Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1992-03-12
2001-09-11
Wortman, Donna C. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S070210, C436S548000, C436S811000, C530S388250
Reexamination Certificate
active
06287793
ABSTRACT:
BACKGROUND OF THE INVENTION
A. General Diagnosis of Diseases
In most cases, diagnosis of a given disease requires a number of agreed-upon concurrent observations made by the attending physician. For some diseases, such as diabetes, a nearly definitive test, such as a glucose tolerance test, is sufficient to make a correct diagnosis. Most diseases, however, such as atherosclerosis, schizophrenia or lupus erytheomatosus require a number of sophisticated tests to arrive at a probable diagnosis. An extreme example is Alzheimer's disease which is currently only confirmed at the time of autopsy by examination of brain tissue.
The reason for the general scarcity of definitive diagnostic tests—particularly those utilizing biological fluids, such as serum plasma, cerebral spinal fluid or urine—is that diagnostic tests are generally found serendipitously. There currently exists no systematic way of arriving at a novel biological fluid-based diagnostic for a given disease.
This problem is very weighty for diseases of unknown etiology, such as Alzheimer's disease. Numerous other diseases with similar diagnostic problems include multiple sclerosis, amyotrophic lateral sclerosis and lupus erytheomatosus.
From a diagnostic standpoint, Alzheimer's disease represents a great challenge for two reasons. First, the disease affects one out of six individuals over the age of 65 or approximately two to five million individuals in the United States alone; and second, virtually nothing is known about distinguishing characteristics of the plasma or other biological fluids of Alzheimer's patients—current knowledge is limited to distinguishing characteristics of the brain tissue itself.
Several specific distinguishing characteristics have been found within the parenchyma of the brain in Alzheimer's disease. One key feature is the presence of amyloid plaques. These can be of neuritic or cerebral vascular origin. The cerebral vascular amyloid is comprised of a small peptide that is 39 to 42 amino acids in length. Molecular cloning studies have shown that this peptide is most likely a product of a larger precursor whose gene maps to chromosome 21 but ultimately does not necessarily segregate with the familial form of Alzheimer's disease. Therefore, this gene does not constitute the locus for the disease, although such was an attractive hypothesis since trisomy 21 or Down's syndrome patients ultimately portray Alzheimer-like pathology. Another'surprising observation is that the amyloid precursor is fairly ubiquitous throughout various organs in the body. This suggests that this protein might be difficult to exploit as a diagnostic tool since its level of expression is already very high in normal healthy individuals.
Attempts have been made to produce monoclonal antibodies to specific brain alterations in Alzheimer's disease and then to utilize these for diagnostic potential. One such antibody, “Alz50,” produced by P. Davies using Alzheimer brain as the antigen, does appear to specifically stain neurofibrillary tangles as seen in Alzheimer's, Picks, and Guam-Parkinson diseased brains. There are some data to suggest that this antibody detects an Alzheimer-specific antigen in Alzheimer cerebral spinal fluid that is 68 kdal in mass compared to 60 kdal in normal cerebral spinal fluid. The reproducibility and usefulness of this observation as a diagnostic for Alzheimer's disease has not yet been confirmed by other laboratories.
Another area of potential diagnostic value for Alzheimer's disease in cerebral spinal fluid is the detection of altered forms of tau and ubiquitin proteins. Both of these proteins are thought to be associated with neurofibrillary tangles. Recently, an abstract describing an enzyme-linked assay against paired helical filaments demonstrated a statistical qualitative difference in the signal generated by Alzheimer cerebral spinal fluid, although the overlap between the two sample groups was large.
Prior to this invention, no distinguishing characteristics have been discovered in the plasma or other biological fluids of Alzheimer patients. Prior efforts to look for Alzheimer specific fragments of the amyloid precursor have thus far produced no concrete discriminating data.
B. Haptoglobin Function
Haptoglobin is a member of the chymotrypsinogen serine protease family. It has, however, lost its protease activity due to the histidine lysine-57 and serine alanine-195 substitutions. Its &agr;
2
&bgr;
2
structural composition closely resembles that of Factor XI.
Haptoglobin has the unique capacity to bind hemoglobin almost irreversibly. Hence, its function in hemolyzed plasma is to conserve hemoglobin in plasma from destruction in the kidney. Haptoglobin-hemoglobin complexes are specifically readsorbed by the liver where the hemoglobin is recycled.
Currently, the only established use of haptoglobin as a diagnostic marker is in the diagnosis of anemia. Haptoglobin levels often fall dramatically in the anemic state. Its usefulness for this purpose is, however, questionable, since high haptoglobin levels can also occur in an anemic state as well.
Haptoglobin is also a member of a class of proteins termed “acute phase reactants.” These proteins are synthesized and released by the liver in response to several types of physiological stress. The hormonal signals involved in the signalling process are thought to include interleukin I and hepatic stimulating factors I and II. Inflammatory processes known to increase levels of acute-phase reactants include arthritis and coronary artery disease.
There are several reports in the literature where an attempt was made to correlate senile dementia of the Alzheimer type with the haptoglobin-1 (Hp-1) allele frequency. The data conflict, however, and the general scientific conclusion is that no such correlation exists.
C. Filamentous Proteins and Alzheimer's Disease
There are two established hallmark brain lesions of Alzheimer's disease: amyloid plaques and neurofibrillary tangles. Amyloid plaques are now known to contain a small peptide termed A
4
. This peptide has been sequenced and based on that sequence a cDNA clone was isolated containing a much larger precursor that contained within it the sequence of the A
4
peptide. Great effort is currently focussed on understanding the role of this precursor protein in the etiology of the disease.
The second hallmark of Alzheimer disease, namely neurofibrillary tangles has proven less amenable to isolation and characterization. The tangles are known to contain at their core paired helical filaments (PHF). These consist of two filaments that twist about each other in an &agr;-helix. Methods have been developed to partially purify paired helical filaments as intact structures and although this has increased our understanding of their structure, it has not been possible to solubilize and sequence the principal core protein(s) See, for example, Iqbal, K., et al.,
Acta Neurophilogica,
62:167-177 (1984) which is incorporated herein by reference. Since PHF might contain proteins that are present in plasma or CSF as well as the brain, this material was used in the present invention as an immunogen. In fact, the antibody 7-C1 is described that has usefulness as an Alzheimer's diagnostic. It appears to recognize an intermediate-filament associated protein which is consistent with the PHF immunogen used to raise it.
SUMMARY OF THE INVENTION
Several objects are attendant to this invention.
For example, a first object of the invention is to provide a method for identifying key diagnostic antigens of, or antibodies to, a disease state. This is particularly important in disease states of unknown etiology, such as Alzheimer's Disease.
Another object of the invention is to provide a method for diagnosing a disease state, wherein a key diagnostic antibody is provided, and wherein said key diagnostic antibody was discovered in accordance with the instant invention.
Yet another object of the invention is to provide a method of diagnosing Alzheimer's Diseas
Barbour Robin M.
Johnson Kelly L.
Schenk Dale B.
Elan Pharmaceuticals Inc.
Murphy Lisabeth F.
Wortman Donna C.
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