Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Patent
1994-09-16
1998-01-06
Knode, Marian C.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
436530, 436531, 436811, G01N 33543, G01N 33544, G01N 33545
Patent
active
057054010
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method of assaying for Alzheimer's disease in a human by determining the relative abundance of one or more forms of amyloid precursor protein (APP) or the enzyme responsible for said forms in circulatory fluid and to a method for treating the disease by modulating divalent cation and/or heparin interaction with APP.
Alzheimer's disease is a progressive dementia characterised by the deposition of amyloid in the intracellular and extracellular compartments of the cerebral cortex (Davies et al, 1988). The extracellular deposits consist of a protein of 4,000 relative molecular mass (M.sub.r =4K) referred to as .beta.A4 (Kang et al, 1987). Molecular cloning and protein sequencing studies have shown that .beta.A4 comprises part of the membrane spanning and extracellular domains of the amyloid precursor protein (APP), which has features of an integral transmembrane cell surface receptor (Kang et al, 1987; Goldgaber et al, 1987).
.beta.A4 appears to result from abnormal cleavage of APP. Normal cleavage occurs at or near a lysine residue within the .beta.A4 sequence (Esch et al 1990; Sisodia et al 1990; Palmert et al 1989). Cleavage at this site would prevent formation of the amyloidogenic .beta.A4 fragment. The enzyme which normally cleaves APP from the membrane surface is designated "APP secretase" although it has never been identified.
At least one form of APP has been shown to have neurotrophic activity, i.e. capable of promoting the survival or outgrowth of nerve processes. A disorder in APP processing may account for the loss of certain populations of neurons seen in Alzheimer's disease. There is now increasing evidence that APP is released from membranes, as part of its normal neurotrophic function, by cleavage at a site within the .beta.A4 sequence (lysine 16) by APP secretase. As .beta.A4 is produced in Alzheimer's disease, this indicates that a failure to cleave at this site might be the cause of Alzheimer's disease or at least contribute to the profession of the disease.
There is a need for an assay which is of predictive and diagnostic value in monitoring Alzheimer's disease and for any therapeutic interventions therein. In accordance with the present invention, it has now been discovered that processing of circulatory. APP is altered in Alzheimer's disease thus providing a basis for an assay for the disease. Furthermore, from work leading up to the present assay, an improved means of treating Alzheimer's disease has been discovered based on modulating the interaction between divalent cations and/or heparin and APP.
Accordingly, one aspect of the present invention provides a method of assaying for Alzheimer's disease in a human said method comprising isolating a sample of circulatory fluid from said human, determining the amount of the 130 kDa form and/or 42 kDa form of APP and/or any derivatives of either form of APP in said fluid relative to a normal control wherein a relative increase in the 130 kDa form and/or its derivative and/or a relative decrease in the 42 kDa form and/or its derivative is indicative of the disease. By "assay" is meant screening or monitoring for the presence of, or a disposition to, Alzheimer's disease and/or to observe the effectiveness, or otherwise, of treatment of the disease following, for example, therapeutic intervention.
In accordance with the assay of the present invention, a sample of circulatory fluid is obtained from a test subject, generally after fasting since the prandial condition alters the level of APP in circulatory fluid, and the relative amounts of the 130 kDa and/or 42 kDa forms and/or their derivatives of APP determined. Fasting is generally for at least four hours but may be longer or shorter depending on the human to be tested. Fasting times may, for example, vary from 3 to 12 hours. Preferably, the circulatory fluid is blood plasma. A number of means exist for determining the relative amounts and such determination may be quantitative or qualitative. Conveniently, a Western blot is performed using an antibody, and pr
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Selkoe et al. (1988) ".beta.-Amyloid precursor protein of Alzheimer disease occurs as 110-to 135-kilodalton membrane-associated proteins in neural and non-neural tissues". Proc. Natl. Acad. Sci., 85:7341-7345.
Horns et al, Neurology 1989; 39:1159-1165 "The Consortum to Establish a Registry fo Alzheimer's Disease".
Bush et al, J. Biol. Chem, Sep. 15, 1990; 265(26):15977-15983 "The Amyloid Precursor Protein of Alzheimer's Disease is Released by Human Platelets".
Beyreuther Konrad Traugott
Bush Ashley Ian
Masters Colin Louis
Duffy Patricia A.
Knode Marian C.
The University of Melbourne
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