, the total cost of CD patients is estimated to be 20000 euro per case per year. In germany, the cost (including indirect costs) for CD patients is estimated to be 2 million (billion) euros, while the socio-economic cost for two IBDs in the united states is reported to be $ 2.6 million (billion).

Anti-tumor necrosis factor alpha preparations are effective against CD, including the remission of chronic diseases. However, these preparations are still unsuitable due to their side effects, which is why they are only possible to use as reserve drugs (dependent on clinical presentation). Only two clinical manifestations in CD patients with active spondyloarthropathies with extra-intestinal complications reflect that they appear to benefit from anti-tnf α therapy.

A clear diagnosis is necessary for adequate treatment and follow-up of these patients. However, this has a number of considerable disadvantages, since no unambiguous tests have been carried out to date and therefore clinical diagnosis of CD has to be carried out, which involves the use of biopsies from the retrocolonoscopic section (ileocoloscopic section) as an essential component, which is also required for carrying out selective intestinal surgery. However, this is not entirely necessary in any acute symptomatic syndrome in the course of the disease, or prior to the performance of new anti-inflammatory therapies. In the basic diagnostic procedure, upper endoscopic diagnosis should be performed in each patient.

Histological studies of highly complex and relatively cost-intensive mucosal biopsies constitute another important element of the diagnostic scope. For this purpose, biopsies are collected, in particular from areas with macroscopic significance and unnoticeability. However, to effectively utilize histopathological differential diagnosis, biopsies from at least five different anatomical parts throughout the colon (including the rectum) from the terminal ileum and upper gastrointestinal tract need to be collected.

Transabdominal ultrasound as an imaging method was used as a sensitive method to detect inflammatory changes in the intestinal wall and in CD patients that develop abscesses, fistulas, and strictures. The increase in blood flow that can be detected in the mesenteric artery and intestinal wall is associated with the presence of acute inflammation. It has been recognized that endorectal ultrasound and Magnetic Resonance Tomography (MRT) of the small pelvis are methods of diagnosis and classification of anorectal fistulae and abscesses with equal sensitivity.

Since no unambiguous testing exists, a large number of laboratory diagnostic parameters for CD were collected. The determination of C-reactive protein (CRP), platelets, hemoglobin (Hb)/red blood cell pressure and white blood cells represents a basic diagnosis. Other parameters, such as differential blood count (differential blood count) and albumin, are additionally used. In the acute phase CD, the above parameters (e.g. CPR, number of leukocytes and acute-phase proteins) increase in many patients, and thus these parameters are recommended for use in the subsequent (following) course of the disease.

During the acute phase, intestinal permeability, clearance of alpha-1-antitrypsin in the feces, and calprotectin secretion are elevated.

However, many clinical and histopathological data fail to make a clear distinction between CD and UC. This deficiency often leads to conclusions about Indeterminate Colitis (IC). In addition, intestinal infections and functional diseases may exhibit similar symptoms, and thus, differential diagnosis is more difficult. In 10-15% of IBD patients, classification as UC or CD from biopsy data is extremely difficult and is also due to some overlap in clinical symptoms in the colon region. Following surgery, it appears that patients with IC are more prone to long-term complications and anastomotic insufficiency (anastomotic insufficiency) than patients with UC. However, whether IC patients in the prognostic period will differentiate in the CD or UC direction has a major impact on prognosis and disease course, as well as in the choice of drug treatment and the time of surgery. At a later point in the course of the disease, determinations can generally only be made based on additional clinical data. For example, differentiation between CD and UC is the basis for determining whether ileoanal pouch anastomosis (ileoanal pouch and anosomis) can be performed in a patient. This surgery is hardly indicative in patients with significant colonic infection (crohn's colitis), while UC is more indicative of the procedure. The incidence of anastomotic insufficiency is much higher in CD patients, and therefore any surgery requires comprehensive consideration.

Many antibodies reactive with endogenous and food antigens have been described in the context of differentiating IBD diagnosis. The disadvantage of these antibodies is that they do not appear to play any pathogenic role and do not reflect the activity of the disease. Nevertheless, serum antibodies are used as a key adjunct to diagnosis and reflect the basis of therapeutic decisions, particularly in the case of indeterminate enteritis.

Autoantibodies against cytoskeletal proteins have been described in CD patients identified by biopsy (maryert et al, 1990)). Autoantibodies to cytokeratin 18, actin, vimentin, desmin, and tropomyosin have been found among other proteins. Although cytokeratin 18 autoantibodies have been found to be associated with disease activity, they are not allowed in the routine diagnosis of IBD, probably due to their low specificity.

For CD patients, autoantibodies against pancreatic exocrine tissue (PAK) and antibodies against mannan from Saccharomyces cerevisiae (ASCA (anti-Saccharomyces cerevisiae antibody)) have been identified as disorders (stakko et al, 1987: (1987))et al, 1987); mein et al, 1988(Main et al, 1988)). Autoantibodies against human neutrophils (ANCA) and goblet cells (BAK) were found in UC patients.

Assays for PAK, ANCA and ASCA are considered useful in diagnosing IC.

However, even today, since the autoantigen that elicits the immune response is unknown, IBD-specific autoantibodies to pancreatic tissue, goblet cells and human neutrophils are still measured using immunofluorescence techniques (IFT). Thus, by using the above techniques, autoantibodies against pancreatic antigens have been found in 27-39% of CD patients. More than half of CD patients with extra-intestinal complications (68%) may have PAK. However, in practice, this technique has been found to be disadvantageous in that it cannot be automated and is therefore cost-intensive and time-consuming.

In contrast, the level of detection of ASCA for CD, which is also specific, in Enzyme Immunoassay (EIA) is increased, since this method is less subjective in evaluation and can be automated.

At present, the assay of isolated antibodies is considered to be too insensitive to be used for serological diagnosis of CD. Binding to different antibody specificities can highly improve the diagnostic sensitivity or specificity of differential diagnosis of IBD and allow prognosis of IC.

The usual technical platforms (e.g. EIA) have an extra advantage in the combination of parameters. However, one such prerequisite is the knowledge of the PAK autoantigens that are specifically recognized by PAK from pancreatic tissue sections of different species.

Various methods have been found in the past to recognize autoantigens in CD, and due to the relatively high sensitivity of PAK mentioned above, attention has been focused on pancreatic antigens. PAK has been detected by means of immunofluorescence techniques (IFT) using tissue sections of different species (human, rat, monkey). From the phylogenetic point of view, conserved epitopes (concervedepipopes) recognized by PAKs are reflected. Frieke et al have described a protein complex consisting of multiple subunits (m.w.) with a molecular weight greater than 800kDa that reacts with PAK (frieke et al 1999.) however, the authors were neither able to sequence to identify the corresponding protein nor to identify the subunits at m.w. 16kDa, 18kDa, 19kDa, 24kDa, 27kDa, 29kDa, 31kDa and 34kDa that react with PAK in immunoblots. It has been postulated that the proteins recognized by PAKs should be large protein complexes comprising a large number of subpopulations. Based on inhibition experiments using various glycoproteins, the reaction of PAK with carbohydrate chains (carbohydrate chains) of a recognized autoantigen has been excluded.

Saibold et al have described PAK and purified pancreatic juice at m.w. 10⁶Reactivity of macromolecular antigens above Da (1000kDa) which lose their PAK reactivity when treated with trypsin. No reactivity with PAK was detected in enzyme-linked immunosorbent assays (ELISA) using various pancreatic proteins (e.g., amylase, lipase, phospholipase a and phospholipase C, enterokinase, carboxypeptidase a and carboxypeptidase B, chymotrypsin a and chymotrypsin B, chymotrypsinogen, elastase, trypsin inhibitors, lactoferrin, and vasotropin (callerclein).

Although the level of GP2 and the severity of IBD are related, as stated by many authors, their physiological cause and effect is unknown. As demonstrated in the knockout mouse model, the deletion of GP2 is neither necessary for secretion by pancreatic exocrine nor for the formation of zymogen granules. Furthermore, the physiological function of the two known subtypes of GP2 is also unclear (foggang (Fukuoca, 2000)). In addition to the short subtype β -GP2, which is 380 amino acids in length, there is also α -GP2, which is 530 amino acids in length. Both subtypes of peptide are detectable in the human pancreas, and among others, the small β -GP2 subtype is much more potent than the large α -GP 2. Furthermore, the detection of the transcription of these two types in pancreatic tissue reflects that β -GP2 is strongly expressed compared to α -GP 2.

The role of the different subtypes is not yet clear. Peptides with sequences that are highly similar to the large α -GP2 subtype are said to be the cause of pancreatic tumor formation. Antibodies to GP2 as analytes and markers are intended for use in the diagnosis of pancreatic cancer, whereas peptides and their nucleic acid sequences are used in the immunotherapy of cancerous pancreatic diseases (WO 01/94409). Antibodies to the small beta-GP 2 subtype have found use as markers for pancreatitis (WO 96/17873). An increase in the concentration of β -GP2 is said to indicate the onset of the disease.

Detailed reference has been made to the identification of the necessary (yet to be discovered) pancreatic autoantibodies to elucidate the role of autoimmune processes in the pathogenesis of CD and to support the differentiation of unclear cases of IBD by appropriate laboratory diagnosis.

Efforts are known from the prior art to use random phage display libraries to obtain peptides that have been used for the detection of CD-specific antibodies. Four different nonamers were detected, which when used in EIA (enzyme immunoassay) gave a positive detection in the serum of 56.5% of CD patients. In this case the control group (UC, duodenal ulcer, healthy) gave no response or only 6% response. However, no conclusions about the native CD autoantigen are known from the known peptide sequences of the nonamer.

However, the corresponding pancreatic antigens have not been successfully identified to date (bosuiett, 2006). Therefore, there is no noninvasive, specific, quantitative, rapid, simple and inexpensive detection assay available for diagnosing crohn's disease and chronic pancreatitis and distinguishing the latter from ulcerative colitis.

Disclosure of Invention

The present invention solves the above problems. Based on the surprising characterization of GP2 as an autoantigen for CD and related chronic pancreatitis, methods for diagnosing or therapeutically controlling IBD using GP2 according to the claims were developed, from which dependent claims advantageous embodiments of the invention may be derived.

The present invention therefore relates to a method for the detection of antibodies from stool and/or body fluids, in particular blood and/or serum, by an immune reaction with GP2, an immunoreactive sequence or an analogue thereof, in particular according to sequence identification number 1(SEQ ID No.1), in the prevention, diagnosis, treatment or post-treatment of autoimmune diseases (aftercare), said SEQ ID No.1 being:

according to the invention, the definition of body fluid also includes human blood, serum, urine, pure pancreatic juice or duodenal juice.

The prior art does not contemplate nor mention the determination of autoantibodies to GP2 or their use as solid phase antigens for ELISA in serological diagnosis of CD or chronic pancreatitis.

The present invention teaches surprisingly that GP2 (particularly a sequence identical to SEQ ID No.1) or a nucleotide encoding the same can be used for the detection and treatment of autoimmune diseases, particularly inflammatory bowel disease, more preferably Crohn's disease, chronic pancreatitis and ulcerative colitis.

In another aspect, the invention relates to a method wherein antibodies to human immunoglobulin a (iga), IgM, and/or IgG are detected for autoimmune disease.

In a preferred embodiment of the method according to the invention, the source of GP2 is human, animal, recombinant or synthetic. GP2 represents a highly conserved peptide, so any source of GP2 can be advantageously used for detection, as long as its sequence is functionally similar to the sequence according to the invention. High binding affinity between GP2 as antigen and autoantibodies is maintained.

In another preferred embodiment of the method according to the invention, the detection of autoantibodies is carried out in an immunoassay, preferably with direct or indirect binding of a reactant to a labeling substance. This allows a flexible adaptation of the method to the potential and requirements of different laboratories and their laboratory diagnostic devices.

In an advantageous embodiment, detection of IBD-specific antibodies is performed in an immunoassay, wherein the antibodies are present dissolved in a liquid phase, preferably diluted in a conventional buffer solution or undiluted body fluid known to the skilled person. According to the present invention, the use of a stool sample for the detection can also be effective.

In another advantageous embodiment, the immunoassay is used to detect an antibody, the binding of the GP2 antigen identical to SEQ ID No.1 to a solid phase being expected at the end of the antibody. As the sample solution increases, the patient's antibodies contained therein bind to the GP2 antigen. Antibodies obtained, for example, from the serum or feces of the patient and bound to GP2 are then detected and optionally quantified using a labeled reagent. This method is a "direct assay" known to those skilled in the art, in which an antigen is bound to a solid phase.

Thus, the detection of the antibody can be achieved in the method according to the invention by using a labeled reagent according to the well-known ELISA (enzyme-linked immunosorbent assay) technique. The labeling according to the invention thus comprises an enzymatically catalyzed chemical reaction which can be detected optically, in particular by means of chromogenic substrates (chemiluminescent substrates), chemifluorescence or fluorescent dyes.

In another preferred embodiment, the autoantibodies are detected in a Radioimmunoassay (RIA) by labeling with a weakly radioactive substance, wherein the radioactivity produced is measured.

In another preferred embodiment of the invention, soluble or solid phase bound GP2 molecules are used to bind to antibodies. In the second reaction step, an anti-human immunoglobulin is used, preferably selected from the group consisting of anti-human IgA, anti-human IgM and/or anti-human IgG antibodies, which is preferably detectably labeled with a conjugate of the two components which is capable of binding to any conventional labeling enzyme, in particular a chromogenic substrate and/or a chemofluorescent substrate, preferably horseradish peroxidase, alkaline phosphatase. An advantage of this embodiment is that the use of ELISA techniques is typically achievable under laboratory facilities conditions, so that the detection according to the invention can be performed in a cost-effective manner.

In another preferred embodiment of the invention, the antibody binding to GP2 is reacted with an anti-human immunoglobulin, preferably selected from the group consisting of anti-human IgA, anti-human IgM, and/or anti-human IgG antibodies, detectably conjugated to Fluorescein Isothiocyanate (FITC). Very similar to the ELISA described above, the FITC technique represents a system that can be utilized in many places and thus allows the detection of the invention to be established smoothly and at low cost under the laboratory route.

The invention also relates to a method for treating inflammatory bowel disease, comprising the steps of:

a) providing a column having GP2 coupled thereto;

b) passing the patient's plasma through the column under conditions that allow the GP2 to effectively bind to antibodies within the patient's plasma, thereby removing a substantial amount of antibodies from the patient's plasma; and

c) the plasma thus obtained is returned to the patient.

In a preferred embodiment of the above method according to the invention, GP2, identical to SEQ ID No.1, recognizes antibodies directed against intestinal tissue. This embodiment has the advantage that diagnosis of IBD can be easily achieved by body fluids or faeces without the need for frequent interventions in the patient, which may cause stress to the patient.

In another preferred embodiment of the invention, GP2 corresponding to SEQ ID NO.1 is bound to the solid phase. GP2 corresponding to SEQ ID NO.1 could be bound to the stationary phase by a spacer (spacer). All chemical compounds with the appropriate structural and functional prerequisites for spacers can be used as spacers, as long as they do not alter the binding behavior in such a way that the binding of GP2 autoantibodies identical to SEQ ID No.1 is adversely affected.

The methods and uses of the present invention allow for the diagnosis or therapeutic management of crohn's disease because the GP2 antigen (preferably identical to SEQ ID No.1) surprisingly allows for the detection of autoantibodies from stool and/or bodily fluids by an immune reaction with GP2, an immunoreactive sequence or an analogue thereof, which can be carried out without the use of tissue sections of animal or human tissue.

In a preferred variant of the above detection method, the detection of the autoantibody is preferably carried out by direct or indirect binding of a reactant to a labeling substance.

According to the invention, it is also preferred to carry out the above-described detection method on a solid phase, in which case the storage capacity of the peptide is advantageously increased, which is a result of the surprisingly stable linkage between the GP2 antigen and the solid phase.

The invention also relates to the use of the GP2 molecule according to SEQ ID No.1 for the production of a medicament for the prophylaxis, diagnosis, treatment and/or post-treatment of autoimmune diseases. In practice, the use according to the invention has the advantage that in many cases more complex and unreliable procedures based on e.g. biopsies, laboratory diagnoses and clinical interventions can be dispensed with or that the above-mentioned measurements can be greatly optimized in the execution in view of the aforementioned detections.

In a preferred embodiment of the invention, the autoimmune disease is selected from the group consisting of Inflammatory Bowel Disease (IBD) and/or autoimmune liver disease, which can be effectively detected using GP2 autoantigen, advantageously in a specific, reproducible and cost-effective manner.

In another more preferred embodiment of the method of the invention, the inflammatory bowel disease is crohn's disease, chronic pancreatitis, and/or ulcerative colitis. So far, the detection or differentiation of the above-mentioned diseases can only be carried out with limited success or with great effort. The above preferred embodiments now allow for easy detection of crohn's disease and chronic pancreatitis, even distinguished from ulcerative colitis by means of differential diagnosis.

Detailed Description

Crohn's disease belongs to the group of chronic inflammatory bowel diseases. It is presumed to be an auto aggressive, chronic granulomatous inflammation that may occur throughout the gastrointestinal tract (i.e., from the mouth down to the anus). Infection is mainly located in the lower small intestine (terminal ileum, infection rate about 40%) and colon, and rarely occurs in the esophagus and mouth. Crohn's disease is characterized by discontinuous, staged infection of the intestinal mucosa (so-called "skip lesions"), i.e. the disease may be present in multiple intestinal sections separated by healthy sections simultaneously. Other names of the disease are regional enteritis, terminal ileitis, regional enterocolitis and sclerosing chronic enteritis (sclerosing chronic enteritis), or the generic terms abbreviated CD (crohn's disease) and IBD (inflammatory bowel disease). Thus, crohn's disease in the sense of the present invention refers to any condition characterized macroscopically by the following changes:

garden hose phenomenon (Garden hose): narrowing of the segment due to fibrosis.

Cobble stone phenomenon (Cobble stone): the inflamed mucosa has alternating deep ulcerations giving a cobblestone-like appearance.

Inflammatory conglomerate-like tumors: many parts of the intestine adhere to each other.

Histopathologically, this is mainly due to the accumulation of lymphocytes (eosinophils), granulocytes and histiocytes that are recognized in biopsies of inflamed intestinal tissue. The size of the lymph nodes around it will generally increase. Granulomas often form, which can be differentiated into two types: epithelial-like cell granulomas and microplasmia (smaller and without central necrosis).

However, the present invention is also directed to Crohn's disease characterized in a diagnostic manner. In this context, the present invention is directed to Crohn's disease in which at least one of the following characteristics is detectable:

appendicitis: pain rapidly develops in the right lower abdomen. The temperature difference measured between the rectum and the armpit is typically > 1 ℃.

Diverticulitis: palpable mass (pain) located in the lower abdomen (usually on the left side), pain.

Yersinia disease: pathogens are detected from stool or biopsy material and antibody titers are increased.

Intestinal tuberculosis: it is now very rare in the central european region. Tuberculosis of the intestine usually has pulmonary tuberculosis. A "cheese-like" epithelioid granuloma was found in the biopsy material.

Any other spreading infectious colitis (Salmonella enteropathy, pseudomembranous colitis, etc.)

Pancreatitis, as referred to herein, refers to inflammation of the pancreas, which may be acute or have a chronic course.

Pancreatitis is usually triggered by the activation of pancreatic enzymes in the organs. The role of these enzymes is to digest protein and fat and thereby cause autodigestion of the organ. Autodigestion leads to inflammation of the pancreas. In severe cases, bleeding, severe tissue damage, infection and cysts may occur. Inflamed glands (gland) may cause enzymes to enter the blood stream and reach the lungs, heart and kidneys, possibly causing further damage. Acute pancreatitis occurs when the pancreas suddenly inflames, but recovers. Some patients suffer from acute pancreatitis many times, but can completely heal each time. Acute pancreatitis occurs suddenly and can be a serious, life-threatening disease that causes a number of complications, but patients are often recovered from acute pancreatitis. The probability of occurrence is about 5-10 new cases per year in every 100000 residents.

There are two types of disease processes:

1. edematous pancreatitis: has a mild course of organ swelling and rarely necroses in the surrounding adipose tissue.

2. Hemorrhagic necrosis pancreatitis: massive necrosis and bleeding in the pancreas and into the surrounding area; it is commonly referred to as pancreatic stroke (pancreatic stroke) due to its fulminant symptoms.

Morphological evaluation of the pancreas, particularly distinguishing between edematous pancreatitis and necrotizing pancreatitis, was most successful when computed tomography with enhanced contrast agents was used. The balthazer score (Balthazar socre) (score 0-10) was found to be favorable in the severity classification.

Acute pancreatitis has multiple etiologies. The most common is the temporary or prolonged obstruction of the biliary tract to the duodenum (also the pancreatic duct) at the orifice of the bile duct (about 45% of acute pancreatitis). The reason for a similar prevalence is long-term alcohol abuse (about 35%). In about 15% of infected individuals, no unique trigger quotation was detected, and these cases were termed idiopathic origin. Furthermore, there are more rare reasons such as the following:

side effects of drugs (e.g., asparaginase, azathioprin, furosemide, glucocorticoids, antibiotics (tetracyclines, sulfamethoxazole, trimethoprim), antispasmodics (sodium 2-propylvalerate, carbamazepine), propofol (propofol), and the like.

Infectious diseases, e.g. mumps, Coxsackie virus, hepatitis, HIV, cytomegalovirus

Elevated serum calcium values, e.g. in hyperparathyroidism

Sharply elevated blood lipids (triglycerides)

Resulting from a doctor's treatment in Endoscopic Retrograde Cholangiopancreatography (ERCP)

Inheritance: cystic fibrosis

Initially, acute pancreatitis is manifested by pain located in the upper abdomen (extragastric region) (to the left of the whole body), which is transmitted chestwardly and spreads over several days. The pain is usually acute and sometimes persists for a long time. The pain can be sudden and concentrated, or begin as mild pain, but will worsen with the ingestion of food (as a result of the stimulation of the pancreas during the synthesis of pancreatin for the digestion of food). The abdomen can swell and be highly sensitive. Abdominal pain to touch and the so-called rubber belly (rubber abdomen) caused by abdominal pneumatosis and (moderate) guarding (guarding) were characterized in physical examinations. Similarly, the lower region of the thoracic spine can also produce pain. Initially, the pain is similar to mild low back pain, but then develops more and more with the sensation of "stabbing" of the back towards the apical area of the ventral pancreas.

Patients with acute pancreatitis often appear severely ill and actually have a feeling of illness. Other symptoms are, for example, nausea, vomiting, constipation, fever, and accelerated pulse. In severe cases, jaundice (jaundice) (jaundice (ichrus) in case of blockage of the bile duct), abdominal edema (ascites) (in case of blockage of the portal system), pleural effusion, shock and signs of sepsis also occur.

In laboratory diagnostics, an increased white blood cell count (leukocytosis) and an increase in the concentration of pancreatin (e.g., trypsin, amylase, lipase) are detected. Furthermore, the value of calcium, magnesium, sodium, potassium, bicarbonate, sugar or fat in the blood may increase.

About 20% of acute pancreatitis cases are severe. Patients may experience dehydration and develop hypotension. Sometimes heart failure, lung failure or renal failure occurs. The most severe acute pancreatitis leads to bleeding, shock and sometimes death.

According to the current Atlanta classification (Atlanta classification), mild acute pancreatitis and severe acute pancreatitis were classified.

The discardable classification divides the phase into the edematous form (early), hemorrhagic with local or global bleeding and acute necrotizing form.

Chronic pancreatitis has multiple etiologies, but 70-80% of them can be attributed to long-term alcohol abuse. Chronic pancreatitis occurs more commonly in men than women and occurs between the ages of 30-40. Chronic pancreatitis can also result from acute inflammation if the source of inflammation is not eliminated or efferent vessels are compromised.

Some chronic pancreatitis has a genetic etiology. They are based on abnormalities in the enzyme formed by insulin and cause tissue damage. Other forms of disease are caused by external causes such as smoking.

In the early stages of pancreatitis, physicians are often unable to determine whether this is an acute or chronic form. Their symptoms may be the same.

Chronic pancreatitis often causes chronic pain. In some cases of chronic pancreatitis, pain is reduced as the disease progresses. This also causes a decline in the function of the pancreas, resulting in weight loss and impaired digestion. Insufficient digestion and absorption results in the excretion of fats and proteins through the feces. Diabetes can occur if endocrine cells (islets) within the pancreas are damaged.

Difficulty in diagnosing chronic pancreatitis; however, some highly developed medical techniques may be used. A pancreatic function blood test may assist in determining whether the pancreas is able to produce sufficient digestive enzymes. However, they are difficult to gain acceptance in practice.

Pancreatic abnormalities can also be detected by using ultrasonography, Endoscopic Retrograde Cholangiopancreatography (ERCP), and computed tomography.

In the more advanced stages of chronic pancreatitis, where diabetes and malabsorption are found to occur, physicians may also test blood, urine, and each for diagnosis.

Treatment of chronic pancreatitis involves prescribing analgesics and dietary changes. The patient can reduce the loss of fat and protein by taking pancreatin-containing drugs. Such a result would improve nutrient intake and weight gain. Insulin or other medication may sometimes be prescribed to control blood glucose levels.

In some cases of chronic pancreatitis, surgery is performed to relieve tension in the enlarged and congested pancreatic ducts to relieve pain.

In another preferred embodiment of the invention GP2 is used to detect liver disease, primary sclerosing cholangitis and/or autoimmune enteritis (enteritides). Surprisingly, the GP2 autoantigen is not only suitable for the specific detection of IBD, but also for the detection of various liver diseases.

Cholangitis in the present invention refers to inflammation of the intrahepatic bile duct. It can be caused by a variety of causes, including (among others) biliary obstruction, stenosis, tumor, or infestation by parasites. It is classified into acute suppurative cholangitis, non-suppurative destructive cholangitis and chronic sclerosing cholangitis.

Acute suppurative cholangitis: acute cholangitis usually develops from infection during colonization by bacteria, mainly of the species Escherichia coli, Enterococcus (enterobacteria) or Klebsiella (Klebsiella). Symptoms are unilateral pain in the upper abdomen, fever and chills. In addition, severe suppurative cholangitis causes shock conditions, central nervous system disorders and renal insufficiency. The treatment employs endoscopic intervention of the bile duct, such as Endoscopic Retrograde Cholangiopancreatography (ERCP) or Percutaneous Transhepatic Cholangiodrainage (PTCD), which restores bile flow and is generally antimicrobial.

Non-suppurative destructive cholangitis: this form of cholangitis manifests as a chronic course and is also known as primary biliary cirrhosis. 95% of infected individuals are women and the peak onset is between 40 and 60 years of age. Diagnostically, anti-mitochondrial antibodies (AMA) are found in the blood in most loops and are therefore presumed to be of autoimmune origin. Clinically, patients are characterized by pruritus, jaundice, and hypercholesterolemia. In a further course of the disease, patients can only be helped by means of liver transplants.

Chronic sclerosing cholangitis: chronic sclerosing cholangitis is the rarest inflammation of the bile ducts and is divided into primary and secondary forms of sclerosis. The primary form develops by infection in the presence of an existing gene array (detected in association with the HLA-B8 antigen), and twice as many males as females are infected. p-ANCA is found in up to 90% of cases. Secondary forms develop on the basis of existing immunodeficiency syndromes. Similar to destructive cholangitis, liver transplantation is necessary in the final stage.

Autoimmune enteritis in the context of the present invention refers to any form of enteritis, in particular caused by chronic inflammatory bowel disease. Furthermore, the autoimmune enteritis in the context of the present invention also relates to those caused by salmonella (salmonella), escherichia coli, cholera or typhus pathogens, or by fungi, protozoa, toxins, and also includes any irritable bowel inflammation or any form of actinic enteritis (inflammatory bowel disease), yersinia enteritis or bacillary dysentery.

In another preferred embodiment of the invention, at least 60%, preferably 70%, more preferably 80%, particularly preferably 90% of the amino acid sequence of the GP2 peptide is homologous to the sequence of SEQ ID NO. 1. That is, the invention relates to all such peptides, preferably 60%, 70%, 80%, particularly preferably 90%, of the sequence of the peptide is homologous to the sequence of SEQ ID NO. 1. It is understood that these homologues may be modified by deletion, addition, substitution, translocation, inversion and/or insertion. More specifically, such modifications relate to homologous peptides having functional similarity. The peptides of the invention are functionally similar when autoantibodies associated with the above-mentioned diseases interact.

It is therefore an object of the present invention to disclose polypeptides and homologues which can be used in a functionally analogous manner. Given homology/functional similarity, it will be understood by those skilled in the art that modifications can be made by addition, deletion or substitution without substantial alteration of the polypeptide. If the modified amino acid sequence fulfills the same function as the sequence of SEQ ID NO.1 in a substantially similar manner, with the same result, the modified amino acid is not substantially changed.

For example, a functionally similar peptide may be SEQ ID No. 2:

MPHLMERMVGSGLLWLALVSCILTQASAVQRGYGNPIEASSYGLDLDCGAPGTPEAHVCF

DPCQNYTLLDEPFRSTENSAGSQGCDKNMSGWYRFVGEGGVRMSETCVQVHRCQTDAP

MWLNGTHPALGDGITNHTACAHWSGNCCFWKTEVLVKACPGGYHVYRLEGTPWCNLRYC

TVPRDPSTVEDKCEKACRPEEECLALNSTWGCFCRQDLNSSDVHSLQPQLDCGPREIKVK

VDKCLLGGLGLGEEVIAYLRDPNCSSILQTEERNWVSVTSPVQASACRNILERNQTHAIYKN

TLSLVNDFIIRDTILNINFQCAYPLDMKVSLQAALQPIVSSLNVSVDGNGEFIVRMALFQDQNY

TNPYEGDAVELSVESVLYVGAILEQGDTSRFNLVLRNCYATPTEDKADLVKYFIIRNSCSNQ

RDSTIHVEENGQSSESRFSVQMFMFAGHYDLVFLHCEIHLCDSLNEQCQPSCSRSQVRSE

VPAIDLARVLDLGPITRRGAQSPGVMNGTPSTAGFLVAWPMVLLTVLLAWLF

or SEQ ID NO. 3:

MPHLMERMVGSGLLWLALVSCILTQASAVQRGYGNPIEASSYGLDLDCGAPGTPEAHVCF

DPCQNYTLLDEPFRSTENSAGSQGCDKNMSGWYRFVGEGGVRMSETCVQVHRCQTDAP

MWLNGTHPALGDGITNHTACAHWSGNCCFWKTEVLVKACPGGYHVYRLEGTPWCNLRYC

TDPSTVEDKCEKACRPEEECLALNSTWGCFCRQDLNSSDVHSLQPQLDCGPREIKVKVDK

CLLGGLGLGEEVIAYLRDPNCSSILQTEERNWVSVTSPVQASACRNILERNQTHAIYKNTLSL

VNDFIIRDTILNINFQCAYPLDMKVSLQAALQPIVSSLNVSVDGNGEFIVRMALFQDQNYTNP

YEGDAVELSVESVLYVGAILEQGDTSRFNLVLRNCYATPTEDKADLVKYFIIRNSCSNQRDS

TIHVEENGQSSESRFSVQMFMFAGHYDLVFLHCEIHLCDSLNEQCQPSCSRSQVRSEVPAI

DLARVLDLGPITRRGAQSPGVMNGTPSTAGF LVAWPMVLLTVLLAWLF

or SEQ ID NO.4

MPHLMERMVGSGLLWLALVSCILTQASAVQRVPRDPSTVEDKCEKACRPEEECLALNSTW

GCFCRQDLNSSDVHSLQPQLDCGPREIKVKVDKCLLGGLGLGEEVIAYLRDPNCSSILQTEE

RNWVSVTSPVQASACRNILERNQTHAIYKNTLSLVNDFIIRDTILNINFQCAYPLDMKVSLQA

ALQPIVSSLNVSVDGNGEFIVRMALFQDQNYTNPYEGDAVELSVESVLYVGAILEQGDTSRF

NLVLRNCYATPTEDKADLVKYFIIRNSCSNQRDSTIHVEENGQSSESRFSVQMFMFAGHYD

LVFLHCEIHLCDSLNEQCQPSCSRSQVRSEVPAIDLARVLDLGPITRRGAQSPGVMNGTPS

TAGFLVAWPMVLLTVLLAWLF

Or SEQ ID NO.5

MPHLMERMVGSGLLWLALVSCILTQASAVQRDPSTVEDKCEKACRPEEECLALNSTWGCF

CRQDLNSSDVHSLQPQLDCGPREIKVKVDKCLLGGLGLGEEVIAYLRDPNCSSILQTEERN

WVSVTSPVQASACRNILERNQTHAIYKNTLSLVNDFIIRDTILNINFQCAYPLDMKVSLQAALQ

PIVSSLNVSVDGNGEFIVRMALFQDQNYTNPYEGDAVELSVESVLYVGAILEQGDTSRFNLV

LRNCYATPTEDKADLVKYFIIRNSCSNQRDSTIHVEENGQSSESRFSVQMFMFAGHYDLVFL

HCEIHLCDSLNEQCQPSCSRSQVRSEVPAIDLARVLDLGPITRRGAQSPGVMNGTPSTAGF

LVAWPMVLLTVLLAWLF

More specifically, the above peptides are required for the diagnosis of the immune diseases described above. The above sequence fulfills substantially the same function and gives substantially the same result in substantially the same manner as SEQ ID NO. 1. Therefore, they are included in the teaching given according to the present invention, i.e. the use of the GP2 molecule as a medicament, in particular in the prevention, diagnosis, treatment and/or post-disease management of inflammatory bowel diseases.

Thus, the amino acid sequence (i.e. the peptide of the invention) may comprise a large number of additional amino acids, spacers or other structures that render them suitable for interaction with autoantibodies, preferably in such a way that they reflect an epitope for the latter. Thus, the sequences according to the invention are not limited to peptides related to antibody epitopes, but also refer to molecules and all fragments thereof that specifically interact with autoantibodies in a way that enables diagnosis of inflammatory bowel disease. Thus in a preferred embodiment the terms epitope, peptide and amino acid sequence are synonymous within the context of the present invention.

Various methods for preparing functionally similar peptides have been disclosed in the prior art. The designed peptides utilize the methods encompassed according to the teachings of the present invention starting with the peptides of the present invention. For example, one way to produce functionally similar peptides has been in the national academy of sciences journal of the United states 1998, 10.31, 9521, 12179-84(PNAS USA 1998, Oct.13, 9521, 12179-84); WO 99/6293 and/or WO 02/38592; the above teachings are incorporated into the present disclosure. That is, all peptides, fragments of peptides or structures containing peptides produced using the above-mentioned methods, starting with the peptides of the invention, achieve the objects of the invention and, more specifically, interact with pathogenic autoantibodies, are peptides according to the invention. For example, these autoantibodies may activate the receptor for a competing autoantibody.

In another preferred embodiment of the invention, the molecule comprises a moiety selected from the group consisting of alpha-amino carboxylic acids and homo-and hetero-oligomers thereof (homooligomers), alpha, omega-amino carboxylic acids and branched homo-or hetero-oligomers thereof, other amino acids and straight and branched homo-or hetero-oligomers thereof; amino-oligoalkoxyalkylamines (amino-oligoakoxyalkylamines); maleimide carboxylic acid (maleinimidocarboxylic acid) derivatives; an alkylamine oligomer; 4-alkylphenyl derivatives; 4-oligoalkoxyphenyl (4-oligoalkoxyxyphenyl) derivatives or 4-oligoalkoxyphenoxy (4-oligoalkoxyxyphenoxy) derivatives; 4-oligoalkylmercaptophenyl (4-oligoalkylmercaptophenyl) derivatives or 4-oligoalkylmercaptophenoxy (4-oligoalkylmercaptophenyloxy) derivatives; 4-oligoalkylaminophenyl derivatives or 4-oligoalkylaminophenoxy derivatives; (oligoalkylbenzyl) phenyl ((oligoalkylbenzyl) phenyl) derivative or 4- (oligoalkylbenzyl) phenoxy derivative, and 4- (oligoalkoxybenzyl) phenyl derivative or 4- (oligoalkoxybenzyl) phenoxy derivative; trityl (trityl) derivatives; a benzyloxyaryl (benzyloxyaryl) derivative or a benzyloxyalkyl derivative; xanthen-3-yloxyalkyl (xanthen-3-yloxylkyl) derivatives; (4-alkylphenyl) alkanoic acid derivatives or ω - (4-alkylphenoxy) alkanoic acid derivatives; an oligoalkylphenoxyalkyl derivative or an oligoalkoxyphenoxyalkyl derivative; a carbamate derivative; an amine; trialkylsilyl (trialkylsilyl) derivatives or dialkylalkoxymethylsilyl (dialkylalkoxysilyl) derivatives; an alkyl or aryl derivative, or a combination thereof.

In another preferred embodiment of the invention, the GP2 molecule is used as a soluble or solid phase-bound antigen for the detection of autoantigens in stool and/or body fluids, in particular blood and/or serum, wherein the GP2 molecule in accordance with SEQ ID No.1 was found to be particularly advantageous.

In another preferred embodiment of the invention, the sequence according to the present application, or a peptide resulting from the sequence, is immobilized. More specifically, the solid phase bound GP2 molecule identical to SEQ ID No.1 is bound to an organic polymer, an inorganic polymer, a synthetic polymer and/or a mixed polymer (preferably agarose, cellulose, silica gel, polyamide and/or polyvinyl alcohol). Immobilization in the context of the present invention refers to various methods and techniques for immobilizing peptides on specific carriers, e.g.according to the teachings of WO99/56126 or WO 02/26292. For example, immobilization may serve to stabilize the peptide so that the activity of the peptide is not reduced or adversely modified by biological, chemical or physical contact, particularly during storage or when used in a single batch. Immobilization of the peptide allows for repeated use under technically or clinically routine conditions; furthermore, the sample (preferably a blood component) can be reacted in a continuous manner with at least one peptide according to the invention. More specifically, this can be achieved by various immobilization techniques, which proceed in such a way that the three-dimensional structure of the corresponding molecule (in particular the peptide) is not altered (in particular the active center mediating the interaction with the autoantibody) by binding the peptide to other peptides or molecules or carriers. Advantageously, the result of this immobilization is that there is no loss of specificity for the patient's autoantibodies. In the context of the present invention, three basic methods can be used for immobilization:

i) a crosslinking method: in the cross-linking method, the peptides are immobilized to each other without adversely affecting their activity. Advantageously, as a result of this crosslinking, they are no longer soluble.

ii) binding to a carrier: the binding to the carrier is carried out by, for example, adsorption, ionic binding or covalent binding. This association may also occur within the microbial cell, or in liposomes or other membranous, closed or open structures. Advantageously, by such immobilization, the peptide is not adversely affected. For example, multiple or sequential uses of a peptide conjugated to a carrier can have clinical diagnostic or therapeutic advantages.

iii) an internal inclusion method: the inclusion method in the context of the present invention occurs especially in semi-permanent films in the form of gels, fibrils or fibers. Advantageously, the encapsulated peptide is separated from the surrounding sample solution by a semi-permanent membrane in a manner that still enables interaction with the autoantibody or fragment thereof. Immobilization can be carried out using various methods, for example adsorption on an inert or dotted inorganic or organic support. Such a support may be, for example, porous gel, alumina, bentonite, agarose, starch, nylon or polyacrylamide. Immobilization by physical binding forces typically involves hydrophobic interactions and ionic bonding. Advantageously, this method is easy to operate and has little effect on the conformation of the peptide. Advantageously, the electrostatic binding force between the peptide and the dotted groups of the support may improve the binding, for example by means of an ion exchanger, in particular sahedex (Sephadex).

Another method is to covalently bond to a support material. In addition, the carrier may have a reactive group that forms a homopolar bond with the amino acid side chain. Suitable groups on the peptide are carboxyl, hydroxyl and thio (sulfide group), especially the terminal amino group of lysine. The aromatic group offers the possibility of diazo coupling. The micro-porous glass particles can be activated by silane treatment and subsequently reacted with peptides. For example, the hydroxyl groups of the natural polymer may be activated by cyanogen bromide and subsequently coupled to the peptide. Advantageously, a large number of peptides can be covalently bound directly to the polyacrylamide resin. The inclusion method in three-dimensional networks involves the inclusion of peptides into ionotropic gels (ionotropic gels) or other structures known to those skilled in the art. More specifically, the pores of the matrix are native so that the peptide is retained, allowing interaction with the target molecule. In the crosslinking process, the peptide is converted to a polymer agglomerate by crosslinking with a bifunctional reagent. This structure is gelatinous, easily deformable and particularly suitable for various reactions. By adding other inactive ingredients (e.g. gels) in the crosslinking process, mechanical and binding properties can be strongly improved. In microencapsulation, the amount of reaction of the peptide is limited by the membrane. For example, microencapsulation may be performed in the form of interfacial polymerization. As the microencapsulation process proceeds, the immobilized peptide becomes insoluble and can therefore be reused. In the context of the present invention, an immobilized peptide is any peptide that is under conditions that allow its reuse. Limiting the mobility and solubility properties of peptides by chemical, biological or physical means strongly reduces the cost of the process, especially when autoantibodies are removed from blood components.

In another preferred embodiment of the invention, in addition to the use of soluble or solid phase bound GP2 molecules according to SEQ ID No.1, non-specific adsorbent molecules selected from the group consisting of protein A, protein G, anti-human immunoglobulins or L-tryptophan may be used.

In another preferred embodiment of the invention, the GP2 molecule identical to SEQ ID No.1 is selected from the group consisting of:

a) a molecule having an amino acid sequence sufficiently homologous to the GP2 molecule of SEQ ID No.1 to be functionally similar thereto;

b) a molecule according to said a), which is modified by deletion, addition, substitution, translocation, inversion and/or insertion and which is functionally similar to a molecule according to said a).

Surprisingly, the use of homologues and modified GP2 molecules in accordance with SEQ ID NO.1 gives rise to a number of advantages. In fact, it was found that the changes made according to b) allow to obtain molecules with improved stability, thus producing beneficial effects in the routine laboratory procedures.

In another preferred embodiment of the invention said molecule described in b) has at least 40% homology with said molecule described in a). In a daily routine in the laboratory, the 40% homologue has a surprising effect that it can be stored not only longer than the molecule according to a), but also has an improved ability to withstand temperature fluctuations.

In another preferred embodiment of the invention, the molecule described under b) has at least 60%, preferably 70%, more preferably 80%, and particularly preferably 90% homology with the molecule described under a). Taking into account all the molecules according to b), this variant has the advantage of having, entirely unexpectedly, the longest pot life.

In another preferred embodiment of the invention, the GP2 molecule identical to SEQ ID No.1 is in linear or cyclic form, cyclisation of the peptide being carried out by amide cyclisation, optionally via side chains, C-and N-termini, or via disulfide bridges when two cysteines are present, or by a combination of these possibilities. In the above method, such a variant of GP2 molecule can improve the stability of GP2 molecule in the presence of various denaturing buffers.

Another aspect of the invention relates to the use of GP2 in accordance with SEQ ID No.1 in the preparation of a column coupled with said GP2 for the treatment of inflammatory bowel disease, in particular crohn's disease, chronic pancreatitis and/or ulcerative colitis, said treatment comprising passing the patient's plasma through said column under conditions selected to allow efficient binding of immune globulin in said patient's plasma to GP2, thereby removing a substantial amount of immune globulin from said patient's plasma, and returning the plasma thus obtained to the patient. The above use has the beneficial effect of being able to remove immune globulin very rapidly from the blood of patients with acute cases of IBD.

Another aspect of the invention relates to the use of immunogenic agents of GP2, an immunoreactive sequence or an analogue thereof in the manufacture of a medicament for the diagnostic or therapeutic management of diseases associated with an immune response to these substances. Surprisingly, the sequence of GP2 described above also enables the production of a readily manageable drug that a patient can follow to know to take or use, so hospitalization is not necessary.

In a preferred embodiment, the invention aims to utilize the above-mentioned immunological reagents in the diagnosis or therapeutic management of chronic inflammatory or autoimmune diseases, in particular inflammatory bowel diseases, such as crohn's disease, chronic pancreatitis and/or ulcerative colitis. An advantage of this embodiment is that even those patients with autoimmune inflammatory bowel disease who have little ability to reach the diagnosis and treatment center can get an easy-to-handle medical treatment and use under the supervision of a home doctor.

In a preferred manner, the sequence of SEQ ID No.1 of GP2, immunoreactive sequence and analogues or fragments thereof according to the present application is used as therapeutically active substance in medical production and in the oral treatment of diseases associated with an immune response against these substances. The expression used as therapeutically active substance in the context of the present invention means that the amino acid sequence or a peptide which can be formed from the amino acid sequence is used throughout the medical field. Advantageously, GP2 is a peptide that is sufficiently stable and absorbable so that it can be administered to a patient as a drug, even by the oral route.

The invention also relates to a pharmaceutical composition comprising at least one molecule of GP2 corresponding to SEQ ID No.1, optionally together with a pharmaceutically acceptable carrier, for the treatment of chronic inflammatory or autoimmune diseases, in particular inflammatory bowel diseases, such as crohn's disease, chronic pancreatitis and/or ulcerative colitis.

More specifically, the pharmaceutical composition may be used as a medicament. For this purpose, for example, the peptide or the entire amino acid sequence can be modified by cyclization or other methods known to the person skilled in the art, so that the structure of the peptide (e.g.serum proteases) is prevented from being cleaved endogenously. By using the peptide or protein (SEQ ID NO.1) according to the present invention, the autoantibody can be neutralized in vivo or ex vivo. In vivo neutralization, administration is directed to the patient, in vitro neutralization, blood is drawn from the body (e.g., by a circuit, such as in a tubular circulation), and then contacted with a drug, which neutralizes the autoantibodies, and then circulates the blood back to the organism (i.e., patient). The medicaments in the context of the present invention are compositions for therapeutic and prophylactic purposes, which together with the pharmaceutical compositions can be used as diagnostic agents.

According to the present invention, pharmaceutical or pharmaceutical compositions (synonymously used herein) are compositions and formulations of matter for curing, alleviating or avoiding diseases, disorders, physical defects or pathological infections by application on or in the human body. According to the invention, medical adjuvants are used for preparing active ingredients of medicaments. Pharmaceutical technical adjuvants serve to stabilize the formulated drug or pharmaceutical composition and they may even be removed later when needed only during manufacture, or they may be part of a pharmaceutical composition as a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers are given below. The configuration of the medicament or the configuration of the pharmaceutical composition may optionally be effected by a combination of pharmaceutically acceptable carriers and/or diluents. Examples of suitable pharmaceutically acceptable carriers are well known to those skilled in the art and include, for example, phosphate buffered saline, water, emulsions such as oil/water emulsions, various types of detergents, sterile solutions, and the like. Pharmaceutical or pharmaceutical compositions containing such carriers can be formulated by conventional methods of salary. These drugs or pharmaceutical compositions can be administered to an individual in a suitable dose, for example, 1. mu.g to 10g of peptide or protein per patient per day. A dose of 1mg to 1g is preferred. Preferably, the amount of drug administered is as small and low in number, preferably in a single dose. Administration can be by various routes, for example, intravenous (i.v.), intraperitoneal (i.p.), intrarectal (i.c.), gastrointestinal (i.e., intestinal), intracoronal (i.e., intraoral), intramuscular (i.m.), topical (i.e., local), subcutaneous (i.e., on the skin or through the mucosa), intradermal (i.e., intradermal). The administration of the nucleic acid encoding the peptide according to the invention can also be carried out in gene therapy, for example by means of viral vectors. The nature of the dose and route of administration may be determined by the attending physician in accordance with clinical practice. As is well known to those skilled in the art, the nature of the dosage will depend on various factors such as the size, body surface area, age, sex or general health of the patient, as well as on the particular agent used, the practice period and type of administration, and on other drugs which may be administered concurrently. The person skilled in the art is also familiar with the fact that the concentration of autoantibodies can first be diagnosed using the peptides according to the invention in order to determine the desired drug concentration.

More specifically, the pharmaceutical composition or medicament comprises a pharmacological substance comprising one or more peptides or proteins according to the invention or/and nucleic acid molecules encoding said peptides or proteins in a suitable solution or administration form. Administration of the above may be carried out alone or together with an appropriate adjuvant linked to the drug or pharmaceutical composition as described, or in combination with one or more adjuvants, such as QS-21, GPI-0100 or other saponins, water-oil emulsions such as Montanide adjuvants, polylysine, polyserine compounds, DNA compounds such as CpG Detox, bacterial vaccines such as typhoid or BCG vaccines, salts such as calcium phosphate and/or other suitable substances which enhance the efficacy of the drug, preferably immunostimulatory molecules such as interleukins, e.g. IL-2, IL-12, IL-4 and/or growth factors such as granulocyte-macrophage colony stimulating factor (GM-CSF). They are mixed with the peptides or recognition molecules of the invention according to known methods and administered in suitable dosage forms and dosages.

Obviously, the pharmaceutical composition or medicament may also be a combination of two or more of the pharmaceutical compositions or medicaments of the invention, as well as in combination with other medicaments, e.g. for simultaneous or spaced-apart administration or administration in antibody therapy, chemotherapy or radiotherapy. The production of the medicaments or pharmaceutical compositions is carried out according to methods known per se.

As envisaged in a preferred embodiment of the present invention, the pharmaceutically acceptable carrier for the pharmaceutically acceptable agent is selected from the group consisting of fillers, disintegrants, binders, humectants, diluents, dissolution retardants, absorption enhancers, wetting agents, absorbents and/or lubricants. Surprisingly, it has been found in practice that a large number of pharmaceutical carriers are advantageously suitable for GP2 so that the administration form of the medicament can be adapted to the needs of the patient to a large extent.

The invention also relates to a diagnostic kit for detecting autoimmune diseases, comprising a GP2 molecule identical to SEQ ID No. 1. The diagnostic kit optionally includes instructions relating to combining the contents of the kit and/or providing a formulation for detecting inflammatory bowel disease, particularly crohn's disease, chronic pancreatitis, and/or ulcerative colitis. The instructions may be, for example, a instructional leaflet or other medium that provides the user with information regarding the type of method in which the referenced material is used. Obviously, such information need not be in the form of an instructional leaflet, and such information can be propagated through a method such as a network. The benefit of such a kit is that for example he or she can determine the actual state of the disease without the need for a direct visit to a physician (even during a trip), and thus can selectively adjust diet and activity accordingly.

The invention also relates to a device for chromatography, in particular for apheresis, comprising a GP2 molecule in accordance with SEQ ID No.1 for the treatment of inflammatory bowel disease, in particular crohn's disease, chronic pancreatitis and/or ulcerative colitis. In general, traditional treatment of acute inflammation requires the use of medication to control intestinal inflammation as quickly as possible. The chromatographic apparatus according to the present invention is advantageous in that it does not require administration to a patient, and instead, the patient's blood is purified from antibodies on a apheresis column, thereby preventing expansion of inflammatory centers and thus avoiding drug interactions and damage to organs.

In a preferred embodiment, a molecule of GP2 identical to SEQ ID No.1 is bound to a solid phase within the chromatography system. The GP2 molecule has a surprisingly high affinity for antibodies and shows good immobilization on a solid phase, so that the device according to the invention can be used to exclude antibodies from a patient's body fluid or to neutralize autoantibodies. This method is known to those skilled in the art of immunoadsorption and apheresis therapy. Immunoglobulins are removed from the patient's blood with the aid of immunoadsorption.

Advantageously, such immunoadsorption therapy can be performed as hospitalization and ambulatory therapy. It is envisaged that the device, in particular the so-called adsorber, is part of an extracorporeal blood circulation. For maintenance purposes, blood is continuously or discontinuously taken from the patient's aorta (more specifically the arm veins) and separated into individual components, such as cellular and bodily fluid components, by filtration or centrifugation. In particular, one essential blood component obtained in this way is plasma. Advantageously, plasma may be passed through the apparatus of the invention and returned to the patient after the autoantibodies have been adsorbed, more particularly via another vein in the arm or leg, with previously separated blood components (particularly cellular components). It is contemplated that the peptide is immobilized on an agarose matrix. The matrix may be placed in a container having a volume of 10-400 ml. Subsequently, the patient's blood stream can be made to flow through the matrix, where the autoantibodies will be bound to allow their removal from the plasma.

The skilled person will be familiar with various ways of providing such solid phase immobilized peptides, for example in the form of (i) a regenerable absorption column, (ii) a double column and (iii) a disposable column. The different wash and elution solutions that provide high efficacy of treatment can be readily determined by one skilled in the art using conventional methods. By providing the teachings in accordance with the present invention, the peptides of the invention are more particularly disclosed to those skilled in the art for the prevention, diagnosis, treatment and post-conditioning (aftercare) of autoantigen-mediated diseases caused by the common cold in various ways of in vivo, ex vivo and in vitro use of the peptides.

Features highlighted in the following according to the teachings of the present application:

detachment from conventional techniques

New problem areas

There is a long-felt, unsatisfied and urgent need to solve the problems solved by the present invention

No effort in the art has been made to date

The simplicity of the solution represents an activity of the invention, in particular it replaces more complex techniques

The development of scientific technology in different directions

Achievement of development

Wrong views of the field on solving the problem of controversy (prejudice)

Advances in technology, such as improvements, performance enhancements, cost reductions, time savings, materials, principals steps, cost or raw material unavailability, enhanced reliability, removal of disadvantages, superior quality, freedom of maintenance, higher efficiency, higher yield, extensions of the technology field, provision of other means, creation of second ways, development of new fields, first time problem solving, conservative devices, interchangeability, implementation, automation or miniaturization scope, or expansion of the range of drugs

Lucky choices (among the possibilities, the choice of one being unpredictable, which makes it a lucky choice for a patent)

Errors in Prior Art files

New technical field

Inventive combinations, i.e. combining a number of known elements to achieve unexpected results

-licensing issues

Praise in the field

Economic success

These characteristics are particularly applicable to the preferred embodiments of the present invention.

The present invention will be explained more specifically with reference to examples, which are not intended to limit the present invention.

Method

GP2 was purified from rat pancreas

Obtaining Zymogen Granules (ZG) and purifying ZG membrane

All working steps below were carried out in an ice bath or cooled to 4 ℃. Four adult Wistar rat pancreases (tissue weighing approximately 2.4g) were mechanically reduced in size by immersion in 10 volumes of ice-cold 0.3M sucrose solution in a POTTER (POTTER) homogenizer (2 strokes at 1000rpm and 2 strokes at 1300 rpm). The impregnated material was then filtered on a gauze. Cell debris and nuclei were removed by centrifugation at 500g for 10 minutes. Centrifugation at 3000g for 10 min resulted in the sedimentation of zymogen granules (lower, white solid globules) and mitochondria (upper, loose brown globules) from the supernatant. Mitochondria were carefully washed off with buffer a (10mM morpholinopropanesulfonic acid (MOPS), pH 6.8) and the zymogen particles were resuspended in 2ml of 0.1M sodium carbonate solution and 1mM Diisopropyl Fluorophosphate (DFP) using a vortex machine. The particles were lysed in an ice bath for 1 hour. The lysed batch was fractionated on a discontinuous sucrose gradient (0.3M/1M) and centrifuged at 200000g for 90 min. The membrane portions accumulate as bands at the density boundaries. The band was absorbed and the resulting solution was adjusted to 0.3M sodium bromide. The membranes were precipitated by centrifugation at 200000g for 60 minutes.

Solubilization of GP2

The resulting membrane pellet was resuspended in 0.5mL of buffer B (20mM morpholinoethanesulfonic acid, MES, pH 7.0; 80mM KCl; 45g/mL saponin) using sonication, and after addition of phosphatidylinositol-specific phospholipase C (Bacillus cereus), GP2 was removed from the membrane by incubation at 37 ℃ for 1 hour, the membrane was pelleted by centrifugation (200,000g, 60min), and the supernatant containing soluble GP2 was concentrated to approximately 1: 5 using ultrafiltration.

Enzyme-linked immunosorbent assay (ELISA) for detecting GP2 antibody

Microtiter plates (Maxisorb, Neuken, Roskilde) were coated with 10. mu.g/ml rat GP2 in coating buffer (100mM sodium carbonate, pH 9.6) at 4 ℃ in 50. mu.l/well and left overnight. After washing the microtiter plates with wash buffer (10mM sodium phosphate, 150mM NaCl, 0.1% Tween 20(Tween 20), pH 7.4), the wells were incubated with 300. mu.l of blocking solution (wash buffer, 1% Bovine Serum Albumin (BSA), pH 7.4) for 30 minutes at Room Temperature (RT). Subsequently, the wells were washed three times with washing buffer, 50. mu.l per well of human serum samples, diluted 1: 100 in dilution buffer (10mM sodium phosphate, 150mM NaCl, 1% murine serum albumin (RSA)), and incubated for 60 minutes at Room Temperature (RT). After three washes with dilution buffer, wells were filled with 50 μ l of conjugate solution (anti-human IgG peroxidase, sheep, 1 μ g/ml, dilution solution) and incubated for 30 min at RT. The wells were then washed 3 more times and 50. mu.l of substrate solution (tetramethylbenzidine) was dispensed into each well. After 10 min incubation at RT, the substrate reaction was stopped by adding 50 μ l of quenching solution (0.3M sulfuric acid). Microtiter plates were subjected to two chromatographic measurements of optical density of the solution in each well at 450nm and 620nm, followed by computer-based analysis using the EIAstar software program.

Indirect immunofluorescence assay (IIF) for pancreatic antigen antibody detection

Pancreatic antigen antibodies were assayed by the method of IIF using commercially available monkey pancreatic sections (omega, Lubeck). Serum samples were diluted 1: 40, 1: 80 and 1: 160 with dilution buffer. 25 μ l of diluted serum was pipetted onto a reaction plate of reagent carrier and incubated with a microscope slide with tissue sections for 30 minutes at RT. Then, the slide was washed with phosphate buffer for 1 minute. 20 μ l of labeled antiserum (anti-human IgG FITC) was pipetted onto each plate of the washed reagent carrier and incubated with the tissue sections on the slide for 30 minutes at RT. After an additional 1 minute wash with phosphate buffer, the coverslips were placed on slides with the aid of a mounting medium. Fluorescence evaluation was performed using fluorescence microscopy.

Reference to the literature

Bosu et x serological markers in inflammatory bowel disease journal of clinical chemistry, 2006, 52 (2): 171-

Molecular cloning and sequence of the protein GP2 associated with the zymogenic membrane of the human pancreas, the alpha (large) subtype and the beta (small) subtype Biochemical and biophysical report 2000, 1491, 376-380(Fukuoka S-I. molecular cloning and sequences of cDNAs encoding alpha (large) and beta (small) iso forms of human pancreatic zymogen granules-associated protein GP2.BBA 2000, 1491, 376-380.)

Friekh, bokhov a, forlowa wackni C, mestra W, skybra PC. from human pancreatic exocrine tissue (Pag) as a representative antigen for autoantibodies in crohn's disease, european journal of clinical research, 1999, 29: 41-45(Fricke H, Birkhofer A, Folwaczny C, Meister W, Scriba PC. Characterisation of antigens from the human exosporic secretory tissue (Pag) innovative as target antigens for autoantibates in Crohn's disease. Eur J Clin Invest, 1999, 29: 41-45.)

WO01/94409 (Corlisha corporation [ US ]; Herster summer K [ US ]; Halake Susan L [ US ]; Dilong (CORXA CORP [ US ]; Hirst Shannon K [ US ]; Harlocker SusanL [ US ]; Dillon)), 12 and 13 months of 2001 (2001-12-13) Compositions and methods for the treatment and diagnosis of pancreatic cancer (Compositions and methods for the same and diagnostics of pancreatic cancer.)

Mehn J, meckenki H, yerman GR, kerma, apocynon D, pennington CR, parlat. brewers yeast (bakers yeast) for crohn's disease. british journal of medical science, 297: 1105 (Main J, McKenzie H, Yeast GR, Kerr MA, Robson D, PenningtonCR, Parrat D. antibody to Saccharomyces cerevisiae (bakers 'year) in Crohn's disease. BMJ, 297: 1105-1106.)

Mayt WJ, Preres AG, Hellman E, Mohr R, Manns M, EvK, antibodies to cytoskeletal proteins in patients with Crohn's disease from Meyer Sumpb Philide KD.. 516-KH.Antibodies to cytoskeletalproteins in patients with Crohn′s disease.Eur J Clin Invest，1990，20：516-524.)

Insulin-sensitive pancreatic antigens in sabo F, weber P, kiensh, vedmann KH. chronic inflammatory bowel disease: specific markers for a subpopulation of patients with crohn's disease the journal of gastroenterology 1991, 32: 1192-119(Seibold F, Weber P, Jens H, Wiedimann K H. additives toa trypta sensitive polymeric antigen in molecular in fluidic plasmid vector diseases: specific markers for a subgroup of substrates with Crohn's disease Gut 1991, 32: 1192-1197.)

WO 96/17873A ((alpha Gene INC. USA) Alphagene INC. US) 1996, 13.6.1996-06-13), Diagnosis of pancreatitis (Diagnosis of pancreas.)

One-page autoimmunity in Stockw, Ottm, Ulrpis, Norman D, Fenberner 'S H, Stockk, Jatteck G, Skliba PC. Crohn' S disease results from autoantibody screening in patients with chronic inflammatory bowel diseaseW，Otte M，Ulrich S，Normann D，Finkbeiner H，K，Jantschek G，Scriba PC.Autoimmunity to pancreatic juice in Crohn′sdisease.Results of an autoantibody screening in patients with chronic inflammatorybowel disease.Scand J Gastroenterol，1987，22(suppl 139)，41-52.)

Claims (33)

1. A method for the detection of antibodies from stool and/or body fluids, in particular blood and/or serum, by immunoreaction with GP2, an immunoreactive sequence or an analogue thereof.

2. The method of claim 1, wherein detection is performed on human IgA, IgM and/or IgG antibodies.

3. The method according to claim 1 or 2, wherein the GP2 is derived from human, animal, recombinant or synthetic material.

4. The method according to any one of claims 1 to 3, wherein the detection is performed in an immunoassay, preferably with direct or indirect coupling of a reactant to a labeling substance.

5. A method of treating inflammatory bowel disease, the method comprising the steps of:

a) providing a column having GP2 coupled thereto;

b) passing the patient's plasma through the column under conditions that allow the GP2 to effectively bind to antibodies within the patient's plasma, thereby removing a substantial amount of antibodies from the patient's plasma; and

c) the plasma thus obtained is returned to the patient.

6. The method of treating inflammatory bowel disease according to claim 5, wherein GP2 in accordance with SEQ ID No.1 recognizes autoantibodies directed against intestinal tissue.

7. A method for treating autoimmune disease by binding and/or removing autoantibodies using a solid phase bound GP2 molecule identical to SEQ ID No. 1.

8. A method for the diagnosis or therapeutic management of crohn's disease, characterized in that an immune reaction is detected against GP2 antibody identical to SEQ ID No.1 from faeces and/or body fluids, in particular from blood and/or serum, by means of said immune reaction with GP2, an immunoreactive sequence or an analogue thereof, said immune reaction being carried out without the use of tissue slices of animal or human tissue.

9. The method according to claim 8, wherein the detection is performed in an immunoassay, preferably using direct or indirect coupling of a reactant to a labeling substance, but not including tissue section-based immunofluorescence assays.

10. The method of claim 8 or 9, wherein the detection is performed on a solid phase.

11. Use of a molecule GP2 identical to sequence SEQ ID No.1 for the preparation of a medicament for the prevention, diagnosis, treatment and/or post-treatment of autoimmune diseases.

12. The use of the aforementioned GP2 molecule,

it is characterized in that

The autoimmune disease is selected from the group consisting of Inflammatory Bowel Disease (IBD) and/or autoimmune liver disease.

13. The use of the GP2 molecule according to claim 11 or 12,

it is characterized in that

The inflammatory bowel disease is Crohn's disease, chronic pancreatitis and/or ulcerative colitis.

14. The use of the GP2 molecule according to any one of claims 11 to 13,

it is characterized in that the preparation method is characterized in that,

the liver disease is primary sclerosing cholangitis and/or autoimmune enteritis.

15. The use of the GP2 molecule according to any one of claims 11 to 14,

it is characterized in that the preparation method is characterized in that,

the GP2 molecule comprises a linker and/or spacer selected from the group consisting of alpha-aminocarboxylic acids and homo-and hetero-oligomers thereof, alpha, omega-aminocarboxylic acids and branched homo-or hetero-oligomers thereof; other amino acids and their linear and branched homo-or hetero-oligomers; amino-oligoalkoxyalkylamines; maleimide carboxylic acid derivatives; an alkylamine oligomer; 4-alkylphenyl derivatives; 4-oligoalkoxyphenyl derivatives or 4-oligoalkoxyphenoxy derivatives; 4-oligoalkylmercaptophenyl derivatives or 4-oligoalkylmercaptophenoxy derivatives; 4-oligoalkylaminophenyl derivatives or 4-oligoalkylaminophenoxy derivatives; (oligoalkylbenzyl) phenyl derivatives or 4- (oligoalkylbenzyl) phenoxy derivatives, and 4- (oligoalkoxybenzyl) phenyl derivatives or 4- (oligoalkoxybenzyl) phenoxy derivatives; a trityl derivative; a benzyloxyaryl derivative or a benzyloxyalkyl derivative; xanthen-3-yloxyalkyl derivatives; (4-alkylphenyl) alkanoic acid derivatives or ω - (4-alkylphenoxy) alkanoic acid derivatives; an oligoalkylphenoxyalkyl derivative or an oligoalkoxyphenoxyalkyl derivative; a carbamate derivative; an amine; a trialkylmethylsilane derivative or a dialkylalkoxymethylsilane derivative; alkyl or aryl derivatives, and/or combinations thereof.

16. The use of the GP2 molecule according to any one of claims 11 to 15,

it is characterized in that the preparation method is characterized in that,

the GP2 molecule is used in soluble form or bound to a solid phase for the direct or indirect detection of autoantibodies in faeces and/or body fluids, in particular blood and/or serum.

17. The use of the GP2 molecule according to any one of claims 11 to 16,

it is characterized in that the preparation method is characterized in that,

the GP2 molecule is used in soluble form or bound to a solid phase for the direct or indirect detection of autoantibodies in faeces and/or body fluids, in particular blood and/or serum.

18. The use of the GP2 molecule according to any one of claims 11 to 17,

it is characterized in that the preparation method is characterized in that,

the solid phase bound GP2 molecule in accordance with SEQ ID No.1 is bound to an organic polymer, an inorganic polymer, a synthetic polymer and/or a mixed polymer, preferably agarose, cellulose, silica gel, polyamide and/or polyvinyl alcohol.

19. The use of the GP2 molecule according to any one of claims 11 to 18,

it is characterized in that the preparation method is characterized in that,

in addition to the use of the soluble or solid phase bound GP2 molecules, non-specific adsorbent molecules selected from the group consisting of protein a, protein G, anti-human immunoglobulin and/or L-tryptophan are used.

20. The use of the GP2 molecule according to any one of claims 11 to 19,

it is characterized in that the preparation method is characterized in that,

the GP2 molecule is selected from the group consisting of:

a) a molecule having an amino acid sequence sufficiently homologous to the GP2 molecule to be functionally similar to the GP2 molecule;

b) the modified molecules of a) are obtained by deletion, addition, substitution, translocation, inversion and/or insertion and are functionally similar to the molecules of a).

21. The use of the GP2 molecule according to any one of claims 11 to 20,

it is characterized in that the preparation method is characterized in that,

the molecule described under b) has at least 40% homology with the molecule described under a).

22. The use of the GP2 molecule according to any one of claims 11 to 21,

it is characterized in that the preparation method is characterized in that,

the molecules described under b) have at least 60%, preferably 70%, more preferably 80%, and particularly preferably 90% homology with the molecules described under a).

23. The use of the GP2 molecule according to any one of claims 11 to 22,

it is characterized in that the preparation method is characterized in that,

the GP2 molecule is in linear or cyclic form, cyclizing the peptide either by amide cyclization, optionally via side chains, C-and N-termini, or by disulfide bridges when two cysteines are present, or by a combination of these possibilities.

Use of GP2 in the manufacture of a column coupled with said GP2 for the treatment of inflammatory bowel disease, in particular crohn's disease, chronic pancreatitis and/or ulcerative colitis, said treatment comprising passing the patient's plasma through said column under conditions selected to allow GP2 to bind effectively to immune globulins in the patient's plasma, thereby removing a substantial amount of immune globulins from the patient's plasma, and returning the plasma thus obtained to the patient, said GP2 preferably being SEQ ID No. 1.

Use of GP2, an immunoreactive sequence or an analogue thereof for the manufacture of a medicament for the diagnostic or therapeutic control of a disease associated with an immune response to such substances, the GP2 preferably corresponding to SEQ ID No. 1.

26. Use according to claim 25, in the diagnostic or therapeutic management of chronic inflammatory or autoimmune diseases, in particular inflammatory bowel disease, such as crohn's disease, chronic pancreatitis and/or ulcerative colitis.

27. Use according to any one of claims 25 or 26 in the diagnosis or therapeutic management of crohn's disease.

Use of GP2, an immunoreactive sequence or an analogue thereof for the manufacture of a medicament for the oral treatment of a disease associated with an immune response thereto, said GP2 preferably corresponding to SEQ ID No. 1.

29. A pharmaceutical composition comprising at least one GP2 molecule, optionally together with a pharmaceutically acceptable carrier, for the treatment of chronic inflammatory or autoimmune diseases, in particular inflammatory bowel disease, such as crohn's disease, chronic pancreatitis and/or ulcerative colitis, said GP2 preferably corresponding to SEQ ID No. 1.

30. A pharmaceutical product according to the preceding claim,

it is characterized in that

The pharmaceutically acceptable carrier is selected from the group consisting of fillers, disintegrants, binders, humectants, diluents, demulcents, absorption enhancers, wetting agents, absorbents and/or lubricants.

31. A diagnostic kit for the determination of an autoimmune disease comprising a GP2 molecule, optionally together with instructions relating to the combination of the contents of the kit and/or providing a preparation for the detection of inflammatory bowel disease, in particular crohn's disease, chronic pancreatitis and/or ulcerative colitis, the GP2 preferably corresponding to SEQ ID No. 1.

32. A device for chromatography, comprising GP2 molecule in accordance with SEQ ID No.1 for the treatment of inflammatory bowel disease, in particular crohn's disease, chronic pancreatitis and/or ulcerative colitis, in particular for apheresis.

33. The apparatus according to the preceding claim, wherein,

it is characterized in that the preparation method is characterized in that,

the GP2 molecule corresponding to SEQ ID NO.1 was bound to a solid phase.