MXPA00002858A - Diagnostic test for alzheimer's disease - Google Patents

Abstract

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. It has been established that approximately 75-95%of the AChE in the CSF of AD patients binds to Concanavalin (Con A) or wheat germ agglutinin (WGA) but with different specificity to each. Accordingly, 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. In an alternative embodiment of the invention a monoclonal antibody specific for AChE with an altered glycosylation pattern is used to detect its presence.

Description

PROOF OF DIAGNOSIS FOR ALZHEIMER'S DISEASE TECHNICAL FIELD The present invention is related to a diagnostic test for Alzheimer's disease.

BACKGROUND OF THE INVENTION Alzheimer's disease (AD) (AD) is a progressive, common dementia that includes memory loss and increased cognitive function. The disease is characterized by the presence of amyloid deposits in the brains of those who suffer from it. These deposits can be extracellular (amyloid plaques) and intracellular (neurofibrillary tangles). The main constituent of the amyloid plaques is the amyloid protein (Aβ) that is produced by dissociation or proteolytic cleavage for the precursor of the amyloid protein (APP) (Evin et al., 1994). The main constituent of neurofibrillary tangles is the cytoskeletal tau protein (Kosik, 1992). One of the characteristic neurochemical changes observed in AD is the loss of acetylcholinesterase activity (AChE) and choline acetyltransferase in regions of the brain such as the cortex, hippocampus, amygdala and bulbar olive (Whitehouse et al., 1981, 1982; Struble et al. 1982; Mesulam and Geula, 1988). The loss of the cholinergic structure and the markers correlate with the number of plaques and lesions of the tangles present, as well as the clinical severity of the disease (Perry et al., 1978; Wilcock et al., 1982; Neary et al. 1986, Perry, 1986). The precise diagnosis of AD during life is essential. However, the clinical evaluation is, at best, only approximately 80% accurate. Therefore, there is a need to identify specific biochemical markers of AD. So far, the analysis of blood or cerebrospinal fluid (CSF) (CSF) has not yet produced a biochemical marker of sufficient diagnostic value (Blass et al., 1998), although detectable differences are reported. in the concentrations of certain proteins (Motter et al., 1995). Studies of the concentrations of AChE activity in the blood and cerebrospinal fluid (LCE) have been proposed as an ante mortem diagnostic test for AD. However, no consensus has been reached as to whether AChE concentrations are consistently affected in these tissues. It has been reported that the concentration of AChE in serum or plasma is 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; Servido et al., 1989) in patients with AD. It has been reported that the concentration of AChE in erythrocytes is not affected (Atack et al., 1985; Perry et al., 1982) or decreases (Chipperfield et al., 1981). The concentration of AChE activity in the LCE of patients with AD, as reported, decreases (more recently by Appleyard and McDonald, 1992; Shen et al., 1993) or does not change (more recently by Appleyard et al., 1987; Ruberg et al., 1987). It has been shown that AChE exists as up to six different molecular isoforms, three of which are the monomeric (Gl), dimeric (G2) and tetrameric isoforms (G4) (Massoulié et al., 1993). The relative proportion of the different isoforms of AChE is 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 regions of the brain for AD including the Brodman areas 9, 10, 11, 21 and 40, as well as the amygdala (Fishman et al., 1986). The asymmetric isoforms with collagen tail (A12) are increased up to 400% in the Brodman area 21, although these represent only a trace amount of the total AChE in the human brain (Younkin et al., 1986). However, the expression of AChE and the distribution of isoforms with sufficient sensitivity or specificity to be useful diagnostic markers of AD have not been found to date. An anomalous isoform of AChE, distinguished by its isoelectric point, has been detected in the LCE of patients with AD ': (Navaratnam et al., 1991; Smith et al., 1991), and the method for detecting EA based on these findings is described in U.S. Patent No. 5,200,324. The method consists of determining, by means of the isoelectric focus, if a patient has an anomalous form of AChE in his LCE. However, the isoform detected by Navaratnam et al., And Smith et al., Has also been detected in the LCE of patients with other neurological diseases (Shen and Zhang, 1993). In fact, in US Pat. No. 5,200,324, in column 7, lines 19-22, this is suggested, where it is established that abnormal AChE "was present in the LCE of four of eight patients with a clinical diagnosis of possible dementia, but those who did not meet the strict histopathological criteria for Alzheimer's disease ". In addition, the text in column 7, lines 60-61 of the U.S. Patent indicates that the detection of AChE-EA in lumbar LCE depends on the amount of LCE analyzed, and column 8, lines 38-40 states that the abnormal band It was often rather weak and the gels that were run were not always ideal. Therefore, a load of 5 mU per pinch was adopted as a normal procedure to scan LCE for the presence of the anomalous form of AChE, and each gel was read independently for four individuals who recorded their interpretation. Thus, there are technical problems associated with the described test, which can only be overcome by adopting a series of arbitrary conditions to avoid false readings, which then make 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 in patients with AD, together with the technical problems associated with the assay described in US Patent No. 5,200,324 suggests that the abnormal electroform of the 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.

DESCRIPTION OF THE INVENTION There is still a need for a diagnostic test for AD based on biochemical analyzes of body fluids such as blood or LCE and the present invention provides this test on the basis that the AChE of patients with AD show a pattern different from glycosylation with respect to the AChE of groups without EA. According to a first aspect of the present invention, a method for the diagnosis of Alzheimer's disease (AD) in a patient is provided, consisting in the steps of: (1) providing a mixture of a suitable patient's body fluid; (2) detect the presence in the sample of acetylcholinesterase (AChE) with an altered glycosylation pattern. In one embodiment of the invention, the relative proportion of the AChE with a first glycosylation pattern and the AChE with a second glycosylation pattern is measured. The measurement of the relative proportions of the AChE with the first and second glycosylation patterns can be performed in any convenient way, for example, using biochemical analysis techniques such as HPLC and mass spectrometry, or immunological techniques such as ELISA or assays. , [sic]. However, a particularly preferred means for measuring the relative proportions of the AChE isoforms involves a lecithin binding assay. It has been established that approximately 75-95% of the AChE in the LCE of patients with AD binds to concanavalin (Con A) or wheat germ agglutinin (WGA) but with different specificity for each. Accordingly, in a particularly preferred embodiment of the invention, to identify the glycosylation pattern of the AChE in the sample, the binding to Con A is determined, then it is determined > the binding to WGA and then the ratio is calculated. The ratio is characteristic of the glycosylation pattern. It is particularly convenient to measure the activity of the unbound AChE in each experiment, hence the relationship of AChE not linked to Con A to the relationship of AChE not linked to WGA. This relationship will be referred to below as the C / W ratio. For patients with AD, the C / W ratio has generally been found above 0.95, while for patients who do not suffer from AD, the C / W ratio is usually below 0.95. For convenience, the total AChE activity is measured and the C / W ratio is plotted against the activity of the AChE. In an alternative embodiment of the invention, a monoclonal antibody specific for AChE with an altered glycosylation pattern is provided and used to detect its presence. Typically, the monoclonal antibody is MA3-042 (clone HR2), available from Chemicon International Inc. of Temecula, California. It is possible to use other suitable monoclonal antibodies, for example, MA304 (clone AE1), also available from Chemicon International Inc.

Although no theory is desired, the abnormal isoform is considered to be the amphilic, monomeric isoform of the AChE and / or the amphilic, dimeric isoform of the AChE. The body fluid analyzed can be the cerebrospinal fluid (CSF), blood or blood plasma. For convenience, when the body fluid is blood, the blood plasma is prepared from the blood for analysis. Blood plasma is treated to separate or inactivate butyrylcholinesterase (BChE) before analysis.

According to a second aspect of the present invention, an abnormal isoform of acetylcholinesterase (AChE) with an altered glycosylation pattern is provided, the amphilic, monomeric isoform of AChE being characterized and having a relatively lower affinity for concanavalin (With A ) and a relatively higher affinity for wheat germ agglutinin (WGA) compared to AChE, with a non-altered glycosylation pattern. According to a third aspect of the present invention there is provided an abnormal isoform of acetylcholinesterase (AChE) with an altered glycosylation pattern, the amphiphilic isoform being dimeric of AChE and characterized because it has a relatively lower affinity for concanavalin A (With A ) and a relatively higher affinity for wheat germ agglutinin (WGA) compared to AChE with an altered glycosylation pattern.

BRIEF DESCRIPTION OF THE DRAWINGS Figure f is a graph 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 other than Alzheimer's disease (AD) and patients with dementia different from the Alzheimer's disease type (DNTA). The circles represent the ventricular LCE; the triangles represent the lumbar LCE; the clear symbols > 60 years old; the dark symbols < 60 years old The average values are expressed as ± S. E. M. * = significantly different from EA (P <0.001). The experiment is described in Example 1. Figure 2 is a graph of the C / W ratio compared to the activity of AChE in post-mortem human LCE. Dashed lines show C / W values and AChE activity that mainly discriminates between groups with AD and non-AD. Approximately 80% of all EA samples were above the cut-off values of C / W = 0.95, while all EA samples were above C / W = 0.60. In the same way, all the samples with EA had less than 15.8 U / ml of AChE activity. The experiment is described in Example 2. Figure 3 shows the activity of the AChE compared to the fraction number for the hydrophobic interaction chromatography of the AChE of the LCE in phenyl agarose. Samples of the LCE from patients with AD (clear circles) or controls (dark circles) were applied to columns of 10 ml of phenyl agarose. The hydrophilic AChE isoforms (HF) were eluted with 50 mM Tris-saline buffer and then the 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 tested for AChE activity. FIGURE 4 is an analysis of the AChE isoforms and glycosylation in the EA LCE and control. The hydrophilic fraction (HF) and an amphiphilic fraction (AF) were obtained from a total fraction of the LCE by hydrophobic interaction chromatography (Figure 2). The C / W ratio was determined in the total CSF, HF and AF fractions, and then the fractions were applied to gradients of 5-20% sucrose density containing 0.5% (w / v) Brij 97 and centrifuged at 150,000 xg during 18 hours. The fractions of the sucrose gradient were collected and tested for • the activity of AChE. The enzymes of known sedimentation coefficient, catalase (C, 11.4S) and alkaline phosphatase (P, 6.1S) were used to determine the approximate sedimentation coefficients of the AChE isoforms. Figure 5 is an analysis of the isoforms of AChE and glycosylation in the frontal cortex and cerebellum of the • controls, people without dementia with diffuse plaques (PD) and patients with AD. The brain samples were homogenized 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 150,000 x g for 18 hours. The fractions were collected and tested for the activity of the AChE. The individual isoforms of the AChE were identified by their sedimentation coefficient using enzymatic markers: catalase (C, 11.4S) and alkaline phosphatase (P, 6.1S). The enzymatic peaks of the AChE G4 and G2 + Gi were selected, concentrated and dialyzed to separate the sucrose. The main peaks G4 and G2 + Gi were then analyzed by binding to lecithin using Con A and WGA and then the C / W ratio was determined from each peak. Figure 6 shows the effect of the monoclonal antibody MA3-042 on the rate of sedimentation of the AChE isoforms from the human frontal cortex, as described in Example 3.

BEST WAY TO CARRY OUT THE INVENTION Abbreviations used: AChE, acetylcholinesterase; ChE, cholinesterase; Aß, ß amyloid protein; EA, Alzheimer's disease; PD, diffuse plates; IN, other neurological diseases; IPM post mortem interval; PBS, phosphate-saline buffer; TB, Tris buffer; TSB, Tris-saline buffer; SS, supernatant soluble in salt; TS, soluble supernatant in Triton X-100; FA, amphiphilic fraction; FH, hydrophilic fraction; Ga, globular amphiphilic isoform; Gna, globular non-amphiphilic isoform; and agglutinins from canavalia ensiformis (Concanavalina A), Con A; Tri tumum Vulgaris (wheat germ) WGA; Ricinus communis, RCA120; Lens culinaris, LCA; Dolichus biflorus, DBA; C7lex europaus, UEA; r .; Glycine max, SBA; and Arachis hypogaea, PNA.

Materials Immobilized lecithins (With A- and LCA-Sepharose, WGA-, RCA120- / DBA-, UEAi-, SBA and PNA-agarose), phenyl-agarose, bovine liver catalase, E. coli alkaline phosphatase, polyoxyethylene- 10-oleyl ether (Brij 97), Triton X-100, tetraisopropyl pyrophosphoramide (iso-OMPA), 1,5-bis (4-alidimethyl-ammonio-phenyl) -pentan-3-1 (BW284c51) dibromide, acetylthiocholine iodide and 5, 5'-dithio-bis-nitrobenzoic acid (DTNB) were all obtained from Sigma-Aldrich Pty. Ltd. (Seven Hills, NSW, Australia). Sepharose CL-4B was purchased from Pharmacia Biotech AB (Uppsala, Sweden).

EXAMPLE 1 Lecithin binding experiments in patients with AD Lumbar or ventricular CSF was obtained post mortem; 18 controls with non-clinical or pathological dementia and without clinical or pathological dementia and without evidence of pathology in the brain, 27 cases of AD, 7 cases of non-EA type dementia (DTNA, 5 with frontal lobe dementia, one with Lewy body dementia / Parkinson's disease and one dementia due to multiple infarctions / congophilic amyloid angiopathy), and 6 cases of other neurological abnormalities (TN, 4 with Huntington's disease, one with schizophrenia and one with corticobasal degeneration). The average age in the control group was 68 + 4 years, there were 10 women and 8 men and the PMI was 40 ± 6. In the EA group the age was 81 ± 2 years, there were 13 women and 14 men and the PMI was 35 ± 6. In the EN group, the age was 65 ± 6, there were 3 women and 3 men and the PMI was 45 ± 12. In the DTNA group the age was 76 ± 3, there were 4 women and 3 men and the PMI was 34 ± 11. The LCE samples were stored at -70 ° C and centrifuged at 1000 xg for 15 minutes before analysis. The activity of the AChE was assayed at 22 ° C by a modified microassay of the Ellman method (Elman et al., 1961) aliquots (0.3 ml) were mixed with 0.1 ml of Sepharose 4B in PBS (control), concanavalin A (Con A) or wheat germ agglutinin (WGA, Tri ticum vulgaris) immobilized on Sepharose. The enzyme-lecithin mixture was incubated overnight at 4 ° C, and then centrifuged (1000 x g, 15 min). The AChE activity was assayed in the fractions of the supernatant. The data were analyzed using a Student's t-test. The total AChE values in the ventricular LCE samples of patients with > 60 years were significantly lower in the EA group (6.98 ± 0.82 nmol / min / ml) compared to the controls (17.24 ± 4.28 nmol / min / ml); P < 0.001). However, as already reported, (Appleyard et al., 1983), the large overlap (40%) between the data prevents the use of total AChE as a significant diagnostic marker. However, the analysis of lecithin binding showed a significant difference in the EA group and the controls. Approximately 75-95% of the AChE in the LCE joined with A or WGA. A relationship (C / W ratio) was defined as AChE not linked to Con A divided by AChE not linked to WGA. The average C / W ratio for the EA group was significantly different from the controls (Figure 1). Of the 27 confirmed LCE of EA, 21 samples had a C / W > 0.95. All 18 control samples had C / W less than 0.95, without significant differences between the samples of the younger patients (n = 5, C / W = 0.37 ± 0.10) and those of the oldest (n = 6, 0.38 ± 0.08). I do not know; observed correlation in the C / W ratio with post mortem interval (MPI). The data is graphically represented in Figure 1. The data indicate that the lecithin-binding assay of the AChE of the LCE can provide a diagnostic test for AD that is 80% sensitive and 97% specific. Thus, it was proposed that the differences observed in the AChE glycosylation pattern in the LCE may be useful as a marker of ante mortem diagnosis for AD, particularly when used in combination with the measurement of other biochemical markers.

EXAMPLE 2 Other lecithin binding experiments Experimental procedures Human brain and LCE samples Samples of ventricular and lumbar LCE, frontal cortex and cerebellum, post mortem, and were stored at -80 ° C. Three groups of non-EA samples were defined, 1) controls without clinical or pathological dementia characteristics (n = 18), 2) individuals who did not present clinical signs of dementia but who were found with a moderate number of diffuse plaques, (PD Ab immunoreactive, not neuritic, but without evidence of neocortical neurofibrillary changes. (n = 6), and 3) individuals with different neurological diseases (EN) containing 7 cases of non-EA dementia (5 dementia of frontal lobe, one dementia of the body of Lewy and one vascular dementia) and 7 cases of other neurological abnormalities (4 Huntington's disease, one Parkinson's disease, one schizophrenia and one corticobasal degeneration). The cases of AD were selected based on their clinical history of dementia and the neuropathological CERAD diagnosis (Mirra et al., 1994). All the LCE samples included in the EA and EN groups were ventricular and only 5 samples of control LCE and one PD (out of a total of 18 and 6 individuals, respectively) were taken for lumbar function. The immunohistochemical examination of the cerebellar samples showed that, unlike the frontal cortex, none of the AD tissues presented a deposit of amyloid plaque, neuritic, compact (data not shown), consistent with previous studies (Mann et al., 1996 ). It has been shown (Gras et al., 1982, Fishman et al., 1986, Sáez-Valero et al., 1993) that for a post-mortem interval (MPI) greater than 72 hours, storage at -20 ° C or cycles Repeated freezing / thawing caused degradation of AChE, which confused the glycosylation analysis. Therefore, only samples with an IPM of less than 72 hours (IMP = 36 ± 4 hours) were used. There was no significant difference in the MPI between each group of samples.

Sample preparation and AChE extraction Samples from the LCE were thawed slowly at 4 ° C and then centrifuged at 1,000 x g for 15 minutes before use. Small pieces (0.5 g) of frontal cortex and cerebellum were thawed slowly at 4 ° C, weighed and homogenized (10% w / v) in Tris-saline buffer (TSB, Tris-HCl, 50 mM, NaCl, and MgCl2 50 mM, pH 7.4) cooled in ice containing a cocktail of proteinace inhibitors (Silman et al., 1978). The tissues were homogenized with glass / Teflon homogenizer and then sonified with 10-15 bursts at 50% intermittence at position 4 using a Branson sonicator. The suspension was centrifuged at 100,000 x g at 4 ° C in a Beckman L8-80M ultracentrifuge using a 70.1 Ti rotor for one hour to recover a fraction of salt soluble (SS) ChE. The package was re-extracted with an equal volume of TSB containing 1% (w / v) Triton X-100, and the suspension was centrifuged at 100,000 xga 4 ° C for one hour to obtain a soluble ChE fraction in Triton X -100 (TS). This method of double extraction recovers 80-90% of the total activity of ChE (Sáez-Valero et al., 1993, Moral-Naranjo et al., 1996).

AChE Assay and Protein Determination AChE activity was determined by a modified microassay method from Ellman (Sáez-Valero et al., 1993). One unit of AChE activity was defined as the number of nmol of acetylthiocholine hydrolyzed per minute at 22 ° C. Protein concentrations were determined using the bicinchoninic acid method with bovine serum albumin as standard (Smith et al, 1985).

Chromatography by hydrophobic interaction on phenylagarose The amphiphilic forms of AChE were separated from the hydrophilic forms by hydrophobic interaction chromatography on phenyl Garosa as already described (Sáez-Valero et al., 1993). The LCE (10 ml combined from 4 samples obtained from 4 different individuals) was applied to a column (10 x 1 cm) of phenyl agarose. A hydrophilic fraction (FH) containing the hydrophilic isoforms of AChE was eluted with 30 ml of TSB, and then an amphiphilic fraction (FA) containing the 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 combined and concentrated using 10 kDa Ultra Free-4 centrifugal concentrators. Filter Device Biomax (Millipore Corporation, Bedford, MA USA).

Sedimentation analysis The molecular isoforms of the AChE were analyzed by ultracentrifugation at 150,000 x g in a continuous gradient of sucrose (5-20% w / v) for 8 hours at 4 ° C in a Beckman SW40 rotor. The gradients contained 10 ml of 50 mM Tris-HCl (pH 7.4) containing 0.5 M NaCl, 50 mM MgCl 2 and 0.5% (w / v) Brij 97. Approximately 40 fractions were collected from the bottom of each tube. Enzymes of known sedimentation coefficient catalase from bovine liver (11. S, S20 Svedberg units) and alkaline phosphatase from E. coli (6.1S) were used in the gradients to determine the approximate sedimentation coefficients of the AChE isoforms. A relation of the AChE species G4 / (G2 + Gi) was defined, which reflects the proportion of the G4 molecules (Gna + G43) against both isoforms of globular, light AChE, G2a and G? A. the estimation of the relative proportions of each molecular form of AChE was performed by adding the activities under each peak (G4 or G2 + Gi) and calculating the relative percentages (recovery> 95%).

Analysis of lecithin binding of AChE Samples (0.3 ml) were added to 0.1 ml (hydrated volume) of Sepharose 4B (control), With A, WGA, CRA? 20 /? LCA, DBA-, UEA, r.-, SBA and PNA immobilized in agarose or Sepharose. The enzyme-lecithin mixture was incubated overnight at 4 ° C with light mixing. The bound and free AChE were separated by centrifugation at 1000 x g for 15 minutes at 4 ° C in a Beckman J2-21 M / E centrifuge using a JA-20 rotor, and the unbound AChE was assayed in the supernatant fraction. The percentage of non-bound AChE in the lecithin incubation was calculated as (AChE not bound to lecithin / AChE not bound to Sepharose) x 100. The C / W ratio was calculated according to the formula, AChE activity not bound in the incubation of Con A divided by the non-bound AChE activity in the WGA incubation. It was observed that this relationship detects a specific alteration in the glycation of AChE that occurs in the LCE of EA.

Lecithin binding of LCE AChE To examine the glycosylation of AChE, LCE samples from 18 controls and 30 cases of AD were incubated with different immobilized lecithins, which recognize different sugars. AChE binds strongly to Con A, WGA and LCA, but weakly to RAC120, PNA, DBA, UEAi, and SBA (Table 1) suggesting that most of the enzyme was devoid of terminal galactose, terminal N-acetyl-galactosamine or fucose. There was a small but significant difference in the binding of AChE to Con A and WGA between the EA group and the controls (Table 1). As the percentage of AChE not bound in the LCE of EA was increased for Con A and decreased for WGA, a relationship was defined (C / W = [% of AChE that does not bind with Con A] / [% of AChE that does not bind to WGA]), which provides greater discrimination between the two groups (Table 1). When using this method it was found that the average C / W ratio for the EA group was significantly higher than for the other control groups, including cases with diffuse plaques (without dementia, PD), and patients with other neurological and neuropsychiatric diseases (EN ) (Figure 2), consistent with the results shown in Example 1. Of the 30 LCE samples from the confirmed EA cases, 24 samples were above a limit value of C / W = 0.95 (Figure 2). Only one sample of 18 controls, one of 6 samples of cases with diffuse plaques, and one of 14 samples of the other groups of neurological diseases, one case of dementia in the frontal lobe were above this value. The 6 EA samples with C / W ratios less than 0.95 had C / W ratios > 0.60, a value greater than the average C / W of the non-EA groups (control = 0.53 ± 0.1, PD = 0.46 ± 0.2, EN = 0.53 ± 0.1). No correlation could be found between the C / W ratio and the MPI that might suggest that the different C / W ratio in the EA group was due to differences in the MPI. In addition, there was no significant difference in the IPM between the EA samples / 33 ± 6 hours) and the non-EA samples (40 ± 6 hours). The LCE samples were also analyzed for the total activity of the AChE (Figure 2). As already reported (Appleyard et al., 1983) Atack et al., 1988), the LCE of patients with AD had significantly lower AChE activity (6.5 ± 0.8 U / ml) compared to the controls (15.8 ± 2.9 U / ml) or of patients with other diseases (12.4 + 2.4 U / ml). However, the C / W ratio was a more reliable index of clinical status than the total concentration of AChE activity in the LCE (Figure 2).

Isoforms of AChE in the LCE To determine if the alteration in glycosylation was due to changes in a specific isoform of AChE, samples of LCE were analyzed by hydrophobic interaction chromatography to separate amphiphilic (Ga) and hydrophilic (Gna) species ( Figure 3), and by centrifugation by sucrose density gradient in 0.5% (w / v) Brij 97 to separate individual isoforms by molecular weight (G4, G2 and Gi) (Figure 3). A decrease in the proportion of the AChE G4 was observed in the LCE EA compared to the controls (Figure 4), upper panels). The ratio of (G4 / (G2 + Gi) was significantly (p <0.01) higher in the controls (1.80 ± 0.12, n = 4) than in the EA cases (1.16 ± 0.12, n = 4). hydrophilic isoforms of the amphiphilic isoforms, the LCE was fractionated by hydrophobic interaction chromatography on phenyl agarose (Figure 3) A smaller percentage of AChE in the normal LCE was bound to phenyl agarose (12 ± 3%, n = 4) in comparison with EA LCE (38 ± 4%, n = 4, p <0.001). The sedimentation analysis of the unbound hydrophilic fraction (FH) showed a main peak of 10.8S, consistent with a tetrameric isoform (G4na) hydrophilic (Atack et al, 1987) as well as a small amount of the lighter AChE isoforms, 5.1S dimers and 4.3S monomers (Figure 4) The bound amphiphilic fraction of the phenyl agarose column contained a smaller peak 9.0-9.5S (probably an amphiphilic tetramer, G43) and a main peak of amphiphilic globular dimer (G2a, 4.2S) and monomer G? A, 3.1S). The concentration of the light amphiphilic isoforms was higher in the EA LCE than in the controls (Figure 4).

Glucosylation of the individual AChE isoforms in LCE Incubation of FH and FA with immobilized A and WGA showed that there was an increase in the C / W ratio in the EA LCE and that the high C / W ratio was associated with an amphiphilic fraction containing dimers and monomers (Figure 4). The data indicate that the contribution of the AChE Gi and Gi in the LCE EA was mainly responsible for the increased C / W ratio of the total AChE in the EA LCE.

Concentrations of AChE in the frontal cortex and cerebellum To determine whether changes in glycosylation of the AChE reflect a change in the expression or glycosylation of AChE isoforms in brain, concentrations of AChE activity in the samples of the frontal cortex and cerebellum were examined. The samples were homogenized with salt and Triton X-100 to extract the soluble and membrane-bound AChE isoforms, and then the AChE activity was determined in both fractions.

(Table 2). Samples from the frontal cortex of patients with AD had significantly lower AChE activity in the fraction soluble in Triton X-100 (TS) (~ 40%), without differences in the concentrations of the salt soluble fraction (SS) in comparison with the controls (Table 3). The results are consistent with previous studies that indicate that the main G4 isoform is decreased only in the TS fraction (Younkin et al., 1986; Siek et al., 1990). A small but significant decrease (~ 15%) in the protein content of the TS fraction of both EA and EN groups was also observed. The concentration of AChE in the samples of the frontal cortex of the EN group was significantly different from the controls in both the SS and TS fractions (Table 2). However, since the EN group was heterogeneous (two frontal lobe dementia, one Huntington's disease and one Parkinson's disease), the significance of changes in AChE concentrations is unclear. The concentrations of AChE in the cerebellum were also significantly decreased in the TF fraction of the EA group (Table 2).

Glycosylation of AChE in frontal and cerebellar cortex To determine if a different glycosylation pattern of AChE in EA LCE is also present in the AD brain, the glycation of the brain AChE was examined by binding to lecithin. The homogenates of frontal cortex and cerebellum were incubated with immobilized Con A or WGA and the amount of unbound activity was calculated. In the frontal cortex EA, the% activity of the AChE that did not bind to A or WGA was significantly different from the controls (Table 3). Like the AChE of the LCE, the C / W ratio of the AChE of the frontal cortex was higher in the SA samples than in the non-EA samples (Table 3). This increase was due to a large increase in the amount of AChE that did not join Con A, and was in spite of an increase in the amount of AChE that did not join WGA (Table 3). There was no increase in the C / W ratio in the PD group or EN (Table 3). No difference in lecithin binding was observed between the EA and non-EA groups in the cerebellar fractions (Table 3).

Isoforms of AChE in the frontal cortex and cerebellum To determine the cause of altered glycosylation in the AD brain, the pattern of AChE isoforms in the frontal cortex and cerebellum was examined. Equal volumes of the SS and ST supernatants (total activity of the AChE) were combined and then analyzed by sedimentation with sucrose density gradient with 0.5% (w / v) Brij 97 to separate the main AChE isoforms (Figure 5) . Based on their sedimentation coefficients (Atack et al, Massoulié et al., 1982) it was possible to identify the hydrophilic tetramers (G4na, 10.7 ± 0.1S) and amphiphilic tetramers (G4a, 8.6 ± 0.1S), the dimers (G2a, 4.7 + 0.1S) and AChE amphiphilic monomers (G? A 3.0 ± 0.1S) (Figure 6). There was no difference in the sedimentation coefficient (S) of the individual isoforms of each group. Due to the overlap in the sedimentation coefficients between AChE G4na and G4a, it was not possible to separate these isoforms. completely (Figure 5). However, G's contribution was greater than G4na. The asymmetric isoforms (A12) of the AChE were identified in trace quantities (2-5%) in some of the fractions. A significant decrease in the AChE G (40% of the average control value, p <0.001) and of the AChE G2 + Gi (60% of the average control value, p = 0.002) was detected in the fractions of the frontal cortex EA. This change in the relative proportion of the AChE isoforms was reflected in the G4 / (G2 + Gi) ratio, which was significantly lower in the EA samples (Table 3). It is interesting to note that a similar and statistically significant decrease was found in the G4 / (G2 + Gi) ratio for PD individuals. This change in the proportion was due to a 25% increase in the concentration of G2 + Gi and a small decrease (10%) in the AChE G4, although no change was statistically significant. No variation was found in the AChE G4 / (G2 + Gi in the cerebellum EA (Table 3), despite a statistically significant decrease (40%) in the AChE in the TS fraction (Table 2) and in the total concentration of AChE G4 (G4 in controls = 380 + 40 U / ml, G4 in EA = 195 ± 70 U / ml, p = 0.008).

Glucosylation of individual AChE isoforms in the frontal cortex and cerebellum Since it was found that the proportion of AChE was altered in the frontal cortex of patients with AD, steps were taken to assess whether the increase in the C / W ratio of brain AChE was due to a change in glycosylation or expression of a specific isoform of AChE. The individual AChE isoforms were separated by centrifugation with sucrose gradient, and then fractions of the G4 or G2 + Gi peaks were combined, dialyzed against TSB-Triton X-100 buffer and concentrated by ultrafiltration. The isoforms of the AChE were then tested by binding to lecithin and a C / W ratio was calculated for each isoform (Figure 5). No differences were observed in the C / W ratio of the AChE G4 between the EA and non-EA groups (Figure 5). However, in all samples of the frontal cortex, the fraction G2 + Gi presented proportions C / W >; 1.00, demonstrating that AChE G2 or Gi is glycosylated differently from the G isoform. In addition, the C / W ratio for the AChE G2 + Gi was higher in the EA group than in the controls or in PD. In the same way, the C / W ratio of the amphilic fraction of the LCE (containing mainly AChE G2 + Gi) was higher in the EA group than in the controls (Figure 3). There was no correlation between the G4 / (G2 + Gi) ratio and the C / W ratio in the PD group in the frontal cortex. In the cerebellum, no differences were observed in the C / W ratios of the AChE G4 or AChE G2 + Gi between the EA and non-EA groups (Figure 4). The G2 + Gi fractions of both EA and non-EA cerebellum groups had a C / W < 0.50, in contrast to the same fraction of the frontal cortex (C / W> 1.00) indicating differences in the glycosylation pattern of AChE G2 + Gi between both areas of the brain. This example shows that AChE is glycosylated differently in the frontal cortex and the LCE of patients with AD compared to the AChE of the groups without AD including patients without dementia type EA. This difference in glycosylation is due to an increase in the proportion of the dimeric and monomeric AChE, amphiphilic, glycosylated differently in the EA samples. The results suggest that abnormally glycosylated AChE in EA LCE may come from the brain as a similar difference in glycosylation in the frontal cortex of patients with AD was also found.

Table 1. Union of AChE lecithin in FCE. AChE lecithin not bound. { %) EA Control With A 5.5 ± 0.8 10.1 ± l.lb WGA 11.3 ± 1.7 7.0 ± 0.6 Coa A / WGA 0.53 0.1 1.37 ± 0.1 * (C / W) LCA 17.2 ± 4.2 15.0 ± 1.3 RCAxao 74.1 ± 3.4 70.8 ± 2.7 SBA 83.0 ± 2.1 82.2 ± 1.9 TEAi 91.6 ± 2.2 87.6 ± 1.9 PNA 92.4 ± 1.7 92.3 ± 1.4 DBA 98.9 ± 0.8 95.8 ± 1.7 All the LCE samples were taken post mortem and the diagnosis was confirmed by pathological examination. The LCE of normal individuals (control group: n = 18, 67 ± 4 years at death, 11 women / 7 men) and patients with AD (group EA: n = 30; 79 + 2 years; 15 women / 15 men) were incubated with an equal volume of different immobilized lecithins, and then centrifuged. The AChE was assayed in the supernatant fractions. The data represents the averages + SEM. a significantly different (p <0.001) of the control group when assessed by the t test Student; significantly different (p <0.05) from the control group when assessed by the Student's t test.

Table 2. AChE activity and protein concentration in human frontal cerebral cortex and cerebellum AChE activity Protein (mg / ml) (U / ml) Group / Source SS S SS S Control Frontal Cortex 3.7 ± 0.4 15.1 ± 2.1 ± 2.4 ± 0.1 (n = 11; 63 ± 5 1.5 0.1 A; 7M / 4H) Cerebellum 64 ± 6 264 ± 25 2.5 ± 1.9 ± 0.1 (n = 7; 66 ± 5A; 0.1 4M / 3H) PD Frontal cortex 5.5 ± 0.9 12.7 ± 2.1 ± 2.2 ± 0.1 (n = 6, 81 ± 2 A, 1.7 0.1 4M / 2H) Cerebellum 49 ± 8 182 ± 46 2.6 ± 0.1 1.9 ± 0.1 (n = 5; 81 ± 3 A; 3M / 2H) EN Front cortex 5.4 ± 9.3 ± 2.1 ± 2.0 ± 0.1b (n = 4; 67 ± 9 A; 0.6 * 1.7b 0.2 2M / 2H) Cerebellum 45 ± 8 160 ± 50 2.7 ± 2.3 ± 0.2 (n = 2; 78 ± 14 0.2 A; 1M / 1H) EA Frontal cortex 3.7 ± 0.3 9.0 ± 2.1 ± 2.1 ± 0.1 * (n = 14; 73 ± 3 0.9 * 0.1 A; 8M / 6H) Cerebellum 48 ± 12 160 + 28b 2.6 ± 0.1 2.0 + 0.1 (n = 7; 73 ± 6 A; 5M / 2H) The tissue of the frontal cortex and the cerebellum was homogenized and extracts soluble in salt (SS) and soluble in Triton X-100 (TS) were obtained. Then, the extracts were assayed for AChE and protein. PD = individuals without dementia, with diffuse plaques; EN = individuals with other neurological diseases and non-EA dementia; EA = individuals with Alzheimer's disease. M = woman; H = man; A = age in years. The values are average ± SEM. significantly different (p <0.005) from the control group when tested by the Student's t-test; significantly different (p <0.05) from the control group when assessed by the Student's t test.

Table 3. Binding to lecithin and AChE isoforms in the frontal cortex and cerebellum Proportion to lecithin binding AChE Group / Source A ACChhE A ACChhEE nnoo uunniiddaa C / W G4 / (Ga + G?) Not linked to WGA (%) a Con A (%) Control Frontal cortex 6.9 ± 0.8 12.3 ± 1.2 0.56 ± 1.90: (n = 11; 63 ± 5 0.03 0.14 A; 7M / 4H) Cerebellum 1.8 ± 0.1 10.7 ± 0.9 0.18 ± 3.02: (n = 7; 66 ± 5 A; 0.02 0.2 4M / 3H) PD Frontal cortex 7.4 ± 0.8 15.0 ± 1.0 0.50 ± 1.32 ± (n = 6; 81 ± 2 A; 0.06 0.12b 4M / 2H) Cerebellum 2.9 * 0.7 12.2 ± 1.3 0.23 ± 2.18 ± 3M / 2H) EN Frontal Cortex 7.0 ± 0.6 13.2 ± 1.2 0.47 ± 2.61 ± (n = 4; 67 ± 9A; 0.05 0.73 2M / 2H) Cerebellum 1.8 ± 0.2 10.1 ± 0.3 0.21 ± 2.50 ± (n = 2; 78 ± 14 0.10 0.70 A; 1M / 1H) EA Frontal cortex 13.1 ± 1.3a 19.7 + 1.4a 0.66 ± 1.34 + (n = 14; 73 ± 3 0.03b 0.18b TO; 8M / 6H Cerebellum 2.4 ± 0.3 13.5 + 2.3 0.19 + 2.33 + (n = 7; 73 ± 6 A; 0.02 0.49 5M / 2H The SS and TS fractions of the frontal cortex and the cerebellum were combined in equal volumes and then analyzed by binding to lecithin using Con A and WGA immobilized. The C / W ratio was calculated as defined in Table 2. The aliquots of the supernatants (SS + TS) were also analyzed by sedimentation with sucrose density gradient to identify the isoforms of the AChE. The values are averages ± SEM. a significantly different (p <0.005) from the control group when assessed by the Student's t-test; significantly different (p <0.05) from the control group when assessed by the Student's t test.

EXAMPLE 3 Union to monoclonal antibody MA3-042 AChE samples solubilized in Triton X-100 (1% w / v). were incubated overnight at 4 ° C without (see left panel of Figure 6) or with (see right panel of Figure 6) MA3-042 (1:50 volume dilution). The isoforms of AChE were separated by centrifugation in 5-20% sucrose gradients prepared in 50 mM Tris-saline buffer solution, pH 7.4, containing 0.5% Triton X-100. The tube was centrifuged at 150,000 x g at 4 ° C, fractions were collected from the bottom and assayed for AChE activity. The markers of sedimentation were catalase (11.4S) and alkaline phosphatase (6.2S). As seen in Figure 6, all peaks are displaced in the presence of MA3-042, indicating the binding of the monoclonal antibody to the particular isoform represented by each peak, except that one peak remains around 4S. The difference between 4. OS and 4.2S is statistically insignificant, suggesting that peak 4.2S represents an isoform with a modified glycosylation pattern, unrecognized by MA3-042. As will be appreciated by those skilled in the art, this peak represents an AChE monomer, which has a molecular weight of about 70,000 kDa.

EXAMPLE 4 Blood analysis using monoclonal antibody The blood is collected and 1 ml of plasma or serum is prepared using the normal techniques. The fluid is passed through 5 ml RCA-agarose (RCA stands for ricinus communis agglutinin) to separate butyrylcholinesterase and the amount of acetylcholinesterase activity eluting from the column is checked using the Ellman assay and the 2 ml peak of the activity collected. This material should then be incubated for 10 minutes at room temperature with 50 micromolar iso-OMPA to inhibit the remaining butylcholinesterase, then passed through a 1 ml MAb-MAb042 column coupled to Sepharose to separate non-specific isoforms from the AChE. The amount of activity eluting from the column is tested using the Ellman test. The amount of activity present in this fraction is greater in steps EA than in non-EA cases. Normally there are less than about 40 mUnit of AChE / ml of original plasma or serum.

INDUSTRIAL APPLICABILITY The present invention provides the diagnostic test for Alzheimer's disease.

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Claims (18)

1. A method for diagnosing Alzheimer's disease (AD) in a patient, comprises the steps of: (1) providing a sample of a patient's adequate body fluid; (2) detect in the sample the presence of acetylcholinesterase (AChE) with an altered glycosylation pattern.

2. The method of claim 1, wherein the relative proportions of the AChE are measured with a first glycosylation pattern and the AChE with a second glycosylation pattern.

3. The method of claim 2, wherein the lecithin binding assay is used to measure the relative proportions of the AChE with the first glycosylation pattern and the AChe with the second glycosylation pattern.

The method of claim 3, wherein the analysis of lecithin binding includes the measurement of concanavalin A (Con A) binding and wheat germ agglutinin (WGA).

5. The method of claim 4, wherein the activity of the unbound AChE is determined.

6. The method of claim 5, wherein the ratio of the AChE not bound to Con A to the AChE not bound to WGA is calculated.

The method of claim 6, wherein the ratio is above 0.95 in patients with AD.

8. The method of any of claims 1 to 7, wherein the total activity of the AChE is also determined.

9. The method of claim 8, wherein the ratio of the AChE not bound to Con A to the AChE not bound to WGA is plotted against the total activity of the AChE.

The method of claim 1, wherein monoclonal antibody is used to detect the presence of AChE with an altered glycosylation pattern.

The method of claim 10, wherein the monoclonal antibody is MA3-042 and AChE is detected with an altered glycosylation pattern by its binding failure.

The method of any of claims 1 to 11, wherein an abnormal isoform of the AChE is detected with an altered glycosylation pattern.

The method of claim 12, wherein the abnormal isoform is the monomeric, amphiphilic isoform of AChE and / or the dimeric, amphiphilic isoform of AChE.

The method of any of claims 1 to 13, wherein the body fluid is cerebrospinal fluid (LCE), blood or blood plasma.

15. The method of claim 14, wherein the body fluid is blood and the blood plasma is prepared from the blood for analysis.

16. The method of claim 14 or claim 15, wherein the body fluid is blood plasma and is separated and / or inactivated butyrylcholinesterase (BChE) prior to analysis for the presence of AChE with an altered glycosylation pattern.

17. An abnormal isoform of acetylcholinesterase (AChE) with an altered glycosylation pattern, being the monomeric, amphiphilic isoform of AChE characterized in that it has a relatively lower affinity for concanavalin A (Con A) and a relatively higher affinity for germ agglutinin of wheat (WGA) compared to AChE with an altered glycosylation pattern.

18. An abnormal isoform of acetylcholinesterase (AChE) with an altered glycosylation pattern, being the dimeric, amphiphilic isoform of AChE characterized because it has a relatively lower affinity for concanavalin A (With A) and a relatively higher affinity for wheat germ agglutinin (WGA) compared to AChE with an altered glycosylation pattern.