Methods to differentiate protein conformers
Inventors
Onisko, Bruce C. • Silva, Christopher J. • Requena, Jesus R. • Carter, John Mark
Assignees
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Publication Number
US-8445642-B1
Publication Date
2013-05-21
Expiration Date
2025-10-13
Abstract
The invention is directed to methods to distinguish among different protein conformers of the same protein such as proteins which form amyloid deposits. Using the methods of the invention, one or more protein conformers in a sample can be detected, differentiated, and quantitated. An example of a protein which is known to exist in at least two conformations is the normal prion protein (PrPC) and its infectious isoform (PrPSc). The invention provides means to distinguish PrPC from PrPSc and allows quantitation of each individually, even when the conformers are present together in a mixture. Thus, the methods of the invention can provide important tools for human and animal health.
Core Innovation
The invention provides methods to detect, distinguish, and quantitate different protein conformers of the same protein, particularly proteins capable of forming amyloid deposits. This includes the example of prion proteins, which exist in at least two conformations: the normal PrPC and the infectious PrPSc isoform. The methods allow detection and quantitation of each conformer individually, even when present together in a mixture, by reacting the sample with a protein-modifying reagent that forms unique covalent bonds with each conformer. Analysis of the modified peptides resulting from protein cleavage then allows the distinction and measurement of each conformer.
The problem addressed arises from the existence of proteins with multiple stable conformations that may be associated with disease, such as prion diseases involving PrPSc. Current structural characterization methods are limited since amyloid proteins like PrPSc are insoluble or non-crystallizable and there are no antibodies known that selectively bind PrPSc and not PrPC. Thus, there is a need for methods to detect and differentiate protein conformers, especially for agents of transmissible spongiform encephalopathies and other amyloid diseases related to human and animal health.
Claims Coverage
The patent discloses two main inventive features disclosed in independent claims related to methods of preparing monoclonal antibodies to differentiate protein conformers and methods for increasing detection sensitivity of protein conformers using these antibodies.
Method for preparing conformation-discriminating monoclonal antibodies
The method involves isolating a macromolecule population containing at least two stable conformations that possess separable physical differences, separating these conformations into distinct homogeneous populations, reacting each with a covalent modifier to generate newly created epitopes unique to each conformation, immunizing a laboratory animal with the modified conformers, monitoring the immune response to determine antibody titers, collecting blood to prepare antisera, and fractionating the antisera to enrich for antibodies specific to the newly created epitopes. This produces antibodies capable of discriminating between at least two stable macromolecular conformations.
Method for increasing sensitivity of macromolecular conformation detection
This method includes preparing monoclonal antibodies as described above that discriminate stable macromolecular conformations, selecting an antibody specific for a newly created epitope unique to the desired protein conformer, preparing biological samples by reacting with a covalent modifier, reacting the antibody with the prepared sample to achieve enhanced detection sensitivity, and interpreting the detection result. The method is applicable to biological tissues or fluids, including brain, muscle, blood, tonsil, spleen, and lymph.
The independent claims provide methods for generating monoclonal antibodies specific to different stable conformers of a macromolecule via covalent modification to create unique epitopes and methods to apply these antibodies for sensitive detection of individual protein conformers in biological samples.
Stated Advantages
Provides important tools for human and animal health by enabling detection, differentiation, and quantitation of protein conformers such as prion proteins.
Fulfills the need for assays that can detect low levels of prions in live animals, which is important for public safety and validation of beef product safety.
Enables identification of asymptomatic carriers of prion diseases, which current methods cannot reliably do.
Allows the differentiation among multiple prion strains, helping to understand etiology of transmissible spongiform encephalopathies (TSE) outbreaks and genesis of new strains.
Offers structural information by exploiting differential reactivity of amino acid residues in different conformers, assisting protein structure modeling not achievable by NMR or X-ray diffraction.
Documented Applications
Detection and quantitation of prion protein conformers PrPC and PrPSc in tissue and biological samples.
Diagnosis and identification of amyloid diseases in humans and animals through detecting amyloid-forming protein conformers.
Demonstration of safety and detection of low levels of prions in live animals, especially in food-producing animals such as cattle and sheep.
Characterization and differentiation of prion strains in agriculture to determine the cause and evolution of transmissible spongiform encephalopathy outbreaks.
Analysis of other protein conformers involved in neurodegenerative diseases and protein regulation, such as cytosolic polyadenylation element binding protein and yeast prions.
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