Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-07-15
2001-07-17
Swartz, Rodney P (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S009100, C424S130100, C424S139100, C424S141100, C424S145100, C424S152100, C435S007100, C435S070100, C435S070210, C436S503000, C436S518000, C436S547000, C436S548000, C530S388100
Reexamination Certificate
active
06261790
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to detection of prion protein (also denoted as PrP-Sc protein) as an indicator of transmissible spongiform encephalopathies (TSEs). In particular, the invention relates to (a) monoclonal antibodies that specifically bind a conserved epitope of prion proteins, and (b) monoclonal antibody cocktail having the monoclonal antibody in combination with a second monoclonal antibody which specifically binds to a second conserved epitope of prion proteins. The new antibodies and antibody cocktail are useful in immunoassays to detect prion proteins in ruminants and other species in which TSEs occur naturally or in related species potentially exposed to TSEs.
2. Description of the Art
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 PrP-Sc), 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), and deposition of the PrP-Sc protein, the abnormal isoform of PrP-C 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 included transmissible mink encephalopathy (TME) in mink and feline spongiform encephalopathy (FSE) of domestic and nondomestic cats. Most recently, a TSE of non-human primates held in zoos in France was reported; this disease probably originated from BSE (Bons et al.,
Proceedings of the National Academy of Sciences of the United States of America
96:4046-4051 (1999)). Prion diseases of humans have also been identified. These include: Creutzfeldt-Jakob Disease (CJD); Gerstmann-Straussler-Scheinker Syndrome (GSS); Fatal Familial Insomnia (FFI), and Kuru.
The transmissible agent in these diseases remains controversial. However, as noted above, an insoluble isoform (prion or PrP-Sc) of a mammalian sialoglycoprotein (PrP-Cellular or PrP-C) is a major component in infectious material. It appears that the scrapie isoform of the prion protein (PrP-Sc) is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans (see S. B. Prusiner,
Science
252:1515-1522 (1991) and S. B. Prusiner,
Proceedings of the National Academy of Sciences of the United States of America
95:13363-13383 (1998)). A leading hypothesis is that prion diseases result from the conversion of PrP-C to PrP-Sc by a nucleation or polymerization event.
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 (M. E. Bruce et al.,
Nature
389:498-501 (1997) and A. F. Hill et al.,
Nature
389:448-450 (1997)) have increased public and scientific awareness of these relatively rare disorders, and have highlighted the need for preclinical detection of TSEs. Although no cases of BSE have been detected in the United States, sensitive immunohistochemical techniques and preclinical detection methods are basic for detection, surveillance, and control of TSEs.
Prion diseases can have a long incubation period. For example, in sheep it can take 3 to 5 years from the time when an animal becomes infected until it first shows disease signs. In bovine spongiform encephalopathy (BSE) it can take two to eight years from the time when an animal becomes infected until it first shows disease signs. Infected animals and humans have neither a disease-specific immune response nor consistent biochemical, hematological and gross pathological abnormalities. The early diagnosis of transmissible spongiform encephalopathies can therefore be dependent on the appearance of clinical signs, electroencephalography, or the invasive method of taking brain biopsies. 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.
Deposition of prion protein (PrP-Sc) in the central nervous system is a reliable marker for the TSEs. Immunohistochemical detection of PrP-Sc is therefore an important adjunct to histopathology in diagnosis, surveillance, and control of TSEs. Monoclonal antibody 263K 3F4 (U.S. Pat. No. 4,806,627) detects PrP-Sc in hamsters and humans, and has received widespread use in diagnostic assays and pathogenesis studies of human TSEs. A major disadvantage is that it fails to react with PrP from sheep and cattle (R. J. Kascsak et al.,
Immunological Investigations
26:259-268 (1997)). Rabbit antisera reactive with ruminant PrP-Sc has the disadvantages that it cannot be standardized for widespread use due to limitations in quantity and specificity. Monoclonal antibodies are preferable to rabbit antisera because quantities are not limited and specificity can be precisely defined at the level of a single epitope. This specificity, however, can be a drawback in species with polymorphic PrP genes. A single base change resulting in an amino acid substitution in the epitope can eliminate binding by the antibody. The human PrP gene has at least 18 pathogenic mutations leading to inherited prion disease (J. Collinge et al.,
Philos. Trans. Royal Soc. Lond. [Biol
.] 343:371-378 (1994)) and a number of non pathogenic mutations, one of which (codon 129) which is associated with predisposition to iatrogenic, sporadic and variant CJD (J. Collinge et al.,
Lancet
337:1441-1442 (1991); M. S. Palmer et al.,
Nature
352:340-342 (1991); M. Zeidler et al.,
Lancet
350:668 (1997)). M. Horiuchi et al. (
Journal of General Virology
76:2583-2587 (1995)) describe a panel of synthetic peptides that generated monoclonal and polyclonal antibodies reactive with the PrP-Cellular, (the non-disease-related protein) in immunoblots of selected sheep and cattle tissue. They did not report effectiveness for detecting the disease-related isoform, PrP-Sc. Additionally, they did not they report effectiveness in detecting either PrP-C or PrP-Sc in formalin fixed tissues.
Post mortem diagnosis of prion diseases is made using histologic and immunohistochemical assays on brain tissue. Ante-mortem testing in humans with suspected CJD is performed by immunohistochemical and histologic examination of brain biopsies. In addition, individuals with the new variant of CJD related to exposure to BSE have PrP-Sc accumulations in lymphoid tissues (A. F. Hill et al.,
Lancet
349:99 (1997)). The presence of PrP-Sc in lym
Connor Margaret A.
Fado John D.
Silverstein M. Howard
Swartz Rodney P
The United States of America as represented by the Secretary of
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