WO1996009388A1

WO1996009388A1 - Novel anti-vh3-15 reagents, vh3-15 polypeptides, cell surface antigens, and methods for their detection and use

Info

Publication number: WO1996009388A1
Application number: PCT/US1995/011789
Authority: WO
Inventors: Jonathan Braun
Original assignee: The Regents Of The University Of California
Priority date: 1994-09-19
Filing date: 1995-09-18
Publication date: 1996-03-28
Also published as: CA2200329A1

Abstract

The present invention provides novel anti-VH3-15 idiotypic antibody materials and hybridomas producing this antibody material. Also provided in the present invention are VH3-15 autoantibodies recognized by anti-VH3-15 idiotypic antibody material and the cell surface antigens which bind VH3-15 autoantibody. The anti-VH3-15 idiotypic antibody material, VH3-15 autoantibodies and cell surface antigens of the present invention can be used in methods for detecting, isolating and purifying VH3-15 polypeptides and in diagnostic assays for IBD and infection by Campylobacter jejuni. Nucleic acid molecules encoding anti-VH3-15 antibody material, VH3-15 autoantibody and cell surface antigens, as well as methods for their use are also provided.

Description

NOVEL ANTI-VH3-15 REAGENTS, VH3-15 POLYPEPTIDES. CELL SURFACE ANTIGENS, AND METHODS FOR THEIR DETECTION AND USE

I. BACKGROUND OF THE INVENTION

A. The Antibody Repertoire

Over a lifetime, a person confronts the possibility of infection with an almost infinite number of unique foreign substances (antigens) . Since it could never be anticipated which of these antigens will ultimately infect a person, it is beneficial that the body possesses an elegant system of producing an equally infinite array of antibodies which recognize, bind and trigger the destruction of antigens.

Antibodies are Y-shaped, tetrameric molecules consisting of a pair of identical, relatively long polypeptide chains called heavy (H) chains and a pair of identical, shorter polypeptide chains called light (L) chains. Each arm of the Y shaped structure is comprised of one light chain and one end of a heavy chain bound together by a single disulfide bond. At the juncture of the arms, the two heavy chains are bound to each other by two disulfide bonds to form the stem of the Y shaped structure.

This architectural description of an antibody, although visually appealing, can be deceptively simplistic. Antibody architecture accommodates a wealth of structural diversity. Both the heavy and light chains contain variable (V) and constant regions. These V regions are responsible for antigen binding. Heavy and light chain variable regions (VH and VL) each consist of B-sheet scaffold, surmounted by three antigen-binding loops (complementarity-determining regions or CDRs) of different lengths which are fleshed with a variety of side chains. The CDRs are the most diverse regions of the antibody molecule; all six associate to to one degree or another in forming the site at which the antibody binds its antigen (antigen-binding site) . The structural diversity of the loops can create binding sites of a variety of shapes, ranging from almost flat surfaces to deep cavities.

Thus the vast array of antibody specificities depends on the diversity of V region structure which in turn depends on the diversity of the primary sequence of the V region. Underpinning the structural diversity of antibodies is a combinatorial genetic diversity. Heavy and light chain polypeptides are each encoded by an ensemble of gene segments selected from immunoglobulin (Ig) gene complexes. During the maturation of B-cell (the cells which produce antibodies) , discontinuous gene segments within these gene complexes undergo a series of somatic rearrangements to form the nucleic acid sequence that ultimately may encode the heavy and light chains of the antibody molecule.

Generally, the first Ig gene rearrangements occur within the Ig heavy chain gene complex. The VH region is generated by the assembly of a VDJ exon from three separate germline DNA segments. One or more diversity (D) gene segments (selected from more than two dozen D germline gene segments) is joined with a single joining (JH) gene segment (selected from about six functional JH germline gene segments) . The resulting DJH complex may then rearrange with a VH gene segment to form a VDJ exon that may encode the variable portion of the antibody heavy chain. About 120 germline VH gene segments (of which only about 80 are potentially functional) are available for Ig gene rearrangement and can be divided into at least six families in the basis of nucleotide homology of 80% or above. After successful VDJ rearrangement, a similar rearrangement occurs to produce the light chain.

Two of the heavy chain CDRs (1 and 2) are encoded by the VH segment. The heavy chain CDR3, the CDR in direct contact with antigen during antigen-antibody binding, is the most variable portion of the antibody molecule and is encoded by the 3'end of the VH gene segment, the D segment and the 5' end of the JH segment. With nucleotide addition (N-region diversity at the VH-D and D-JH junctions) the use of different reading frames in the D segment, and the combination of different rearranged heavy and light chains, the diversity of primary antibody libraries is huge. During an immune response, the antibody variable regions are further diversified by somatic hypermutation, leading to higher affinity binding of the antigen.

Contrary to what was first believed, that which may define the uniqueness of the antibody, idiotype, includes not only the antigen binding site (as was once believed) but also includes portions of the variable region that can be bound by other antibodies. It follows then that an idiotype of a given antibody molecule can be described as a collection of "idiotopes" as mapped by a panel of monoclonal anti-idiotypic antibodies, a conventional cross-absorbed polyclonal anti-idiotype antibody, binding of defined antigens or any combination of these.

B. Autoantibodies

The monumental repertoire of the adaptive immune system has evolved to allow it to recognize and ensnare virtually any shaped microbial molecule either at present in existence or yet to come. However, in doing so it has been unable to avoid the generation of autoantibodies, antibodies that bind with the body's own constituents and trigger an immunological path of destruction.

Natural immunological tolerance mechanisms prevent the expanded production of autoantibodies. After antibody gene rearrangement, virgin B-cells (the cells that generate antibodies) that display autoantibodies are destroyed or supressed by the bodies tolerance mechanisms. Despite this safety-net, autoantibodies are still produced and for many people create no recognizable pathogenic disorder. It has been estimated that 10-30% of B cells in normal, healthy individuals are engaged in making autoantibodies. Production of autoantibodies is not only the result of an exceptionally diverse immune system, an immune response against one's self can also arise in autoimmune disease or after infections, but tolerance mechanisms can lead to their deletion or to their anergy.

C. Anti-Idiotypic Markers

Despite the tremendous potential for diversity, evidence is accumulating which would suggest that the use of V gene segments is not random. For example, expression of different VH gene families does not reflect the size of the family, nor are functional members of any given family expressed equally. Although some VH gene segments are polymorphic, certain genes appear to be remarkably conserved among unrelated individuals.

Recently a great deal of attention has been directed at investigating the hypothesis that there is programmed use of certain V gene segments in normal human B cell ontogeny and in autoimmune and neoplastic B cell disorders. However, the present techniques of screening cDNA is cumbersome and suffers the shortcomings of the PCR.

Novel clonal markers that are specific for human immunoglobulins of defined VH or VL gene products would provide a reliable and quick means for screening B cell populations for the expression of specific VH and VL idiotypes. They would also readily permit the identification of trends in the use of particular VH segment in pathogenic immune responses as compared to healthy immune responses. Such clonal markers might ultimately prove useful as diagnostic markers of disease states associated with a particular use of VH segment. Even more advantages would be the development of clonal markers that do not compete or sterically interfere with binding of antigen to the antibody. If we are to move forward with our understanding of the immunological system, compositions and methods must be developed to allow for convenient, reliable and efficient purification of antibodies on the basis of variable segment utilization.

D. Inflammatory Bowel Disease and Ca pylobacter i eiuni Enterocolitis 1. Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is the collective term used to describe two gastrointestinal disorders: ulcerative colitis (UC) and Crohn's disease (CD) . Although the diseases have distinct pathophysiological characteristics, they are frequently considered together due to several clinical and therapeutic similarities. Excluded from this category, however, are gastrointestinal inflammatory disorders of known infectious, toxic or ischemic etiology which may mimic IBD acutely, but do not cause a chronic relapsing and remitting syndrome.

IBD occurs world-wide and is reported to afflict as many as two million people. The course and prognosis of IBD is widely variable. Onset has been documented at all ages; however, IBD predominately begins in young adulthood. The three most common presenting symptoms of IBD are diarrhea, abdominal pain, and fever. The diarrhea may range from mild to severe and is often accompanied by urgency and frequency. In UC, the diarrhea is usually bloody and may contain mucus and purulent matter as well . Anemia and weight loss are additional common signs of IBD. 10% to 15% of all patients with IBD will require surgery over a 10-year period. The risk for the development of cancer is increased in patients with IBD as well, particularly in those with ulcerative colitis. The longer the duration of disease, the higher the risk of developing carcinoma. Patients with ulcerative colitis regularly undergo cancer surveillance by endoscopy after ten years of disease. Reports of an increasing occurrence of psychological problems, including anxiety and depression, are perhaps not surprising secondary effects of what is often a debilitating disease that occurs in people in the prime of life.

2. Methods of Diagnosing IBD A battery of laboratory, radiological, and endoscopic evaluations are combined to derive a diagnosis of IBD and to assess the extent and severity of the disease. Nevertheless, differentiating UC from CD, as well as other types of inflammatory conditions of the bowel, such as irritable bowel syndrome, infectious diarrhea, rectal bleeding, radiation colitis, and the like, is difficult, because the mucosa of the small and large intestines reacts in a similar way to a large number of different insults. Once infectious-types of bowel disorders have been ruled out, the final diagnosis of IBD is often made on the basis of the progression of the disease. In many patients, though, the diagnosis of IBD must still be regarded as indeterminate because of the overlapping features of UC and CD, particularly with CD of the colon.

3. Campγlobacter ieiuni Enterocolitis Can

Mimic IBD but Has Not Been Shown to be a Causative Factor

Infection with Campylobacter jejuni has been reported to be the most common bacterial cause of acute diarrheal illness in developed countries. Campylobacter may cause a spectrum of intestinal diseases ranging from acute gastroenteritis to toxic megacolon, lymphadenitis mesenterialitis, and even appendicitis and cholecystitis. Usually infection with this organism results in acute gastroenteritis with fever and frequent loss of often bloody stools. The disease resolves spontaneously (although antibiotic-treatment is recommended) within about one week, but in 20% of patients it runs a more prolonged course and relapses resembling chronic IBD. Although Campylobacter jejuni enterocolitis may be indistinguishable from UC by endoscopic examination, and histological examination of rectal biopsies can range from normal to inflammatory changes suggestive of acute infectious colitis or CD, it can be distinguished from IBD by any one of several serological tests or by culturing fecal specimens.

This mimicry of the pathological features of IBD, has spurred investigation into whether Campylobacter jejuni infection may be the cause or be a causative factor of IBD. Although these investigations do not unequivocally eliminate Campylobacter jejuni as pathogenically important to IBD, defined populations of IBD patients show that Campylobacter jejuni is not normally present in the intestinal flora and could not be found in CD patients during exacerbation. The reported conclusion from the data is that Campylobacter jejuni cannot be implicated as a cause of IBD.

4. The Cause (s) of IBD are Unknown Although the etiology of IBD is unknown, a number of studies have suggested that genetics is important in a person's susceptibility to IBD and that the immune system is responsible for mediating the tissue damage in these diseases. Generally speaking, a failure to down regulate the normal self-limited inflammatory response of the bowel is characteristic of IBD, but it remains unclear what initiates the pathogenic processes.

It has also been suggested that a primary abnormality of the immune system and its regulation might serve as primary initiating factors, or that the disease process might be initiated by an infectious agent and the injury is then perpetuated through immune-mediated or other processes. Although the mucosal injury observed during episodes of acute disease can resemble the effects of any of a number of recognized infectious agents, such as CampyloJbacter jejuni as discussed above, no transmissible infectious agent has been consistently identified with IBD.

Autoimmunity has been suggested in the pathogenesis of IBD. Evidence to suggest this hypothesis is based on the existence of circulating antibodies that react with unknown alimentary tract antigens of both human and animal origin. For example, human fetal and adult colonic, biliary, skin and vascular epithelial cells, epithelial cell associated components from murine small intestine, rat and human colonic epithelial glycoproteins, intestinal bacterial polysaccharide, and antigens from germ-free rat feces have been described to react with sera from patients with IBD. Other studies demonstrated an increased local IgG response in the colonic mucosa of patients with IBD and other colonic inflammations. The mechanism of this IgG response, the specific local antigens involved, and the role of these antibodies are unknown. While a wide range of immunologic abnormalities have been reported in these disorders, none other than the detection of perinuclear anti-neutrophil cytoplasmic antibodies ("pANCAs") in a biological sample appear to be sufficiently reliable to be of diagnostic value.

5. Need for Objective Diagnostic Tools Inflammatory bowel disease poses a clinical and scientific challenge to physicians and researchers. To date most of the diagnostic tools for IBD are quite subjective. Diagnosis depends upon a host of procedures aimed at confirming the suspected diagnosis. The initial symptoms are often confused for non-chronic bowel disorders by physicians unfamiliar with IBD. Consequently, IBD often goes mistreated and undiagnosed until the disease shows its chronicity which results in referral of the patient to a specialist. The imprecise and subjective nature of endoscopic and radiologic examination can result in a misdiagnosis or indeterminate diagnosis even when the IBD is suspected. Unfortunately, the patient must often suffer as the disease progresses before a definitive diagnosis can be made.

IBD and quite often its treatment affects the lifestyle and functional capabilities of those afflicted. Treatment courses often result in adverse physiologic manifestations which must be balanced against the therapeutic benefit. Any intervention which can improve patients' toleration of their disease and therapeutic program is welcome.

The availability of anti-idiotypic antibodies, especially those that could completely or partially distinguish IBD from non-chronic inflammatory diseases of the bowel, would represent a major clinical advance which would aid in therapeutic management of IBD and the design of more specific treatment modalities. In addition specific detection of the disease in prospective parents can be useful in genetic counseling. Accordingly, there has existed a need for convenient and reliable methods of screening for IBD for diagnostic, prognostic and therapeutic purposes.

II. BRIEF DESCRIPTION OF THE INVENTION

Autoantibodies encoding the rarely used VH3-15 gene sub-family have been discovered, as well as cell surface antigens recognized by these autoantibodies. These autoantibodies, referred to as VH3-15 autoantibodies, are easily detected using the methods and reagents of the present invention.

It has also been discovered for the first time that these VH3-15 autoantibodies are associated with IBD and with infection by Campylobacter jejuni . This unexpected association provides the basis for convenient and reliable diagnostic assays for IBD and infection by Campylobacter jejuni enterocolitis.

Novel anti-VH3-15 idiotypic antibody material and hybridomas capable of producing anti-VH3-15 idiotypic antibody material have also been created. This novel antibody material has specificity for the variable heavy chain region of a VH3-15 polypeptide and may take the form of antibody molecules or portions thereof. The antibody material may be polyclonal or monoclonal. Monoclonal anti-VH3-15 idiotypic antibodies are specifically provided and hybridomas for their product are taught.

These novel anti-VH3.-15 idiotypic antibody material, surface antigens, and VH3-15 autoantibodies can be used in the diagnostic assays, the kits and the methods for detecting, quantifying and isolating VH3-15 polypeptides provided in the present invention. These diagnostic assays and methods exploit the immunoreactivity of anti- idiσtypic antibody with autoantibody, and autoantibody with antigen to provide a wealth of different strategies for detection, isolation and purification of these immunological reagents.

Also provided are isolated nucleic acid molecules encoding the anti-VH3-15 idiotypic antibody material, the cell surface antigens and VH3-15 autoantibody of the present invention. These nucleic acid molecules may be used as probes for detecting the presence, absence or amount of nucleic acid encoding the inventive antibody material or antigens in a sample. These nucleic acid molecules may also be used in the kits of the present invention.

IV. BRIEF DESCRIPTION OF THE DRAWING

Figure 1 is a graph reporting the results of sera from 101 subjects (see Table 1) which were analyzed for VH3-15 autoantibodies. Values are relative fluorescence intensities (the ratio of mean fluorescence for staining with anti-VH3-15 idiotypic antibody and buffer alone) for each subject in a disease group determined by flow cytometry as described in Example IV. Black bars are the mean values for all subjects in each disease group. The vertical stippled bar is the 90% confidence limits for values of the control group (healthy adults) . P values compare each disease group to the control group.

Figure 2 is a graph reporting the results of two Crohn's disease patients, two ulcerative colitis patients and one normal tested for serum levels of VH3-15 autoantibody by fixed Campylobacter jejuni cell ELISA as described in Example VII.

V. DETAILED DESCRIPTION OF THE INVENTION

The monumental repertoire of the adaptive immune system has evolved to allow it to recognize and ensnare virtually any shaped microbial molecule either at present in existence or yet to come. Underpinning the structural diversity of antibodies is an elegant system of combinatorial genetic diversity in which an ensemble of gene segments are joined together and subject to mutation to produce a unique binding site with high affinity for one or more antigens. Although great strides have been made in recent years at understanding this mechanism of diversity, the tools of research have limited investigators' progress.

The present invention provides powerful immunological tools useful for screening large populations or particular antibodies, B cells or other polypeptides for the VH3-15 idiotope. Although the VH3- 15 idiotope is often found in the early stages of B cell ontogeny,' it is rarely found in the mature immune repertoire as compared, for example, to VH3-26. Presently, there is only one reported antibody that possesses the VH3-15 idiotope and has known antigenic specificity: ant i-Haemophil us influenzae type b capsular polysaccharide antibody. These tools will allow investigators to associate the use of particular VH segments with particular pathogenic immune responses as compared to healthy immune responses and can serve as markers for locating and isolating antigen. The present invention also provides powerful tools for the isolation and purification of VH3-15 polypeptides.

Traditionally, VH gene segments that cross-hybridize by Southern filter hybridization under standard conditions (0.1X saturated sodium citrate, 0.1% sodium dodecyl sulfate, 65 degrees celsius) are considered members of the same VH gene family, whereas those VH gene segments that do not cross hybridize under these conditions are members of a distinct VH gene family. In practical terms this means approximately 80% nucleotide sequence homology places two genes within the same family and less than 70% nucleotide sequence homology classifies molecules as belonging to separate VH families. The VH3 gene family is presently considered to have the largest membership. Recently, VH gene families have been subdivided into sub-families based upon homology to a germline sequence within a VH gene family. VH germline sequences have been mapped and the nomenclature for the sub-families reflects the locus of the germline gene segment. Thus, VH3-15 refers to the fifteenth VH segment from the 3' end of the human Ig heavy chain locus. See, Matsuda, et. al., Nature Genetics, 3:88-94 (1993) incorporated herein by reference. The nucleic acid sequence of the VH3-15 gene segment is available on Genbank. The VΗ3-15 gene segment is also known as M26, 20pl, DP-38 and 9-1.

The term "VH3-15 nucleic acid sequence" as used herein refers the nucleic acid sequence of a 'member of the VH3-15 sub-family. A nucleic acid sequence is a member of the VH3-15 sub-family if it has at least 92% nucleotide sequence homology with the VH3-15 germline gene segment. SEQ ID NO. 1, the nucleic acid sequence of 9-1 as reported in Pascual, et al, Adv. Immun. , 49:1-74 (1991) incorporated herein by reference, is provided as a representative example of a VH3-15 nucleic acid sequence. The nucleic acid sequence encoding LJ86 (SEQ ID NO. 4) is available on Genbank Accession No. M82929 and is also representative of a VH3-15 nucleic acid sequence.

Additional VH3-15 nucleic acid sequences can be found in the published literature. See for example, Braun, et al., J. Clin. Invest., 89:1395-1402 (1992) , incorporated herein by reference. A "VH3-15 polypeptide" refers to a polypeptide sequence that encodes a member of the VH3-15 sub-family. A polypeptide sequence is a member of the VH3-15 sub¬ family if it is encoded by a VH3-15 nucleic acid sequence or if its CDRl and CDR2 regions share at least 90% amino acid sequence homology with the CDRl and CDR2 regions of SEQ ID NO. 3. SEQ ID NO. 2, 3 and 4 are representative of VH3-15 polypeptides.

Thus, VH3-15 nucleic acids and VH3-15 polypeptides may be encoded as part of larger sequences. For example, a VH3-15 polypeptide may take the form of an antibody

("VH3-15 antibody"), an antibody fragment ("VH3-15 (Fab') " ^or "VH3-15 Fab") or the like. LSF2 anti- Haemophilus influenzae type b capsular polysaccharide antibody is another polypeptide representative of a VH3- 15 sub-family.

In accordance with the present invention there is provided a purified antibody of predominately IgA, IgG₁₍ and IgG₄ isotype, characterized in vivo as a serum antibody in humans diagnosed with IBD and/or infection with Campylobacter jejuni . This antibody is also characterized by immunoreactivity with proteinaceous antigen on the surface of erythrocyte cell membrane and Campylobacter jejuni cell membrane, referred to collectively as cell surface antigen. More specifically, this antibody is capable of binding a surface-exposed erythrocyte cell membrane protein having a molecular weight of about 22,000 daltons on 12.6% SDS-PAGE, a surface-exposed erythrocyte cell membrane protein having a molecular weight of about 28,000 daltons on 12.6% SDS- PAGE, a surface-exposed Campylobacter jejuni cell membrane protein having a molecular weight of about 29,000 daltons on 10% SDS-PAGE, a surface-exposed Campylobacter jejuni cell membrane protein having a molecular weight of about 50,000 daltons on 10% SDS-PAGE and a surface-exposed Campylobacter jejuni cell membrane protein having a molecular weight of about 63,000 daltons on 10% SDS-PAGE. This antibody is further characterized as encoding a VH3-15 polypeptide and binds the monoclonal antibody produced by the hybridoma having ATCC Accession No. HB11720. For convenience, this antibody will be referred to herein as VH3-15 autoantibody. As used herein, the term "purified" means that the molecule is substantially free of contaminants normally associated with a native or natural environment. For example, VH3-15 autoantibody can be purified of other constituents of serum or of cell surface antigen by a number of methods, preferably those which employ the anti-VH3-15 idiotypic antibody material of the present invention. Methods of purifying antibodies are well known in the art and include precipitation, affinity chromatography, solid or soluble phase immunoassays, and the like. These and other well known methods are described in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory 1988) incorporated herein by reference.

In accordance with the present invention, there is also provided, antibody material having immunoreactivity with a variable heavy chain segment of a VH3-15 polypeptide, for example a variable heavy chain segment of VH3-15 autoantibody, LSF2 and the like. Such antibody material may be referred to as "anti-VH3-15 idiotypic" antibody material and includes, for example, antibody material and monoclonal antibody molecules produced by hybridomas specifically identified in Example I as BK1, BK2, BK3, BK4, BK5, and BK7, as well as antibody material that bind the same idiotope as the monoclonal antibody molecules produced by these hybridomas.

The term "antibody" or "antibody material" in its various grammatical forms is used herein as a collective noun that refers to an antibody molecule and immunologically active portions of an antibody molecule, i.e., molecules that contain an idiotope.

The term "antibody molecule" in its various grammatical forms as used herein refers to an intact immunoglobulin molecule.

An "idiotope" in its various grammatical forms is used herein to refer to any portion of the variable region (heavy and light chain variable and hypervariable regions) of an antibody molecule that is capable of binding an antibody or an antigen. An "epitope" in its various grammatical forms is used herein refers to any portion of an antigen that is capable of binding an antibody. The word "epitope" will be reserved for use herein only to refer to antigenic determinants on non- immunoglobulin antigens.

Exemplary antibody material useful in the compositions and methods of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain an idiotope, including those portions known in the art as Fab, Fab' , F(ab')₂ and F(v) . Fab and (Fab')₂ portions of antibodies are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibodies by methods that are well known in the art. See, for example, U.S. Patent No. 4,342,566 to Theofilopolous and Dixon incorporated herein by reference. Fab' antibody portions are also well known and are produced from F(ab')₂ portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules are preferred, and are utilized as illustrative herein. Immunoreactivity of antibody material with VH3-15 polypeptides can be measured by a variety of immunological assays known in the art, as described for example in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory 1988) incorporated herein by reference. Exemplary immunoreactivity of an anti-VH3-15 idiotypic antibody with a VH3-15 polypeptide is described in Example II.

Anti-VH3-15 idiotypic antibodies of either monoclonal or polyclonal form can be produced using techniques presently known in the art. For example, polyclonal and monoclonal antibodies can be produced as described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory 1988) , which is incorporated herein by reference. Altered antibodies, such as chimeric, humanized, CDR-grafted or bifunctional antibodies can also be produced by methods well known to those skilled in the art. Such antibodies can also be produced by hybridoma, chemical or recombinant methodology described, for example in Ausubel et al., Current Protocols in Molecular Biology (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. 1993) , also incorporated herein by reference. Exemplary methods of making and isolating monoclonal anti-VH3-15 idiotypic antibodies are provided in Examples I and II. The phrase "monoclonal antibody" in its various grammatical forms refers to a population of antibody molecules that contain only one species of idiotope capable of immunoreacting with a particular epitope on an antigen or idiotope on an antibody. A monoclonal antibody typically displays a single binding affinity for an epitope or idiotope with which it immunoreacts; however, a monoclonal antibody may be a molecule having a plurality of idiotopes, each immunospecific for a different epitope or idiotope, e.g., a bispecific monoclonal antibody.

Monoclonal antibodies are typically composed of antibodies produced by clones of a single cell called a hybridoma that secretes (produces) but one kind of antibody molecule. In accordance with the present invention hybridomas capable of producing antibody material having specific immunoreactivity with the variable heavy chain segment of a VH3-15 polypeptide is provided. Such hybridomas include, for example, the hybridoma line producing anti-VH3-15 idiotypic monoclonal antibody which was deposited with American Type Culture Collection ("ATCC"), Rockville Maryland, on September 26, 1994 and assigned ATCC Accession No. HB11720 (referred to herein as BK2) . Additional hybridomas of the present invention, include, BK1, BK3, BK4, BK5, and BK7, specifically described in Example I. One of skill in the art will recognize that the hybridomas disclosed herein can be used to produce other immortal cell lines that produce antibody material of the present invention.

A hybridoma cell is formed by fusing an antibody-producing cell and a myeloma or other self- perpetuating cell line. The preparation of such hybridomas was first described by Kohler and Milstein, Nature, 256:495-497 (1975), which description is incorporated by reference. Polypeptide-induced hybridoma technology is also described by Niman et al. , Proc. Natl . Sci. , U.S.A. , 80:4949-4953 (1983), which description is also incorporated herein by reference.

To obtain an antibody-producing cell for fusion with an immortalized cell, a mammal is inoculated with an immunogen. The word "immunogen" in its various grammatical forms is used herein to describe a composition containing a VH3-15 polypeptide as an active ingredient used for the preparation of the antibodies against VH3-15 polypeptides. When a polypeptide is used in an immunogen to induce antibodies, it is to be understood that the polypeptide can be used in various embodiments, e.g., alone or linked to a carrier as a conjugate, or as a polypeptide polymer or as a fusion protein for ease in purification. For a VH3-15 polypeptide that contains fewer than about 35 amino acid residues, the peptide may be bound to a carrier, for the purpose of inducing the production of antibodies.

The amount of VH3-15 polypeptide immunogen used to inoculate the mammal should be sufficient to induce an immune response to the immunizing polypeptide. This amount depends, among other things, on the species of animal inoculated, the body weight of the animal and the chosen inoculation regimen as is well known in the art. Inocula typically contain about 10 micrograms of immunogen per inoculation for mice and may contain up to about 500 milligrams of immunogen per inoculation for larger mammals.

The spleen cells of the mammal immunized with a VH3- 15 polypeptide are then harvested and can be fused with myeloma cells using polyethylene glycol (PEG) 1500. Fused hybrids are selected by their sensitivity to HAT. Hybridomas producing an anti-VH3-15 idiotypic monoclonal antibody can be identified by screening hybridoma supernates for the presence of antibody molecules that immunoreact with VH3-15 polypeptide. Such screening methods include for example, radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA) .

A monoclonal antibody of the present invention can also be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes anti-VH3-15 idiotypic antibody molecules. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well known techniques.

Other methods of producing a monoclonal antibody, a hybridoma cell, or a hybridoma cell culture are also well known. See, for example, the method of isolating monoclonal antibodies from an immunological repertoire as described by Sastry et al. , Proc. Natl. Acad. Sci.. 86:5728-5732 (1989) ; and Huse et al. , Science. 246:1275-1281 (1981), both of which are incorporated herein by reference.

Media useful for the preparation of these compositions are well known in the art and commercially available and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. , 8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mm glutamine, and 20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

In another aspect of the present invention, there is provided purified cell surface antigen that immunoreacts with VH3-15 autoantibody. These antigens are characterized in vivo as proteinaceous material exposed on the surface of human erythrocyte cells or Campylobacter jejuni cells. Thus, in one embodiment there is provided purified cell surface antigen characterized in vivo as proteinaceous material bound to the cell membrane of human erythrocytes. These antigens are further characterized as having a molecular weight of about 22,000 daltons or about 28,000 daltons on 12.6% SDS-PAGE and immunoreactivity with VH3-15 autoantibody.

In an alternative embodiment of the invention, there is provided purified cell surface antigen characterized in vivo as being proteinaceous material bound to the cell membrane of Campylobacter jejuni . These cell surface antigens are further characterized as having a molecular weight of about 29,000 daltons, 50,000 daltons or 63,000 daltons on 10% SDS-PAGE and immunoreactivity with VH3-15 autoantibody.

These cell surface antigens can be purified of cell membrane components, for example, by a number of methods well known in the art including, for example, precipitation, affinity chromatography, solid or soluble phase immunoassays, and the like. Alternatively, purified cell surface antigen of the present invention, and immunoreactive fragments thereof, can be obtained by well-known recombinant methods as described, for example, in Ausubel et al., Current Protocols in Molecular Biology (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. 1993) , also incorporated herein by reference. An example of how these purified cell surface antigens and biologically active fragments can be obtained through recombinant means is to produce human reticulocyte cDNA library in the form of a surface expression vector such as pcDNA-1 (Invitrogen, San Diego, California) cloned following the manufacture's directions. The library could then be expressed in COS cells and screened by flow cytometry or autoradiography using labeled VH3-15 autoantibody or anti-VH3-15 idiotypic antibody, such as for example, the monoclonal antibody produced by hybridoma having ATCC Accession No. HB11720. See, H. Lin, et al. , Cell 68:775-785 (1992) , incorporated herein by reference. Cell surface antigen can also be purified and isolated through recombinant means by producing a Campylobacter jejuni cDNA library using the lambda gtll vector (Stratagene, LaJolla, CA) . Phage produced from this library could then be induced to express the gene insert and nitrocellulose filter contact replicas of plated phage plaques could be screened using labeld VH3- 15 autoantibody or anti-VH3-15 idiotypic antibody. These cell surface antigens and immunoreactive fragments thereof can also be produced by chemical synthesis. Synthetic proteins can be produced using Applied Biosystems, Inc. Model 430A or 431A automatic polypeptide synthesizer and chemistry provided by the manufacturer.

The present invention also encompasses nucleic acid molecules encoding any one of the following: anti-VH3-15 idiotypic antibody material, VH3-15 autoantibody, and cell surface antigen or immunoreactive fragments thereof. This invention also encompasses nucleic acid molecules characterized by conservative changes in coding regions that do not alter the phenotype of the polypeptide produced therefrom when compared to the nucleic acid molecule described hereinabove. This invention further encompasses nucleic acid probes of at least 30 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid encoding any one of the following: anti-VH3-15 idiotypic antibody material, VH3-15 autoantibody material, cell surface antigen, and immunoreactive fragments of cell surface antigen.

As used herein, "specifically hybridizing" means the ability of a nucleic acid to recognize a sequence of nucleic acids complementary to its own and to form double-helical segments through hydrogen bonding between the complementary base pairs. Nucleic acid probe technology is well-known to those skilled in the art, who readily appreciate that such probes may vary greatly in length, and accordingly, can hybridize under both non- stringent and stringent conditions to the nucleic acid molecules of the subject invention. One example of stringent hybridization includes incubation of the nucleic acid(s) with the probe in a solution comprising 50% formamide, 5x SSPE (NaCI, NaH₂P0₄, EDTA) , lx Denhardt's, 0.1% SDS and single stranded salmon sperm DNA at 42°C. Non-stringent hybridization is performed similarly, using a lower concentration, i.e., 35%, of formamide. Alternatively, the utilization of formamide can be obviated, by modifications well known to a skilled artisan, for example, increasing the temperature at which the hybridization is performed. A person of skill in the art is familiar with the various manipulations which can be applied to hybridization conditions in order to obtain optimal results. As used herein, the term "nucleic acid" encompasses RNA as well as single and double-stranded DNA and cDNA. In addition, as used herein, the term "polypeptide" encompasses any naturally occurring allelic variant thereof as well as man-made recombinant forms. This invention provides an isolated nucleic acid molecule encoding an one of the following: anti-VH3-15 idiotypic antibody material, VH3-15 autoantibody, cell surface antigen and immunoreactive fragments of cell surface antigen. As used herein, the term "isolated nucleic acid molecule" means a nucleic acid molecule that is in a form that does not occur in nature. One means of isolating nucleic acid molecules of the present invention is to probe a mammalian cDNA expression library with a natural or artificially designed antibody to the polypeptide encoded by the nucleic acid molecule sought, using methods well known in the art (see, for example, Ausubel et al. , supra 1993) . For example, nucleic acid encoding cell surface antigen can be isolated using human reticulocyte cDNA library and labeled VH3-15 autoantibody or serum and labeled anti-VH3-15 idiotypic antibody material. DNA and cDNA molecules which encode anti-VH3- 15 idiotypic antibody material, VH3-15 autoantibody, cell surface antigen or immunoreactive fragments of cell surface antigen can be used to obtain complementary genomic DNA, cDNA or RNA from human or other mammalian sources.

The invention further provides the above-described isolated nucleic acid molecules operatively linked to a promoter, as well as other regulatory sequences. As used herein, the term "operatively linked" means positioned in such a manner that the promoter will direct the transcription of RNA from the nucleic acid molecule. Examples of such promoters are SP6, T4 and T7.

Vectors which contain both a promoter and a cloning site into which an inserted piece of DNA is operative linked to that promoter are well known in the art.

Preferably, these vectors are capable of transcribing RNA or cDNA in vi tro or in vivo. Examples of such vectors are the pGEM series (Promega Biotech, Madison, WI) and pcDNA-1 (Invitrogen, San Diego, CA) cloned following the manufacture's directions.

This invention also provides a vector comprising an isolated nucleic acid molecule such as DNA, cDNA or RNA encoding any one of the following: an anti-VH3-15 idiotypic antibody material, VH3-15 autoantibody material, cell surface antigen and immunoreactive fragments of cell surface antigen. Examples of additional vectors useful herein are viruses, such as bacteriophages, baculoviruses and retroviruses, cosmids, plasmids, and the like. Nucleic acid molecules are inserted into vector genomes by methods well known in the art. For example, insert and vector DNA can both be exposed to a restriction enzyme to create complementary ends on both molecules that base pair with each other and which are then joined together with a ligase. Alternatively, synthetic nucleic acid linkers that correspond to a restriction site in the vector DNA, can be ligated to the insert DNA which is then digested with a restriction enzyme that recognizes a particular nucleotide sequence. Additionally, an oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following: a selectable marker gene, such as neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vi tro transcription of sense and antisense RNA. Other means are available and can readily be accessed by those of skill in the art.

Also provided are expression vectors comprising a DNA molecule encoding an anti-VH3-15 idiotypic antibody material, VH3-15 autoantibody material, or cell surface antigen or immunoreactive fragments thereof adapted for expression in a bacterial cell, a yeast cell, a mammalian cell and other animal cells. The vectors additionally comprise the regulatory elements necessary for expression of the DNA in the bacterial, yeast, mammalian or animal cells so located relative to the DNA encoding the antibody material as to permit expression thereof. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation, the Shine-Dalgarno sequence and the start codon AUG (Ausubel et al. , supra 1993) . Similarly a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors can be obtained commercially or assembled by the sequences described in methods well known in the art, for example, the methods described above for constructing vectors in general. Expression vectors are useful to produce cells that express the polypeptide.

This invention provides a mammalian cell containing cDNA encoding a mammalian anti-VH3-15 antibody material, VH3-15 autoantibody material, or cell surface antigen or immunoreactive fragments thereof. An example is a mammalian cell comprising a plasmid adapted for expression in a mammalian cell. The plasmid contains cDNA encoding antibody material and the regulatory elements necessary for expression of the polypeptide. Various mammalian cells may be utilized as hosts, including for example, mouse fibroblast cell NIH3T3, CHO cells, HeLa cells, Ltk- cells, etc. Expression plasmids such as those described above can be used to transfect mammalian cells by methods well known in the art, for example, calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, lipofection, and the like.

The anti-VH3-15 idiotypic antibody materials VH3-15 autoantibody and cell surface antigens of the present invention can be used in the methods of the present invention to detect the presence, absence or amount of VH3-15 polypeptide in a sample or for the immunoaffinity or affinity chromatographic purification of VH3-15 polypeptides from serum or from other biological materials. More specifically, the various immunoassay methods of the present invention employ the use of anti-VH3-15 idiotypic antibody materials and or cell surface antigen as immunochemical reagents to form an immune complex with any VH3-15 polypeptide which might be present in a sample. In this manner the presence, absence or amount of VH3-15 polypeptide in a sample is easily detected by detecting immune complex. Alternatively, the formation of immune complex can be exploited as a technique for purification of VH3-15 polypeptides. Similarly, VH3-15 autoantibody can be used as an immunochemical reagent to form immune complex with cell surface antigen or anti-VH3-15 idiotypic antibody material that might be present in a sample. The presence, absence or amount of cell surface antigen or anti-VH3-15 idiotypic antibody material can easily be detected by detecting immune complex. The methods of present invention can also include comparison of the resulting complex to a control to assure accuracy. For example, a composition containing VH3-15 autoantibody can be used as a positive control to confirm or quantify the results of the method.

Of course, one of skill in the art will appreciate that there are various heterogenous and homogenous protocols, either competitive or noncompetitive, solution-phase or solid-phase, which can be employed in performing an assay method of this invention. Thus, while exemplary assay methods are described herein, the invention is not so limited.

In one embodiment of the present invention there is provided a method of detecting the presence, absence or amount of a VH3-15 polypeptide, for example VH3-15 autoantibody, in a sample comprising (a) contacting a sample with anti-VH3-15 idiotypic antibody material under conditions suitable to form an immune complex comprising anti-VH3-15 idiotypic antibody material and VH3-15 polypeptide, and (b) detecting the presence or amount of the immune complex formed. Preferably, anti-VH3-15 idiotypic antibody material is labeled with a detectable marker and the immune complex is detected by detecting the presence, absence or amount of complexed anti-VH3-15 idiotypic antibody material. The presence of VH3-15 polypeptide, for example VH3-15 autoantibody, may thus be determined on a qualitative or quantitative basis depending upon the manner of detection employed and the type of marker used, if any, to label the anti-VH3-15 idiotypic antibody material.

The terms "immune complex" and "immunocomplex" as used herein refers to the product of a specific binding reaction such as for example that between an eptiope and an antigen binding site, between idiotope and an anti- idiotypic antibody, and the like.

In yet another embodiment of the invention, the presence, absence or amount of VH3-15 autoantibody in a sample can be detected by (a) contacting a sample with anti-VH3-15 idiotypic antibody material and cell surface antigen under conditions suitable to form an immune complex comprising cell surface antigen, VH3-15 autoantibody and anti-VH3-15 idiotypic antibody material, and (b) detecting the presence, absence or amount of immune complex. The anti-VH3-15 idiotypic antibody material and the cell surface antigen can be sequentially contacted with the sample or simultaneously contacted with the sample.

Preferably, anti-VH3-15 idiotypic antibody material and/or cell surface antigen is labeled with a detectable marker and the immune complex is detected by detecting the presence, absence or amount of complexed anti-VH3-15 idiotypic antibody material and/or cell surface antigen. The presence of VH3-15 autoantibody can also be determined on a qualitative or quantitative basis depending upon the manner of detection employed and the type of marker used, if any, to label the anti-VH3-15 idiotypic antibody material and/or cell surface antigen. When using cell surface antigen in the methods of the present invention, it can be bound to human erythrocyte cell membrane or Campylobacter jejuni cell membrane and the cells, or more preferably the membranes, can be immobilized on a substrate. Preferably, the erythrocyte cell membrane is pre-treated with bromelase to remove extraneous antigens. In still another embodiment of the invention, the presence, absence or amount of VH3-15 autoantibody in a sample can be detected by (a) contacting a sample with cell surface antigen under conditions suitable to form an immune complex comprising cell surface antigen and VH3-15 autoantibody and (b) detecting the presence, absence or amount of immune complex. Of course, cell surface antigen can be labeled with a detectable marker and the immune complex can then be detected by detecting the presence absence or amount of complexed cell surface antigen. The present invention also provides methods of detecting IBD and infection by Campylobacter jejuni . Infection by Campylobacter jejuni can cause Campylobacter jejuni enterocolitis. In a presently prefered embodiment, UC, CD and infection by Campylobacter jejuni can be detected by contacting cell surface antigen and detectable anti-VH3-15 idiotypic antibody material with a biological sample from a patient under conditions suitable to form a complex of cell surface antigen, VH3- 15 autoantibody and detectable anti-VH3-idiotypic antibody material, and then assaying for the amount of VH3-15 autoantibody by detecting the amount of complexed anti-VH3-15 idiotypic antibody material. The presence of complexed anti-VH3-15 idiotypic antibody material in excess of a control indicates one of these disease states.

A control for purposes of detecting IBD or infection by Campylobacter jejuni represents the average amount of VH3-15 autoantibody detected in a sample from a patient without UC, CD and infection by Campylobacter jejuni

("normals") . Most preferably, that patient is healthy. To increase the accuracy of identifying IBD and Campylobacter jejuni infection in human, the control should represent the average amount of VH3-15 autoantibody in the same type of sample from a normal human which has been subjected to the same test procedures and parameters as the sample of the subject being assayed. For example, when the amount of VH3-15 autoantibody in a sample of human blood serum is detected by flow ctyometry as described in Example IV, the control is most preferably defined as the average amount of VH3- 15 autoantibody detected by flow cytometry in samples of human blood serum from normals. Thus, as that detection method is described in Example IV, the control is 2.5 and IBD or infection by Campylobacter jejuni is detected by the presence of an amount of VH3-15 autoantibody in a human serum sample that exceeds 2.5. As a general guideline, the greater the amount VH3- 15 autoantibody by which the test sample exceeds the control, the greater the assurance that the method accurately detects IBD or infection by Campylobacter jejuni . Thus, one can increase the assurance that the method accurately identifies IBD or infection by Campylobacter jejuni by requiring that the amount of VH3- 15 autoantibody exceeds the control by at least a given amount. For example, when the amount of VH3-15 autoantibody in a sample of human blood serum is detected by flow cytometry as described in Example IV and the control is defined as 2.5, the sensativity of the method approaches 100% for CD, 90% for UC and 70% for infection by Campylobacter jejuni when a sample is considered positive only if it exceeds the control by at least 0.75. Likewise, when an ELISA format is used to detect the amount of VH3-15 autoantibody in a sample, as described for example in Example VI and VII, absorbance for a panel of sera from normal subjects is determined. From this data, the mean (e.g., control) and preferably, the

.standard error of the mean ("SEM"), is calculated. If the amount of VH3-15 autoantibody in a test sample, defined by absorbance in the ELISA format, exceeds the control by one SEM for the control, then the sensativity for correctly detecting IBD or infection by Campylobacter jejuni reaches 85%.

The control, as provided in the kits of the present invention for example, may take on many different physical forms. For example, a control can simply be a written expression of the average amount of VH3-15 autoantibody detected by a particular method in samples from normals or alternatively a statement that an amount in excess of a given amount detected in normals indicates a disease state. Alternatively, the control can be photograph of the results of the method performed on a normal sample using a visually detectable marker which can be observed in a photograph. Preferably, the control is the same type of sample as the test sample that has been taken from a normal patient known to contain an average amount of VH3-15 autoantibody for normals and the control sample is subjected to the same procedure as the test sample.

A sample can be obtained from any biological fluid or tissue containing or suspected of containing antibodies, for example, whole blood, plasma, biopsies of the colon, and the like, preferably serum. The methods of the present invention are typically performed at or below room temperature at about physiological pH. Because the methods involve the use of proteins, substantially higher temperatures acidity or alkalinity which would substantially modify the tertiary and quaternary structures of the proteins should be avoided. Accordingly, conditions suitable for performing the methods of the present invention generally range from about 1°C to about 37°C, at about physiological pH. The time for performing the methods, of course, will decrease in relation to the increase in temperature at which the methods are performed.

Thus, for example, "conditions suitable to form" an immune complex of cell surface antigen and VH3-15 autoantibody comprise contacting cell surface antigen with human blood serum sample at about physiological pH at a temperature in the range of about 4°C to about 37°C for about 5 minutes to about 120 minutes, or preferably at a temperature in the range of about 20°C to about 37°C for about 5 minutes to about 60 minutes, and even more preferably at about room temperature for about 25 minutes to about 35 minutes. "Conditions suitable to form" an immune complex comprising cell surface antigen, VH3-15 autoantibody and anti-VH3-15 idiotypic antibody material further comprise, for examle, then contacting the cell surface antigen/VH3-15 autoantibody immune complex with anti-VH3-15 idiotypic antibody material at about physiological pH, at a temperature in the range of about 1°C to about 15°C for about 5 minutes to about 120 minutes, or more preferably at a temperature in the range of about 5°C to about 10°C for about 10 minutes to about 60 minutes, or even more preferably by contacting them on ice for about 30 minutes

In a presently preferred embodiment, UC, CD or infection by Campylobacter jejuni in a human may be detected by contacting bromelase-treated human type O erythrocyte cell membrane with human blood serum at about room temperature for about 25 minutes to about 35 minutes at about physiological pH and then contacting the cell membrane and serum with detectably labeled monoclonal antibody produced by hybridoma having ATCC Accession No. HB11720, on ice at about physiological pH for about 30 minutes.

In still another embodiment of the invention, any immune complex formed in the methods of the present invention is separated from any uncomplexed anti-VH3-15 idiotypic antibody material and/or from the remaining sample and VH3-15 polypeptide prior to assaying for the presence or amount of VH3-15 polypeptide-containing immune complex.

In yet another embodiment of the present invention, there is provided a method of purifying or isolating VH3- 15 polypeptide comprising contacting a sample containing VH3-15 polypeptide with anti-VH3-15 idiotypic antibody material under conditions suitably to form an immune complex comprising VH3-15 autoantibody and anti-VH3-15 idiotypic antibody material, and then separating any unbound sample from any immune complex that formed.

In accordance with the compositions and methods of the present invention, anti-VH3-15 idiotypic antibody material can be directly or indirectly labeled with a detectable marker to create detectable anti-VH3-15 idiotypic antibody material. In this manner, the presence of a VH3-15 polypeptide, for example VH3-15 autoantibody, which has complexed with anti-VH3-15 idiotypic antibody material can be detected by detecting enzymatic conversion, radioactivity, fluorescence, color and the like. Similarly, VH3-15 autoantibody, cell surface antigen, nucleic acid molecules and probes of the present invention can be labeled with a detectable marker.

Methods of labeling the compositions of the present invention (antibodies, nucleic acids and the like) are well known in the art and contemplated as within the scope of the present invention. For instance, antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. See, for example, Galfre et al . , Meth. Enzvmol.. 73:3-46 (1981) incorporated herein by reference. The techniques of protein conjugation or coupling through activated functional groups are applicable (See, for example, Aurameas et al. , Scand. J. Immunol. , Vol. 8, Suppl. 7:7-23 (1978), Rodwell et al. , Biotech.. 3:889-894 (1984), and U.S. Patent No. 4,493,795 all of which are incorporated herein by reference.) and the specific use of biotin/avidin for labeling the compositions of the present invention is exemplified.

As used herein, the word "marker" in its various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any marker can be linked to or incorporated in an expressed protein, polypeptide fragment, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention, or used separately. These atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins, methods, and/or systems.

The detectable marker can be a fluorescent labeling agent that chemically binds to antibodies of antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC) , fluorescein isothiocyante (FITC) , 5-dimethylamine-l-naphthalenesulfonyl chloride (DANSC) , tetramethylrhodamine isothiocyanate (TRITC) , lissamine, rhodamine 8200 sulphonyl chloride (RB-200-SC) , phycoerythrin and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis et al. , eds. , John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incorporated herein by reference.

Radioactive elements are also useful detectable markers. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions.

Elements which themselves emit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁶I, ¹³¹I and ⁵¹Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is ¹²⁵I . Another group of useful labeling means are those elements such as ¹¹C, ¹⁸F, ¹⁵0 and ¹³N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such as ¹¹¹indium or ³H. In one embodiment, the detectable marker is an enzyme, such as horseradish peroxidase ("HRP") , glucose oxidase, and the like. In such cases where the detectable marker is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the fact that an immune complex has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine, o- phenylenediamine dihyrochloride ("OPD") and the like. An additional reagent useful with glucose oxidase is 2, 2' -azino-di- (3-ethyl-benzthiazoline-G-sulfonic acid) . Depending on the nature of the label or catalytic signal producing system used, a signal can be detected by irradiating the complexed test sample with light and observing the level of fluorescence; by contacting the complexed sample with a substrate which can be catalytically converted by the label to produce a dye, fluorescence or chemiluminescence, in which the formation of dye can be observed visually or in a spectrophotometer; fluorescence can be observed visually or in a fluorometer; or, in the case of chemiluminescence or a radioactive label, by employing a radiation counter such as a gamma counter or gamma emitting markers such as iodine-125. For detection of enzyme-catalyzed markers when the presently preferred combination of HRP is used as the enzyme and o-phenylenediamine dihydrochloride as the substrate, a quantitative analysis of complex can be made using a spectrophotometer, for example a EMAX

Microplate Reader (available from Molecular Devices, Menlo Park, California) , at 492 nm in accordance with the manufacturer's instructions.

Specific binding agent are also useful as detectable markers. A "specific binding agent" is a molecular entity capable of selectively binding anti-VH3-15 idiotypic antibody material, VH3-15 polypeptides or the nucleic acids of the present invention or a complex containing these, but which is not itself a polypeptide or antibody molecule composition of the present invention. Exemplary specific binding agents are secondary antibody molecules (e.g., anti-Ig antibodies), complement proteins or fragments thereof, and the like which may themselves be labeled with a detectable marker. If one or more specific binding agents, in the form of secondary antibody molecules are used, each secondary antibody molecule is preferably species-specific for the antibody or antigen it binds. Accordingly, in a preferred embodiment of the present invention, anti-VH3- 15 idiotypic antibody material are detectably labeled by contacting them with specific binding agent, preferably labeled, specie-specific antibody molecule under conditions suitable to form a complex of anti-VH3-15 idiotypic antibody material and the specific binding agent. For example, when the monoclonal antibody produced by the hybridoma having ATCC Accession No. HB11720 is employed as the anti-VH3-15 idiotypic antibody material, labeled goat anti-mouse IgG can be used as a specific binding agent.

In preferred embodiments of the present invention, anti-VH3-15 idiotypic antibody material, cell surface antigen or VH3-15 autoantibody is immobilized on a solid matrix. The solid matrix can be any support useful in immunometric assays. The matrix can be made from natural or synthetic material which is insoluble in water and can be rigid or non-rigid. However, the matrix should not significantly affect the desired activity of the antigen antibody material. Preferred matrices include glass slides, test wells made from polyethylene, polystyrene, nylon, nitrocellulose, glass and the like. Also useful are test tubes, filter paper, filtering devices such as glass membranes, beads, and particulate materials such as agarose, cross-linked dextran and other polysaccharides, and the like.

The separation steps for the various assay formats described herein, including, for example, removing any unbound sample from the immune complex or separating complexed anti-VH3-15 idiotypic antibody material from uncomplexed anti-VH3-15 idiotypic antibody material, can be performed by methods known in the art. When appropriate, a simple washing with a suitable buffer followed by filtration or aspiration is sufficient. If anti-VH3-15 idiotypic antibody material, cell surface antigen or VH3-15 autoantibody is immobilized on a particulate support, as in the case of microparticles for example, it may be desirable to centrifuge the particulate material, followed by removal of wash liquid. If the anti-VH3-15 idiotypic antibody material, cell surface antigen or VH3-15 autoantibody is immobilized on membranes or filters, applying a vacuum or liquid absorbing member to the opposite side of the membrane or filter allows one to draw the wash liquid through the membrane or filter. The present invention also provides kits for detecting the presence, absence or amount of VH3-15 polypeptide in a sample. A suitable kit includes, in an amount sufficient for at least one assay, anti-VH3-15 idiotypic antibody material, preferably the monoclonal antibody produced by the hybridoma having ATCC Accession No. HB11720 as a separately packaged reagent. Preferably the kits also include a detectable marker and a VH3-15 polypeptide useful as a positive control. Instructions for use of the packaged reagent are also typically included.

Kits for detecting the presence of VH3-15 autoantibody are also provided in the present invention. A suitable kit includes, in an amount sufficient for at least one assay, anti-VH3-15 idiotypic antibody material and cell surface antigen, preferably as a separately packaged reagents . Cell surface antigen included in these kits can be bound to the cell membrane of Campylobacter jejuni cells or erythrocytes. If erythrocyte cell membrane is used, preferably it is pre- treated with bromelase. The kits may also include a detectable marker and a control, preferably in the form of a normal serum sample. Instructions for use of the packaged reagents are also typically included.

In yet another embodiment of the present invention, kits for detecting the presence, absence or amount of nucleic acid encoding anti-VH3-15 idiotypic antibody material in a sample are provided. A suitable kit includes, in an amount sufficient for at least one assay, nucleic acid probe for nucleic acid encoding anti-VH3-15 idiotypic antibody material as a separately packaged reagent. Preferably the kits also include a detectable marker and a nucleic acid molecule encoding anti-VH3-15 idiotypic antibody material useful as a positive control .

Instructions for use of the packaged reagent are also typically included.

Kits for detecting the presence, absence or amount of nucleic acid encoding cell surface antigen in a sample are provide. A suitable kit includes, in an amount sufficient for at least one assay, nucleic acid probe for nucleic acid encoding cell surface antigen as a separately packaged reagent . Preferably the kits also include a detectable marker and a nucleic acid molecule encoding cell surface antigen useful as a positive control. Instructions for use of the packaged reagent are also typically included.

Also provided in the present invention are kits for detecting the presence, absence or amount of nucleic acid encoding VH3-15 autoantibody in a sample. A suitable kit includes, in an amount sufficient for at least one assay, nucleic acid probe for nucleic acid encoding VH3-15 autoantibody as a separately packaged reagent. Preferably the kits also include a detectable marker and a nucleic acid molecule encoding VH3-15 autoantibody useful as a positive control. Instructions for use of the packaged reagent are also typically included.

As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil and the like capable of holding within fixed limits antibody material, polypeptide, nucleic acid probe, or nucleic acid sequence of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated protein or polypeptide fragment, or it can be a microtiter plate well to which microgram quantities of a contemplated protein or polypeptide fragment have been operatively affixed, i.e., linked so as to be capable of being immunologically bound by an antibody.

"Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like. The kits can also include, preferably as a separately package reagent, a specific binding agent as defined above.

The kits can be used in an "ELISA" format. "ELISA" refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen or antibody present in a sample. A description of the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D.P. Sites et al . , published by Lange Medical Publications of Los Altos, CA in 1982 and in U.S. Patents No. 3,654,090, No. 3,850,752; and No. 4,016,043, which are all incorporated herein by reference. Thus, anti-VH3-15 idiotypic antibody, VH3-15 autoantibody, cell surface antigen or even nucleic acid probe can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems. A reagent is typically affixed to a solid matrix by adsorption from an aqueous medium although other modes of affixation applicable to polypeptides and nucleic acids well known to those skilled in the art can be used.

Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark from Pharmacia Fine Chemicals (Piscataway, NJ) ; agarose; beads of polystyrene about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, IL; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride.

The anti-VH3-15 idiotypic antibodies, labeled specific binding agent, VH3-15 polypeptides, nucleic acid probes, cell surface antigens, VH3-15 autoantibody or

VH3-15 nucleic acid molecules of any kit described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form. Where the indicating means is an enzyme, the enzyme's substrate can also be provided in a separate package of a system. A solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this kit. The invention will now be described in greater detail ]by reference to the following non-limiting examples.

EXAMPLES

Statistical analysis. Two-tailed paired Student's t-test values were calculated using Statview™ on a Macintosh II™ computer.

EXAMPLE I Production of Anti-VH3-15 Hybridomas Monoclonal antibodies specific for VH3-15 polypeptides can be produced using standard hybridoma techniques, i.e., immunizing a mammal with a VH3-15 polypeptide, fusing B lymphocytes from the immunized animal with immortalized cells to produce hybridomas and then screening the hybridomas for antibodies that bind the immunogen. Representative VH3-15 polypeptides are provided in SEQ ID NO. 2 through 4. SEQ ID NO. 1 represents an example of a VH3-15 nucleic acid sequence. The germline VH3-15 nucleic acid sequence is available from Genbank. Any one or all of these polypeptides may be used as an immunogen.

Alternatively, other VH3-15 polypeptides may be created for use as immunogens from the given sequences by substitution, addition or deletion of one or more amino acids. Another alternative is to use other known VH3-15 amino acid sequence as immunogens such as, for example LJ11, LJ67. LJ23 as described in Braun, et al. , J. Clin. Invest. , 89:1395-1402 (1992) .

The nucleic acid sequence encoding LJ86 (Genbank Accession No. M82929) was subcloned into the pGEX bacterial expression system (catalog no. 27-4570-01, Pharmacia Biotech, Inc., Piscataway, NJ) to produce a VH3-15/glutathione-S-transferase fusion protein and the fusion protein purified all in accordance with the manufacturer's instructions. This material was then used to immunize Balb/c mice, from which hybridomas were produced and screened for IgM and IgG antibodies reacting with the VH3-15 fusion protein immunogen. More specifically, primary immunization of Balb/c mice was carried out with 10 micrograms purified VH3-15 immunogen by intrasplenic injection. See, Spitz, et al. J. Immunol. Methods. 70:39-43 (1984) , incorporated herein by reference in its entirety. Four days later, spleen cells were harvested for fusion. Spleenocytes from immunized animals were prepared and fused with NS-1 cell (ATCC Accession No. TIB18) as described, for example in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory 1988) . Hybrids were selected by use of medium containing hypoxanthine, aminopterin and thymidine ("HAT medium") two days after fusion. Surviving hybrids were transferred to micotitre culture plates and medium supernates assessed for specific reactivity with the VH3- 15 immunogen. Positive hybridomas were subcloned twice in microtitre plates and selected by ELISA for VH3-15 reactivity using peroxidase-anti-mouse Ig. (Southern Biotech Assoc, Birmingham, Al. ) Among 600 hybridomas screened, nine produced monoclonal antibodies specific for the VH3-15 immunogen. Six of these nine hybridoma cell lines are stored in liquid nitrogen by Dr. Jonathan Braun in room 4-557 of the McDonald Research Medical Laboratory located at 675 Circle Drive South Los Angeles, California 90024. These six hybridoma cell lines are labeled and identified by the following laboratory names: NS2B9D7E6F5 (and also known as BK1) NS5A4D3F4F9 (and also known as BK2) NS5B7F3F6E1 (and also known as BK3)

NS1H6C1D4B3 (and also known as BK4)

NS1H6B9D6 (and also known as BK5) NS5B7F3F6E1 (and also known as BK7) Of these, a number are IgM and IgG producing hybridomas and BK2 is an IgG,kappa producing hybridoma.

Larger amounts of monoclonal antibody were obtained by inoculating Balb/c mice interperitoneally with lxlO⁷ cells per animal. Ascites protein was purified by ammonium sulphate precipitation and DEAE-Sephacel chromatography, and in some cases biotinylated with NHS- LC-biotin (Pierce, Rockford, IL) following manufacturer's recommendations.

EXAMPLE II

ELISA FOR SCREENING HYBRIDOMAS FOR ANTI-VH3-15 IDIOTYPIC MONOCLONAL ANTIBODY PRODUCTION

Specificity of monoclonal antibodies for VH3-15 polypeptides can be determined by a standard ELISA method described in Berberian, et al. , Science. 261:1588-1591

(1993) , incorporated herein by reference in its entirety. Briefly, ELISA plate wells were coated with various concentrations of either a known VH3-15 antibody or a known non-VH3-15 antibody. For each hybridoma being screened, monoclonal antibodies derived from the hybridoma were added to VH3-15 antibody and non-VH3-15 antibody coated wells. Specificity was detected by enzymatic digestion of substrate using peroxidase anti- mouse Ig.

More specifically, LSF2 is a human ant ± -Hemophil us influenza monoclonal antibodies encoding VH3-15 and is described in Adderson, et al. , J. Immunol .. 147:1667-1674 (1991) . In the following ELISA, LSF2 was used as an VH3- 15 antibody. 477 is a human monoclonal antibody to Waldenstrom' s paraproteins encoding VH3-30 and is described in Axelrod, et al. , Blood. 77:1484-1490 (1991) ) . In the following ELISA, 477 was used as the non-VH3-15 antibody. 1 to 10,000 ng of VH3-15 antibody or non-VH3-15 antibody per well (or preferably 10 to 1000 ng, or even more preferably 20 ng) were diluted in 50 μL carbonate- bicarbonate buffer, pH 9.6 (Sigma, St. Louis, MO), added to microtiter plates (Costar, Pleasanton, CA) , and incubated overnight at 4° C. The plates were washed 3 times for 15 minutes each with phosphate-buffered saline + 0.5% Tween-20 (ELISA buffer) and blocked for 30 minutes in ELISA buffer.

Monoclonal antibodies from each hybridoma being screened were reacted against VH3-15 antibody and against non-VH3-15 antibody by adding 50 μl of monoclonal antibody (diluted 1:1000 in ELISA buffer) to sample wells and incubated for 1 hour at 4° C. Plates were washed five times with PBS-Tween 20 (0.05%) at room temperature for one minute per wash.

Monoclonal antibody specificity was detected by enzymatic digestion of substrate. Each well was incubated for one hour at 4° C with 1:10,000 goat anti- mouse IgG horseradish peroxidase (Caltag, San Francisco, CA) and washed five times with PBS-Tween 20 (0.05%) at room temperature for one minute per wash. Each well was then incubated with o- phenylenediamine dihydrochloride (Sigma) for 30 minutes at 37° C. 3 N H₂S0₄ was added to stop the reaction. Optical density was determined by absorbance at 492 nm and ranged from 0 to 0.8 optical density units. (OD range in correspondence with 1-10,000 ng Ab used.) It is recommended that an absorbance reading two times the background be considered positive binding to antibody. Non-coated wells were used as the control. Monoclonal antibodies which bind VH3-15 antibody are considered an anti-VH3-15 idiotypic monoclonal antibody. Their specificity is confirmed by their lack of binding with non-VH3-15 antibody.

Using the foregoing assay, it can be shown that monoclonal antibodies produced in accordance with the present invention distinguish between the following VH3 gene products: VH3-30, VH3-23, and VH3-15. BK2 produced mAb which were strongly reactive with the VH3-15 antibody (LSF2) , but were unreactive with non-VH3-15 antibody (477) . Conversely, B6 and D12 (known anti-VH3- 30 idiotypic monoclonal antibodies) were reactive with non-VH3-15 antibody (477) , but unreactive with VH3-15 antibody (LSF2) . 16/6 (a known anti-VH3-23 idiotypic monoclonal antibody, Young, et al. , J. Immunol. , 145:2545-2553 (1990)) reacted with neither VH3-15 antibody (LSF2) nor non-VH3-15 antibody (477) . BK2 lacked detectable reactivity with polyclonal human IgM and IgG.

These findings demonstrated that BK2 selectively bound to VH3-15 antibody (versus other closely related VH3 family gene products) , and lacked reactivity with prevalent Ig sequences such as heavy or light chain constant regions. The apparent paucity of serum VH3-15 was consistent with the infrequent use of this gene, compared to other VH3 genes, in cDNA libraries prepared from polyclonal human B cell populations. EXAMPLE I I I

SUBMUCOSAL STAINING OF COLONIC BIOPSY BY MONOCLONAL ANTIBODIES SPECIFIC FOR VH3-15 POLYPEPTIDES

Five colonic mucosal biopsy specimens were obtained from diagnostic colonic endoscopy specimens of patients seen at UCLA. Three of these specimens were from patients diagnosed with CD, two were from patients diagnosed with UC and one was from a normal patient without either disease. Procedures for subject recruitment, consent, and specimen procurement were in accordance with protocols approved by the Institutional Human Subject Protection Committees at UCLA and Cedars- Sinai Medical Center.

Paraffin sections of normal, UC, and CD colonic biopsies were deparaffinized and blocked for endogenous peroxidase by incubating 20 minutes in H₂0₂ in methanol . To rehydrate sections, slides were immersed in Coplin jars with decreasing concentrations of ethanol, and prepared for staining by washing slides in Tris-buffered saline (TBS) twice, followed by a 30 minute wash in TBS supplemented with 3% goat serum.

Anti-VH3-15 idiotypic monoclonal antibody (diluted to 1 μg/ml in TBS) was applied and incubated 2 hours at room temperature in a humidified chamber, followed by the series of washes with 200 ml TBS for 1 minute at room temperature. Sections were then incubated with 0.2 ml goat anti-mouse Ig peroxidase (Caltag) for 30 minutes at room temperature and then washed with TBS as described above. Finally, binding of anti-VH3-15 idiotypic monoclonal antibody was visualized by the production of a brown precipitate using 3-amino-9-ethylcarbazole (Sigma) substrate. Control slides were stained in the same fashion with the substitution of an isotype-matched mouse IgG. The anti-VH3-15 idiotypic monoclonal bound to intravascular erythrocytes in biopsies from UC patients and CD patients but not in normals. EXAMPLE IV FLOW CYTOMETRIC ASSAY FOR VH3-15 AUTOANTIBODIES Blood serum samples from normal, UC, CD, and other acute and chronic enterocolitis patients were assayed by flow cytometry to detect and quantify VH3-15 autoantibody activity. Samples were obtained from 101 subjects among patients seen at the Inflammatory Bowel Disease and Gastroenterology clinics of UCLA Center for Health Sciences and Cedars-Sinai Medical Center. These samples were segregated into disease groups, as set forth in Table 1 below, by standard clinical criteria.

Table 1. Numbers of subjects studied for serum VH3-15 autoantibody by flow cytometry.

Non-transfused healthy adults 17 Ulcerative colitis 22

Post-Colectomy UC 7

Crohn's disease 17

Other colitis 38

Campylobacter enterocolitis 10 Collagenous colitis 8

Acute infectious colitis 3

Shigella colitis 1

Acute self-limited diarrhea 2

Pseudomembranous colitis 5 Acute schistosomiasis 2

Amoebic colitis 2

Eosinophilic diarrhea 1

Neutropenic colitis 1

Irritable bowel syndrome diarrhea 1

Radiation proctitis 1

Laxative abuse 1

Total 101

A. VH3-15 Autoantibody Associated With CD and UC Confirmed As Encoding VH3-15 Polypeptide

1 X 10^s 0 ^ne9^ative, Rh ⁿa^tive _{blood bank} reagent red blood cells (Dade, Miami, FL) in 100 μl physiological saline were incubated with 100 μl of UC and CD serum samples at room temperature for 30 minutes. The cells were washed in flow buffer (Hanks balanced salt solution, 2% fetal calf serum, and 10 mM HEPES, pH 7.4) . After washing, 50 μl cell aliquots in flow buffer were incubated on ice for 30 minutes with 0.5 μg of a biotinylated anti-VH3-15, anti-VH3-23 or anti-VH3-30 idiotypic monoclonal antibody (BK2, 16/6 and D12 or B6, respectively) . The cells were washed with flow buffer, incubated on ice for another 30 minutes with 5 μl of streptavidin-phycoerythrin (Becton-Dickinson, Burlingham, CA) , and finally washed again with flow buffer. Fluorescence was detected by cytofluorography using a Becton-Dickinson FACSCAN™ instrument. Histograms of log fluorescence were generated and analyzed by Lysis II™ software.

UC and CD samples incubated with anti-VH3-23 or anti-VH3-30 idiotypic monoclonal antibodies exhibited a fluorescent intensity and pattern equivalent to that of the control (serum and streptavidin-phycoerythrin alone) . However, UC and CD samples incubated with anti-VH3-15 idiotypic monoclonal antibody exhibited a uniform shift in fluorescent intensity and pattern to one 4-fold brighter than the control . The marked increase in fluorescent intensity demonstrated by the anti-VH3-15 idiotypic antibody in UC and CD samples indicates that the VH3-15 autoantibody encodes a VH3-15 polypeptide, but not a VH3-23 or VH3-30 polypeptide. This distinction is notable, since B cells expressing the latter two gene subfamilies are actually much more prevalent than VH3-15 expressing B cells.

The uniform fluorescent pattern seen in UC and CD samples indicates that the VH3-15 autoantibody recognizes a common red blood cell antigen. Reagent red cells varying in minor blood group antigens did not differ in reactivity with the VH3-15 autoantibody. This indicated that the autoantigen could not be correlated with a conventionally-defined blood group antigen.

B. VH3-15 Autoantibody Levels Are Elevated In UC and CD Blood serum samples from normal, UC and CD patients were assayed by flow cytometry (as described in Example IVA above) to detect, quantify and compare VH3-15 autoantibody levels. For numerical comparison between samples, values for relative fluorescence intensity were calculated: (fluorescence with serum, anti-VH3-15 idiotypic monoclonal antibody, and phycoerythrin) / (mean fluorescence with anti-VH3-15 idiotypic monoclonal antibody and phycoerythrin) . Relative fluorescence intensity values for each sample are given in Figure 1. Mean values for subjects in each disease group are depicted by a black bar. Levels of VH3-15 autoantibody were significantly elevated in CD patients (mean group value of 8.5) and UC patients (mean group value 6) as compared to healthy normal patients (mean group value 2.5) .

C. VΗ3-15 Autoantibody Is Unique to UC, CD and C. iei uni

Two other patient groups were tested to confirm that elevated VH3-15 autoantibody levels were specific to patients with UC and CD, and were not elicited as part of general response to mucosal injury. First, blood serum samples from seven UC patients who had previously undergone colectomy (between six months and three years earlier) were tested for VH3-15 autoantibody levels. For numerical comparison between samples, values for relative fluorescence intensity were calculated: (fluorescence with serum, anti-VH3-15 idiotypic monoclonal antibody, and phycoerythrin)/ (mean fluorescence with anti-VH3-15 idiotypic monoclonal antibody and phycoerythrin) . Relative fluorescence intensity values for each sample are given in Figure 1. Mean values for subjects in each disease group is depicted by a black bar. Although the mean level of the post-colectomy UC subgroup (3.8) was apparently reduced compared to pre-colectomy UC patients (6) , VH3-15 autoantibody levels were still significantly elevated above healthy normal controls (2.5) . The second patient group tested were patients with other acute or chronic colitis. Of the sera tested, 32/38 had VH3-15 autoantibody levels comparable to normals. An interesting and unexpected finding was that the 6 sera with elevated VH3-15 autoantibody levels (mean group value of 5) corresponded to the group of patients with Campylobacter jejuni enterocolitis. The association of these three disparate gastrointestinal diseases with VH3-15 autoantibody is unexpected. The VH3-15 autoantibody is selectively expressed among individuals with CD, UC and Campylobacter jejuni enterocolitis. Thus, detection of VH3-15 autoantibody levels above the upper 90% confidence limit dor normals (relative fluorescence > 3.25) is a sensitive indicator of these diseases: Crohn's disease (17/17, 100%) , ulcerative colitis (26/29, 90%) , and C. jejuni enterocolitis (7/10, 70%) . In this range, the detection of VH3-15 autoantibody is also specific for this subset (50/53, 94%), since positive sera were detected in only 3/28 (11%) of individuals with other gastrointestinal diseases.

EXAMPLE V

Immunoprecipitation of Erythrocyte Membrane Cell Surface Antigen Immunoprecipitation was used to isolate the erythrocyte proteins recognized by VH3-15 autoantibody. To focus analysis on the surface-exposed antigens accounting for the flow cytometry findings, detection was restricted to surface-displayed proteins in two ways: erythrocytes were chemically surface-labeled, and sera were reacted with intact erythrocytes prior to cell lysis.

Surface-exposed erythrocyte membrane proteins were first labeled by surface biotinylation. Intact 0 ^ne9«lve, Rh ^ne9^atlve blood bank reagent red blood cells (Dade-Baxter, Miami, Fl. ) were pre-treated with bromelase (Dade-Baxter, Miami, Fl. ) following manufacturer's instructions and then washed in PBS three times, resuspended at 1% (I X I0^ε cells/ml) in PBS with 50 μg/ml NHS-LC-biotin (Pierce) , and rotated for 15 minutes at room temperature. Biotinylated cells were then washed with PBS, and aliquots (3 X IO⁶ cells in PBS) were combined with 300 μl test serum. Test sera were selected from 11 healthy controls and 9 patients (5 CD and 4 UC patients) to represent a range of VH3-15 autoantibody levels identified above by flow cytometry. The biotinylated cell/test serum were rotated for 1 hour at room temperature.

Cells were again washed with PBS, incubated with 1.5 μg anti-VH3-15 idiotypic monoclonal antibody for 30 minutes at 4 °C, and washed with PBS again. The cells were then treated with 100 μl lysis buffer (2% Triton-X in PBS) for one hour on ice. Lysates were then diluted with 0.9 ml PBS and incubated with 20 μL goat anti-mouse IgG(H+L) sepharose beads (Zymed, San Francisco, CA) for 30 minutes on ice. Beads were washed three times with PBS, and eluted by 5 minutes at 100°C with 50 μl Laemmli sample buffer including 5% 2-mercaptoethanol.

Sample aliquots (10 μl) were run on 12.6% SDS-PAGE gels as described in Cleveland et al. , J. Biol. Chem. 252:1102 (1977) incorporated herein by reference, and electro-transferred to nitrocellulose filters following standard procedures. See, Towbin, et al . , Proc. Natl. Acad. Sci. USA. 76:4350 (1979), incorporated herein by reference. To detect the surface-labeled immunoprecipitated proteins, the blots were stained with streptavidin-peroxidase (diluted 1:5000) and visualized by enhanced chemiluminescence (ECL) (Amersham, Chicago, IL) following the supplier's recommendations. Lanes stained either in the absence of serum or with an anti- VH3-15 idiotypic monoclonal antibody matched isotype control immunoglobulin were controls for nonspecific binding. Two protein species (22 and 28 kD) were detected by positive sera (sera which produced any detectable proteins after immunoprecipitation) and the same pattern was observed for sera from 9/9 patients with UC or CD. In contrast, no surface proteins were detected by sera from 11/11 healthy subjects. No detectable non-specific bands were observed with immunoprecipitations in the absence of serum or anti-VH3-15 idiotypic monoclonal antibody. It was also determined that the autoantigen does not correspond to commonly analyzed blood group antigens, since the different typing panel red cell samples were all autoantigen positive.

Thus, VH3-15 autoantibody from different individuals shared a common protein antigen specificity. The autoantigen is surface-exposed on the erythrocyte, and is at least in part protein-expressed, since immunoprecipitation of surface-labeled erythrocytes with positive sera detected the same pair of protein species (22 and 28 kD) .

EXAMPLE VI

Fixed Erythrocyte ELISA An alternative method for detecting and quantifying serum VH3-15 autoantibody was also designed. 1 X IO⁶ 0 negative, Rh negative blood bank reagent red blood cells (Dade-Baxter, Miami, FL) were prepared for bromelase digestion by spinning them down at 1500 RPM for 10 minutes at room temperature and then washing them twice with saline 0.9% at 1500 RPM for 10 minutes. Cells were digested with Bromelase Enzyme Reagent (Dade-Baxter, Miami, FL) in accordance with the manufacture's directions (normally 1 ml of bromelase per 1 X IO⁶ red blood cells) . Then the cells were washed twice with saline 0.9% at 1500 RPM for 10 minutes.

The red blood cells were then lysed and cell membrane purified as follows: 1. The total volume of RBC pellet was divided into four microfuge tubes (-50 μl each) after the last saline wash.

2. Eight times the pellet volume of 60 mOsm buffer made from stock solution buffer containing

0.200 m NaCI, 0.075 mNa₂HP0₄, and 0.025 mKH₂P0₄ was added.

3. The microfuge tubes were shaken for 30 minutes at 30°C. 4. The tubes were centrifuged at 10,000Xg for 20 minutes.

5. The supernatant very carefully aspirated.

6. Steps 2, 3 and 4 were performed with 30 and 20 mOsm. 7. The protein on the ghost was measured by Lowry protein quantitation assay (Biorad, Richmond, CA) using a protein standard (e.g. bovine serum albumin) . 8. Cells may be stored at -70°C for up to about 1 week.

An ELISA was performed using the purified erythrocyte cell membrane described above:

1. EIA/RIA Plate form costar (Sigma) were coated with 200 ng erythrocyte cell membrane in 50 μl of

0.05 M sodium carbonate buffer, pH 9.6 per well using the instructions of SIGMA.

2. Coated plates were incubated over night at 4°C.

3. Coated wells were washed three times with Tween-PBS.

4. 50 μl of serum from normals, CD patients or UC patients diluted with Tween-PBS to a preferred concentration of 1/2500 was added per well. (Alternative concentrations useful in the present assay include, for example 1/1000,

1/2000, 1/5000 and 1/10,000) . 5. Plates were incubate for 1 hour at room temperature.

6. Wells were washed three times with Tween-PBS.

7. 50 μl of anti-VH3-15 idiotypic monoclonal antibody was added per well at 1/1000 dilution.

8. Plates were incubated for 1 hour at room temperature.

9. Wells were washed twice with Tween-PBS.

10. 50 μl goat anti-mouse IgG-horseradish peroxidase was added per well at 1/1000 dilution.

11. Plates were incubated for 1 hour at room temperature.

12. Wells were washed three times with Tween-PBS.

13. An OPD tablet (Sigma) was diluted in OPD buffer following instructions from SIGMA and 50 μl was added per well.

14. The plates were incubated at 37° C for 20 minutes.

15. The reaction was stopped using 50 μl of 3N

H₂S0₄

16. Absorbencies were determined by a microtiter plate ELISA reader at 492 nm for twenty seven normal, CD and UC samples each and are provided in Table 2.

Table 2. Mean absorbancies corrected for background, standard error of mean and p values, as compared to normal group for reported absorbancies at 492 nm of serum samples tested for VH3-15 autoantibody by fixed erythrocyte ELISA.

Normal CD UC

MEAN 0.035 0.088 0.056

SEM 0.007 0.016 0.013

P VALUE - β.004 0.15 Table 2, cont,

Sample Normal CD UC

1 0.120 0.160 0.066

2 0.040 0.119 0.012

3 0.011 0.256 0.056

4 0.032 0.066 0.140

5 0.021 0.030 0.076

6 0.011 0.010 0.056

7 0.005 0.036 0.003

8 0.016 0.015 0.025

9 0.016 0.258 0.019

10 0.014 0.311 0.002

11 0.006 0.012 0.326

12 0.005 0.057 0.008

13 0.001 0.010 0.012

14 0.001 0.012 0.021

15 0.023 0.077 0.041

16 0.072 0.006 0.013

17 0.002 0.146 0.012

18 0.016 0.123 0.047

19 0.125 0.079 0.087

20 0.000 0.056 0.071

21 0.032 0.058 0.097

22 0.042 0.047 0.018

23 0.088 0.058 0.063

24 0.083 0.100 0.089

25 0.052 0.084 0.098

26 0.012 0.088 0.026

27 0.089 0.016 Table 2 , cont ,

Table 3. Number of positive and negative samples by disease group using fixed erythrocyte ELISA tested for VH3-15 autoantibody. Positive sample is a sample having an amount of VH3-15 autoantibody that exceeds control.

DISEASE GROUP # POSITIVE # NEGATIVE

NORMAL 9 18

UC 15 12

CD 19 8

Table 4. Number of positive and negative samples by disease group using fixed erythrocyte ELISA tested for VH3-15 autoantibody. Positive sample is a sample that exceeds control by at least one SEM for control providing 85% confidence.

DISEASE GROUP # POSITIVE # NEGATIVE

NORMAL 7 20

UC 13 14

CD 18 9

The titre of VH3-15 autoantibody was also quantified in antibody units. An antibody unit is the percent of the absorbance ratio (test serum/positive reference serum) of samples at 1:2500 read at 492 nm. The positive test serum was a high titre CD sample having an absorbance of 0.392 after subtracting the background, in which coated wells were reacted with reagents but no serum. A reading of seventy-five (75) antibody units or greater (2.5 times the mean antibody units for normals) was considered positive for IBD with 90% confidence limits. The mean antibody units for normal samples was 29±6 (standard error of mean, "SEM") . The mean antibody units for UC was 54±18 (SEM) . The mean antibody unit for CD was 164±20 (SEM) . The results of the assay performed on 27 samples each for UC, CD and healthy normal patients are reported in Table 3. Table 5. Number of positive and negative samples by disease group using fixed erythrocyte ELISA tested for VH3-15 autoantibody. Positive sample is a sample that has seventy-five (75) antibody units or greater (2.5 times the mean antibody units for normals) providing 90% confidence limits.

DISEASE GROUP # POSITIVE # NEGATIVE

NORMAL 2 25

UC 9 18

CD 23 4

EXAMPLE VII Fixed Campylobacter ieiuni ELISA As an alternative to the ELISA of Example VI, an ELISA using fixed Campylobacter jejuni cells was also developed.

1. EIA/RIA Plate form costar (Sigma) were coated with 5 X IO⁴ Campylobacter jejuni (ATCC Accession No. 29428) in 50 μl 0.05 M sodium carbonate buffer, pH 9.6 per well following the procedure described in Horwitz, M.A. and

Schlesinger, L.S., Infection Immunity. 62: 280- 289 (1994), incorporated herein by reference. Alternatively wells may be coated with bacteria in the range of about 5 X IO² to about 5 X 10^s. 2. Coated plates were incubated overnight at 4°C.

3. Coated wells were washed three times with Tween-PBS.

4. 50 μl of serum diluted with Tween-PBS to a preferred concentration of 0.01% was added per well. (Alternative concentrations useful in the present assay range from about 0.1% to about 0.0001%.)

5. Plates were incubate for 1 hour at room temperature. 6. Wells were washed three times with Tween-PBS. 7. 50 μl of anti-VH3-15 idiotypic monoclonal antibody was added per well at 1/1000 dilution.

8. Plates were incubated for 1 hour at room temperature. 9. Wells were washed twice with Tween-PBS.

10. 50 μl goat anti-mouse IgG was added per well at 1/1000 dilution.

11. Plates were incubated for 1 hour at room temperature. 12. Wells were washed three times with Tween-PBS.

14. The plates were incubated at 37° C for 20 minutes.

15. The reaction was stopped using 50 μl of 3N

H₂S0₄

16. Absorbencies were determined by a microtiter plate ELISA reader at 492 nm.

Optical density readings are for two serum samples from patients diagnosed Crohn's disease, two serum samples from patients diagnosed with ulcerative colitis and one sample from a normal patient are provided in Figure 2. Once again, high titre VH3-15 autoantibody was detected in IBD patients as compared to normals. In this assay, VH3-15 autoantibody concentrations exceed normals by up to about eight times.

Although the invention has been described with reference to presently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: The Regents of the University of California

(B) STREET: 300 Lakeside Drive, 21st Floor

(C) CITY: Oakland

(D) STATE: California

(E) COUNTRY: US

(F) POSTAL CODE (ZIP) : 94612-3550

(ii) TITLE OF INVENTION: NOVEL ANTI-VH3-15 REAGENTS, VH3-25

POLYPEPTIDES, CELL SURFACE ANTIGENS, AND METHODS FOR THEIR DETECTION AND USE

(iii) NUMBER OF SEQUENCES: 4

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/320,200

(B) FILING DATE: 07-OCT-1994

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/309,025

(B) FILING DATE: 19-SEP-1994

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 501 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 161..460

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: group (47..149, 470..492) (ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 247..261

(D) OTHER INFORMATION: /function= "Structural domain of protein product" /product= "Complement Determing Region I - CDRl"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 308..364

(D) OTHER INFORMATION: /function= "Structural domain of protein product"

/product= "Complement Determining Region II - CDR II"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

ATGGAGTTTG GGCTGAGCTG GATTTTCCTT GCTGCTATTT TAAAAGGTGA TTTATGGAGA 60

ACTAGAGAGA TTAAGTGTGA GTGAACGTGA GTGAGAGAAA CAGTGGATAT GTGTGGCAGT 120

TTCTGAACTT AGTGTCTCTG TGTTTGCAGG TGTCCAGTGT GAG GTG CAG CTG GTG 175

Glu Val Gin Leu Val 1 5

GAG TCT GGG GGA GGC TTG GTA AAG CCT GGG GGG TCC CTT AGA CTC TCC 223 Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser 10 15 20

TGT GCA GCC TCT GGA TTC ACT TTC AGT AAC TCC TCG ATG AGC TGG GTC 271 Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser Ser Met Ser Trp Val 25 30 35

CGC CAG GCT CCA GGG AAA GGG CTG GAG TGG GTT GGC CGT ATT AAA AGC 319 Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg lie Lys Ser 40 45 50

AAA ACT GAT GGT GGG ACA ACA GAC TAC GCT GCA CCC GTG AAA GGC AGA 367 Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro Val Lys Gly Arg 55 60 65

TTC ACC ATC TCA AGA GAT GAT TCA AAA AAC TCA CTG TAT CTG CAA ATG 415 Phe Thr lie Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gin Met 70 75 80 85

AAC AGC CTG AAA ACC GAG GAC ACA GCC GTG TAT TAC TGT ACC ACA 460

Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr 90 95 100

GACACAGCGA GGGGAGGTCA GTGTGAGCCC GGACACAAAC C 501

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 100 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser 20 25 30

Ser Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Arg lie Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60

Pro Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80

Leu Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95

Tyr Cys Thr Thr 100

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 100 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 31..36

(D) OTHER INFORMATION: /label= CDRl

/note= "Complement Determining Region I"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 50..69

(D) OTHER INFORMATION: /label= CDRII

/note= "Complement Determining Region II" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30

Trp Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Arg lie Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60

Pro Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80

Leu Tyr Leu Gin Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr 85 90 95

Tyr Cys Thr Thr 100

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 125 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 28. -33

(D) OTHER INFORMATION: /label= CDRl

/note= "Complement Determining Region I"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 47..66

(D) OTHER INFORMATION: /label= CDRII

/note= "Complement Determining Region II"

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 98..125

(D) OTHER INFORMATION: /label= CDRIII

/note= "Complement Determining Region III" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Leu Val Glu Ser Arg Gly Gly Leu Val Lys Pro Gly Arg Ser Leu Arg 1 5 10 15

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala Trp Met Ser 20 25 30

Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg lie 35 40 45

Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro Val Lys 50 55 60

Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80

Gin Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95

Thr Trp Tyr Pro Asp lie Leu Asp Ser Cys Tyr Ala Ser Tyr Phe Asp 100 105 110

Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125

Claims

I claim :

1. The BK2 hybridoma having ATCC accession number HB11720.

2. Antibody material produced by the hybridoma of claim 1.

3. An isolated nucleic acid molecule encoding the antibody material of claim 2.

4. A probe comprising at least 30 nucleotides capable of specifically hybridizing with a sequence included within the nucleic acid molecule of claim 3.

5. A method of detecting a nucleic acid sequence encoding an anti-VΗ3-15 idiotypic antibody material, said method comprising: contacting a sample of cells suspected of containing anti-VH3-15 idiotypic antibody material with a nucleic acid probe according to claim 4; and detecting hybridization of the probe to the nucleic acid sequence.

6. Antibody material that specifically binds the same idiotope as the antibody material produced by the hybridoma of claim 1.

7. Antibody material having immunoreactivity with a variable heavy chain segment of a VH3-15 polypeptide.

8. A hybridoma cell capable of producing the antibody material of claim 7.

9. The antibody material of claim 7, wherein the VH3-15 polypeptide comprises an LSF2 an i-Haemophil us influenzae type b capsular polysaccharide antibody.

10. The antibody material of claim 7, wherein the antibody material is selected from a group consisting of an antibody molecule, a (Fab')₂ and a Fab.

11. A method of detecting the presence or amount of a VH3-15 polypeptide in a sample, comprising:

(a) contacting a sample with the detectable anti-VH3-15 idiotypic antibody material under conditions suitable to form a complex of anti-VH3-15 antibody and VH3-15 polypeptide, and (b) assaying for the presence or amount of VH3-15 polypeptide-containing complex by detecting bound anti- VH3-15 antibody material.

12. The method of claim 11, wherein the anti-VH3-15 antibody material is selected from a group consisting of an antibody molecule, a (Fab')₂ and a Fab.

13. The method of claim 11, wherein the anti-VH3-15 antibody material is the monoclonal antibody produced by the BK2 hybridoma having ATCC accession number HB11720.

14. The method of claim 11, further comprising separating any complex resulting from step (a) prior to assaying for the presence or amount of VH3-15 polypeptide-containing complex.

15. A kit for detecting the presence of VH3-15 polypeptide in a sample, comprising the anti-VH3-15 idiotypic antibody material of claim 7 and packaging material .

16. The kit of claim 15, wherein the anti-VH3-15 idiotypic antibody material is a monoclonal antibody that binds the same idiotope as the monoclonal antibody produced by the BK2 hybridoma.

17. Antibody material having immunoreactivity with a variable heavy chain segment of VΗ3-15 autoantibody associated with Inflammatory Bowel Disease and infection by Campyl oba cter j ej uni .

18. A method of detecting the presence of VH3-15 autoantibody in a sample, comprising:

(a) contacting a sample with the anti-VH3-15 idiotypic antibody material of claim 17 under conditions suitable to form an immune complex comprising VH3-15 autoantibody and anti-VH3-15 idiotypic antibody material, and

(b) detecting the presence of immune complex.

19. The method of claim 18, wherein the anti-VH3-15 idiotypic antibody material is labeled with a detectable marker and the sample comprises a colonic biopsy.

20. The method of claim 19, wherein the sample is also contacted with cell surface antigen under conditions suitable to form an immune complex comprising cell surface antigen, VH3-15 autoantibody, and anti-VH3-15 idiotypic autoantibody material prior to detecting the presence of immune complex.

21. The method of claim 20, further comprising separating any uncomplexed cell suface antigen from complexed cell surface antigen prior to detecting immune complex and wherein the cell surface antigen is labeled with a detectabel marker.

22. The method of claim 20, further comprising separating any uncomplexed anti-VH3-15 idiotypic antibody material from complexed anti-VH3-15 idiotypic antibody material prior to detecting immune complex and wherein the anti-VH3-15 idiotypic antibody material is labeled with a detectabel marker.

23. A method of detecting a disease state in a human, comprising:

(a) contacting cell surface antigen and the anti-VH3-15 idiotypic antibody material of claim 17 with a sample from a human under conditions suitable to form an immune complex comprising cell surface antigen, VH3-15 autoantibody and anti-VH3-15 idiotypic antibody material, and

(b) determining the amount of VH3-15 autoantibody by detecting the amount of immune complex, wherein the presence of VH3-15 autoantibody in the sample in excess of a control indicates a disease state selected from the group consisting of UC, CD and infection by Campylobacter jejuni .

24. The method of claim 23, wherein the anti-VH3-15 idiotypic antibody material is labeled with a detectable marker.

25. The method of claim 23, wherein the anti-VH3-15 idiotypic antibody material is a monoclonal antibody molecule .

26. The method of claim 25, wherein the anti-VH3-15 idiotypic monoclonal antibody is the monoclonal antibody produced by hybridoma having ATCC Accession No. HB11720.

27. The method of claim 24, wherein the cell surface antigen is bound to Campylobacter jejuni cell membrane .

28. The method of claim 24, wherein the cell surface antigen is bound to human erythrocyte cell membrane.

29. The method of claim 24, wherein the cell surface antigen is immobilized.

30. The method of claim 29, wherein the conditions suitable to form a complex of cell surface antigen, VH3- 15 autoantibody and anti-VH3-15 idiotypic antibody material comprise contacting human blood serum sample with immobilized cell surface antigen for about 5 minutes to about 120 minutes at a temperature in the range of about 4°C to about 37°C and at about physiological pH, and then contacting the anti-VH3-15 idiotypic antibody material with the immobilized cell surface antigen for about 5 minutes to about 120 minutes at a temperature in the range of about 1°C to about 15°C and at about physiological pH.

31. The method of claim 30, wherein human blood serum sample is contacted with immobilized cell surface antigen for about 5 minutes to about 60 minutes at a temperature in the range of about 20°C to about 37°C and at about physiological pH.

32. The method of claim 31, wherein human blood serum sample is contacted with cell surface antigen for about 25 minutes to about 35 minutes at about room temperature and at about physiological pH.

33. The method of claim 32, wherein the immobilized cell surface antigen is bound to erythrocyte cell membrane that has been pre-treated with bromelase.

34. The method of claim 33, wherein anti-VH3-15 idiotypic antibody material is contacted with the immobilized cell surface antigen for about 10 minutes to about 60 minutes at a temperature in the range of about 2°C to about 10°C and at about physiological pH.

35. The method of claim 34, wherein anti-VH3-15 idiotypic antibody material is contacted with cell surface antigen for about 30 minutes on ice at about physiological pH.

36. The method of claim 21, wherein the immobilized cell surface antigen is bound to human type 0 erythrocyte cell membrane which has been pre-treated with bromelase, wherein the anti-VH3-15 idiotypic antibody material is monoclonal antibody produced by hybridoma having ATCC Accession No. HB11720, wherein the biological sample is human blood serum and wherein the conditions suitable to form an immune complex comprise contacting the cell surface antigen with the sample for about 25 minutes to about 35 minutes at about room temperature and at about physiological pH, and then contacting the cell surface antigen with anti-VΗ3-15 idiotypic monoclonal antibody for about 30 minutes on ice at about physiological pH.

37. Purified antibody characterized by immunoreactivity with erythrocyte cell membrane protein having a molecular weight of about 22,000 daltons on SDS- PAGE and by binding an erythrocyte cell membrane protein having a molecular weight of about 28,000 daltons on SDS- PAGE.

38. The antibody of claim 37 further characterized as encoding a VH3-15 polypeptide.

39. The antibody of claim 38, further characterized by immunoreactivity with the monoclonal antibody produced by the hybridoma having ATCC Accession No. HB11720.

40. Purified cell surface antigen characterized by a molecular weight of about 22,000 daltons on SDS-PAGE and by immunoreactivity with the antibody of claim 39.

41. The cell surface antigen of claim 40 further characterized in vivo as being bound to the membrane of human erythrocyte cells.

42. A nucleic acid sequence encoding the cell surface antigen of claim 41.

43. Purified cell surface antigen characterized by a molecular weight of about 28,000 daltons on SDS-PAGE and binding specificity for the antibody of claim 39.

44. A kit for detecting the presence of VH3-15 polypeptide in a sample, comprising an anti-VH3-15 idiotypic antibody material and cell surface antigen.

45. A kit for detecting a UC, CD and infection by Campylobacter jejuni in a human, comprising anti-VH3-15 idiotypic antibody material, cell surface antigen and a control.