University of California

Other

The Scripps Research Institute

United States of America, As Represented by the Secretary of Agriculture

Stanley B Prusiner

Jiri G Safar

Surachai Supattapone

Michael R Scott

Dennis R Burton

Fred Ehrenkranz Cohen

R Anthony Williamson

Glenn C Telling

Michael Glenn Scott

Thomas L James

Kiyotoshi Kaneko

Leroy E Hood

Larry H Stanker

Richard L Moore

Shenheng Guan

Robert M Hnasko

Carsten Korth

Sina Ghaemmaghami

David Westaway

Inyoul Lee

Patrick Bosque

John C Price

Al Burlingame

Patrick Tremblay

Tamaki Muramoto

Kivotoshi Kaneko

An entire complement or plurality of isotopically labeled amino acids are introduced into the diet of a test subject. Sufficient amounts of the isotopically labeled amino acids are provided to the subject in order to ensure that the subject incorporates a large percentage of isotopically labeled amino acids into newly synthesized proteins. Tissue samples are removed from the subject at different points in time and proteins are extracted and separated so that different proteins of different tissues can be individually analyzed and their amount and pattern of isotopic labeling can be determined. In a preferred embodiment, the methodology can be combined with proteolytic digestion to peptides and analysis by mass spectrometry in order to measure rates of protein turnover in vivo relating to thousands of different proteins.

Isotopic labeling for the measurement of global protein levels and turnover in vivo

Peptide sequences that specifically bind infectious prion protein for the generation of antibodies and therapeutic agents are disclosed herein.

High-affinity monoclonal anti-prion antibodies

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.

Sodium dodecyl sulfate compositions for inactivating prions

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 PrP, 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.

Muscle sample prepared for prion assay

An assay method is disclosed which isolates and detects the presence of a disease related conformation of a protein (e.g., PrP) present in a sample also containing the non-disease related conformation of the protein (e.g., PrP). 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 PrP) and the occurrence of binding provides and indication that PrPis present. Alternatively the PrPof the treated sample is denatured (e.g., contacted with guanadine) or unfolded. The unfolded PrPis contacted with a binding partner and the occurrence of binding indicates the presence of PrPin the sample. In another embodiment, PrPand PrPare reacted with a labeled antibody that binds both conformations and a conformation that binds only the disease related conformation, and the presence of the disease related conformation is determined by comparing the two.

Method of preparing cow brain homogenate

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.

Somatic cells with ablated PrP gene and methods of use

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.

Method of concentrating proteins from serum

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

Antibodies specific for ungulate PrP

A method of isolating and detecting the presence of a disease related conformation of a protein (e.g., PrP) is disclosed. The sample is treated such as by contacting it with a protease and then contacting the treated sample with a binding partner which binds the PrPwhich can be isolated and separated from the sample.

Antibodies are disclosed which specifically bind to native PrPin situ. Preferred antibodies bind only to the native PrPof a particular species e.g., human, cow, sheep, pig, etc. Particularly preferred antibodies bind specifically to a particular isoform of human PrP. Preferred antibodies of the invention are (1) produced by phage display methodology, (2) bind specifically to native PrP, (3) neutralizes the infectivity of prions, (4) bind to PrPin situ and (5) bind 50% or more of PrPin a liquid flowable sample. Antibodies of the invention can be bound to a substrate and used to assay a sample (which has any PrPdenatured via proteinase K) for the presence of PrPof a specific species which PrPis associated with disease. Antibodies which specifically bind to human PrPcan be labeled and injected carrying out an in vivo diagnostic test to determine if the human is infected with prions associated with disease. The antibodies are preferably produced using phage display technology wherein the genetic material in the phage expressing the antibody is obtained from a mammal with an ablated endogenous PrP protein gene and an endogenous chimeric PrP gene which mammal had been inoculated with PrPto induce antibody production.

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The invention provides prion protein standards for use as reference materials for prion detection. The standard may be species specific, i.e. the standard is comprised of a preparation for detection of a single strain prion or it may be prepared to allow detection of multiple prion strains simultaneously. The invention also provides methods of preparing the prion protein standards using a group of non-human host mammals which have their genome manipulated with respect to genetic material related to a PrP gene such that the mammals are susceptible to infection with a prion which generally only infects an animal which is genetically diverse from the host.

Prion protein standard and method of making the same

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.

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San Francisco, CA, United States of America

Stanley B Prusiner