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WO1996009388A1 - Nouveaux reactifs anti-vh3-15, polypeptides de vh3-15, antigenes de surface de cellule et procedes servant a les detecter et a les utiliser - Google Patents

Nouveaux reactifs anti-vh3-15, polypeptides de vh3-15, antigenes de surface de cellule et procedes servant a les detecter et a les utiliser Download PDF

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Publication number
WO1996009388A1
WO1996009388A1 PCT/US1995/011789 US9511789W WO9609388A1 WO 1996009388 A1 WO1996009388 A1 WO 1996009388A1 US 9511789 W US9511789 W US 9511789W WO 9609388 A1 WO9609388 A1 WO 9609388A1
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Prior art keywords
antibody
cell surface
surface antigen
idiotypic
antibody material
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PCT/US1995/011789
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English (en)
Inventor
Jonathan Braun
Original Assignee
The Regents Of The University Of California
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Publication date
Priority claimed from US08/309,025 external-priority patent/US5738847A/en
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Publication of WO1996009388A1 publication Critical patent/WO1996009388A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4233—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-bacterial Ig
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/121—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Helicobacter (Campylobacter) (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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.
  • VH and VL variable regions
  • CDRs complementarity-determining regions
  • 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.
  • V region structure which in turn depends on the diversity of the primary sequence of the V region.
  • 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.
  • Ig immunoglobulin
  • 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.
  • 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.
  • 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.
  • 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.
  • the antibody variable regions are further diversified by somatic hypermutation, leading to higher affinity binding of the antigen.
  • 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.
  • VH gene segments are 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.
  • 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.
  • IBD Inflammatory bowel disease
  • UC ulcerative colitis
  • CD Crohn's disease
  • 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.
  • Campylobacter jejuni 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.
  • 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.
  • Campylobacter jejuni infection may be the cause or be a causative factor of IBD.
  • 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.
  • 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.
  • VH3-15 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.
  • 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.
  • 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.
  • diagnostic assays and methods exploit the immunoreactivity of anti- idi ⁇ typic antibody with autoantibody, and autoantibody with antigen to provide a wealth of different strategies for detection, isolation and purification of these immunological reagents.
  • 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.
  • 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.
  • 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.
  • 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.
  • ant i-Haemophil us influenzae type b capsular polysaccharide antibody 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.
  • VH gene segments that cross-hybridize by Southern filter hybridization under standard conditions 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.
  • the VH3 gene family is presently considered to have the largest membership.
  • 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.
  • 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 V ⁇ 3-15 gene segment is also known as M26, 20pl, DP-38 and 9-1.
  • VH3-15 nucleic acid sequence 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.
  • 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.
  • VH3-15 nucleic acids and VH3-15 polypeptides may be encoded as part of larger sequences.
  • a VH3-15 polypeptide may take the form of an antibody
  • VH3-15 antibody an antibody fragment
  • VH3-15 (Fab') an antibody fragment
  • VH3-15 Fab an antibody fragment
  • LSF2 anti- Haemophilus influenzae type b capsular polysaccharide antibody is another polypeptide representative of a VH3- 15 sub-family.
  • a purified antibody of predominately IgA, IgG 1( and IgG 4 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an antibody-producing cell for fusion with an immortalized cell a mammal is inoculated with an immunogen.
  • 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.
  • 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.
  • the peptide may be bound to a carrier, for the purpose of inducing the production of antibodies.
  • 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) .
  • RIA radioimmunoassay
  • ELISA enzyme linked immunosorbent assay
  • 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.
  • DMEM Dulbecco's minimal essential medium
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, NaH 2 P0 4 , 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.
  • 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.
  • nucleic acid encompasses RNA as well as single and double-stranded DNA and cDNA.
  • 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.
  • isolated nucleic acid molecule means a nucleic acid molecule that is in a form that does not occur in nature.
  • 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) .
  • 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.
  • a promoter as well as other regulatory sequences.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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.
  • a composition containing VH3-15 autoantibody can be used as a positive control to confirm or quantify the results of the method.
  • 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.
  • 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.
  • VH3-15 polypeptide for example VH3-15 autoantibody
  • 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.
  • immunocomplex 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.
  • 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.
  • 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.
  • 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.
  • the erythrocyte cell membrane is pre-treated with bromelase to remove extraneous antigens.
  • 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.
  • 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 .
  • 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
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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
  • control may take on many different physical forms.
  • 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.
  • 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.
  • 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.
  • condition 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.
  • compositions 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • VH3-15 autoantibody, cell surface antigen, nucleic acid molecules and probes of the present invention can be labeled with a detectable marker.
  • compositions of the present invention are well known in the art and contemplated as within the scope of the present invention.
  • 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.
  • 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.
  • fluorochromes such as fluorescein isocyanate (FIC) , fluorescein isothiocyante (FITC) , 5-dimethylamine-l-naphthalenesulfonyl chloride
  • 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 represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is 125 I .
  • Another group of useful labeling means are those elements such as 11 C, 18 F, 15 0 and 13 N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body.
  • a beta emitter such as 111 indium or 3 H.
  • the detectable marker is an enzyme, such as horseradish peroxidase (“HRP") , glucose oxidase, and the like.
  • additional reagents are required to visualize the fact that an immune complex has formed.
  • 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) .
  • 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.
  • a radiation counter such as a gamma counter or gamma emitting markers such as iodine-125.
  • a quantitative analysis of complex can be made using a spectrophotometer, for example a EMAX
  • Microplate Reader available from Molecular Devices, Menlo Park, California
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • labeled goat anti-mouse IgG can be used as a specific binding agent.
  • 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 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.
  • kits for detecting the presence, absence or amount of VH3-15 polypeptide in a sample 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • kits for detecting the presence, absence or amount of nucleic acid encoding VH3-15 autoantibody in a sample 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.
  • 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.
  • a 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.
  • 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.
  • kits 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)
  • BK7F3F6E1 (and also known as BK7)
  • IgM and IgG producing hybridomas are IgM and IgG producing hybridomas and BK2 is an IgG,kappa producing hybridoma.
  • ELISA plate wells were coated with various concentrations of either a known VH3-15 antibody or a known non-VH3-15 antibody.
  • 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.
  • 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) .
  • 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.
  • VH3-15 antibody or non-VH3-15 antibody per well were diluted in 50 ⁇ L 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 ⁇ l 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 H 2 S0 4 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.
  • VH3-30, VH3-23, and VH3-15 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) .
  • B6 and D12 known anti-VH3- 30 idiotypic monoclonal antibodies
  • 16/6 a known anti-VH3-23 idiotypic monoclonal antibody, Young, et al. , J. Immunol.
  • Paraffin sections of normal, UC, and CD colonic biopsies were deparaffinized and blocked for endogenous peroxidase by incubating 20 minutes in H 2 0 2 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.
  • TBS Tris-buffered saline
  • Anti-VH3-15 idiotypic monoclonal antibody (diluted to 1 ⁇ g/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.
  • Table 1 Numbers of subjects studied for serum VH3-15 autoantibody by flow cytometry.
  • VH3-15 Autoantibody Associated With CD and UC Confirmed As Encoding VH3-15 Polypeptide
  • Rh n a tive blood bank reagent red blood cells (Dade, Miami, FL) in 100 ⁇ l physiological saline were incubated with 100 ⁇ l 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) .
  • flow buffer Hanks balanced salt solution, 2% fetal calf serum, and 10 mM HEPES, pH 7.4
  • 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) .
  • 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.
  • 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) .
  • 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.
  • 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.
  • VH3-15 autoantibody is selectively expressed among individuals with CD, UC and Campylobacter jejuni enterocolitis.
  • detection of VH3-15 autoantibody levels above the upper 90% confidence limit dor normals 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%) .
  • 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.
  • Immunoprecipitation of Erythrocyte Membrane Cell Surface Antigen 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.
  • erythrocyte membrane proteins were first labeled by surface biotinylation. Intact 0 ne9 «lve , Rh ne 9 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 ⁇ cells/ml) in PBS with 50 ⁇ g/ml NHS-LC-biotin (Pierce) , and rotated for 15 minutes at room temperature.
  • Biotinylated cells were then washed with PBS, and aliquots (3 X IO 6 cells in PBS) were combined with 300 ⁇ l 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.
  • 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) .
  • 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 ⁇ l each) after the last saline wash.
  • the microfuge tubes were shaken for 30 minutes at 30°C. 4. The tubes were centrifuged at 10,000Xg for 20 minutes.
  • Steps 2, 3 and 4 were performed with 30 and 20 mOsm.
  • the protein on the ghost was measured by Lowry protein quantitation assay (Biorad, Richmond, CA) using a protein standard (e.g. bovine serum albumin) .
  • Cells may be stored at -70°C for up to about 1 week.
  • EIA/RIA Plate form costar (Sigma) were coated with 200 ng erythrocyte cell membrane in 50 ⁇ l of
  • OPD tablet (Sigma) was diluted in OPD buffer following instructions from SIGMA and 50 ⁇ l was added per well.
  • 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.
  • Positive sample is a sample that exceeds control by at least one SEM for control providing 85% confidence.
  • 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) .
  • Table 3 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.
  • EIA/RIA Plate form costar (Sigma) were coated with 5 X IO 4 Campylobacter jejuni (ATCC Accession No. 29428) in 50 ⁇ l 0.05 M sodium carbonate buffer, pH 9.6 per well following the procedure described in Horwitz, M.A. and
  • OPD tablet (Sigma) was diluted in OPD buffer following instructions from SIGMA and 50 ⁇ l was added per well.
  • 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.
  • high titre VH3-15 autoantibody was detected in IBD patients as compared to normals.
  • VH3-15 autoantibody concentrations exceed normals by up to about eight times.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • Gly Arg lie Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60
  • MOLECULE TYPE peptide
  • FRAGMENT TYPE N-terminal
  • 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
  • MOLECULE TYPE peptide
  • FRAGMENT TYPE N-terminal
  • Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg lie 35 40 45

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Abstract

L'invention concerne de nouveaux matériaux d'anticorps idiotypiques anti-VH3-15, ainsi que des hybridomes produisant ces matériaux. Elle concerne également des auto-anticorps de VH3-15 reconnus par le matériau d'anticorps idiotypique anti-VH3-15, ainsi que les antigènes de surface de cellule se fixant à l'auto-anticorps de VH3-15. Ce matériau d'anticorps idiotypique anti-VH3-15, les auto-anticorps de VH3-15 et les antigènes de surface de cellule peuvent s'utiliser dans des procédés de détection, d'isolation et de purification de polypeptides de VH3-15, ainsi que dans des procédés servant à effectuer le diagnostic de colites inflammatoires et d'infection par Campylobacter jejuni. L'invention concerne également des molécules d'acide nucléique codant le matériau d'anticorps anti-VH3-15, l'auto-anticorps de VH3-15 et les antigènes de surface de cellule, ainsi que des procédés servant à les utiliser.
PCT/US1995/011789 1994-09-19 1995-09-18 Nouveaux reactifs anti-vh3-15, polypeptides de vh3-15, antigenes de surface de cellule et procedes servant a les detecter et a les utiliser WO1996009388A1 (fr)

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US320,200 1989-03-07
US309,025 1994-09-19
US08/309,025 US5738847A (en) 1994-09-19 1994-09-19 Anti-VH3-15 reagents and methods for their use
US32020094A 1994-10-07 1994-10-07

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999052551A1 (fr) * 1998-04-15 1999-10-21 King's College London Traitement de l'atherosclerose

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
E. ADDERSON ET AL.: "Restricted Ig H chain V gene usage in the human antibody response to Haemophilus influenzae type b capsular polysaccharide.", THE JOURNAL OF IMMUNOLOGY, vol. 147, no. 5, 1 September 1991 (1991-09-01), BALTIMORE, MD, USA, pages 1667 - 1674 *
E. ADDERSON ET AL.: "The human VH3b gene subfamily is highly polymorphic.", THE JOURNAL OF IMMUNOLOGY, vol. 151, no. 2, 15 July 1993 (1993-07-15), BALTIMORE, MD, USA, pages 800 - 809 *
F. MATSUDA ET AL.: "Structure and physical map of 64 variable segments in the 3' 0.8-megabase region of the human immunoglobulin heavy-chain locus.", NATURE GENETICS, vol. 3, no. 1, NEW YORK, NY, USA, pages 88 - 94 *
J. BRAUN ET AL.: "Restricted use of fetal VH3 immunoglobulin genes by unselected B cells in the adult. Predominance of 56p1-like VH genes in common variable immunodeficiency.", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 89, no. 5, NEW YORK, NY, USA, pages 1395 - 1402 *
L. BERBERIAN ET AL.: "Expression of a novel autoantibody defined by the VH3-15 gene in inflammatory bowel disease and Campylobacter jejuni enterocolitis.", THE JOURNAL OF IMMUNOLOGY, vol. 153, no. 8, 15 October 1994 (1994-10-15), BALTIMORE, MD, USA, pages 3756 - 3763 *
L. BERBERIAN ET AL.: "Immunoglobulin VH3 gene products: Natural ligands for HIV gp120.", SCIENCE, vol. 261, no. 5128, 17 September 1993 (1993-09-17), WASHINGTON, DC, USA, pages 1588 - 1591 *
Y. VALLES-AYOUB ET AL.: "Characterization of a common VH3-15 autoantibody relating inflammatory bowel disease and C. jejuni enterocolitis.", THE FASEB JOURNAL, vol. 8, no. 5, 8 March 1994 (1994-03-08), BETHESDA, MD, USA, pages A1010 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999052551A1 (fr) * 1998-04-15 1999-10-21 King's College London Traitement de l'atherosclerose

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