Abstract: The present invention provides a novel PrP protein, and nucleic acids encoding this protein, where the PrP protein is characterized in vivo by 1) incomplete glycosylation relative to glycosylation of wild-type PrPC and 2) proper cellular localization, i.e. an ability to be transported to the cell surface. This novel, under-glycosylated PrP, unlike its normal cellular counterpart, can easily be converted into a protease-resistant isoform by incubation with infectious prions. The invention further provides systems for the study of prion disorders and methods of using these systems, e.g. the study of the mechanical processes in progression of prion-mediated disease or the identification of new therapeutic agents for treatment of prion-mediated disorders. In such systems, protease-resistant under-glycosylated PrP is generated de novo and can be detected by standard immunoblot techniques.

Abstract: The present invention comprises a method for producing mammalian therapeutics free from prion contamination and cells for use in such methods. Such therapeutics are produced in somatic cells having a genome with an artificially altered PrP gene. The PrP gene in these cells may be ablated, or replaced by an exogenous inducible form of the PrP gene. The endogenous gene in the host cells may be disrupted, or disrupted and replaced by an exogenous PrP gene.

Abstract: An assay method is disclosed which isolates and detects the presence of a disease related conformation of a protein (e.g., PrPSc) present in a sample also containing the non-disease related conformation of the protein (e.g., PrPC). The sample is treated (e.g., contacted with protease) in a manner which hydrolyzes the disease related conformation and not the non-disease related conformation. The treated sample is contacted with a binding partner (e.g., a labeled antibody which binds PrPSc) and the occurrence of binding provides and indication that PrPSc is present. Alternatively the PrPSc of the treated sample is denatured (e.g., contacted with guanadine) or unfolded. The unfolded PrPSc is contacted with a binding partner and the occurrence of binding indicates the presence of PrPSc in the sample.

Abstract: An antiseptic composition useful in destroying the infectivity of infectious proteins such as prions is disclosed. The antiseptic composition is preferably maintained at either a low pH of 4.0 or less or a high pH of 10.0 or more either of which allows for an environment under which the active component (which is preferably sodium dodecyl sulfate) destroys infectivity. The composition may be added to blood, blood products, collagen, tissues and organs prior to transplantation. The composition also may be added to livestock feed to denature any prions in the livestock. Methods of denaturing infectious proteins are also disclosed which method can use but do not require higher temperatures and long period of exposure.

Abstract: An antiseptic composition useful in destroying the infectivity of infectious proteins such as prions is disclosed. The antiseptic composition is preferably maintained at either a low pH of 4.0 or less or a high pH of 10.0 or more either of which allows for an environment under which the active component (which is preferably sodium dodecyl sulfate) destroys infectivity. The composition may be added to blood, blood products, collagen, tissues and organs prior to transplantation. The composition also may be added to livestock feed to denature any prions in the livestock. Methods of denaturing infectious proteins are also disclosed which method can use but do not require higher temperatures and long period of exposure.

Abstract: An antiseptic composition useful in destroying the infectivity of infectious proteins such as prions is disclosed. The antiseptic composition is preferably maintained at either a low pH of 4.0 or less or a high pH of 10.0 or more either of which allows for an environment under which the active component (which is preferably sodium dodecyl sulfate) destroys infectivity. The composition may be added to blood, blood products, collagen, tissues and organs prior to transplantation. The composition also may be added to livestock feed to denature any prions in the livestock. Methods of denaturing infectious proteins are also disclosed which method can use but do not require higher temperatures and long period of exposure.

Abstract: An antiseptic composition useful in destroying the infectivity of infectious proteins such as prions is disclosed. The antiseptic composition is preferably maintained at a pH of 4.0 or less which allows for an environment under which the active component destroys infectivity. The composition may be added to blood, blood products, collagen, tissues and organs prior to transplantation. The composition also may be added to livestock feed to denature any prions in the livestock. Methods of denaturing infectious proteins are also disclosed.

Abstract: An antiseptic composition useful in destroying the infectivity of infectious proteins such as prions is disclosed. The antiseptic composition is preferably maintained at either a low pH of 4.0 or less or a high pH of 10.0 or more either of which allows for an environment under which the active component (which is preferably sodium dodecyl sulfate) destroys infectivity. The composition may be added to blood, blood products, collagen, tissues and organs prior to transplantation. The composition also may be added to livestock feed to denature any prions in the livestock. Methods of denaturing infectious proteins are also disclosed which method can use but do not require higher temperatures and long period of exposure.

Abstract: The present invention provides assays for identifying the levels of both protease sensitive and protease resistant conformers of PrPSc in a sample. In a preferred embodiment, the assay comprises determining levels of total PrPSc in a sample, subjecting the PrPSc fraction to treatment with a protease that selectively hydrolyzes the protease sensitive PrPSc (sPrPSc) conformers, and quantifying the levels of sPrPSc in the sample. The ability to detect sPrPSc allows early detection of prions, since the PrPSc in easily accessible biological samples such as blood is predominantly sPrPSc. The ratio of sPrPSc to rPrPSc also allows the identification of a particular prion strain in an infected sample.

Abstract: An assay method is disclosed which isolates and detects the presence of a disease related conformation of a protein (e.g., PrPSc) present in a sample also containing the non-disease related conformation of the protein (e.g., PrPC). The sample is treated (e.g., contacted with protease) in a manner which hydrolyzes the disease related conformation and not the non-disease related conformation. The treated sample is contacted with a binding partner (e.g., a labeled antibody which binds PrPSc) and the occurrence of binding provides and indication that PrPSc is present. Alternatively the PrPSc of the treated sample is denatured (e.g., contacted with guanadine) or unfolded. The unfolded PrPSc is contacted with a binding partner and the occurrence of binding indicates the presence of PrPSc in the sample.

Abstract: Antibodies are disclosed which specifically bind to native PrPSc in situ. Preferred antibodies bind only to the native PrPSc of a particular species e.g., human, cow, sheep, pig, etc. Particularly preferred antibodies bind specifically to a particular isoform of human PrPSc. Preferred antibodies of the invention are (1) produced by phage display methodology, (2) bind specifically to native PrPSc, (3) neutralizes the infectivity of prions, (4) bind to PrPSc in situ and (5) bind 50% or more of PrPSc in a liquid flowable sample. Antibodies of the invention can be bound to a substrate and used to assay a sample (which has any PrPc de-natured via proteinase K) for the presence of PrPSc of a specific species which PrPSc is associated with disease. Antibodies which specifically bind to human PrPSc can be labeled and injected carrying out an in vivo diagnostic test to determine if the human is infected with prions associated with disease.

Abstract: A sample is prepared from blood in a manner which makes it possible to further analyze proteins in the sample, e.g. to detect prions in the sample. Blood is extracted, allowed to clot and subjected to separation processing (e.g. centrifugation) to obtain serum. The serum is treated with a complexing agent which agent binds prions in the sample forming an agent/protein complex which makes it possible to concentrate the complex. Concentration of the complex results in a sample which can be successfully analyzed, e.g. assayed using a range of different types of assay methodologies for detecting prions.

Abstract: Devices such as flow through columns, substrates such as spherical polymer beads, and methods of using such to remove prions from any liquid sample are disclosed. A surface of a substrate is coated with a prion complexing agent, such as a salt of phosphotungstic acid. Blood or plasma passing through a column containing beads coated with prion complexing agent are rendered prion free.

Abstract: The present invention provides assays for identifying the levels of both protease sensitive and protease resistant conformers of PrPSc in a sample. In a preferred embodiment, the assay comprises determining levels of total PrPSc in a sample, subjecting the PrPSc fraction to treatment with a protease that selectively hydrolyzes the protease sensitive PrPSc (sPrPSc) conformers, and quantifying the levels of sPrPSc in the sample. The ability to detect sPrPSc allows early detection of prions, since the PrPSc in easily accessible biological samples such as blood is predominantly sPrPSc. The ratio of sPrPSc to rPrPSc also allows the identification of a particular prion strain in an infected sample.

Abstract: Assay methodology of the invention allows for: (1) determining if a sample contains a conformation of a protein which is associated with disease and the concentration and amount of such if present; (2) determining the amount of protease resistant disease related protein in a sample and by subtracting that amount from the total amount of disease related protein present determining the amount of protease sensitive disease protein in the sample; and (3) determining the strain and incubation time of a disease related protein by (i) relating the relative amounts of protease resistant and protease sensitive protein to known strains to thereby determine the strain; and (ii) plotting the concentration of protease sensitive protein on a graph of incubation time versus concentration of protease sensitive protein for known strains to predict the incubation time of an unknown strain of pathogenic protein in a sample.

Abstract: DNA constructs are provided of epitope-tagged proteins or protein fragments which are conveniently purified with immunoaffinity chromatography such as epitope-tagged prion proteins (PrP). Transgenic animals expressing an epitope-tagged protein are provided, including transgenic animals expressing epitope-tagged PrP. Methods for distinguishing between the conformational shapes of a protein and a convenient method for isolating a tagged protein by immunoaffinity chromatographic methods are provided.

Abstract: The present invention provides antibodies that specifically bind with a high degree of binding affinity to a native ungulate PrPC and/or a denatured ungulate PrPSc, but not to a native ungulate PrPSc. Preferred antibodies find native bovine PrPC and treated PrPSc but not native bovine PrPSc and can be used in an assay to determine if a sample is infected with infectious prions, i.e. PrPSc.

Abstract: A method of preparing a sample of muscle tissue and of assaying the prepared sample to determine the presence of prions in the sample is disclosed. The muscle tissue is homogenized and mixed with a complexing agent which forms a complex with a higher specific gravity than PrPSc, the complexing agent or other components of the homogenate. Gravity is then used (e.g. ultra centrifugation) to concentrate the complex and the concentrate is assayed to detect prions. The muscle tissue is preferably extracted from a muscle or group of muscles such as hind limb muscle which have a higher or more preferably the highest concentration of prions as compared to other muscle in the mammal.

Abstract: Antibodies are disclosed which specifically bind to native PrPSc in situ. Preferred antibodies bind only to the native PrPSc of a particular species e.g., human, cow, sheep, pig, etc. Particularly preferred antibodies bind specifically to a particular isoform of human PrPSc. Preferred antibodies of the invention are (1) produced by phage display methodology, (2) bind specifically to native PrPSc, (3) neutralizes the infectivity of prions, (4) bind to PrPSc in situ and (5) bind 50% or more of PrPSc in a liquid flowable sample. Antibodies of the invention can be bound to a substrate and used to assay a sample (which has any PrPc denatured via proteinase K) for the presence of PrPSc of a specific species which PrPSc is associated with disease. Antibodies which specifically bind to human PrPSc can be labeled and injected carrying out an in vivo diagnostic test to determine if the human is infected with prions associated with disease.

Abstract: The present invention provides antibodies that specifically bind with a high degree of binding affinity to a native ungulate PrPC and/or a denatured ungulate PrPSc, but not to a native ungulate PrPSc. Preferred antibodies find native bovine PrPC and treated PrPSc but not native bovine PrPSc and can be used in an assay to determine if a sample is infected with infectious prions, i.e. PrPSc.