Transmissible spongiform encephalopathies (TSEs) are a heterogeneous group of fatal neurodegenerative disorders that occur in humans, ruminant herbivores, mink, and cats. Sheep scrapie is the prototype of this group. TSEs are characterized by deposition of prion proteins (also denoted as PrP-Scrapie or PrPSc, the infectious form of the proteins), in the central nervous system of affected individuals. Prions have been defined as small proteinaceous infectious particles which resist inactivation by procedures that modify nucleic acids. The term “prion” is a contraction of the words “protein” and “infection,” and prions are comprised largely if not exclusively of PrP-Sc molecules encoded by a PrP gene. Prion diseases are often called spongiform encephalopathies because of the post mortem microscopic or histopathologic appearance of the brain of an infected animal with large vacuoles in the cortex and cerebellum. Prion proteins are insoluble, protease-resistant glycoproteins resulting from post translational modification of normal mammalian glycoproteins (PrP-Cellular or PrP-C). Deposition of the prion protein, an abnormal isoform of a native cellular sialoglycoprotein, in the central nervous system is a reliable marker of TSE infection.
The most widely studied TSEs in food-producing animals include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle (also known as “Mad Cow” disease), and chronic wasting disease (CWD) in mule deer and elk. Other TSEs in animals include transmissible mink encephalopathy (TME) in mink and feline spongiform encephalopathy (FSE) in cats. Prion diseases of humans have also been identified, which include: Creutzfeldt-Jakob Disease (CJD); Gerstmann-Straussler-Scheinker Syndrome (GSS); Fatal Familial Insomnia (FFI); Alper's Syndrome, and Kuru.
The transmissible agent in these diseases remains controversial. It appears that the scrapie isoform of the prion protein (PrP-Sc or PrPSc) is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans (see Prusiner, Science, 252, 1515-1522, 1991).
A new clinical version of CJD in humans is believed to be the result of transfer of BSE from cattle to humans. This finding led to the conclusion that variant CJD was caused by infection of humans with prions from BSE infected cattle. Furthermore, the incidence and timing of the appearance of variant CJD cases opened the possibility that a considerable number of humans, presently free of clinical symptoms, could be latent for the disease. Given that CJD might pass from human to human in infected blood, it can be assumed that humans infected with variant CJD contain the infectious agent in their blood. A potentially infectious species specific agent has been discovered in blood of humans with CJD, cattle with BSE, and sheep with scrapie. ELISA tests have been developed that detect these TSE specific proteins that are associated with PrPSc (“Prion associated proteins”) in the blood of animals and humans. Prion associated proteins are expressed in a disease specific manner in all subjects with clinical symptoms of BSE (“BSAS”), scrapie (“SCRAPAS”) and CJD (“CJD”). Prion associated proteins appear to have the chemical characteristics for binding to PrP-C and converting it to PrPSc, and thus are most likely the infectious agents in TSE diseases. Furthermore, expression of prion associated proteins in a subject is accompanied by expression of specific anti-prion associated protein endogenous antibody. Detecting this endogenous antibody in human blood and blood products is the basis of some TSE ELISA tests (U.S. Pat. No. 6,350,854).
The occurrence of novel transmissible spongiform encephalopathies in cattle in the United Kingdom and Europe, and in mule deer and elk in parts of the United States has emphasized the need for reliable diagnostic tests. Further, the epizootic of a TSE in cattle and its postulated relationship to a new variant of human Creutzfeldt Jakob Disease have increased public and scientific awareness of these relatively rare disorders, and have highlighted the need for preclinical detection of TSEs. Accordingly, sensitive immunohistochemical techniques and preclinical detection methods are necessary for the detection, surveillance, and control of TSEs.
Confirmation of TSEs is accomplished by postmortem microscopic or histological examination of brain tissue of suspected cases. Postmortem histopathologic diagnosis of the ruminant TSEs is based on the appearance of neuronal vacuolation, spongiform changes, gliosis, and astrocytosis. However, these can vary in intensity and anatomic location depending on the host species, the individuals, host genetics, stage of disease, and infectious source. Thus, diagnosis by histopathology alone may be equivocal in early cases and usually not possible in autolyzed tissue.
Monoclonal antibody 263K 3F4 (U.S. Pat. No. 4,806,627) detects PrPSc in hamsters and humans, and has received use in diagnostic assays and pathogenesis studies of human TSEs. Ante-mortem testing in humans with suspected CJD is performed by immunohistochemical and histologic examination of brain biopsies. Because brain biopsy in ruminant animals is not feasible, an alternative approach has been to biopsy selected lymph nodes.
Therefore, there exists a need for a practical, inexpensive, and more rapid method for detection of prion proteins, prion-associated proteins and peptides and/or their respective host antibodies in live animals or humans. In addition, there exists a need for sensitive diagnostic assays to detect prion and prion associated proteins and peptides and/or their respective host antibodies in animal tissues and animal by-products in a most rapid fashion to minimize quarantine time.
Aptamers are functional synthetic nucleic acids useful for high-affinity binding to targets (e.g., nucleic acids, proteins, and chemical compounds). Unlike naturally occurring nucleic acids, which transfer genetic information, aptamers are selected on the basis of their ability to specifically bind their ligand. The specificity of binding is defined in terms of the dissociation constant Kd of the aptamer for its ligand. Aptamers can have high affinity with Kd range similar to antibody (pM to nM) and specificity similar/superior to antibody (Tuerk and Gold, Science, 249:505, 1990; Ellington and Szostak, Nature, 346:818, 1990).
Many aptamers have a stem-loop structure in which the bases in the loop and the stem are intimately involved in interaction with the ligand. RNA aptamers have been isolated against the protease-sensitive, N-terminus of PrP (Weiss et al., J. Virol. 71:8790-8797, 1997) but these do not discriminate between PrPC and PrPSc and are sensitive to nucleases. Therefore, there is a need in the art to design and utilize aptamers for binding to specifically folded prions, specifically those prions that are infectious and disease-causing in animals/mammals, in order to prevent the transmission and spread of such diseases in the food supply. The present disclosure provides improved aptamers for detecting the presence of PrPSc where the aptamers are not sensitive to nucleases.