Use of monoclonal antibodies to distinguish protein conformational isoforms
First Claim
1. A method of detecting prion disease in a sample, said method comprising:
- contacting said sample with a diagnostically effective amount of an antibody characterized by its ability to bind to PrPCTM in situ; and
determining whether said antibody binds specifically to any material in said sample under conditions whereby differential binding of said antibody to PrP isoforms associated with prion disease occurs, wherein antibody binding is indicative of prion disease.
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Abstract
Methods of preparing monoclonal antibodies that differentially bind to a single conformer of a protein of interest are described. Passive immunization using these antibodies as well as use of conformer-specific antibodies as diagnostic reagents for the purpose of stratification of patient populations with regards to disease outcome, drug efficacy or drug sensitivity is also disclosed as well as active immunization with the protein conformer. In the screening techniques, detection can be for example by tissue immunostaining, western blotting or solution IP. A specific mab termed 7VC which shows conformation specificity to CtmPrP, a prion protein conformer that triggers neurodegeneration under specific assay conditions of pH and copper concentration, is described. A second specific antibody termed 19B10 shows conformation specificity for NtmPrP, a prion protein conformer that downregulates total PrP expression and effects cell differentiation.
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Citations
40 Claims
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1. A method of detecting prion disease in a sample, said method comprising:
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contacting said sample with a diagnostically effective amount of an antibody characterized by its ability to bind to PrPCTM in situ; and
determining whether said antibody binds specifically to any material in said sample under conditions whereby differential binding of said antibody to PrP isoforms associated with prion disease occurs, wherein antibody binding is indicative of prion disease. - View Dependent Claims (2, 3, 4, 5, 29, 30)
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6. A composition comprising:
a hybridoma cell culture producing monoclonal antibodies which bind specifically to one conformer of PrP selected from the group consisting of PrpCTM and PrPNTM and have low or no cross reactivity with a second conformer of PrP. - View Dependent Claims (7)
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8. A composition comprising:
a monoclonal antibody which binds to an antigenic determinant of a PrP conformer on the surface of a neuroblastoma cell and has low or no cross reactivity with non cell surface antigenic determinants of PrP conformers.
- 9. A hybridoma which expresses monoclonal antibodies having the same antigenic specificity as that of monoclonal antibodies 7VC or 19B10.
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13. A method for promoting cell differentiation, said method comprising:
providing a cell with a sufficient amount of a monoclonal antibody that binds to an antigenic determinant on PrPNTM but not other conformers of mature PrP, whereby said cell undergoes differentiation. - View Dependent Claims (14)
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15. A method of inhibiting PrP expression in a cell, said method comprising:
providing a cell with a sufficient amount of a monoclonal antibody that binds to an antigenic determinant on PrPNTM but not other conformers of mature PrP, whereby PrP expression in said cell is inhibited. - View Dependent Claims (16)
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17. A method for obtaining prion protein conformer-specific monoclonal antibodies, said method comprising:
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contacting prion protein conformers each prepared using a cell-free translation system, with a composition comprising one or more monoclonal antibodies that bind to a prion protein conformer;
detecting binding of said monoclonal antibodies to one or more of said prion protein conformers by solution immunoprecipitation; and
isolating monoclonal antibodies that bind substantially specifically to a single prion protein conformer;
whereby conformer-specific monoclonal antibodies are obtained.
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18. A method for screening hybridomas for those that express conformer-specific monoclonal antibodies said method comprising:
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combining a panel of radiolabelled conformer-enriched translation products obtained using a cell-free translation system comprising microsomal membranes derived from the endoplasmic reticulum with individual samples of monoclonal antibodies expressed by said hybridomas; and
immunopreciptating any antibody-conformer complexes, whereby detection of complexes with a single conformer from said panel of radiolabelled conformer-enriched translation products is indicative of a hybridoma that expresses conformer-specific monoclonal antibodies. - View Dependent Claims (19, 20, 21, 22)
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23. A specific antibody (7VC) that recognizes prion protein in a copper-dependent fashion.
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25. A specific antibody (SS) that recognizes the signal sequence of PrP
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26. A monoclonal antibody designated 19B10.
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27. A monoclonal antibody designated 7VC.
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31. An epitope recognized by monoclonal antibody 7VC, wherein said epitope occurs in an amino acid sequence selected from the group consisting of an octarepeat sequence of a prion protein, WGQPHGGGWGQPHGG, GWGQPHGGGWGQPH, and GGGEGQGGGTHNQWN.
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32. A kit for detecting prion disease in a sample, said kit comprising:
an antibody that exhibits differential binding to PrPCTM in situ as compared to other PrP conformers under conditions that promote said differential binding. - View Dependent Claims (33, 34, 35, 36, 39, 40)
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37. Prion protein conformer-specific monoclonal antibodies, obtainable by a method comprising:
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contacting a panel of prion protein conformers, wherein each member of said panel is prepared using a cell-free translation system, with a composition comprising one or more monoclonal antibodies that bind to a prion protein conformer;
detecting binding of said monoclonal antibodies to one or more of members of said panel of prion protein conformers by solution immunoprecipitation; and
isolating monoclonal antibodies that bind substantially specifically to a single prion protein conformer;
whereby conformer-specific monoclonal antibodies are obtained. - View Dependent Claims (38)
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Specification