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WO1992006105A1 - Anticorps et procedes de diagnostic et de traitement du diabete - Google Patents

Anticorps et procedes de diagnostic et de traitement du diabete Download PDF

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Publication number
WO1992006105A1
WO1992006105A1 PCT/US1991/007052 US9107052W WO9206105A1 WO 1992006105 A1 WO1992006105 A1 WO 1992006105A1 US 9107052 W US9107052 W US 9107052W WO 9206105 A1 WO9206105 A1 WO 9206105A1
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Prior art keywords
autoantigen
cells
monoclonal antibody
beta
cell
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PCT/US1991/007052
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English (en)
Inventor
Hun-Chi Lin
Toha Thai
Shau-Ping Lei
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Trigen Incorporated
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Publication of WO1992006105A1 publication Critical patent/WO1992006105A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • 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
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to antibodies useful in the diagnosis and treatment of insulin dependent diabetes mellitus (IDDM) , and more particularly to a hybridoma cell line producing a novel monoclonal antibody, reactive with beta cell autoantigen on pancreatic islet cells.
  • IDDM insulin dependent diabetes mellitus
  • the invention also relates to isolation of the beta cell autoantigen reactive with the monoclonal antibody, and to methods of diagnosing and treating IDDM patients using the antibodies and autoantigen produced in accordance with the present invention.
  • IDDM Insulin Dependent Diabetes Mellitus
  • IDDM Insulin Dependent Diabetes Mellitus
  • the beta cells produce insulin, and are clustered together in the pancreas in structures called the Islets of Langerhans. Islet tissue is composed of approximately 80 percent beta cells.
  • the affected individual's immune system produces autoantibodies against the antigens associated with pancreatic islet beta cells ("beta cell autoantigens") .
  • the islet cell autoantibodies attack and progressively destroy all the pancreatic islet beta cells, resulting in the loss of the ability to produce insulin.
  • IDDM In the United States, from 30,000 to 60,000 new cases of IDDM occur annually. The disease presently affects 600,000 Americans. Thus, for this disease there is a critical need for an early diagnostic test to determine the presence of beta cell autoantibodies before beta cell destruction takes on clinical significance, and for treatment strategies. IDDM is currently diagnosed by the abrupt and sometimes fatal clinical symptoms of ketoacidosis. This represents an inability in a thinner, younger patient to adjust blood glucose levels without receiving a therapeutic dose of insulin. Other than ketoacidosis, there are a variety of methods currently used by physicians to make a positive diagnosis, such as the glucose tolerance test and measurement of endogenous insulin level. However, these tests are able to detect IDDM only after 90% of the beta cells are already destroyed by the islet cell autoantibodies.
  • IDDM is currently treated through multiple, daily subcutaneous injections of insulin. Recently, immunological methods and tissue culture techniques have been developed which permit transplantation of pancreatic islets into animals to treat diabetes
  • Anti-id antibodies can bind to that site on the idiotypic antibodies which stimulated their production, thereby blocking the antibodies from binding to their natural target. Idiotypic interactions have been proposed for therapy of a wide variety of disease conditions ranging from cancer to autoimmune diseases. (Geha, Advances in Immunology. 39:255-297 (1986)). It would be desirable to provide anti-id antibodies reactive with autoantibodies against beta cell autoantigen for use in an immunotherapeutic plasma treatment system, such as plasmapheresis, to specifically and quickly remove the autoantibodies from patient blood, without affecting other immunoglobulins of the patient.
  • an immunotherapeutic plasma treatment system such as plasmapheresis
  • anti-id antibodies conjugated with a toxin, or beta cell autoantigen conjugated with a toxin, or combinations thereof may be used to bind and destroy specifically these T and B lymphocytes.
  • anti-beta cell autoantigen antibodies, substantially purified beta cell autoantigen, and anti-id antibodies in the diagnosis, prevention, and treatment of IDDM.
  • the invention also provides substantially purified beta cell autoantigen.
  • the monoclonal antibody of the invention can be used to isolate beta cell autoantigen.
  • the autoantigen may also be obtained from body tissues such as pancreatic islet cells or from body fluids such as blood serum.
  • the beta cell autoantigen of the invention has a molecular weight of approximately 64,000 daltons as determined by polyacrylamide gel electrophoresis and has an immunodominant epitope associated with the presence of IDDM.
  • the autoantigen may also be isolated from cell lines, including insulinoma D115 or HEp2.
  • the monoclonal antibody of the invention may be used to prepare a vaccine composition for preventing pathogenesis of IDDM. This method prevents destruction of pancreatic beta cells by introducing a vaccine comprising the monoclonal antibody into a human.
  • Fig. 1 is a restriction map for plasmid pUH14 indicating a DNA insert comprising about 1400 base pairs coding for the beta cell autoantigen specifically reactive with the DM monoclonal antibody and the anti-64 kd autoantibody.
  • the present invention concerns hybridoma cell lines and novel monoclonal antibodies produced by the hybridoma cell lines.
  • One of the novel monoclonal antibodies designated DM monoclonal antibody, is specifically reactive with an autoantigen bound to the plasma membrane of pancreatic beta cells.
  • Hybridoma fusion techniques were first introduced by Kohler and Milstein (see Kohler and Milstein, Nature, 256:495-97 (1975); Brown et al., J. Immunol.. 127 (2):539-45 (1981); Brown et al., J. Biol. Chem.. 255:4980-83 (1980); Yeh et al., Proc. Nat'l. Acad. Sci. (USA) . 76 (6):927-31 (1976); and Yeh et al., Int. J. Cancer. 29:269-75 (1982)).
  • These techniques involve the use of an animal which is primed to produce a particular antibody.
  • the animal can be primed by injection of an immunogen (e.g.
  • a primed animal is also one which is expressing a disease.
  • Lymphocytes derived from the lymph nodes, spleens or peripheral blood of primed, diseased animals can be used to search for a particular antibody. For example, when either lymphocytes from prediabetic BB rats or lymphocytes from patients with Type I diabetes are fused with myeloma cells, hybridomas can be selected which produce antibodies that preferentially bind to beta cells.
  • the lymphocyte chromosomes encoding desired immunoglobulins are immortalized by fusing the lymphocytes with myeloma cells, generally in the presence of a fusing agent such as polyethylene glycol (PEG) .
  • a fusing agent such as polyethylene glycol (PEG) .
  • PEG polyethylene glycol
  • Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques; for example, the P3-NSl/l-Ag4-l, P3-X63- Ag8.653, Sp2/0-Agl4, or HL1-653 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC) , Rockville, Maryland.
  • ATCC American Type Culture Collection
  • the resulting cells which include the desired hybridomas, are then grown in a selective medium such as HAT medium, in which unfused parental myeloma or lymphocyte cells eventually die. Only the hybridoma cells survive and can be grown under limiting dilution conditions to obtain isolated clones.
  • the supernatants of the hybridomas are screened for the presence of the desired specificity, e.g. by immunoassay techniques using the antigen that has been used for immunization. Positive clones can then be subcloned under limiting dilution conditions, and the monoclonal antibody produced can be isolated. Novel antibodies of mouse or human origin can be also made to the beta cell autoantigen having the appropriate biological functions.
  • human monoclonal antibodies may be made by using the antigen, e.g. the beta cell autoantigen of the present invention, to sensitize human lymphocytes in vitro followed by EBV transformation or hybridization of the antigen- sensitized lymphocytes with mouse or human lymphocytes, as described by Borrebaeck et al. (Proc. Nat'l. Acad. Sci. (USA). 85:3995-99 (1988)).
  • Various conventional methods can be used for isolation and purification of the monoclonal antibodies so as to obtain them free from other proteins and contaminants.
  • Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascites fluid) using techniques known in the art (see generally Fink et al., Prog. Clin. Pathol.. 9:121-33 (1984), Fig. 6-1 at p. 123).
  • the individual cell line may be propagated in vitro, for example, in laboratory culture vessels, and the culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration, or centrifugation.
  • chimeric (mouse-human) or human monoclonal antibodies may be preferable to murine antibodies because patients treated with mouse antibodies generate human anti-mouse antibodies.
  • Chimeric mouse-human monoclonal antibodies reactive with the beta autoantigen bound to the plasma membrane of pancreatic beta cells can be produced, for example, by techniques recently developed for the production of chimeric antibodies (Oi et al., Biotechnologies. 4(3):214-221 (1986); Liu et al., Proc. Nat'l. Acad. Sci. (USA) .
  • genes coding for the constant regions of the murine DM monoclonal antibody molecule are substituted with human genes coding for the constant regions of an antibody with appropriate biological activity (such as the ability to activate human complement and mediate antibody dependent complement- mediated cytotoxicity) .
  • recombinant antibodies include fusion proteins where the antibody is combined with a second protein having a desired biological function (Neuberger et al., "Recombinant Antibodies Possessing Novel Effector Functions", Nature 312:604-608 (1984)). Fragments of the monoclonal antibodies containing the active binding region, such as Fab, F(ab) ' 2 and Fv fragments may be produced from the monoclonal antibody using techniques well-established in the art (Rousseaux et al., Methods Enzvmol. 121:663-669, Academic Press (1986)) .
  • a typical method for producing the monoclonal antibody by recombinant means involves a recombinant gene expression system situated in E. coli bacteria, yeast, or in a mammalian cell system.
  • the gene expression system is constructed with a DNA sequence taken from hybridoma cell line ATCC No. HB 10502 and encodes at least a portion of the immunoglobulin molecule.
  • the recombinant means can be a recombinant gene expression system situated in E. coli bacteria.
  • the gene expression system can be constructed to express a DNA sequence containing the DM monoclonal antibody.
  • Such a DNA sequence which can be derived at least in part from hybridoma cell line ATCC No. HB 10502, encodes at least a portion of the DM immunoglobulin molecule.
  • the antibody of this invention designated DM monoclonal antibody
  • DM monoclonal antibody was produced via hybridoma techniques as described by Kohler and Milstein, with minor modifications as described in Example 1, infra. Spleen cells from a spontaneously diabetic mouse were used as a source of cells producing an antibody against the autoantigen located on the mouse beta cells.
  • the DM hybridoma, producing the DM monoclonal antibody has been deposited with the ATCC, Rockville, Maryland. The deposit was received by the ATCC July 11, 1990, and has been there identified as follows: DM Monoclonal Antibody Accession No.: HB 10502
  • the DM monoclonal antibody is of the IgM class. As described below in Example 2, the antibody displays a very strong reactivity with pancreatic beta cells and cell lines which express a specific plasma membrane bound antigen similar to the 64 kd autoantigen located on pancreatic beta cells.
  • the DM monoclonal antibody shows no detectable binding to human stomach or kidney cells, nor to rat stomach, heart, liver and kidney cells.
  • the present invention encompasses the DM monoclonal antibody described above and any fragments thereof containing the active binding region of the antibody, such as Fab, F(ab) '2 and Fv fragments. Such fragments can be produced from the DM monoclonal antibody using techniques well established in the art (see e.g. Rousseaux et al., in Methods Enzymol. f 121:663-69, Academic Press (1986)).
  • the DM monoclonal antibody recognizes and binds to an immunodominant epitope of the approximately 64 kd beta cell autoantigen.
  • the immunodominant epitope is the antigenic determinant site on an immunogen to which most of the immune response is directed.
  • the immunodominant epitope is the antigenic determinant site to which the major reactivity of autoantibodies are targeted.
  • the antigenic determinant is that portion of an antigen that makes contact with a particular antibody or T cell receptor. In a typical protein antigen, it is likely that any residue accessible from the surface is part of one or another antigenic determinant. A protein therefore has sequential and/or conformational determinants.
  • the molecular weight of the beta cell autoantigen is approximately 64,000 daltons, as determined by polyacrylamide gel electrophoresis.
  • This autoantigenic protein component of beta cell plasma membrane is the primary target for autoantibodies, namely anti-64 kd autoantibodies, responsible for immune destruction of islet beta cells.
  • This autoantigen is considered to be primarily involved in the beta cell autoimmunity underlying IDDM based on the concurrence of the anti-64 kd autoantibody with the onset of IDDM in man, mouse and rat, by the early appearance of anti-64 kd autoantibody in the natural history of islet cell inflammation in these species, and by the restriction of this protein autoantigen to beta cells and perhaps to cell membranes (Atkinson et al.. Lancet. 335:1357-60, (1990)).
  • the specificity of the DM monoclonal antibody for the immunodominant epitope of the beta cell autoantigen is indicated by the observation, as illustrated in Example 2, infra. that the DM monoclonal antibody and the autoantibody reactive with the beta cell autoantigen exclude each other from binding to the beta cell autoantigen. Accordingly, the DM monoclonal antibody inhibits the binding of human islet cell autoantibody positive serum to human pancreatic beta cells. It should be understood that the specificity of the DM monoclonal antibody encompassed by the present invention includes recognizing and binding functional equivalents of the immunodominant epitope of the beta cell autoantigen.
  • the specificity of the DM monoclonal antibody for the 64,000 dalton beta cell autoantigen is further indicated, as shown in Example 3 infra. by the DM monoclonal antibody's capability of precipitating antibody-dependent complement-mediated cytotoxicity of rat insulinoma cells.
  • the DM monoclonal antibody specifi ⁇ ally mediates cytotoxicity of these insulinoma cells.
  • the present invention further embodies the conjugation of the DM monoclonal antibody or purified beta cell autoantigen to a label capable of producing a detectable signal for use in various types of quantitative immunoassays.
  • These immunoassays include monoclonal competitive immunoassays, monoclonal "sandwich" direct immunoassays and indirect immunoassays to carry out the method of the invention involving detection of beta cell autoantigen.
  • the three most common quantitative immunoassays are radioimmunoassay (both soluble phase and solid phase) , quantitative fluorescence immunoassays, and quantitative enzyme- linked assays. Any of these may be used to practice the invention.
  • label is intended to include moieties that may be detected directly such as fluorochromes and radiolabels, as well as moieties like enzymes that must be reacted or derivatized to be detected.
  • the particular label that is used will depend on the type of quantitative immunoassay used.
  • the assay should be such as to provide sensitivities sufficient for detection by labeled DM monoclonal antibody of beta cell autoantigen or detection of anti-64 kd autoantibodies by labeled beta cell autoantigen.
  • coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succini ides, bisdiazotized benzidine, and the like may be used to tag the antibodies or antigens with fluorescent, chemi-luminescent, or enzyme labels.
  • the label detection means used in the immunoassay will depend upon the particular label involved. For instance, enzyme labels may be read with colorimeters that detect the magnitude of color change caused by the reaction between the enzyme and the substrate or spectrophotometers (e.g. microtiter plate readers) , radiolabels may be read with scintillation counters or gamma counters, and fluorescent labels with fluorescent microscopes (Fluorimeters) .
  • enzyme labels may be read with colorimeters that detect the magnitude of color change caused by the reaction between the enzyme and the substrate or spectrophotometers (e.g. microtiter plate readers)
  • radiolabels may be read with scintillation counters or gamma counters
  • fluorescent labels with fluorescent microscopes (Fluorimeters) .
  • An indirect immunoassay such as enzyme linked immunosorbent assay (ELISA) is a common method for detecting reactivity between antigen and antibody.
  • an antigen or antibody is typically bound (immobilized) to a solid phase, such as a tube, bead or well surface.
  • a solid phase such as a tube, bead or well surface.
  • an antigen may be immobilized to a solid phase.
  • a patient's serum sample containing a first antibody reactive with the bound antigen is added, and a second enzyme-labeled antibody against the retained antibody is added.
  • Immobilized antigen-antibody complexes are then detected via reaction of the enzyme with an appropriate substrate.
  • the enzyme horseradish peroxidase may be used along with its substrate orthophenylene diamine as a color indicator.
  • anti-id antibodies can be developed which exhibit selectivity toward antigenically distinct features of immunoglobulins, in particular, the DM monoclonal antibody of the present invention.
  • Anti-id antibodies are prepared according to Rivas et al., Proc. Nat. Acad. Sci. USA. 85:6092-6096 (1988) and Miller et al. New Engl. J. Med.. 321: 851-857 (1989) .
  • Small and active fragments of the DM monoclonal antibody for immunizing mice are prepared according to the method of Mathew and Reichardt in . Immunol. Methods. 50:239-253 (1982).
  • one part of 500 mM Tris, pH 8.0, lOmM CaCl 2 is added to nine parts of purified DM monoclonal antibody in PBS.
  • the final concentration of monoclonal antibody is adjusted to 0.3 mg/ml with 50 mM Tris, pH 8.0, 150 mM NaCl, l mM CaCl 2 .
  • the sample is digested with 0.005 mg/ml trypsin (Sigma) for two hours at 30 ⁇ C. 10 mg/ml of soybean trypsin inhibitor is added to a final concentration of 0.1 mg/ml and incubated for 5 minutes at 37"C.
  • Iodoacetamide is added to a final concentration of 60 mM and incubated at room temperature for ten minutes. The samples are then dialyzed against four changes of 1 liter of 10 mM phosphate buffer, pH 7.4 at 4 ⁇ C over 48 hours. Active small fragments are further purified by ion exchange chromatography. One hundred micrograms of purified F(ab') 2 are used to immunize a rabbit or mouse. Sera are collected after boosting twice. Anti-id antibodies are purified from the sera by immunoaffinity column containing purified DM monoclonal antibody ligand.
  • DM monoclonal antibodies are coupled to a CNBr activated Sepharose 4B gel (Pharmacia) according to the manufacturer's recommendation.
  • the anti-id antibodies are precipitated with ammonium sulfate at 50 percent of saturating concentration. The precipitant is dissolved in PBS and dialyzed extensively against PBS. The preparation is then incubated with mouse DM monoclonal antibodies affinity column overnight at 4°C.
  • Anti-id antibodies from the sera specifically bound to the column is eluted with elution buffer purchased from Bio ⁇ Rad and dialyzed extensively against PBS.
  • Immunoaffinity purified anti-id antibodies are then coupled to the CNBr-activated Sepharose 4B gel or Affigel HZ (Bio-Rad Laboratories, Richmond, CA) .
  • the anti-id antibodies produced are characterized using standard techniques (Miller et al.. New Enql J.
  • Development of a hybridoma cell line that produces anti-id antibodies is accomplished using conventional techniques described above with modifications according to Miller et al., New England J. Med. 321:851-857(1989).
  • the major difference involved in selecting for a hybridoma producing anti-id antibodies is that the screening procedures intails excluding selections for variable regions other than the active site.
  • the invention further concerns the beta cell autoantigen, recombinant methods for producing the beta cell autoantigen, diagnostic methods for the early detection of IDDM using the beta cell autoantigen and DM monoclonal antibody, and immunotherapeutic methods for preventing destruction of pancreatic beta cells in a subject using the beta cell autoantigen, the anti-id antibodies, or combinations of the autoantigen and anti- id antibodies.
  • the beta cell autoantigen is recognized and bound by the DM monoclonal antibody of the present invention.
  • the monoclonal antibody of the present invention provides an advantage because its specificity of binding to the beta cell autoantigen makes possible the specific isolation of the autoantigen from very small amounts of tissue or fluids.
  • the beta cell autoantigen is further recognized by anti-64 kd autoantibodies, a component of human islet cell positive serum.
  • An epitope on the beta cell autoantigen is the primary target of anti-64 kd autoantibodies that mediate pancreatic islet cell destruction in the pathogenesis of IDDM.
  • the same or a different epitope on the beta cell autoantigen is the primary target of the DM monoclonal antibody.
  • the invention also provides a method for purifying the beta cell autoantigen from cells.
  • the method employs a cellular source for the beta cell autoantigen, as described in Example 5, infra.
  • the cellular source is preferably any tissue or cell line rich in the approximately 64,000 dalton beta cell autoantigen.
  • a most preferred source of the autoantigen is human epidermoid carcinoma (Hep2) cells.
  • Hep2 human epidermoid carcinoma
  • Other preferred sources which are also unexpected, are neuronal cell lines, such as human glioblastomas U87MG (ATCC No. HTB14) (Ponten, J. et al., Acta Path. Microbiol. Scand. 74: 465:486 (1968)) and U138MG (ATCC No. HTB16) (ibid) , human neuroblastomas SKNMC (ATCC No.
  • Rat insulino a D115 cells (derived from CRI-D11, Carrington, CA. et al., ⁇ Endocrinol. 109: 193-200 (1986)) are another preferred source.
  • the invention embodies a further method for the production of recombinant beta cell autoantigen, as described in Example 7, infra.
  • the method involves construction of a cDNA library encoding the beta cell autoantigen.
  • the source of nucleic acid for the cDNA library can be a cell line of non- pancreatic origin. It is preferably a cell line which has a high constitutive rate of producing the approximately 64,000 dalton beta cell autoantigen, such as HEp2 cells.
  • Other preferred sources include the neuronal cell lines described above, and rat insulinoma cells.
  • Isolated DNA encoding the beta cell autoantigen of the invention is analyzed by restriction and/or sequenced by the dideoxy method of Sanger et al. , Proc.
  • the DNA sequences encoding the beta cell autoantigen are obtained from a cDNA library prepared from mRNA isolated from cells expressing the beta cell autoantigen according to procedures described in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, pp. 198-198 (1982) , with particular reference to Young et al.. Nature. 316:450-452 (1988).
  • the cDNA insert from the successful clone, excised with a restriction enzyme such as EcoRI, is then used as a probe of the original cDNA library to obtain the additional clones containing inserts encoding other regions of the autoantigen which, together with this probe, span the nucleotides containing the complete coding sequence of the beta cell autoantigen.
  • Double-stranded cDNA is synthesized and prepared for insertion into a lambda vector using the technique of Okayama and Berg, Molecular and Cellular Biology. 2:263 (1982).
  • First strand cDNA is synthesized by the RNA-dependent DNA polymerase (reverse transcriptase) from a source such as Avian Myeloblastosis virus by priming with oligo (dT) 12-18 or random primer on 5 ⁇ g mRNA.
  • Second strand DNA is synthesized by using E. coli DNA polymerase I and the nicked RNA as primers. Any nicks in the double-stranded cDNA were repaired by E. coli ligase. These molecules were further blunt-ended by adding T4 DNA polymerase. Linkers containing EcoRI and NotI sites were added. The blunt-ended, double stranded cDNA is then fractionated by agarose gel electrophoresis. cDNA roughly greater than 500 base pairs is recovered for further library construction.
  • Vector Construction Construction of a suitable lambda vector containing the desired coding and control sequences employs standard ligation and restriction techniques which are well understood in the art. (Young et al., Nature 316:450-452 (1988)). Site specific DNA cleavage is performed by treating with the suitable restriction enzyme, such as EcoRI, (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog.
  • phage DNA seguence is cleaved by one unit of enzyme in about 20 ⁇ l of buffer solution; in the examples herein, typically, an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37 ⁇ C are workable, although variations can be tolerated. After each incubation, protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction and the nucleic acid recovered from aqueous fractions by precipitation with ethanol.
  • vector construction employing "vector fragments”
  • the vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase (CIP) in order to remove the 5* phosphate and prevent religation of the vector.
  • Digestions are conducted at pH 8 in approximately 150 mM Tris, in the presence of Na+ and Mg+ 2 using about 1 unit of BAP or CIP per ⁇ g of vector at 60°C or 37°C, respectively, for about one hour.
  • the preparation is extracted with phenol/chloroform and ethanol precipitated.
  • religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion of the unwanted fragments.
  • Ligations are performed in 15-50 ⁇ l volumes under the following standard conditions and temperatures: 20 mM Tris-Cl, pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 ⁇ g/ml BSA, 10 mM-50 mM NaCl, and either 40 ⁇ M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C. (for "sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14 ⁇ C (for "blunt end” ligation).
  • Intermolecular "sticky end” ligations are usually performed at 33-100 ⁇ g/ml total DNA concentrations (5- 100 nM total end concentration) .
  • Intermolecular blunt end ligations (usually employing a 10-30 fold molar excess of linkers) are performed at 1 ⁇ M total ends concentration.
  • the nucleotide sequences encoding the beta cell autoantigen may be expressed in a variety of systems as set forth below.
  • the cDNA may be excised by suitable restriction enzymes and ligated into procaryotic or eucaryotic expression vectors for such expression.
  • procaryotic and eucaryotic systems may be used to express the DNA sequences encoding the beta cell autoantigen; procaryotic hosts are the most convenient for cloning procedures. Procaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used.
  • procaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al., Nature 198: 1056 (1977)) and the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake et al., Nature 292:128 (1981)).
  • promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al., Nature 198: 1056 (1977)) and the tryptophan (trp) promoter system (Goeddel et al., Nucleic
  • eucaryotic microbes such as yeast
  • yeast may also be used as hosts.
  • Laboratory strains of Saccharomvces cerevisiae. Baker's yeast are most used although a number of other strains are commonly available.
  • Vectors employing, for example, the 2 ⁇ origin of replication of Broach, Meth. Enz. 101:307 (1983) , or other yeast compatible origins of replications (see, for example, Stinchcomb et al., Nature 282:39 (1979)); Tschempe et al., Gene 10:157
  • Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149 (1968); Holland et al., Biochemistry 17:4900 (1978)).
  • promoters known in the art include the promoter for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073 (1980)), and those for other glycolytic enzymes.
  • Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and enzymes responsible for maltose and galactose utilization. It is also believed terminator sequences are desirable at the 3' end of the coding sequences. Such terminators are found in the 3' untranslated region following the coding sequences in yeast-derived genes.
  • eucaryotic host cell cultures derived from multicellular organisms.
  • These systems have the additional advantage of the ability to splice out introns and thus can be used directly to express genomic fragments.
  • Useful host cell lines include VERO and HeLa cells, and Chinese hamster ovary (CHO) cells.
  • Expression vectors for such cells ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and late promoters from Simian Virus 40 (SV 40) (Fiers, et al..
  • transformation is done using standard techniques appropriate to such cells.
  • transfeetion methods include DEAE-dextran mediated transfection techniques, lysozyme fusion or erythrocyte fusion, scraping, direct uptake, osmotic or sucrose shock, direct microinjection, indirect microinjection such as via erythrocyte-mediated techniques, and/or by subjecting host cells to electric currents.
  • DEAE-dextran mediated transfection techniques lysozyme fusion or erythrocyte fusion, scraping, direct uptake, osmotic or sucrose shock, direct microinjection, indirect microinjection such as via erythrocyte-mediated techniques, and/or by subjecting host cells to electric currents.
  • the beta cell autoantigen may be produced either as a mature protein or a fusion protein, or may be produced along with a signal sequence in cells capable of processing this sequence for secretion. It is advantageous to obtain the protein in its native conformation by obtaining it as it is secreted from the endoplasmic reticulum membrane to the membrane compartment. This minimizes the difficulties in purification; thus, it is preferred to express the beta cell autoantigen gene which includes the codons for native signal sequence in cells capable of appropriate processing. It has been shown that cultured mammalian cells are able to cleave and process heterologous mammalian proteins containing signal sequences, and to secrete them into the membrane compartment. (McCormack et al., Mol. Cell. Biol. 4:166 (1984)).
  • the protein When secreted into the medium, the protein is recovered using standard protein purification techniques. However, while the procedures are more laborious, it is within the means known in the art to purify this protein from sonicates or lysates of cells in which it is produced intracellularly in fused or mature form.
  • the beta cell autoantigen so produced by recombinant means has a determinant site containing the immunodominant epitope associated with IDDM. Accordingly, the beta cell autoantigen is specifically recognized and bound by (a) the DM monoclonal antibody of the present invention, and (b) anti-64 kd autoantibodies produced in a subject in response to the beta cell autoantigen.
  • the beta cell autoantigen so produced by recombinant means can be used to produce monoclonal antibodies to the autoantigen by immunizing suitable animals, such as rats or mice, and then applying the monoclonal antibody techniques discussed above.
  • Human monoclonal antibodies can also be made to the recombinant autoantigen by sensitization of human lymphocytes in vitro followed by EBV transformation or hybridization of the antigen-sensitized lymphocytes with mouse or human lymphocytes.
  • a diagnostic method for detecting autoantibodies reactive with the beta cell autoantigen in a sample from a human.
  • the method can be useful in the diagnosis of IDDM, and in particular, the diagnosis of the early onset of IDDM.
  • the method involves reacting the beta cell autoantigen of the present invention, labeled, for example with a fluorescent label, with a sample of tissue or fluid from a suspected IDDM patient.
  • the sample is fluid, such as serum.
  • the beta autoantigen, fragments or equivalents thereof can be derived from purification of a cell line rich in the beta autoantigen, such as HEp2 cells.
  • the beta autoantigen can be derived from recombinant DNA techniques, preferably a cDNA library.
  • the beta autoantigen derived from cDNA methods can be a fusion protein comprised of a carrier protein and the DM antigen or DM antigen fragment.
  • the carrier protein can be beta-galactosidase or arabinose isomerase.
  • Standard technigues such as direct or indirect immunofluorescence, immunoprecipitation, or enzyme linked immunoabsorbent assay (ELISA) are used to detect reaction between the beta cell autoantigen and autoantibodies reactive with the beta cell autoantigen. These techniques also apply to staining tissue sections to localize antigens or antibodies on the surfaces of or inside cells.
  • a method for diagnosing IDDM by detecting endogenous beta cell autoantigen is a serologic diagnostic technique for detecting endogenous beta cell autoantigen that has been secreted or "shed” into the serum or other biological fluids of patients thought to be suffering from IDDM.
  • the method involves reacting the purified, labeled DM monoclonal antibody of the invention with a sample of human tissue or fluid.
  • the tissue sample is preferably pancreatic islet tissue.
  • the fluid sample is preferably serum.
  • Standard immunological techniques are used to then detect the interaction of the DM monoclonal antibody with endogenous beta cell autoantigen, fragments or equivalents of endogenous beta cell autoantigen.
  • the method employs the DM antibody conjugated to a label capable of producing a detectable signal.
  • the label can be selected from the group consisting of a radionuclide, an enzyme, a fluorescer and a chromophore.
  • a vaccine and methods for treating IDDM patients with the vaccine contains an effective amount of the DM monoclonal antibody in association with a pharmaceutically acceptable carrier vehicle.
  • the vaccine is effective because the DM monoclonal antibody contains the same idiotypic determinant site as the beta cell autoantigen autoantibody.
  • a method of the present invention comprises introducing into individuals the vaccine comprising the monoclonal antibody, stimulating the production of anti-id antibodies directed toward the autoantibodies.
  • the anti-id antibodies prevent or retard destruction of pancreatic islet cells by anti-64 kd autoantibodies.
  • This method comprises introducing the vaccine preferably into subjects who are either already producing islet cell autoantibody or who are likely to begin producing islet cell autoantibody.
  • islet cell autoantibodies are those who are first degree relatives of probands with IDDM.
  • Vaccines may be prepared froir one or more immunogenic polypeptides encoded within cDNA sequences isolated by utilizing the cDNA sequences encoding the DM monoclonal antibody or from the genome of hybridoma ATCC No. HB 10502 to which they correspond.
  • the preparation of vaccines which contain an immunogenic polypeptide(s) as active ingredients, is known to one skilled in the art. (See, for example, Voller and Friedman, Eds. New Trends and Developments in Vaccines. University Park Press, Baltimore, MD (1978)).
  • Administration to an IDDM patient of a vaccine which comprises immunogenic polypeptide(s) associated with the DM monoclonal antibody stimulates in vivo the production of anti-id antibodies against anti 64 kd autoantibodies.
  • Such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection, may also be prepared. The preparation may also be emulsified, or the protein encapsulated in liposomes.
  • the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP) , N-acetyl-nor-muramyl- L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP) , N-acetylmuramyl-L-alanyl-D-sialoglutaminyl-L- alanine-2-(1•-2•-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton
  • the effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing DM monoclonal antibody antigenic sequence resulting from administration of this polypeptide in vaccines which are also comprised of the various adjuvants.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols, e.g., polyethylene glycol, or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25%-70%.
  • the autoantigen may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine, and the like.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
  • the quantity to be administered which is generally in the range of 5 micrograms to 250 micrograms of antigen per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient reguired to be administered may depend on the judgment of the practitioner and may be peculiar to each subject.
  • the vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reenforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • the dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.
  • the vaccine containing the immunogenic DM monoclonal antibody antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, immunoglobulins.
  • the antibodies of the invention are also used in therapeutic procedures for treating IDDM. Falling within the scope of the embodiments of the present invention is a method of immunotherapy to treat IDDM that involves two steps.
  • a first step employs an immunoaffinity column designed to remove autoantibodies specifically reactive with beta cell autoantigen from a patient's plasma.
  • the immunoaffinity columns loaded with the anti-id antibodies and/or autoantigen of the present invention can remove a clinically significant percentage of autoantibodies specifically reactive with beta cell autoantigen from the plasma of an IDDM patient.
  • Plasmapheresis which is a method for the treatment of autoimmune diseases, can be used for carrying out this procedure.
  • plasma is removed from an individual and passed through columns containing ligands for specifically removing from the plasma the autoantibodies responsible for attacking and destroying specific tissue (Jones et al. Plasma Therapy Transfusion & Technology. 7:333-349 (1986).
  • This method typically involves extracorporeal immunoadsorption for the reduction of plasma factors known to be associated with enhancement of immune response in IDDM.
  • An immunoadsorptive column can have protein ligands covalently bound to an inert silica matrix.
  • the patient's whole blood is separated using either an apheresis machine or centrifuge. A specified volume of the plasma is perfused through the column and then returned to the patient.
  • the method of the present invention accordingly employs a plasmapheresis column loaded with anti-id antibodies and/or autoantigen.
  • the column is tested with one patient's plasma at a predefined dilution.
  • An individual preparation of anti-idiotypic antibody is initially immobilized on CNBr-activated Sepharose 4B gel.
  • Plasma from an individual is run through the column using conventional plasmapheresis protocols.
  • This first step involves treating the peripheral circulation of a patient for a sufficiently long time to remove substantially all of the circulating anti-64 kd autoantibodies (Jones et al., Plasma Therapy Transfusion & Technology. 7:333-349 (1986)).
  • the peripheral circulation is that portion of the blood circulating from the heart through the arteries, capillaries, and veins back again to the heart.
  • a typical plasmapheresis apparatus is made by Prosorba(TM) , (Imre Corp. Seattle, WA) .
  • the ligand is preferably the anti-id antibodies that specifically recognize and bind autoantibody specifically reactive with beta cell autoantigen. Beta- cell autoantigen may also be used as the ligand.
  • an immunoassay such as ELISA, the peripheral circulation can be assayed after various treatment periods for the presence of autoantibodies by reacting a sample of plasma with anti-id antibody.
  • the second step of destroying lymphocytes involves introducing to a human a sufficient amount of anti-id antibody reactive with the autoantibody.
  • the invention uses anti-id antibodies to recognize and bind to lymphocytes that present anti-beta cell autoantigen autoantibody on their surface. Autoantibodies presented on the surface of these lymphocytes combine with the anti-id antibodies to form an antigen-antibody complex. This complex reacts with complement and results in destruction, that is, lysis of the cell.
  • the anti-id antibody suppresses the generation of the immune response to beta cell autoantigen.
  • anti-id antibodies or beta cell autoantigen are linked with toxins and carry the toxins to the surfaces of B-lymphocytes that are producing autoantibody. At the surfaces of these cells, the anti- id antibodies or autoantigen react and bind specifically with autoantibodies. The toxin molecule is then absorbed into the B-cell, killing it.
  • toxins include, but are not limited to, abrin and gelanin (Thorpe, P.E. et al., Eur. J. Biochem.. 116:447- 454 (1981)), and diphtheria toxin A chains and ricin A chains (Oeltmann, T.N. and Forbes, J.T., Arch. Biochem. Biophys.
  • Anti-id antibodies also cause destruction of a specific population of T-cells which had been previously presented beta cell autoantigen by a macrophage. These T-cells have receptors on their surfaces that specifically react with anti-id antibody of the present invention. Accordingly, anti-id antibody, by itself or conjugated with a toxin, will cause toxicity to these T-cells. Because autoantigen specifically reacts with autoantibody, autoantigen linked with a toxin will destroy B- lymphocytes and does so by specifically reacting with and binding to autoantibody located at the surface of these B-lymphocytes (Kernan et al., JAMA. 259:3154-3157 (1988), Blythman et al.. Nature. 290:145-146 (1981)).
  • the spleen was removed and the cells obtained therefrom obtained were fused with myeloma line HL1-653 (Ventrex Laboratories, Inc., Portland, Maine) according to the procedure described in the Ventrex product brochure, following the method of Taggart, R. et al. Science 219:1228 (1983).
  • the myeloma cells were washed twice with complete medium without fetal calf serum (FCS) .
  • FCS fetal calf serum
  • the spleen cells were washed twice with serum free medium.
  • 10 8 spleen cells were combined with 10 7 myeloma cells in a 50 cc conical tube. This mixture was washed again with serum free medium then spun down. The supernatant was discarded and the pellet loosened.
  • the tube containing the cell mixture was immersed in a 37° water bath. 1 ml of 50% PEG is added with gentle shaking over a minute. Over the next 7 minutes, with gentle shaking of the tube, 12 ml of RPMI medium without serum was added. The cell mixture was then centrifuged at 1000 rpm for 5 minutes.
  • the pellet was gently resuspended in enough AAT medium (Adenine, 7.5 x 10 "5 M, Aminopterin, 8 x 10 "7 M, Thymidine, 1.6 x 10 "5 M) to yield about 3 x 10 6 spleen cells per ml. About 150 microliters of cell mixture was plated per well. Fused cells were plated on 96-well culture plates. Selection of hybridoma clones was accomplished by testing the binding of supernatants of the clones to frozen human pancreas sections using the immunofluorescence assay procedure described in Example 2 below. Positive clones were further subcloned by limited dilution. Hybridoma ATCC No.
  • HB 10502 was subcloned from the limited dilution of one of these positive clones. Binding of antibodies produced by hybridoma ATCC No. HB 10502 to frozen pancreas sections was confirmed according to the procedures in Example 2, infra. (Bonifacio et al., . Immunol. Methods. 106:83- 88 (1988)). Adaptation of Hybridoma Cell Line ATCC No. HB 10502 to Serum Free Medium
  • Hybridoma cell line ATCC No. HB 10502 was adapted to serum free medium to simplify subsequent purification of the monoclonal antibody free of contaminating proteins, specifically other contaminating serum- originating immunoglobulins.
  • Hybridoma line ATCC No. HB 10502 was originally grown in HL1 medium (Ventrex, Portland, Maine) plus 1% calf bovine serum.
  • HL1 medium is optimized for the growth of hybridoma lines derived from myeloma lines HL1-653.
  • the adaptation procedure was initiated by changing the growth medium to WRC935 medium (Amicon, Danvers, MA) plus 1% calf bovine serum. After one to two weeks of adaptation, the serum concentration was gradually reduced to 0.5%, then to
  • DM monoclonal antibody by the hybridoma of the present invention was doubled as estimated by an ELISA method using the goat anti-mouse IgM (mu chain specific)-alkaline phosphatase conjugate as secondary antibody. (Engvall and Perlmann, Immunochemistry 8:871-879 (1971); Van Weeman and Schuurs, FEBS Lett. 15:232-236 (1971)).
  • the serum-free cultured medium was collected by centrifugation and concentrated 60 to 100 fold.
  • the concentrate was treated with DNase I and RNase A at concentrations of 1 microgram/ml respectively at 4°C for 2 hours.
  • Six milliliters of clarified concentrate was loaded on a Sephacryl S-300 column (1.5 cm x 120 cm) (Pharmacia, Piscataway, NJ) and eluted with PBS buffer.
  • the fractions containing the antibody were identified by SDS-PAGE and pooled.
  • the purity of the DM monoclonal antibody was examined on SDS-PAGE. Purity was greater than approximately 95% and the preparation was free of bovine IgM.
  • Tissues were obtained from different organs. These organs and the cell lines tested are listed in Table 1 ("Characterization of the specificity of DM Monoclonal Antibody to different tissues and cell lines by IFA Method") . These tissues were islets from human, rat and dog; human pancreas and kidney, rat stomach, heart, liver and kidney. The following cell lines were obtained: myeloma cells HL1-653 (Taggart, R.T. et al.. Science 219:1228-1230 (1983)) from mouse; rat insulinoma cells D11D5 (Carrington, CA. et al., J. Endocrin. 109:190-200 (1986)); and human epidermoid carcinoma cells - HEp2 (Moore, A.E. et al. Cancer Res. 15:598 (1955) ) .
  • the tissues were prepared according to Bonifacio et al., . Immunol. Methods, 106: 83-88 (1988). Tissue sections were cut at 4 microns thickness by cryostat and stored in a sealed plastic bag at -80°C until used. The 4-micron thick tissue sections were thawed at room temperature and incubated with purified DM monoclonal antibody serially diluted in phosphate buffered saline (PBS) , produced as described above in Example 1. The incubations were carried out in a humidified chamber for two hours (or overnight) at room temperature. The sections were washed once with PBS - 0.05% Tween 20 and three times with PBS alone with gentle shaking.
  • PBS phosphate buffered saline
  • the sections were incubated with goat anti-mouse (IgG and IgM)-FITC conjugate (at 1:80 dilution) for one hour. After repeating the washing procedure as described above, the sections were coated with mounting buffer, covered with a cover slide and examined under a fluorescent microscope for binding of the DM monoclonal antibody to the various tissues.
  • goat anti-mouse IgG and IgM
  • FITC conjugate at 1:80 dilution
  • the DM monoclonal antibody stained the frozen human pancreas sections but not sections of other organs, such as human kidney and stomach, and rat stomach, heart, liver and kidney. As shown in Table 1, these results demonstrate the specificity of the DM monoclonal antibody for pancreatic tissue, and for islet beta cells which are present in the pancreas. DM monoclonal antibody staining of the pancreas sections also was observed in the epidermal cell layers of the pancreas. For that reason, an epidermoid cell line was tested for reactivity with DM monoclonal antibody. Neuronal cell lines (Table 2, below) were also tested for reactivity with DM monoclonal antibody as neuronal cells and epidermal cells have a common embryonic origin.
  • Purified islet cells less than three days after harvesting were maintained in RPMI-1640 medium supplemented with 10% bovine serum.
  • the cell lines e.g. rat insulinoma and mouse myeloma
  • the cell lines were grown to confluency in RPMI medium supplemented with 10% calf serum.
  • Cells were detached from the culture flask using trypsin-EDTA (Irvine Scientific, CA) and reseeded on non-treated culture flasks. After 24 hours of cultivation, the cells were harvested by gentle suction and washed one time with PBS.
  • DM Monoclonal antibody was serially diluted with PBS and incubated with cell suspensions at 10 5 cells/ml for two hours at room temperature.
  • the cells were resuspended in PBS containing goat anti-mouse (IgG and IgM)-FITC conjugate at 1:80 dilution, and incubated at room temperature for another hour. After the cells were washed, they were examined under the microscope.
  • IgG and IgM goat anti-mouse
  • Table 1 shows that the DM monoclonal antibody was able to bind to the surface of live human, rat and dog islet cells. Binding to insulinoma cells was also observed.
  • the DM monoclonal antibody was not able to bind to the mouse myeloma line HL1-653, from which hybridoma cell line ATCC No.HB 10502 was derived. As shown in
  • DM monoclonal antibody was able to bind to the cytoplasmic area of fixed human epidermoid carcinoma (HEp2) cells. These binding studies were conducted using the Fluorescent ANA Test, Immunoconcepts, Sacramento, CA.
  • Beta Cell Autoantigen-Rich Cells Different cell lines listed in Table 2 were grown in RPMI-1640 medium supplemented with 10% calf bovine serum. One day before harvesting, the cells were treated with trypsin-EDTA to detach them from the petri dish or culture flask walls. After being washed with RPMI medium the cells were resuspended in the same medium and grown in the non-treated petri dishes in which attachment of the cells to the plastic walls was reduced. Twenty million cells collected from the overnight culture were resuspended in 1 ml of lysis buffer.
  • This buffer contained 20 mM HEPES, pH 7.4, 5 mM benzamidine, 10 mM N-ethylmaleimide, 0.1 mM Na vanadate, 5 mM Na pyrophosphate, 2.5 mM EDTA, 2.5 mM NaF, 0.01% trypsin inhibitor, and 2% Triton X-114.
  • the cells were disrupted by continuously rocking for 4 hours at 4 ⁇ C
  • the extracted proteins were obtained after removing the cell debris by centrifugation at 100,000 x g for 10 minutes at 4° C. 75 micrograms of extracted protein from each cell line was separated on 7.5% or 11.25% SDS-PAGE and electroblotted to the nitrocellulose paper.
  • nitrocellulose paper was then incubated with 10 micrograms/ml of purified DM monoclonal antibody in PBS buffer with 1% nonfat milk at room temperature overnight. After 4 brief washings with PBS, the nitrocellulose paper was incubated with goat-anti-mouse IgM horseradish peroxidase conjugate (at 1:500 dilution) for 2 hours and then developed.
  • Table 2 shows that binding of the DM monoclonal antibody to HEp2 cells occurred with greater intensity than to other cell lines tested, including rat insulinoma. (See Example 5 for use of HEp2 cells as source of the beta cell autoantigen.)
  • the first experiment involved the ability of either the DM monoclonal antibody or autoantibodies to block the other from binding to the beta cell autoantigen in situ.
  • This blocking experiment had two parts. In the first part, the ability of DM monoclonal antibody derived from mouse cells to block binding of human islet cell autoantibody to beta cell autoantigen was tested. In the second part, the ability of the human autoantibody to block the binding of the DM monoclonal antibody to beta cell autoantigen was tested.
  • the second experiment involved the ability of a mixture of DM monoclonal antibody and autoantibodies to simultaneously exclude each other from binding to the beta cell autoantigen.
  • This simultaneous exclusion experiment had two parts. In the first part, autoantibody concentration was held constant and mixed with various concentrations of DM monoclonal antibody. In the second part, DM monoclonal antibody concentration was held constant and mixed with various concentrations of autoantibodies.
  • Table 3 indicates that DM monoclonal antibody was able to partially inhibit human islet cell antibody (ICA) positive serum from binding to human pancreas sections. Inhibition of ICA binding by DM monoclonal antibody was more effective at lower ICA concentrations. These results demonstrate that occupation of a binding site on beta cells by the DM monoclonal antibody precludes binding of human ICA positive serum to the same or nearby site.
  • ICA islet cell antibody
  • ICA positive serum reflects the production of islet cell autoantibodies in response to an immunodominant epitope, and since such autoantibodies bind to this immunodominant epitope, it follows that either (a) the DM monoclonal antibody recognizes and binds to an epitope; that is the immunodominant epitope of human ICA positive serum or (b) an epitopic site on the autoantigen which, when occupied by the DM monoclonal antibody, precludes binding of the human ICA positive serum to the autoantigen.
  • human ICA positive sera can block mouse DM monoclonal antibody from binding to islet beta cells in pancreas sections, a similar procedure was carried out as described above.
  • the cells were first incubated with human ICA positive sera, and then incubated with mouse DM monoclonal antibody. The cells were then treated with goat anti-mouse (IgG and IgM)- FITC conjugate. Further blocking studies revealed that human ICA positive sera were able to block mouse DM monoclonal antibodies from binding to islet beta cells.
  • human ICA positive sera were able to block mouse DM monoclonal antibodies from binding to islet beta cells.
  • DM monoclonal antibody Different concentrations of DM monoclonal antibody were mixed with 40 Juvenile Diabetes Foundation (JDF) units of pooled human ICA positive sera and incubated with human pancreas sections overnight at room temperature in a humidified chamber. The sections were then washed and incubated with anti-human (IgG and IgM)- FITC conjugate for one hour as described above in this
  • the isolated beta cell autoantigen is critically important for development of a diagnostic reagent for the early detection of the early onset of IDDM.
  • the autoantigen is of further importance for the development of therapeutic agents for the removal of islet cell autoantibodies and for the destruction of IDDM-causing lymphocytes.
  • Rat insulinoma D11D5 cells (Carrington, CA. et al., J. Endocrin. 109:193-200 (1986)) were used to determine the antibody-dependent complement-mediated cytotoxicity of DM monoclonal antibody.
  • D11D5 cells were trypsinized and reseeded in non-treated petri dishes. The cells were harvested, resuspended in R10 medium (10% calf serum in RPMI medium) , counted and adjusted to a final cell concentration of 10 6 cells/ml. 60 microcuries of 51 Cr (specific activity 421 mC/mg) per 10 6 cells were added and incubated at 37°C for one hour.
  • the cells were centrifuged at 1500 rpm for 5 minutes and resuspended in the R10 medium and incubated for another one hour. After washing with 20 ml of PBS twice, the cells were resuspended at a concentration of 1.8 x 10 6 cells/ml.
  • 0.1 ml of cell suspension (1.8 x 10 5 cells) was delivered to each tube and 0.1 ml of the DM monoclonal antibody solution-TBS buffer was added to make final concentrations of 0, 0.5, 1, 2, 10 micrograms/ml.
  • the mixture was then incubated at 37 ⁇ C 0.2 ml of rabbit complement (Lot 10, One Lambda Company, Los Angeles, CA) was added to each tube and incubated for another one hour at 37 "C.
  • the released 51 Cr was collected by centrifugation at 4000 rpm for five minutes.
  • the residual 51 Cr in the cells was released by lysing the cells with 0.1% SDS and counted.
  • the specific cytotoxicity was calculated by the formula as described in Table 5) .
  • DM monoclonal antibody has antibody-dependent complement- mediated cytotoxicity to rat insulinoma D11D5 cells.
  • the best killing effect of DM monoclonal antibody was achieved at a concentration of 1 microgram/ml.
  • the results of Table 5 are consistent with the hypothesis that the humoral immune response to the approximately 64,000 dalton beta cell autoantigen may be in part responsible for the destruction of islet cells.
  • Humoral immunity is mediated by antibodies that are in the body fluids.
  • the humoral immune response to the beta cell autoantigen is also discussed in Example 5.
  • mice Twenty three Balb/C mice weighing from 20 g to 27 g were divided into two groups.
  • the fasting blood glucose level was measured at day 38 by removing food 14 hours prior to collecting blood samples from the tail vein.
  • the glucose concentration was determined by a commercial kit (Sigma Chemical Co., St. Louis, MO).
  • Table 6 shows the in vivo effects of the DM monoclonal antibody on blood glucose level of Balb/C mice during the glucose tolerance test.
  • mice developed acute paralysis after one to two months of injecting the DM monoclonal antibody.
  • Baekkeskov et al. .Nature. 347:151-156 have demonstrated that autoantibodies associated with the development of IDDM specifically react with a 64 kd antigen associated with a subpopulation of central nervous system neurons.
  • neuronal tissues e.g. human neuroblastoma and human glioblastoma react specifically with DM monoclonal and are a rich source of the beta cell autoantigen.
  • Example 2 After the unexpected finding in Example 2 of relatively strong reactivity between DM monoclonal antibody and human epidermoid carcinoma cells and neuronal cells, the cell lines listed in Table 2 were evaluated to determine the richest source of beta cell autoantigen for subsequent isolation.
  • Six different cell lines as listed in Table 2 were grown in RPMI-1640 medium supplemented with 10% calf bovine serum.
  • the cells were treated with trypsin-EDTA to detach them from the petri dish or culture flask wells. After being washed with RPMI medium, the cells were resuspended in the same medium and grown in the non- treated petri dishes where attachment of the cells to the plastic wells was reduced.
  • HEp2 cells, glioblastoma, and neuroblastoma cells were tested for their reactivities with the DM monoclonal antibody.
  • Table 2 shows that all of the cell lines contain an approximately 64,000 dalton immunoreactive protein band, each with different relative intensities.
  • HEp2 and D11D5 have an additional approximately 52,000 dalton protein that can react with this antibody. The ratio between the approximately
  • HEp2 cells were grown as described in Example 5. After being trypsinized and regrown on non-treated petri dished for 8 to 16 hours at 37"C, the cells were collected by repeatedly pipetting using a 10 ml pipet and transferred to a 50 ml centrifuge tube. The cells were pelleted by centrifugation at 1000 x g for 5 minutes. The cell pellets were resuspended in one-fifth original volume of PBS. 1 ml of the resuspension was transferred to a 1.5 ml Eppendorf tube. The cells were repelletted at 1000 x g for two minutes at room temperature. After the supernatant was removed, the pellets were frozen at - 80 ⁇ C
  • the frozen pellet was added, at a ratio of 1 ml per 20 to 40 million frozen cells, a solution of 20 mM phosphate buffer, pH 7, 10 mM N-ethylmaleimide, 0.01% trypsin inhibitor, and 2.5 mM EDTA.
  • the pellets were thawed and resuspended by repeatedly pipetting at room temperature.
  • the suspended cells were then rocked for 30 minutes at 4°C
  • the aqueous soluble fraction was collected by centrifugation at 1500 x g for 2 minutes at room temperature.
  • the pellets (Pellet 1) were washed once with half volume of the same buffer and pooled.
  • the combined supernatant was further centrifuged at 100,000 x g for 2 hours at 4 ⁇ C
  • the pellet (Pellet 2) was resuspended in 1 ml of 10 mM phosphate buffer, pH 7, 2% Triton X-114 (Buffer T) and rocked at 4°C for 2 to 16 hours.
  • the extract was collected by centrifugation at 100,000 x g for 30 minutes at 4"C
  • the Triton X-114 detergent condensation was then performed as described in Example 5.
  • the aqueous phase of the extract (supernatant 2 or SS2) was stored at 4°C or frozen at - 80°C until further purification.
  • the SS2 was centrifuged at 100,000 x g for 10 minutes to remove the aggregates.
  • a MonoQ (Pharmacia) column was employed to further purify the 64 kd autoantigen.
  • the clarified SS2 was directly loaded onto the MonoQ column pre- equilibrated with 10 mM phosphate buffer, ph &, 0.01% Triton X-114. The gradient extended from 0 to 0.5 M NaCl in the same buffer. The majority of 64 kd autoantigen was detected between 0.15 and 0.2 M NaCl.
  • the peak fractions were pooled for further analysis. It was estimated that the 64 kd autoantigen had been enriched by at least 400-fold from the total cell extract. The purity of aqueous, soluble 64 kd autoantigen was estimated at 10% to 30 %.
  • Pellet 1 prepared as previously described, was used to further purify the aqueous-insoluble form of 64 kd autoantigen. 1 ml of 10 mM phosphate buffer, pH 7, with 2% Triton X-114 was added per amount of lysate equivalent to that derived from 20 to 40 million cells, i.e.., equivalent to Pellet 1, and rocked for 2 hours at 4"C The lysate was clarified at 100,000 x g for 30 minutes at 4°C The detergent condensation procedure was performed to remove excess detergent as described previously.
  • the aqueous phase of the extract (pellet- supernatant 1 or PSI) was stored at 4°C or frozen at - 80"C until further purification.
  • the next purification step also employed the MonoQ column chromatography in a manner as described above. It was estimated that the 64 kd autoantigen was enriched more than 100-fold after this step, and was deemed suitable for serving as a reagent for diagnosing anti-64 kd autoantibodies in the sera of IDDM patients. (See Example 7).
  • the partially purified autoantigen was also suitable for undergoing protein sequencing or peptide mapping.
  • Beta cell autoantigen extracted from HEp2 cells and partially purified as described in Example 6 was used to develop a diagnostic reagent for specific reactivity with islet cell autoantibody.
  • an enzyme- linked immunoassay (EIA) method was developed to detect anti-64 kd autoantibodies in patients' sera.
  • EIA enzyme- linked immunoassay
  • Approximately 10 micrograms/ml of the partially purified autoantigen was coated on an Immunolon II plate (Dynatech Laboratories, Inc., Chantilly, VA) overnight at 4°C.
  • Aqueous phase prepared from Buffer T (see Example 6) after detergent condensation was used as a non-antigen background control.
  • TBS-2% Tris Buffer: 10 mM Tris, pH 7.4, 150 mM NaCl
  • BSA 0.05% azide
  • 100 microliters of human sera at 1:100 dilution in the same buffer were added to each well and incubated for 30 minutes at room temperature with shaking.
  • 100 microliters of goat anti-human Ig-alkaline phosphatase conjugate (1:1000 dilution) in TBS-2% BSA and 5% azide buffer were added to each well and incubated for another 30 minutes. The washing step was repeated and the antibody binding was determined using phosphatase substrate (1 mg/ml buffer, Sigma) .
  • IDDM patient sera could immunoreact with a protein band at approximately 64 kd in size.
  • the intensity of the signal was considerably weaker than the immunoreactivity of the DM monoclonal antibody.
  • the autoantigen would be suitable for use as an immunodiagnostic for IDDM in methods employing, for example, immunoprecipitation or immunoblot.
  • mRNA Isolation A general procedure for obtaining total cellular RNA encoding beta cell autoantigen was followed according to the method of Chirgwin et al., Biochemistry 18:5294 (1979). While selection of polyadenylated mRNA from total cellular RNA may be accomplished with a procedure described in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, pp. 197-198, (1982), a Fast Track mRNA Kit (Invitrogen, San Diego, CA) was employed to isolate mRNA directly from HEp2 cells without having to isolate total cellular RNA as an intermediate. The cells were grown to confluency in RPMI medium supplemented with 10% calf serum.
  • Cells were detached from the culture flask using trypsin-EDTA (Irvine Scientific, Irvine, CA) and reseeded on culture flask. After 24 hours of cultivation, the cells were harvested by gentle suction and washed one time with PBS. The harvested cells were frozen under liquid nitrogen until used. 10 8 cells were used to isolate the mRNA according to the manufacturer's protocol (Invitrogen, San Diego, CA) . Approximately 20 micrograms of purified mRNA was obtained.
  • DNA polymerase I DNA polymerase I, RNase, and E. coli DNA ligase.
  • Reverse transcriptase was used to synthesize a cDNA from an RNA template in the presence of oligo dT primer and random primer.
  • the E. coli RNase then cleaved RNA while in a hybrid RNA-DNA form.
  • the enzyme E. coli DNA polymerase I then used the fragments of nicked RNA as primers to synthesize the second strand of cDNA. Any nicks in the double-stranded cDNA were repaired by E ⁇ coli ligase.
  • a modification of this protocol was used involving the addition of T4 DNA polymerase to the reaction after the E. coli DNA polymerase I reaction was completed.
  • a LibrarianTM cDNA Library Construction system (Invitrogen, San Diego, CA) was used to construct a bacteriophage lambda gtll cDNA library. (Young et al.. Nature, 316:450-452 (1988)). Approximately 5 micrograms of purified mRNA from HEp2 cells were used to prepare the lambda gtll library according to the manufacturer's procedure. It is estimated that approximately 1.24 x 10 6 primary phage clones were obtained from one preparation and among them 9 x 10 5 primary clones (72%) contained inserts as determined by a lack of beta-galactosidase activity. The primary library was divided into four parts and amplified separately. The insert frequency dropped from 72% to 62% after amplification.
  • IPTG isopropyl beta-D thiogalactopyranoside
  • nitrocellulose papers were incubated with PBS containing 1% skim milk and 5 micrograms/ml DM monoclonal antibody for four to five hours, or overnight, at room temperature with shaking. After washing with PBS four times, the papers were incubated with goat anti-mouse IgM-horse radish peroxidase conjugate (1:500 dilution) in the same buffer for two hours. After washing with
  • beta-galactosidase-fusion proteins were produced. Fusion of the beta cell autoantigen nucleotide sequence with the beta galactosidase nucleotide sequence is believed to be essential for efficient transcription and translation in a procaryotic cell host, such as E. coli.
  • Beta-galactosidase-fusion proteins were produced as follows. Cell lysates from several clones were collected from plates after soaking with phage buffer (10 mM Tris, pH 7.5, 10 mM MgS0 A , 100 mM NaCl), or were prepared from the lysogen lysate, as described in Maniatis, et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, p. 254, (1982). The lysates were separated on a 7.5% SDS-PAGE gel and electrotransf rred to the nitrocellulose paper according to the methods of Burnette, Anal. Biochem.. 112:195-203 (1981) and Towbin et al, Proc. Natl.
  • the fusion proteins consisting of the beta galactosidase gene and the gene for the beta cell autoantigen were detected with the DM monoclonal antibody or with mouse anti-beta galactosidase monoclonal antibodies. Most clones tested thus far contained a protein detectable using the DM monoclonal antibody. Except for one clone, the protein bands observed were larger than the intact beta- galactosidase. In addition to a top band, other protein bands, which were presumably degradation products of beta-galactosidase, also were detected by anti-beta galactosidase monoclonal antibodies.
  • the eighteen positive clones were identified at low stringency conditions as described above. These clones were further characterized at high stringency conditions as follows: E. coli strain Y1090, which was infected with approximately 5 to 200 phage forming units (pfu) , was plated on an 82 mm TYE agar plate and overlaid with IPTG-treated nitrocellulose paper as described previously. The nitrocellulose papers, which absorbed the E. coli lysate, were then incubated with TBS containing 1% skim milk and 10 micrograms/ml of DM monoclonal antibody for 4 hours or overnight at room temperature.
  • the filter papers were washed twice with TBS-0.5% Tween 20 and twice with TBS for five minutes each and then incubated with goat anti-mouse IgM- horseradish peroxidase conjugate (1:1000 dilution) for 2 hours.
  • the nitrocellulose papers were washed under the same conditions as described above and developed with DBA (3,3'-diaminobenzidine) substrate.
  • DBA 3,3'-diaminobenzidine
  • the cDNA inserts comprising sequences coding for the beta cell autoantigen, and in particular, the immunodominant epitope specifically reactive with the DM monoclonal antibody and the anti-64 kd autoantibody, were isolated from recombinant phage clones 4 and 14 with EcoRI digestion and cloned into the EcoRI site of plasmid pUC13. Restriction mapping was accomplished using standard techniques as specified in Maniatis, et al.. Molecular Cloning; A Laboratory Manual- Cold Spring Harbor Press, (1982) .
  • the restriction map for plasmid pUH14 which is a subclone of clone 14, is illustrated in Figure 1. The map indicates a DNA insert sequence comprising about 1400 bases.
  • Each terminus is marked by an EcoRI site.
  • Two Pst restriction sites are located at about 180 bases and at about 670 bases respectively from one of the EcoRI termini. Located at about 1000 bases and at about 1200 bases from this EcoRI terminus are, respectively, a Hindlll site and a Sad site.
  • the fusion protein that clone 14 produced had a molecular weight of approximately 110-115 kd which is slightly larger than the authentic beta-galactosidase as judged by the mobility of the immunoreactive band on the Western blot.
  • Clone 4 contained an approximately 0.75 kb cDNA insert.
  • the fusion protein that clone 4 produced had a molecular weight of approximately 120-130 kd as determined by immunoblot using either DM monoclonal antibody or anti-beta galactosidase monoclonal antibody.

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Abstract

L'invention concerne une lignée cellulaire continue produisant un anticorps monoclonal du diabète sucré réagissant avec l'auto-antigène de cellules bêta dans des cellules bêta pancréatiques humaines ayant une masse moléculaire d'approximativement 64.000 daltons et associé à l'apparition du diabète sucré insulino-dépendant (IDDM). L'anticorps du diabète sucré sert dans des procédés de dosages diagnostiques ainsi que dans des procédés de traitement et de prévention du diabète sucré insulino-dépendant. L'invention concerne également l'auto-antigène des cellules bêta d'une masse moléculaire d'approximativement 64.000 daltons associé à l'apparition du diabète sucré insulino-dépendant, réagissant avec l'anticorps monoclonal du diabète sucré. On utilise l'auto-antigène des cellules bêta afin de diagnostiquer l'apparition précoce de diabète sucré insulino-dépendant. De plus, l'invention concerne des procédés immunothérapeutiques employant des anticorps monoclonaux anti-idiotypiques ainsi que l'auto-antigène des cellules bêta. On utilise ces procédés dans l'inhibition de la liaison d'auto-anticorps des cellules des ilôts de Langerhans du pancréas contre des cellules des ilôts pancréatiques, et afin d'éliminer des auto-anticorps et des lymphocytes des cellules des ilôts de Langerhans du pancréas réagissant avec l'auto-antigène des cellules bêta de la circulation périphérique d'un sujet.
PCT/US1991/007052 1990-10-02 1991-09-25 Anticorps et procedes de diagnostic et de traitement du diabete WO1992006105A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5674978A (en) * 1990-09-21 1997-10-07 The Regents Of The University Of California Peptides derived from glutamic acid decarboxylase
US5998366A (en) * 1990-09-21 1999-12-07 The Regents Of The University Of California Method for ameliorating glutamic acid decarboxylase associated autoimmune disorders
EP2478364A4 (fr) * 2009-09-15 2013-02-20 Gemini Research Limited Détection d'anticorps
CN111175518A (zh) * 2020-01-08 2020-05-19 杭州北角医学检验所有限公司 一种用于ⅰ型糖尿病联合抗体检测试剂盒及其制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990007117A1 (fr) * 1988-12-13 1990-06-28 University Of Florida Procedes et compositions servant a la detection precoce et au traitement du diabete sucre dependant de l'insuline
EP0383129A2 (fr) * 1989-02-17 1990-08-22 Bayer Corporation Antigènes de cellules islet pancréatiques obtenus par clonage moléculaire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990007117A1 (fr) * 1988-12-13 1990-06-28 University Of Florida Procedes et compositions servant a la detection precoce et au traitement du diabete sucre dependant de l'insuline
EP0383129A2 (fr) * 1989-02-17 1990-08-22 Bayer Corporation Antigènes de cellules islet pancréatiques obtenus par clonage moléculaire

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ANNUAL REVIEW OF IMMUNOLOGY, Volume 4, issued 1986, GAULTON et al., "Idiotypic Mimicry of Biological Receptors", pages 255-280. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5674978A (en) * 1990-09-21 1997-10-07 The Regents Of The University Of California Peptides derived from glutamic acid decarboxylase
US5846740A (en) * 1990-09-21 1998-12-08 The Regents Of The University Of California Methods and kits useful for detecting autoantibody to glutamic acid decarboxylase
US5998366A (en) * 1990-09-21 1999-12-07 The Regents Of The University Of California Method for ameliorating glutamic acid decarboxylase associated autoimmune disorders
US6011139A (en) * 1990-09-21 2000-01-04 The Regents Of The University Of California Hybridomas and monoclonal antibodies that specifically bind to glutamic acid decarboxylase peptides
US6455267B1 (en) 1990-09-21 2002-09-24 Diamyd Medical Ab Methods and kits useful for determining the status of and detecting pancreatic B-cell associated autoimmune diseases
US8926966B1 (en) 1990-09-21 2015-01-06 Regents Of The University Of California Cloned glutamic acid decarboxylase
EP2478364A4 (fr) * 2009-09-15 2013-02-20 Gemini Research Limited Détection d'anticorps
US8883517B2 (en) 2009-09-15 2014-11-11 Gemini Research Limited Detection of antibodies
CN111175518A (zh) * 2020-01-08 2020-05-19 杭州北角医学检验所有限公司 一种用于ⅰ型糖尿病联合抗体检测试剂盒及其制备方法

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