KR20010015620A - Diagnostic test for alzheimer's disease - Google Patents

Abstract

(1) providing a suitable body fluid sample from a patient;

(2) A method of diagnosing Alzheimer's disease in a patient is provided, comprising detecting the presence of acetylcholinesterase (AChE) in the sample with a modified glycosylation pattern. Approximately 75-95% of AChE in CSF of AD patients binds to concanavalin (Con A) or wheat germ aglutinin (WGA) but has been established to have different specificities, respectively. Therefore, in order to confirm the glycosylation pattern of AChE in the sample, the binding to Con A is measured, then the binding to WGA is measured, and the ratio is calculated. The ratio is characteristic of the glycosylation pattern. In a separate embodiment of the present invention a monoclonal antibody specific for AChE having a modified glycosylation pattern is used to detect its presence.

Description

Diagnostic test method of Alzheimer's disease {DIAGNOSTIC TEST FOR ALZHEIMER'S DISEASE}

Alzheimer's disease (AD) is a common progressive dementia that loses memory and advanced cognitive function. The disease is characterized by the presence of amyloid deposits in the patient's brain. Such deposits are found both extracellularly (amyloid plaques) and intracellularly (neurofibrillary tangles). The main component of amyloid plaques is amyloid protein (Aβ) produced by proteolysis of amyloid protein precursor (APP) (Evin et al., 1994). The main component of the neurofibrous concentrate is the cytoplasmic protein tau (Kosik, 1992).

One of the characteristic neurochemical changes found in AD is the loss of acetylcholinesterase (AChE) and choline acetyltransferase activity in brain regions such as cortex, hippocampus, tonsils and haoliocytes (Whitehouse et al. , 1981, 1982; Struble et al., 1982; Mesulam and Geula, 1988). The loss of cholinergic structures and markers correlates with the number of plaque and fibroconcentrate lesions present, as well as 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 most about 80% accurate. Therefore, there is a need to identify specific biochemical markers of AD. To date, detectable differences have been reported at the level of specific proteins, but analysis of blood or cerebrospinal fluid (CSF) does not yield sufficient diagnostic values of biochemical markers (Motter et al., 1995).

Assessment of AChE activity levels in blood and cerebrospinal fluid (CSF) has been proposed as a pre-death diagnostic test for AD. There is no consensus, however, whether the level of AChE consistently affects these organizations. Increased serum or plasma AChE levels in AD patients (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). Erythrocyte AChE levels have been reported to be unaffected (Atack et al., 1985; Perry et al., 1982) or decreased (Chipperfield et al., 1981). The level of AChE activity in CSF in AD patients was reduced (most recently reported by Appleyard and McDonald, 1982; Shen et al., 1993) or unchanged (most recently Appleyard et al., 1987; Ruberg et al. , 1987).

AChE has been found to exist in six different molecular isoforms, three of which are monomeric (G1), dimeric (G2) and tetrameric (G4) isoforms (Massoulie et al., 1993). Relative proportions of different isoforms of AChE significantly affect AD, with decreased G4 isoforms in the cortical side membranes (Atack et al., 1983) and increased G1 isoforms (Arendt et al., 1986). Similar changes have been identified in the amygdala as well as other AD brain regions, including Broderman regions 9, 10, 11, 21 and 40 (Fishman et al., 1986). Asymmetric collagen-tail isoforms (A12) increase by 400% in broadman region 21, but they correspond to traces of the total AChE in the human brain (Younkin et al., 1986).

However, to date, no change in AChE expression and homozygous distribution is known to have sufficient sensitivity or specificity to be a useful diagnostic marker of AD.

Anomalous isotypes of AChE, identified by isoelectric points, have been detected in CSF of AD patients (Navaratnam et al., 1991; Smith et al., 1991), and AD screening methods based on these findings are described in US Pat. No. 5,200,324. It is. The method is characterized by using isoelectric focusing to determine whether a patient has anomalous isoform AChE in his CSF. However, isoforms detected by Navaratnam et al. And Smith et al. Were also detected in CSF of patients with other neurological diseases (Shen and Zhang, 1993). It is also shown in US Pat. No. 5,200,324, column 7, lines 19-22, where anomalous AChE is "clinically diagnosed to be possible dementia, but of eight patients who do not meet the strict pathological criteria for Alzheimer's disease. In four CSFs ".

In addition, detection of AChE-AD in lumbar spine CSF in line 60-61 of column 7 of the US patent is dependent on the amount of CSF to be analyzed, and in line 38-40 of column 8 anomalous bands are typically faint and gel It is mentioned that movement is not always ideal. Thus, a load of 5 mU per track is adopted as the standard process for screening CSFs for the presence of anomalous forms of AChE, and each gel is read independently by four people recording their interpretations. Thus, there is a technical problem with the above assay, which can be avoided only by adopting an arbitrary set of conditions to avoid erroneous readings, making the interpretation of the results difficult.

With the suggestion that the anomalous form of AChE detected by Navaratnam et al. And Smith et al. Is not specific for AD patients, the technical problem associated with the assay described in US Pat. No. 5,200,324 is the abnormality of AChE found by Navaratnam et al. And Smith et al. It is suggested that the electronic form does not form the basis of a diagnostic test of AD suitable for clinical use.

The present invention relates to a diagnostic test for Alzheimer's disease.

1 is a plot of C / W ratios for the number of control patients, Alzheimer's disease (AD) patients, other neurological disease patients distinguished from Alzheimer's disease (AD), and non-Alzheimer's disease type dementia (DNAT) patients. The circles represent ventricular CSF; The triangles represent the lumbar spine CSF; Open symbols are ≥ 60 years old; Black symbols are ≤ 60 years old. The mean value is expressed as ≠ S.E.M. * Indicates significantly different from AD (p <0.001). This experiment is described in Example 1.

2 is a plot of C / W ratio AChE activity in post-human CSF. Comb lines represent the levels of C / W and AChE activity that are maximally identified between AD and non-AD groups. Approximately 80% of all AD samples have a cutoff value of C / W = 0.95 or more, while all AD samples have C / W = 0.60 or more. Similarly, all AD samples have an AChE activity of less than 15.8 U / ml. The experiment is described in Example 2.

Figure 3 shows the fraction number for AChE activity versus hydrophobic interaction chromatography of CSF AChE on Phenyl-Agarose. CSF samples from AD patients (open ring) or control (closed ring) are applied to 10 ml column of phenyl-agarose. The hydrophilic AChE isoform (HF) was eluted with 50 mM Tris- saline buffer and the bound amphoteric (AF) was bound to 50 mM Tris-HCl (TB) containing 2% (w / v) Triton X-100 (pH) Elute with 7.4). 1.4 ml fractions are harvested and assayed for AChE activity.

4 is an analysis of AChE isoforms and glycosylation in AD and control CSF. Hydrophilic fractions (HF) and amphoteric fractions (AF) are obtained from the total CSF fractions by hydrophobic interaction chromatography (FIG. 2). The C / W ratio, HF and AF fractions in the total CSF were measured and then the fractions were subjected to a 5-20% sucrose density gradient containing 0.5% (w / v) Brij 97 and centrifuged at 150,000xg for 18 hours. Isolate. Fractions from the sucrose gradient are collected and assayed for AChE activity. Catalase (C, 11.4S) and alkaline phosphatase (P, 6.1S), enzymes of known sedimentation coefficients, are used to determine approximate sedimentation coefficients of AChE isoforms.

FIG. 5 is an analysis of AChE isoforms and glycosylation in the prefrontal cortex and cerebellum from control, non-dementia patients with diffused plaques and AD patients. Brain samples are homogenized and extracted to obtain SS and TS fractions. Equal volumes of SS and TS fractions are mixed and applied to a 5-20% sucrose density gradient containing 0.5% (w / v) Brij 97 and centrifuged at 150,000 × g for 18 hours. Fractions are harvested and assayed for AChE activity. Each AChE isoform is identified by its sedimentation coefficient using enzyme markers: catalase (C, 11.4S) and alkaline phosphatase (P, 6.1S). Enzyme peaks of G ₄ and G ₂ + G ₁ AChE are selected, concentrated and dialyzed to remove sucrose. The main peaks of G ₄ and G ₂ + G ₁ are analyzed by lectin binding using Con A and WGA and the C / W ratio is determined from each peak.

6 shows the effect of monoclonal antibody MA3-042 on the sedimentation rate of AChE isoforms from the human frontal cortex as described in Example 3. FIG.

Abbreviations used:

AChE, acetylcholinesterase; ChE, cholinesterase; Aβ, amyloid β protein; AD, Alzheimer's disease; DP, widely spread plaque; ND, other neurological diseases; PMI, interval after death; PBS, phosphate-saline buffer; TB, Tris buffer; TSB, Tris-Brine Buffer; SS, salt-soluble supernatants; TS, Triton X-100-soluble supernatant; AF, amphoteric fraction; HF, hydrophilic fraction; G ^a , spherical amphoteric isoform; G ^na , spherical non-bilateral isoforms; And aglutinin from Canavalia ensiformis (Concanavalin A), Con A; Triticum vulgaris (wheat embryo), WGA; Ricinus communis, RCA ₁₂₀ ; Lens culinaris, LCA; Dolichus biflorus, DBA; Ulex europaeus, UEA _I ; Glycine max, SBA; And Arachis hypogaea, PNA.

material

Immobilized Lectin (Con A- and LCA-Sepharose, WGA-, RCA _120- , DBA-, UEA _I- , 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-allyldimethyl-ammoniumphenyl ) -Pentane-3-1 dibromide (BW284c51), acetylthiocholine iodide and 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) are all Sigma Aldrich Pty. Ltd. From Seven Mills, NSW, Australia. Sepharose CL-4B is purchased from Pharmacia Biotech AB (Uppsala, Sweden).

There remains a need for AD diagnostic assays based on biochemical analysis of body fluids such as blood or CSF and the present invention is based on the fact that AChE in AD patients exhibits different glycosylation patterns for AChE in non-AD groups. Provide test methods.

According to the first aspect of the present invention

(1) providing a sample of appropriate body fluid from a patient;

(2) there is provided a method of diagnosing Alzheimer's disease (AD) in a patient, comprising detecting the presence of acetylcholinesterase (AChE) in the sample with the presence of modified acetylcholinesterase (AChE). .

In an embodiment of the present invention the relative ratio of AChE having a first glycosylation pattern and AChE having a second glycosylation pattern is measured.

Determination of the relative proportions of AChE with the first and second glycosylation patterns can be performed using convenient methods such as biochemical assays such as HPLC and mass spectroscopy, or immunological techniques such as ELISA, and the like. . However, a particularly preferred method for determining the relative proportion of isoforms of AChE is the lectin-binding assay.

Approximately 75-95% of AChE in CSF of AD patients binds to concanavalin (Con A) or wheat germ aglutinin (WGA) but each has different specificities. Thus, in a particularly preferred embodiment of the present invention, in order to identify the glycosylation pattern of AChE in the sample, the binding to Con A is measured and then the binding to WGA is measured to calculate the ratio. The ratio is characteristic of the glycosylation pattern. It is particularly convenient to measure the activity of unbound AChE during each experiment, thus measuring the ratio of AChE not bound to Con A to AChE not bound to WGA. This ratio is hereinafter referred to as the C / W ratio. For AD patients, the C / W ratio is found to be typically above 0.95, whereas for AD-non-patients the C / W ratio is typically below 0.95.

It is advantageous to measure total AChE activity and plot the C / W ratio against AChE activity.

Detecting its presence using monoclonal antibodies specific for AChE having a modified glycosylation pattern in a separate aspect of the present invention. Typically the monoclonal antibody is MA3-042 (clone HR2) available from Chemicon International Inc of Temecula, California. Other suitable monoclonal antibodies can be used, for example MA304 (clone AE1; available from Chemicon International Inc.).

Without wishing to be bound by theory, it is contemplated that the abnormal isoforms are the monomeric isoforms of AChE and / or the dimer isoforms of AChE, which are amphoteric.

The fluids analyzed may be cerebrospinal fluid (CSF), blood or plasma. If the body fluid is blood, it is advantageous to prepare plasma from blood for analysis. Plasma is treated to remove or inactivate butyrylcholinesterase (BChE) prior to analysis.

According to the second aspect of the present invention, the affinity of monomers of AChE is relatively small for affinity of concanavalin (Con A) and for wheat germ aglutinin (WGA) than AChE having a monomeric homomorphic and unmodified glycosylation pattern. Abnormal isoforms of acetylcholinesterase (AChE) with modified glycosylation patterns are provided which are characterized by having a relatively greater affinity.

According to a third aspect of the present invention, AChE has a relatively lower affinity for concanavalin A (Con A) and wheat germ aglutinin (AChE) than AChE having an amphoteric, dimeric homomorphic and modified glycosylation pattern. Aberrations of acetylcholinesterase (AChE) with modified glycosylation patterns are provided, characterized by relatively greater affinity for WGA).

Example 1

Lectin binding experiment in AD patients

Lumbar or ventricular CSF is obtained after death; 18 controls without clinical or pathological dementia and no brain pathological evidence, 27 AD patients, 7 patients with dementia or non-AD type (DNAT, 5 patients with prefrontal dementia, 1 with Lewy body dementia / Parkinson's disease) Persons and one patient with multi-infarction dementia / Chilegophilic amyloid vasculature), and six other neurological disorders (ND, four Huntington's disease patients, one schizophrenic patient and one cortical basal degeneration). The mean age of the control group was 68 ± 4 years old with 10 females and 8 males with a PMI of 40 ± 6. Age in the AD group was 81 ± 2, 13 women and 14 men, and PMI was 35 ± 6. Age in the ND group was 65 ± 6, 3 women and 3 men, and PMI was 45 ± 12. Age in the DNAT group was 76 ± 3, 4 women and 3 men, and PMI was 34 ± 11. Samples of CSF are stored at −70 ° C. and centrifuged at 1,000 × g for 15 minutes prior to analysis. AChE activity is assayed at 22 ° C. by an improved microanalysis of the Ellman method (Ellman et al. 1961). Aliquots (0.3 mL) are mixed with 0.1 mL of Sepharose 4B (control) in PBS, Concanavalin A (Con A) or wheat germ aglutinin (WGA, Triticum vulgaris) immobilized on Sepharose. The enzyme-lectin mixture is incubated overnight at 4 ° C. and then centrifuged (1,000 × g, 15 min). AChE activity is assayed in the supernatant fractions. Analyze the data using Student's t-test.

The total AChE level in ventricular CSF samples of patients> 60 years was much lower in the AD group (6.98 ± 0.82 nmol / min / ml) than in the control group (17.24 ± 4.28 nmol / min / ml; p <0.001). However, as reported earlier (Appleyard et al, 1983), the large overlap between the data (40%) limits the use of the entire AChE as a reliable diagnostic marker.

However, lectin-binding assays revealed a distinct difference between the AD and control groups. Approximately 75-95% of AChE in CSFs bound to Con A or WGA. The mean C / W ratio for the AD group is significantly different from the control (FIG. 1). Of 27 CSFs from patients identified with AD, the C / W ratio of 21 samples is> 0.95. All 18 control samples had C / W <0.95, with no significant difference between younger patients (n = 5, C / W = 0.37 ± 0.10) and older patients (n = 6, 0.38 ± 0.08). There was no correlation to post-interval (PMI) in C / W ratio. The data is shown graphically in FIG. 1.

The data indicate that the lectin-binding assay of CSF AChE provides a diagnostic test for AD with 80% sensitivity and 97% specificity. Thus, the differences observed in glycosylation patterns of AChE in CSF may be useful as pre-death diagnostic markers for AD, particularly when used in combination with other biochemical markers.

Example 2

Additional Lectin Binding Experiments

Experiment process

Human Brain and CSF Samples

Ventricular and lumbar CSF, frontal cortex and cerebellum samples are obtained after death and stored at -80 ° C. Three non-AD group samples were: 1) control group without clinical or pathological features for dementia (n = 18), 2) non-neuroinflammatory Ab-immunoreactive diffuse plaque (DP) with no clinical signs of dementia Patients found to have (n = 6), and 3) 7 for non-AD type dementia (5 prefrontal dementia, 1 Levitic dementia and 1 vascular dementia) and 7 for other neurological diseases (hunting 4 patients, 1 Parkinson's disease, 1 schizophrenia and 1 cortical basal degeneration). AD is chosen based on their dementia clinical history and neuropathological CERAD diagnosis (Mirra et al., 1994). All CSF samples included in the AD and ND groups are from the ventricle and only 5 controls and one DP CSF sample (from a total of 18 and 6 patients, respectively) are taken by lumbar puncture. Immunohistochemical examination of cerebellar samples revealed that, unlike the frontal cortex, none of the AD tissues had full neuronal amyloid plaque deposition, consistent with previous studies (Mann et al., 1996).

If the mortality interval (PMI) is greater than 72 hours, regression of AChE occurs with repeated cycles of storage or freeze-thaw at −20 ° C., which is confused with glycosylation analysis. Therefore, only samples with PMI less than 72 hours (PMI = 36 ± 4 hours) are used. There is no significant difference in PMI between samples of each group.

Preparation of Samples and Extraction of AChE

The CSF samples are thawed slowly at 4 ° C. and then centrifuged at 1,000 × g for 15 minutes before use. Small pieces of the frontal cortex and cerebellum (0.5 g) were thawed slowly at 4 ° C., weighed and ice-cold Tris-saline buffer (TSB; 50 mM) containing a cocktail of proteinase inhibitors (Silman et al., 1978). Homogenized in Tris-HCl, 1M NaCl, and 50 mM MgCl ₂ , pH 7.4) (10% w / v). Tissues are homogenized with a glass / teflon homogenizer and then sonicated to 10-15 branches at 50% intermittent with 4 using a Branson sonicator. The suspension is centrifuged for 1 hour at 100,000 × g at 4 ° C. in a Beckman L8-80M ultracentrifuge using a 70.1 Ti rotor to recover the salt-soluble ChE fraction (SS). The pellet was re-extracted with an equal volume of TSB containing 1% (w / v) Triton X-100, and the suspension was centrifuged at 100,000 × g for 4 hours at 4 ° C. to Triton X-100-soluble ChE fraction (TS ). The double-extraction method recovers 80-90% of the total ChE activity (Saez-Valero et al., 1993; Moral-Naranjo et al., 1996).

AChE Assay and Protein Measurement

AChE activity is measured by Elman's improved microassay (Saez-Valero et al., 1993). One unit of AChE activity is defined as the number of acetylthiocholine nanomoles hydrolyzed per minute at 22 ° C. Protein concentrations are measured using the non-shinkoninic acid method using bovine serum albumin as a standard (Smith et al., 1985).

Hydrophobic Interaction Chromatography on Phenyl-Agarose

Amphiphilic AChE forms are separated from hydrophobic forms by hydrophobic interaction chromatography on phenyl-agarose as described above (Saez-Valero et al., 1993). CSF (10 mL blue from 4 samples obtained from 4 different individuals) is applied to a column of phenyl-agarose (10 × 1 cm). The hydrophilic fraction (HF) containing the hydrophilic isoform of AChE was eluted with 30 ml of TSB, and then the amphoteric fraction (AF) containing the bound amphoteric isoform contained 2% (w / v) Triton X-100. Eluate with 50 mM Tris-HCl (TB, pH 7.4). Peak fractions with high AChE activity are pooled and concentrated using an Ultrafree-4 Centrifugal Filter Device Biomax 10 kDa concentrator (Millipore Corporation, Bedford, Mass., USA).

Sedimentation method

Molecular isoforms of AChE are analyzed by ultracentrifugation at 150,000 × g with a continuous sucrose gradient (5-20% w / v) for 18 hours at 4 ° C. in a Beckman SW40 rotor. The gradient contains 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 are collected from the bottom of each tube. Enzymes of known sedimentation coefficient, bovine liver catalase (11.4S, S _{20, W} , Svedberg Units) and E. coli. Coli alkaline phosphatase (6.1S) is used in the gradient to determine the approximate settling coefficient of the AChE isoform. The ratio of Gh molecules (G ₄ ^na + G ₄ ^a ) to AChE species G ₄ / (G ₂ + G ₁ ) reflecting the ratio of G ₂ ^a and G ₁ ^a , both of which are light spherical AChE isoforms, is defined. Estimation of the relative proportion of AChE in each molecular form is performed by adding the activity under each peak (G ₄ or G ₂ + G ₁ ) and calculating the relative percentages (recovery rate> 95%).

Lectin-binding assay of AChE

Sample (0.3 mL) was added to 0.1 mL (hydrated volume) of Sepharose 4B (control), Con A, WGA, RCA ₁₂₀ , LCA, DBA, DEAr, SBA or PNA immobilized in agarose or Sepharose. do. The enzyme-lectin mixture is incubated overnight at 4 ° C. with gentle mixing. Bound AChE and free AChE are separated by centrifugation at 1000 × g for 15 min at 4 ° C. in a Beckman J2-21M / E centrifuge using a JA-20 rotor, and unbound AChE is assayed in the supernatant fractions. The percentage of unbound AChE in the lectin culture is calculated as (AChE not bound to lectin / AChE not bound to Sepharose) x 100. The C / W ratio is calculated according to the above formula, and divides unbound AChE activity in Con A culture by unbound AChE activity in WGA culture. This ratio was observed to detect specific modifications in AChE glycosylation occurring in AD CSF.

Lectin binding of CSF AChE

To investigate glycosylation of AChE, CSF samples from 18 control and 30 AD patients are incubated with different immobilized lectins, recognizing different sugars. AChE (Table 1), which binds to Con A, WGA and LCA but weakly binds to RCA ₁₂₀ , PNA, DBA, UEA _I and SBA, shows that most enzymes have terminal galactose, terminal N-acetyl-galactosamine or fructose. It suggests a lack of lactose.

There is a small but significant difference in binding of AChE to Con A and WGA between the AD group and the control group (Table 1). The percentage of unbound AChE in AD CSF increases for Con A and decreases for WGA and is defined by the ratio (C / W = [AChE% not bound to Con A] / [AChE% not bound to WGA]]. This makes the distinction between the two groups larger (Table 1) .. Using this method, the average C / W ratio for the AD group has diffused plaques (non-dementia, DP), and other nerves and It was found to be much larger than the ratio to other controls, including neuropsychiatric disease patients (ND) (Figure 2), which is consistent with the results shown in Example 1. 30 CSF samples from cases identified as AD patients Among them, 24 samples had a cut-off value of C / W = 0.95 or greater (Figure 2): 1 of the samples from 18 controls, 1 of 6 samples with diffused plaques, and other nerves Only 1 out of 14 samples from the prefrontal dementia group, The C / W ratio of six AD samples with C / W ratios greater than 0.95 was> 0.60, which is greater than the C / W mean of the non-AD group (control = 0.53 ± 0.1; DP = 0.46 ± 0.2; ND). = 0.53 ± 0.1).

No correlation was found between the C / W ratio and the PMI, which may suggest that different C / W ratios in the AD group are due to differences in PMI. There was also no significant difference in PMI between AD (33 ± 6 hours) and non-AD samples (40 ± 6 hours).

CSF samples were further analyzed for total AChE activity (FIG. 2). As reported earlier (Appleyard et al., 1983; Atack et al., 1988), CSF from AD patients was associated with AChE activity (15.8 ± 2.9 U / ml) of control or patients with other diseases (12.4 ± 2.4 U / Ml) has much lower AChE activity (6.5 ± 0.8 U / ml). However, the C / W ratio was a more reliable indicator of clinical status than the total level of AChE in CSF (FIG. 2).

AChE homomorphic in CSF

To investigate whether the modification in glycosylation is due to changes in specific isoforms of AChE, hydrophobic interaction chromatography (FIG. 3) to separate CSF samples from amphoteric (G ^a ) and hydrophilic species (G ^na ) And sucrose density gradient centrifugation (FIG. 3) in 0.5% (w / v) Brij 97 to separate each molecular weight isoform (G ₄ , G ₂ and G ₁ ). A reduction in the ratio of G ₄ AChE in AD CSF compared to the control (FIG. 4, top panel) was observed. The ratio of (G ₄ / (G ₂ + G ₁ )) was significantly (P <0.01) higher in the control group (1.80 ± 0.12; n = 4) than in the AD case (1.16 ± 0.12; n = 4). To separate the hydrophilic isoforms from the amphoteric isoforms, CSF was fractionated by hydrophobic interaction chromatography on phenyl-agarose (FIG. 3). AChE in normal CSF (12 ± 3%, n = 4) bound less to phenyl-agarose than in AD CSF (38 ± 4%, n = 4, P <0.001). Sedimentation analysis of the unbound hydrophilic fraction (HF) revealed a 10.8S main peak, 5.1S dimer, consistent with hydrophilic tetramer (G ₄ ^na ) isoforms (Atack et al., 1987), as well as smaller amounts of lighter AChE isoforms. And 4.3S monomers (FIG. 4). The combined amphoteric fraction from the phenyl-agarose column has a small peak of 9.0-9.5S (amido amphoteric tetramer, G ₄ ^a ) and the main peak of amphoteric spherical dimer (G ₂ ^a , 4.2S) and It represents a monomer (G ₁ ^a, 3.1S). The level of amphoteric isomorphism is greater in AD CSF than in control (FIG. 4).

Glycosylation of Each AChE Isomorphism in CSF

Incubation of HF and AF with immobilized Con A and WGA increases the C / W ratio for AD CSF, which appears to be associated with the amphoteric fraction containing dimers and monomers (FIG. 4) . The data indicate that the distribution of G ₂ and G ₁ AChE in AD CSF are mainly based on the increased C / W ratio of total AChE in AD CSF.

Levels of AChE in the Frontal Cortex and Cerebellum

To determine if the change in AChE glycosylation reflects the expression of brain AChE isoforms or changes in glycosylation, the level of AChE activity in the frontal cortex and cerebellum samples was examined. Samples were homogenized with salt and Triton X-100 to extract soluble and membrane-bound AChE isoforms and then measured AChE activity in both fractions (Table 2). The AChE activity of the frontal cortex samples from AD patients was much smaller than the AChE activity in the Triton X-100-soluble (TS) fraction (-40%) and the level in the salt-soluble (SS) fraction compared to the control. Was not different (Table 3). The results are in agreement with previous studies (Younkin et al., 1986; Siek et al., 1990), in which the major G ₄ isoforms are reduced only in the TS fraction. A small but significant reduction (-15%) in the protein content of the TS fractions of both AD and ND groups was observed. The level of AChE in the frontal cortex samples of the ND group was clearly different from the control in both the SS and TS fractions (Table 2). However, since the ND group is heterogeneous (two prefrontal dementia, one Huntington's disease, and one Parkinson's disease), the significance of changes in AChE levels is not clear. The level of AChE in the cerebellum was also significantly reduced in the TS fraction from the AD group (Table 2).

Glycosylation of the Frontal Cortex and Cerebellum AChE

To determine if different glycosylation patterns of AChE in AD CSF are also present in the AD brain, glycosylation of brain AChE was investigated by lectin binding. Homogenates from the frontal cortex and cerebellum were incubated with immobilized Con A or WGA and the amount of unbound activity was calculated. In the AD frontal cortex, the percentage of AChE activity that did not bind to Con A or WGA was significantly different from the control (Table 3). Similar to CSF AChE, the C / W ratio of frontal cortical AChE is greater in AD than in non-AD samples (Table 3). This increase is due to a large increase in the amount of AChE not bound to Con A, despite an increase in the amount of AChE not bound to WGA (Table 3). There is no increase in the C / W ratio among the DP and ND groups. No difference in lectin binding was observed between AD and non-AD groups in cerebellar fraction (Table 3).

AChE isoforms in the frontal cortex and cerebellum

To determine the cause of modified glycosylation in the AD brain, the patterns of AChE isoforms in the frontal cortex and cerebellum are examined. Equal volumes of SS and ST supernatants (total AChE activity) are pooled and then analyzed by sucrose density gradient sedimentation using 0.5% (w / v) Brij 97 to isolate the major AChE isoforms (FIG. 5). Based on their sedimentation coefficients (Atack et al., 1986; Massoulie et al., 1982), the hydrophilicity of AChE (G ₄ ^na , 10.7 ± 0.1S) and the amphoteric tetramer (G ₄ ^a , 8.6 ± 0.1 S), amphoteric dimers (G ₂ ^a , 4.7 ± 0.1S) and monomers (G ₁ ^a , 3.0 ± 0.1S) can be identified (FIG. 6). There was no difference in the sedimentation coefficient of each homomorphism from each group. Due to the overlap in the settling coefficients between AChE G ₄ ^na and G ₄ ^a , these isoforms cannot be completely separated (FIG. 5). However, G ₄ ^a is present more than G ₄ ^na . Asymmetric (A ₁₂ ) AChE isoforms were found in traces (2-5%) in some of the fractions.

Significant decreases in G ₄ (40% of mean control value, P <0.001) and G ₂ + G ₁ AChE (60% of mean control value, P = 0.002) were detected in fractions from the AD frontal cortex. This change in the relative ratio of AChE isomorphism is reflected in the G ₄ / (G ₂ + G ₁ ) ratio, which is much lower in the AD sample (Table 3). Interestingly, a similar and statistically significant decrease was found in the G ₄ / (G ₂ + G ₁ ) ratio for DP individuals. Although the change itself is not statistically significant, the change in this ratio is due to a 25% increase in the level of G ₂ + G ₁ and a small decrease in G ₄ AChE (10%). Statistical at the overall levels of AChE (Table 2) and G ₄ AChE in the TS fraction (G ₄ = 380 ± 40 U / mL in the control, G ₄ = 195 ± 70 U / mL in the ADs; P = 0.008) Despite a significant decrease (40%), no change in AChE G ₄ / (G ₂ + G ₁ ) was found in the AD cerebellum (Table 3).

Glycosylation of Each AChE Isomorphism in the Frontal Cortex and Cerebellum

Since the ratio of AChE has been found to be altered in the frontal cortex of AD patients, a step is performed to determine whether the increase in the C / W ratio of brain AChE is due to changes in glycosylation or specific isoforms of AChE. Each AChE isoform is isolated by sucrose gradient centrifugation and then fractions from G ₄ or G ₂ + G ₁ peaks are dialyzed against TSB-Triton X-100 buffer and concentrated by ultrafiltration. AChE isoforms are then assayed by the lectin binding method and the C / W ratio calculated for each isoform (FIG. 5).

No difference was observed in the C / W ratio of G ₄ AChE between AD and non-AD groups (FIG. 5). However, the G ₂ + G ₁ fraction in all frontal cortex samples has a C / W ratio of> 1.00, demonstrating that G ₂ or G ₁ AChE is glycosylated differently from the G ₄ isoform. Also the C / W ratio for G ₂ + G ₁ AChE is higher in the AD group than in the control or DP. Similarly, the C / W ratio of the amphoteric fraction from CSF (containing mainly G ₂ + G ₁ AChE) is higher in the AD group than in the control group (FIG. 3). There was no correlation between the G ₄ / (G ₂ + G ₁ ) and C / W ratios in the DP group in the frontal cortex. In the cerebellum, no difference was observed in the C / W ratio of G ₄ AChE or G ₂ + G ₁ AChE between AD and non-AD groups (FIG. 4). G ₂ + G ₁ fraction from AD and non-cerebellum -AD group are both in contrast to the same fraction (C / W> 1.00) from the frontal cortex has a C / W <0.50, which G ₂ between the two brain areas + The difference in the glycosylation pattern of G ₁ AChE is shown.

This example shows that AChE is glycosylated differently in the frontal cortex and CSF of AD patients compared to AChE from non-AD groups, including non-AD dementia patients. This difference in glycosylation is due to the increase in the ratio of differently glycosylated amphoteric dimers and monomolecular AChE in AD samples. The results suggest that abnormally glycosylated AChE in AD CSF can be derived from the brain because similar differences in glycosylation are found in the frontal cortex of AD patients.

Lectin-binding of AChE in CSF Lectin Unbound AChE (%) Control AD Con A 5.5 ± 0.8 10.1 ± 1.1 ^b WGA 11.3 ± 1.7 7.0 ± 0.6 ^b Con A / WGA 0.53 ± 0.1 1.37 ± 0.1 ^a (C / W) LCA 17.2 ± 4.2 15.0 ± 1.3 RCA ₁₂₀ 74.1 ± 3.4 70.8 ± 2.7 SBA 83.0 ± 2.1 82.2 ± 1.9 UEA ₁ 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 CSFs are collected after death and the diagnosis is confirmed by pathological examination. CSFs from normal subjects (control: n = 18; 67 ± 4 years of death; 11 females / 7 males) and AD patients (AD group: n = 30; 79 ± 2; 15 females / 15 males) were recruited Incubation with an enemy different immobilized lectins is followed by centrifugation. AChE is analyzed in the supernatant fractions. Data represent mean ± SEM. ^a indicates significantly (P <0.001) difference from the control as assessed by the Student's t test; ^b is significantly different (P <0.05) from the control as assessed by the Student t test.

AChE Activity and Protein Levels in the Human Frontal Cortex and Cerebellum County / Supply Source AChE activity (U / ml) Protein (µg / ml_ SS TS SS TS Control Frontal cortex (n = 11; 63 ± 5y; 7F / 4M) 3.7 ± 0.4 15.1 ± 1.5 2.1 ± 0.1 2.4 ± 0.1 Cerebellum (n = 7; 66 ± 5y; 4F / 3M) 64 ± 6 264 ± 25 2.5 ± 0.1 1.9 ± 0.1 DP Frontal cortex (n = 6; 81 ± 2y; 4F / 2M) 5.5 ± 0.9 12.7 ± 1.7 2.1 ± 0.1 2.2 ± 0.1 Cerebellum (n = 5; 81 ± 3y; 3F / 2M) 49 ± 8 182 ± 46 2.6 ± 0.1 2.2 ± 0.1 ND Frontal Cortex (n = 4; 67.9 ± y; 2F / 2M) 5.4 ± 0.6 ^a 9.3 ± 1.7 ^b 2.1 ± 0.2 2.0 ± 0.1 ^b Cerebellum (n = 2; 78 ± 14y; 1F / 1M) 45 ± 8 160 ± 50 2.7 ± 0.2 2.3 ± 0.2 AD Frontal Cortex (n = 14; 73 ± 3y; 8F / 6M) 3.7 ± 0.3 9.0 ± 0.9 ^a 2.1 ± 0.1 2.1 ± 0.1 ^a Cerebellum (n = 7; 73 ± 6y; 5F / 2M) 48 ± 12 160 ± 28 ^b 2.6 ± 0.1 2.0 ± 0.1

Tissues from the frontal cortex or cerebellum are homogenized and salt-soluble (SS) and Triton X-100-soluble (TS) extracts are obtained. The extract is assayed for AChE and protein. DP = non-dementia patients with widespread plaques; ND = patients with other neurological diseases and non-AD dementia; AD = patients with Alzheimer's disease. F = female; M = male; y = age. The figures are mean ± SEM. ^a indicates significantly (P <0.005) difference from the control as assessed by the Student's t test; ^b is significantly different (P <0.05) from the control as assessed by the Student t test.

Lectin Binding and AChE Isomorphism in the Frontal Cortex and Cerebellum County / Supply Source Lectin binding AChE Rain AChE (%) not bound to Con A AChE (%) not bound to WGA C / W G ₄ / (G ₂ + G ₁ ) Control Frontal cortex (n = 11; 63 ± 5y; 7F / 4M) 6.9 ± 0.8 12.3 ± 1.2 0.56 ± 0.03 1.90 ± 0.14 Cerebellum (n = 7; 66 ± 5y; 4F / 3M) 1.8 ± 0.1 10.7 ± 0.9 0.18 ± 0.02 3.02 ± 0.2 DP Frontal cortex (n = 6; 81 ± 2y; 4F / 2M) 7.4 ± 0.8 15.0 ± 1.0 0.50 ± 0.06 1.32 ± 0.12 ^b Cerebellum (n = 5; 81 ± 3y; 3F / 2M) 2.9 ± 0.7 12.2 ± 1.3 0.23 ± 0.05 2.18 ± 0.33 ND Frontal Cortex (n = 4; 67 ± 9y; 2F / 2M) 7.0 ± 0.6 13.2 ± 1.2 0.47 ± 0.05 2.61 ± 0.73 Cerebellum (n = 2; 78 ± 14y; 1F / 1M) 1.8 ± 0.2 10.1 ± 0.3 0.21 ± 0.10 2.50 ± 0.70 AD Frontal Cortex (n = 14; 73 ± 3y; 8F / 6M) 13.1 ± 1.3 ^a 19.7 ± 1.4 ^a 0.66 ± 0.03 ^b 1.34 ± 0.18 ^b Cerebellum (n = 7; 73 ± 6y; 5F / 2M) 2.4 ± 0.3 13.5 ± 2.3 0.19 ± 0.02 2.33 ± 0.49

SS and TS fractions from the frontal cortex and cerebellum are isotopically pooled and then analyzed by lectin binding using immobilized Con A and WGA. The C / W ratio is calculated as defined in Table 2. Aliquots of supernatant (SS + TS) are also analyzed by sucrose density gradient sedimentation to confirm AChE isomorphism. The figures are mean ± SEM. ^a indicates significantly (P <0.005) difference from the control as assessed by the Student's t test; ^b is significantly different (P <0.05) from the control as assessed by the Student t test.

Example 3

Binding to monoclonal antibody MA3-042

Samples of Triton X-100 (1% w / v) solubilized AChE were at 4 ° C. without MA3-042 (diluted to 1:50 volume) (see left panel of FIG. 6) or together (see right panel of FIG. 6). Incubate overnight. AChE isoforms are isolated by centrifugation on a 5-20% sucrose gradient prepared in 50 mM Tris saline buffer pH 7.4 with 0.5% Triton X-100. The tube is centrifuged at 150,000 × g at 4 ° C. and fractions are collected from the bottom and assayed for AChE activity. Sedimentation markers are catalase (11.4S) and alkaline phosphatase (6.1S). As shown in FIG. 6, all peaks migrate in the presence of MA3-042, indicating that the monoclonal antibody binds to the particular isoform represented by each peak, except for the peaks held around 4S. The difference between 4.0S and 4.2S is not statistically significant, suggesting that the 4.2S peak represents an isomorphism with a modified glycosylation pattern that is not recognized by MA3-042. As will be apparent to those skilled in the art, the peaks represent AChE monomers, the molecular weight of which is about 70000 kDa.

Example 4

Analysis of Blood Using Monoclonal Antibodies

Blood is collected to prepare 1 ml of plasma or serum by standard techniques. Pass the fluid through 5 ml RCA-agarose (RCA stands for ricinus communis agglutinin) to remove butyrylcholinesterase and elutate the amount of acetylcholinesterase eluted from the column (Ellman assay). Monitor by assay and collect 2 ml of active peak. The material was then incubated with 50 mM iso-OMPA at ambient temperature for 10 minutes to inhibit the remaining butyrylcholinesterase and then passed through 1 ml column of MAb MA3-042 coupled to Sepharose. Eliminate non-specific AChE isoforms. The amount of activity eluted from the column is assayed using the Elman assay. The amount of activity present in this fraction is greater in the AD case than in the non-AD case. Typically about 40 mUnits is less than AChE / ml of original plasma or serum.

The present invention provides a diagnostic test for Alzheimer's disease.

references

The following references are incorporated herein by reference:

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Claims (18)

(1) providing a suitable body fluid sample from a patient; (2) detecting the presence of acetylcholinesterase (AChE) in the sample with a modified glycosylation pattern. The method of claim 1, wherein the relative ratio of AChE having a first glycosylation pattern and AChE having a second glycosylation pattern is measured. The method of claim 2, wherein the relative ratio of AChE having the first glycosylation pattern to AChE having the second glycosylation pattern is determined using a lectin-binding assay. The method of claim 3, wherein the lectin-binding assay comprises measuring binding to Concanavalin A (Con A) and wheat germ aglutinin (WGA). The method of claim 5, wherein the activity of unbound AChE is measured. The method of claim 5, wherein the ratio of AChE not bound to Con A to AChE not bound to WGA is calculated. The method of claim 6, wherein said ratio is at least 0.95 in AD patients. The method of claim 1, wherein the total AChE activity is also measured. The method of claim 8, wherein the ratio of AChE not bound to Con A to AChE not bound to WGA is plotted against total AChE activity. The method of claim 1, wherein a monoclonal antibody is used to detect the presence of AChE having a modified glycosylation pattern. The method of claim 10, wherein said monoclonal antibody is MA3-042 and has AChE having a modified glycosylation pattern as its binding failure. The method of claim 1, wherein the method detects abnormal isoforms of AChE having a modified glycosylation pattern. The method of claim 12, wherein the abnormal isoforms are amphoteric, monomeric isoforms of AChE and / or amphoteric dimeric isoforms of AChE. The method of claim 1, wherein said body fluid is cerebrospinal fluid (CSF), blood or plasma. The method of claim 14, wherein said body fluid is blood and plasma is prepared from analytical blood. The method of claim 14 or 15, wherein the body fluid is plasma and the butyrylcholinesterase (BChE) is removed and / or inactivated prior to the assay for the presence of AChE having a modified glycosylation pattern. The affinity of monomeric, monomeric isoforms and unmodified glycosylation patterns of AChE are relatively lower in affinity for concanavalin A (Con A) and for wheat germ aglutinin (WGA). Abnormal isoforms of acetylcholinesterase (AChE) with a modified glycosylation pattern, characterized by larger. The affinity, dimeric isomorphism of AChE is relatively lower in affinity for concanavalin A (Con A) than AChE with a modified glycosylation pattern and affinity for wheat germ aglutinin (WGA) Abnormal isoforms of acetylcholinesterase (AChE) with a modified glycosylation pattern, characterized by large.