Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-09-21
2002-10-08
Gitomer, Ralph (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S019000, C435S827000, C435S007100
Reexamination Certificate
active
06461831
ABSTRACT:
TECHNICAL FIELD
The present invention is concerned with a diagnostic test for Alzheimer's disease.
BACKGROUND ART
Alzheimer's disease (AD) is a common progressive dementia involving loss of memory and higher cognitive function. The disease is characterized by the presence of amyloid deposits in the brains of sufferers. These deposits are found both extracellularly (amyloid plaques) and intracellularly (neurofibrillary tangles). The principal constituent of amyloid plaques is the amyloid protein (A&bgr;) which is produced by proteolytic cleavage for the amyloid protein precursor (APP) (Evin et al., 1994). The principal constituent of neurofibrillary tangles is the cytoskeletal protein tau (Kosik, 1992).
One of the characteristic neurochemical changes observed in AD is the loss of acetylcholinesterase (AChE) and choline acetyltransferase activity in regions of the brain such as the cortex, hippocampus, amygdala and nucleus basalis (Whitehouse et al., 1981, 1982; Struble et al., 1982; Mesulam and Geula, 1988). The loss of cholinergic structure and markers correlates with the number of plaque and tangle lesions present, as well as with the clinical severity of the disease (Perry et al., 1978; Wilcock et al., 1982; Neary et al., 1986; Perry, 1986).
Accurate diagnosis of AD during life is essential. However, clinical evaluation is at best only about 80% accurate. Therefore, there is a need to identify specific biochemical markers of AD. So far, analysis of blood or cerebrospinal fluid (CSF) has not yielded a biochemical marker of sufficient diagnostic value (Blass et al., 1998), although detectable differences are reported in the levels of certain proteins (Motter et al., 1995).
The assay of levels of AChE activity in the blood and the cerebrospinal fluid (CSF) has been proposed as an ante mortem diagnostic test for AD. However, no consensus has been reached as to whether the levels of AChE are consistently affected in these tissues. The level of serum or plasma AChE has been reported to be increased (Perry et al., 1982; Atack et al., 1985), decreased (Nakano et al., 1986; Yamamoto et al., 1990) or unchanged (St. Clair et al., 1986; Sirvio et al., 1989) in AD patients. The level of erythrocyte AChE has been reported as either unaffected (Atack et al., 1985; Perry et al., 1982) or decreased (Chipperfield et al., 1981). The level of AChe activity in the CSF of AD patients has been reported to be decreased (most recently by Appleyard and McDonald, 1992; Shen et al., 1993) or unchanged (most recently by Appleyard et al., 1987; Ruberg et al., 1987).
AChE has been shown to exist as up to six different molecular isoforms, three of which are the monomeric (G1), dimeric (G2) and tetrameric (G4) isoforms (Massoulie et al., 1993). The relative proportion of the different isoforms of AChE are markedly affected in AD, with a decrease in the G4 isoform in the parietal cortex (Atack et al., 1983), and an increase in the G1 isoform (Arendt et al., 1992). Similar changes have been identified in other AD brain regions including Brodman areas 9, 10, 11, 21 and 40, as well as the amygdala (Fishman et al., 1986). Asymmetric collagen-tailed isoforms (A12) are increased by up to 400% in Brodman area 21, although they represent only a trace amount of the total AChE in the human brain (Younkin et al., 1986).
However, to date changes in AChE expression and isoform distribution have not been found to be of sufficient sensitivity or specificity to be useful diagnostic markers of AD.
An anomalous isoform of AChE, distinguished by its isoelectric point, has been detected in the CSF of AD patients (Havaratnam et al., 1991; Smith et al., 1991), and a method for screening for AD based on these findings is described in U.S. Pat. No. 5,200,324. The method comprises determining, by means of isoelectric focusing, if a patient has an anomalous form of AChE in his CSF. However, the isoform detected by Navaratnam et al and Smith et al has also been detected in the CSF of patients with other neurological diseases (Shen and Zhang, 1993). Indeed, this is suggested in U.S. Pat. No. 5,200,324 at column 7 lines 19-22, where it is stated that the anomalous AChE “was present in the CSF of four out of eight patients with a clinical diagnosis of possible dementia, but who did not satisfy strict histopathological criteria for Alzheimer's disease”.
Moreover, the passage at column 7 lines 60-61 of the US patent indicates that the detection of AChE-AD in lumbar CSF depends upon the amount of CSF analysed, and column 8 lines 38-40 state that the anomalous band was often rather faint and the gels run were not always ideal. Accordingly, a loading of 5 mU per track was adopted as a standard procedure for screening CSF for the presence of the anomalous form of AChE, and each gel was read independently by four individuals who recorded their interpretation. Thus, there are technical problems associated with the assay described which can only be overcome by adopting an arbitrary set of conditions to avoid false readings, which then makes interpretation of the results difficult.
The suggestion that the anomalous form of AChE detected by Navaratnam et al and Smith et al is not unique to AD patients, together with the technical problems associated with the assay described in U.S. Pat. No. 5,200,324 suggests that the abnormal electroform of AChE discovered by Navaratnam et al and Smith et al will not form the basis of a diagnostic test for AD suitable for clinical use.
DISCLOSURE OF THE INVENTION
There remains a need for a diagnostic test for AD based on a biochemical analysis of body fluids such as blood or CSF and the present invention provides such a test on the basis that the AChE of AD patients shows a different glycosylation pattern to the AChE of non-AD groups.
According to a first aspect of the present invention there is provided a method for the diagnosis of Alzheimer's disease (AD) in a patient, comprising the steps of:
(1) providing a sample of an appropriate body fluid from said patient;
(2) detecting the presence of acetylcholinesterase (AChE) with an altered glycosylation pattern in said sample.
In one embodiment of the invention the relative proportion of AChE with a first glycosylation pattern and AChE with a second glycosylation pattern is measured.
Measurement of the relative proportions of AChE with first and second glycosylation patterns may be carried out in any convenient manner, for example, by using biochemical analysis techniques such as HPLC and mass spectrometry, or immunological techniques such as ELISA or, assays. However, a particularly preferred means of measuring the relative proportions of the isoforms of AChE involves a lectin-binding analysis.
It has been established that approximately 75%-95% of the AChE in the CSF of AD patients binds to Concanavalin A (Con A) or to wheat germ agglutinin (WGA), but with different specificity to each. Accordingly, in a particularly preferred embodiment of the invention, in order to identify the glycosylation pattern of AChE in the sample, the binding to Con A is determined, then the binding to WGA is determined, and a ratio calculated. The ratio is characteristic of the glycosylation pattern. It is particularly convenient to measure the activity of unbound AChE in each experiment, hence the ratio of AChE unbound to Con A to the ratio of AChE unbound to WGA is determined. This ratio is referred to hereinafter as a C/W ratio. For patients with AD, the C/W ratio has generally been found to be above 0.95, whereas for non-sufferers of AD the C/W ratio is typically below 0.95.
In an alternative embodiment of the invention a monoclonal antibody specific for AChE with an unaltered glycosylation pattern is used to detect its presence. Typically the monoclonal antibody is MA3-042 (clone HR2), available from Chemicon International Inc of Temecula, Calif. Other suitable monoclonal antibodies may be used, for example, MA304 (clone AE1) also available from Chemicon International Inc.
While not wishing to be bound by theory, it is believed that the abnormal isoform is the
Saez-Valero Javier
Sberna Gian
Small David Henry
Chaudhry Mahreen
Clark & Elbing LLP
Gitomer Ralph
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