Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-12-01
1998-09-15
Housel, James C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
424 91, 424 92, 436 91, 436 99, G01N 3353, G01N 3300, A61K 4900
Patent
active
058076875
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to techniques for the detection of disease of the central nervous system, particularly bovine spongiform encephalopathy, scrapie, Jacob-Creuzfeld syndrome and related states.
It has been noted that serotonin levels are reduced in the blood of scrapie infected sheep (Chatelain et al (1984) CR Soc. Biol. 178, 664-670, and that some electrochemical changes occur in the urine of such sheep (Combrisson et al (1991) Bull. Acad. Vet. de France 64, 257-266; Banissi-Sabourdy et al (1992) Bioelectrochem & Bioenergetics 28, 127-147, but no reliable test has resulted from these findings.
At present there is no diagnostic test available for the screening of animals, including humans, for spongiform encephalopathy type disease and the confirmation of its presence is usually by direct observation of tissues in autopsy. There remains a particular need for a method of screening live animals, particularly prior to slaughter for consumption purposes, and preferably before their milk is consumed, and most preferably for a non-invasive test. The present inventor has now identified a factor present in the biological fluids of affected animals, particularly their urine, the amount of which correlates well with the occurence of these diseases.
The present invention provides a method for determining the presence of central nervous system disease in a human or animal body by analysis of one or more of its biological fluids characterised in that the fluid is analysed for the presence of a factor associated with a fraction obtainable from an ethanol extract of urine from animals having the disease, that fraction being elutable from a 0.2M Tris buffer pH 7.0 equilibrated DEAE-Sepharose Cl ion exchange column (particularly a CL6B) increasing gradient elution using from 0 to 0.2M sodium chloride in 0.02M Tris buffer pH 7.0; the fraction being elutable with between 0.08M and 0.015M sodium chloride, particularly between 0.08M and 0.09M sodium chloride in Tris buffer pH 7.0; wherein the amount of this factor is related to presence of the disease state.
The most convenient method for detecting this `disease` factor may of course vary depending upon the fluid to be tested, eg. whether CSF fluid, whole blood or a fraction thereof, or urine. The sampling of urine renders the test suitable for routine screening by electro-chemical detection means and can avoid the need for invasive methods. Other tests, eg. colorimetric and immunological, will also be applicable.
A preferred aspect of the present invention relates the amount of the `disease factor` with the amount of one or more reference factors present in the fluid, particularly in urine, and uses the ratio of these to indicate disease presence. These reference factors are a reference factor (2) that is associated with a fraction elutable with between 0.09 and 0.13M sodium chloride, and a reference factor (3) associated with a fraction elutable with between 0.13 and 0.2M sodium chloride; both using the ion-exchange column technique described above and the same ethanol extract of disease animal's urine.
The second reference factor (3) has been found to be uric acid, the first reference factor (2) has been found to have properties associated with tyrosine or a precursor or derivative thereof having at least one, and possibly two or three, phenolic groups including one phenol group para to a substitute side chain. It should be realised that the amount of material isolatable from urine is very small and has so far proven difficult to identify.
The disease factor (1) has been found to have properties consistent with having a substituted phenyl or histidine nucleus, also with a para-hydroxy substituent and at least one methyl substituent.
Both disease factor (1) and reference factor (2) have similar physical properties. Both are water soluble and soluble in methanol and ethanol, both are bound to aminopropyl solid phase extraction columns but only reference factor (2) can be desorbed using 0.5M HCl in 50:50 methanol/water; thus in this way reference factor can be separ
Everest Sally J.
Jackman Roy
Housel James C.
Minister of Agriculture, Fisheries and Food in Her Britannic Maj
Swartz Rodney P.
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