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  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2814—Dementia; Cognitive disorders
  • G01N2800/2821—Alzheimer
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/827—Actinoplanes

Definitions

• the present invention is concerned with a diagnostic test for Alzheimer's disease.
• AD Alzheimer's disease
• A/ amyloid protein
• APP amyloid protein precursor
• cytoskeletal protein tau The principal constituent of neurofibrillary tangles is the cytoskeletal protein tau (Kosik, 1992) .
• AD Alzheimer's disease
• AChE acetylcholinesterase
• choline acetyltransferase activity in regions of the brain such as the cortex, hippocampus, amygdala and nucleus basalis
• 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).
• AD Alzheimer's disease
• 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.
• CSF cerebrospinal fluid
• 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
• AChE has been shown to exist as up to six different molecular isoforms, three of which are the monomeric (Gl), 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 Gl 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).
• An anomalous isoform of AChE distinguished by its isoelectric point, has been detected in the CSF of AD patients (Navaratnam et al . , 1991; Smith et al . , 1991), and a method for screening for AD based on these findings is described in US patent number 5,200,324.
• the method comprises determining, by means of isoelectric focusing, if a patient has an anomalous form of AChE in his CSF.
• 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) .
• AD Alzheimer's disease
• 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.
• biochemical analysis techniques such as HPLC and mass spectrometry
• immunological techniques such as ELISA or. assays.
• a particularly preferred means of measuring the relative proportions of the isoforms of AChE involves a lectin-binding analysis.
• 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.
• 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.
• the total AChE activity is measured and the C/W ratio plotted against AChE activity.
• a monoclonal antibody specific for AChE with an altered glycosylation pattern is used to detect its presence.
• the monoclonal antibody is MA3-042 (clone HR2), available from Chemicon International Inc of Temecula, California.
• MA3-042 clone HR2
• Other suitable monoclonal antibodies may be used, for example, MA304 (clone AEl) also available from Chemicon International Inc.
• the abnormal isoform is the amphiphilic, monomeric isoform of AChE and/or the amphiphilic, dimeric isoform of AChE.
• the body fluid analysed can be cerebrospinal fluid (CSF), blood or blood plasma.
• CSF cerebrospinal fluid
• blood plasma is prepared from the blood for analysis .
• the blood plasma is treated to remove or inactivate butyrylcholinesterase (BChE) prior to analysis .
• an abnormal isoform of the acetylcholinesterase (AChE) with an altered pattern of glycosylation being the amphiphilic, monomeric isoform of AChE and characterised in that it has a relatively lesser affinity for Concanavalin (Con A) and a relatively greater affinity for wheat germ agglutinin (WGA) than AChE with an unaltered glycosylation pattern.
• Con A Concanavalin
• WGA wheat germ agglutinin
• an abnormal isoform of acetylcholinesterase (AChE) with an altered glycosylation pattern being the amphiphilic, dimeric isoform of AChE and characterised in that it has a relatively lesser affinity for Concanavalin A (Con A) and a relatively greater affinity for wheat germ agglutinin (WGA) than AChE with an altered glycosylation pattern.
• Con A Concanavalin A
• WGA wheat germ agglutinin
• Figure 1 is a plot of the C/W ratio for a number of patients in a control group, patients with Alzheimer's disease (AD) , patients with other neurological disorders distinct from Alzheimer's disease (AD) and patients with non-Alzheimer's disease type dementia (DNAT) .
• Figure 3 shows AChE activity vs fraction number for hydrophobic interaction chromatography of CSF AChE on phenyl-agarose.
• Samples of CSF from AD patients (open circles) or controls (closed circles) were applied to 10 ml columns of phenyl-agarose.
• Hydrophilic AChE isoforms (HF) were eluted with 50 mM Tris-saline buffer and then bound amphiphilic isoforms (AF) were eluted with 50 mM Tris-HCl (TB) (pH 7.4) containing 2% (w/v) Triton X-100. Fractions of 1.4 ml were collected and assayed for AChE activity.
• HF Hydrophilic AChE isoforms
• AF amphiphilic isoforms
• Figure 4 is an analysis of AChE isoforms and glycosylation in AD and control CSF.
• a hydrophilic fraction (HF) and an amphiphilic fraction (AF) were obtained from a total CSF fraction by hydrophobic interaction chromatography (Fig. 2) .
• the C/W ratio in the total CSF, HF and AF fractions was determined, and then fractions were applied to 5-20% sucrose density gradients containing 0.5% (w/v) Brij 97 and centrifuged at 150,000xg for 18 hr. Fractions from the sucrose gradient were collected and assayed for AChE activity.
• Enzymes of known sedimentation coefficient, catalase (C, 11.4S) and alkaline phosphatase (P, 6. IS) were used to determine the approximate sedimentation coefficients of AChE isoforms.
• Figure 5 is an analysis of AChE isoforms and glycosylation in frontal cortex and cerebellum from controls, non-demented individuals with diffuse plaques (DP) and AD patients.
• Samples of brain were homogenised and extracted to obtain SS and TS fractions. Equal volumes of SS and TS fractions were mixed and applied to 5-20% sucrose density gradients containing 0.5% (w/v) Brij 97 and centrifuged at 150,000x for 18 hr. Fractions were collected and assayed for AChE activity.
• Individual AChE isoforms were identified by its coefficient of sedimentation using enzyme markers: catalase (C, 11.4S) and alkaline phosphatase (P, 6. IS).
• MA3-042 on the sedimentation velocity of AChE isoforms from human frontal cortex, as described in Example 3.
• Immobilised leetins Con A- and LCA-Sepharose, WGA-, RCA 120 -, DBA-, UEAj;-, SBA and PNA-agarose
• phenyl- agarose phenyl- agarose
• bovine liver catalase E.
• Sepharose CL-4B was purchased from Pharmacia Biotech AB (Uppsala, Sweden) .
• Lumbar or ventricular CSF was obtained post mortem; 18 controls with no clinical or pathological dementia and no clinical or pathological dementia and no evidence of brain pathology, 27 cases of AD, 7 cases of dementia non-AD type (DNAT, 5 frontal lobe dementia, 1 Lewy body dementia/Parkinson's disease and 1 multi-infarct dementia/congophilic amyloid angiopathy), and 6 cases of other neurological disorders (ND, 4 Huntington's disease, 1 schizophrenia and 1 corticobasal degeneration) .
• the average age in the control group was 68 ⁇ 4years, there were 10 females and 8males and the PMI was 40 ⁇ 6.
• the age was 81 ⁇ 2 years, there were 13 female and 14 males and the PMI was 35 ⁇ 6.
• the enzyme-lectin mixture was incubated overnight at 4°C, and then centrifuged (1,000 xg, 15 min) .
• AChE activity was assayed in the supernatant fractions. Data were analysed using a Student's t-test.
• Ventricular and lumbar CSF, frontal cortical and cerebellar samples were obtained post mortem and stored at -80°C.
• DP non-neuritic Ab-immunoreactive diffuse plaques
• ND neurological diseases
• Samples of CSF were thawed slowly at 4°C and then centrifuged at l,000xg for 15 min prior to use.
• Small pieces (0.5 g) of frontal cortex and cerebellum were thawed slowly at 4°C, weighed and homogenised (10% w/v) in ice- cold Tris-saline buffer (TSB; 50 mM Tris-HCl, 1 M NaCl, and 50 M MgCl 2 , pH 7.4) containing a cocktail of proteinase inhibitors (Silman et al., 1978).
• TTB Tris-saline buffer
• Tissues were homogenised with a glass/Teflon homogeniser and then sonicated with 10- 15 bursts at 50% intermittency at setting 4 using a Branson sonifier.
• the suspension was centrifuged at 100,000x at 4 2 C in a Beckman L8-80M ultracentrifuge using a 70.1 Ti rotor for 1 hr to recover a salt-soluble ChE fraction (SS) .
• SS salt-soluble ChE fraction
• the pellet was re-extracted with an equal volume of TSB containing 1% (w/v) Triton X-100, and the suspension centrifuged at 100,000xg at 4°C for 1 hr to obtain a Triton X-100-soluble ChE fraction (TS) .
• TS Triton X-100-soluble ChE fraction
• AChE activity was determined by a modified microassay method of Ellman (Saez-Valero et al., 1993). One unit of AChE activity was defined as the number of nmoles of acetylthiocholine hydrolysed per min at 22°C. Protein concentrations were determined using the bicinchoninic acid method with bovine serum albumin as standard (Smith et al . , 1985). Hydrophobic interaction chromatography on phenyl-agarose
• Amphiphilic AChE forms were separated from hydrophilic forms by hydrophobic interaction chromatography on phenyl-agarose as previously described (Saez-Valero et al., 1993). CSF (10 ml-pooled from four samples obtained from four different subjects) was applied to a column (10x1 cm) of phenyl-agarose. A hydrophilic fraction (HF) containing hydrophilic isoforms of AChE was eluted with 30 ml of TSB, and then an amphiphilic fraction (AF) containing bound amphiphilic isoforms was eluted with 50 mM Tris-HCl (TB, pH 7.4) containing 2% (w/v) Triton X-100. Peak fractions with high AChE activity were pooled and concentrated using Ultrafree-4 Centrifugal Filter Device Biomax 10 kDa concentrators (Millipore Corporation, Bedford, MA, USA) .
• G 4 /(G 2 +G ⁇ ), that reflected the proportion of G 4 molecules (G 4 n +G 4 a ) versus both light globular AChE isoforms, G 2 a and G ⁇ a was defined.
• Estimation of the relative proportions of each molecular form of AChE was performed by adding the activities under each peak (G 4 or G 2 +G ⁇ ) and calculating the relative percentages (recovery >95%) .
• Samples (0.3 ml) were added to 0.1 ml (hydrated volume) of Sepharose 4B (control), Con A, WGA, RCA ⁇ 0 , LCA, DBA, UEA Ir SBA or PNA immobilised in agarose or Sepharose.
• the enzyme-lectin mixture was incubated overnight at 4°C with gentle mixing. Bound and free AChE were separated by centrifugation at lOOOxg for 15 min at 4°C in a Beckman J2- 21M/E centrifuge using a JA-20 rotor, and the unbound AChE was assayed in the supernatant fraction.
• Percentage of unbound AChE in the lectin incubation was calculated as (AChE unbound to lectin / AChE unbound to Sepharose) x 100.
• the C/W ratio was calculated according to the formula, AChE activity unbound in the Con A incubation divided by the AChE activity unbound in the WGA incubation. It was observed that this ratio detects a specific alteration in AChE glycosylation that occurs in AD CSF.
• AChE glycosylation of AChE
• CSF samples from 18 controls and 30 cases of AD were incubated with different immobilised lectins, which recognise different sugars .
• AChE bound strongly to Con A, WGA and LCA but weakly to RCA ⁇ 20 , PNA, DBA, UEAi and SBA (Table 1), suggesting that most of the enzyme was devoid of terminal galactose, terminal N-acetyl-galactosamine or fucose.
• CSF was fractionated by hydrophobic interaction chromatography on phenyl-agarose (Fig. 3) .
• AChE glycosylation reflect a change in the expression or glycosylation of brain AChE isoforms
• the levels of AChE activity in samples of frontal cortex and cerebellum were examined. Samples were homogenised with salt and Triton X- 100 to extract soluble and membrane-bound AChE isoforms, and then the AChE activity determined in both fractions (Table 2) .
• the frontal cortex samples from AD patients had significantly less AChE activity in the Triton X-100- soluble (TS) fraction ( ⁇ 40%), with no difference in levels in the salt-soluble (SS) fraction compared with controls (Table 3) .
• AChE isoforms in the frontal cortex and cerebellum were examined. Equal volumes of SS and ST supernatants (total AChE activity) were pooled and then analysed by sucrose density gradient sedimentation with 0.5% (w/v) Brij 97 to separate the major AChE isoforms (Fig. 5) .
• G 4 AChE between the AD and non-AD groups (Fig. 5) .
• the G 2 +G ⁇ fraction possessed C/W ratios >1.00, demonstrating that G 2 or G_ AChE is glycosylated differently from the G 4 isoform.
• the C/W ratio for G 2 +G ⁇ AChE was higher in the AD group than controls or DP.
• the C/W ratio of the amphiphilic fraction from CSF was higher in the AD group than in controls (Fig. 3). There was no correlation between the G 4 /(G 2 +G ⁇ ) ratio and the C/W ratio in the DP group in frontal cortex.
• This Example shows that AChE is glycosylated differently in the frontal cortex and CSF of AD patients compared with AChE from non-AD groups including patients with non AD-type dementias. This difference in glycosylation is due to an increase in the proportion of differentially glycosylated amphiphilic dimeric and monomeric AChE in the AD samples .
• the results suggest that the abnormally glycosylated AChE in AD CSF may be derived from the brain as a similar difference in glycosylation was also found in the frontal cortex of AD patients.
• CSFs were taken post mortem and the diagnosis confirmed by pathological examination.
• AChE was assayed in the supernatant fractions.
• the data represent the means ⁇ SEM. a Significantly different (P ⁇ 0.001) from the control group as assessed by Student's t test; b significantly different (P ⁇ 0.05) from the control group as assessed by Student's t test.
• AChE activity Protein (mg/ml)
• Tissue from frontal cortex or cerebellum was homogenised and salt-soluble (SS) and Triton X-100-soluble (TS) extracts obtained. The extracts were then assayed for AChE and protein.
• DP non-demented subjects with diffuse plaques
• ND individuals with other neurological diseases and dementias of non-AD type
• AD individuals with Alzheimer's disease.
• SS and TS fractions from frontal cortex and cerebellum were pooled in equal volumes and then analysed by lectin binding using immobilised Con A and WGA.
• the C/W ratio was calculated as defined in Table 2.
• Aliquots of the supernatants (SS+TS) were also analysed by sucrose density gradient sedimentation to identify AChE isoforms . Values are means ⁇ SEM. a Significantly different (P ⁇ 0.005) from the control group as assessed by Student's t test; b significantly different (P ⁇ 0.05) from the control group as assessed by Student's t test.
• Triton X-100 (1 % w/v) solubilized AChE were incubated overnight at 4°C without (see left panel of Fig 6) or with (see right panel of Fig 6) MA3-042 (dilution 1:50 by vol.).
• AChE isoforms were separated by centrifugation on 5-20% sucrose gradients made in 50 mM Tris saline buffer pH 7.4 containing 0.5% Triton X-100. The tube was centrifuged at 150,000 xg at 4°C, fractions were collected from the bottom and assayed for AChE activity. Sedimentation markers were catalase (11.4S) and alkaline phosphatase (6. S).
• Blood is collected and 1 ml of plasma or serum prepared using standard techniques.
• the fluid is passed across a 5 ml RCA-Agarose (RCA stands for ricinus communis agglutinin) to remove butyrylcholinesterase and the amount of acetylcholinesterase activity eluting from the column is monitored using the Ellman assay and the peak 2 ml of activity collected.
• This material would then be incubated for 10 min at ambient temperature with 50 micromolar iso- OMPA to inhibit the remaining butyrylcholinesterase, then passed across a 1 ml column of MAb MA3-042 coupled to Sepharose to remove non-specific AChE isoforms.
• the amount of activity eluting from the column is assayed using the Ellman assay.
• the amount of activity present in this fraction is greater in AD cases than in non-AD cases. There is normally less that about 40 mUnits of AChE / ml of original plasma or serum.
• the present invention provides a diagnostic test for Alzheimer's disease.
• Acetylcholinesterase accelerates assembly of amyloid-b-peptides into Alzheimer's fibrils - possible role of the peripheral site of the enzyme. Neuron 16, 881-891.
• Acetylcholinesterase is a senile plaque component that promotes assembly of amyloid beta-peptide into Alzheimer's filaments. Mo 1 . Psychiatry 1, 359-361. Kang J., Lemaire H.-G., Unterbeck A., Salbaum J. M., Masters C. L., Grzeschik K.-H., Multhaup G., Beyreuther K., and Muller-Hill B. (1987) The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature 325, 733-736.
• amyloid b-protein of Alzheimer's disease increases acetylcholinesterase expression by increasing intracellular calcium in embryonal carcinoma P19 cells. J. Neurochem. 69, 1177-1184.
• Acetylcholinesterase is increased in the brains of transgenic mice expressing the C-terminal fragment (CTlOO) of the b-amyloid protein precursor of Alzheimer's disease. J. Neurochem. 71, 723-731.

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