Patent EA-005239-B1 (EA)
Title: Monoclonal antibodies and antibody cocktail foe detection of prion protein as an indication of transmissible spongiform encephalopathies
Inventor: O'ROURKE KATHERINE I
IPC: C12N5/10
Filed: 2000-07-14
Abstract: 1. A monoclonal antibody which specifically binds to a conserved epitope of prion proteins, designated as Gln-Tyr-Gln-Arg-Glu-Ser (SEQ ID NO. 1), and capable of binding PrP-Sc protein in fixed or unfixed tissue that has been treated to unmask said epitope to PrP-Sc protein and eliminate availability of a corresponding epitope of PrP-Cellular. 2. The monoclonal antibody of claim 2 designated as F99/97.6.1 produced by a hybridoma cell line, ATCC HB-12696. 3. A hybridoma cell line, ATCC HB-12696, which produces and secretes monoclonal antibody F99/97.6.1, which specifically binds to a conserved epitope of prion proteins, designated as Gln-Tyr-Gln-Arg-Glu-Ser (SEQ ID NO. 1), and binds PrP-Sc protein in fixed or unfixed tissue that has been treated to unmask said epitope to PrP-Sc protein and eliminate availability of a corresponding epitope of PrP-Cellular. 4. A monoclonal antibody cocktail which comprises a first monoclonal antibody comprising the monoclonal antibody of claim 1 in combination with a second monoclonal antibody comprising a monoclonal antibody which specifically binds to a second conserved epitope of prion proteins, designated as Ile-His-Phe-Gly (SEQ ID NO. 2), and is capable of binding PrP-Sc protein in fixed or unfixed tissue that has been treated to unmask said epitope to PrP-Sc protein and eliminate availability of a corresponding epitope of PrP-Cellular. 5. The monoclonal antibody cocktail of claim 4 wherein said first monoclonal antibody is F99/97.6.1 produced by a hybridoma cell line ATCC HB-12696 and said second monoclonal antibody is F89/160.1.5 or F89/193.1.5. 6. An immunoassay method for detecting PrP-Sc protein in an animal or human which comprises: (1) obtaining tissue from an animal or human to be tested; (2) fixing or freezing said tissue; (3) treating said fixed or frozen tissue to unmask epitopes to PrP-Sc and eliminate availability of a corresponding epitope of PrP-Cellular; (4) contacting said treated tissue with monoclonal antibodies which comprises (a) monoclonal antibodies of claim 1 or (b) monoclonal antibody cocktail of claim 5, in an amount and under conditions such that said antibodies bind PrP-Sc protein if said protein is present in said tissue, and (5) detecting the presence of said bound antibodies. 7. The method of claim 6 wherein said tissue is fixed tissue and said treating is hydrated autoclaving, treatment with formic acid with or without subsequent hydrated autoclaving, and digestion with trypsin. 8. The method of claim 6 wherein said tissue is frozen tissue and said treating comprises treatment with proteinase K to eliminate a 35KPrP-C band and reveal characteristic multiple 28-32K bands of proteinase K-resistant PrP-Sc. 9. The method of claim 6 wherein said monoclonal antibody is F99/97.6.1 produced by a hybridoma cell line, ATCC HB-12696. 10. The method of claim 6 wherein said first monoclonal antibody is F99/97.6.1 produced by a hybridoma cell line ATCC HB-12696 and said second monoclonal antibody is F89/160.1.5 or F89/193.1.5. 11. The method of claim 6, wherein said detecting comprises contacting said antibodies with a detectably labeled antimouse immunoglobulin reagent under conditions such that said reagent binds to said antibodies, and detecting said bound reagent. 12. The method of claim 6, wherein said detecting comprises: (a) contacting said antibodies with labeled antimouse immunoglobulin reagent under conditions such that said reagent antibody binds to said antibodies; (b) contacting said reagent with an enzyme-ligand complex such that said complex binds to said label on said reagent, and (c) detecting said enzyme-ligand complex with a chromogenic substrate. 13. The method of claim 6 wherein said animal is selected from the group consisting of ruminant livestock, cats, mink, and non-human primates. 14. The method of claim 13 wherein said ruminant livestock is selected from the group consisting of sheep, goat, cattle, mule deer and elk. 15. The method of claim 6 wherein said tissue is third eyelid-associated lymphoid tissue, brain necropsy tissue, lymph node biopsy or necropsy tissue or spleen biopsy or necropsy tissue.