IPG was performed according to the protocol of the manufacturer (Amersham Pharmacia Biotech, Buckinghamshire, UK) using dry strips (linear pH gradient: 4–7, length: 7 cm). Dry strips were rehydrated overnight at room temperature to 0.5 mm gel height, using the following rehydration solution: 8 m urea, CHAPS 0.5% (w/v), DTT 0.2% (w/v), Serdolit MB-1 1% (w/v), pharmalyte 3–10 0.8% (v/v). Thirty microlitres of sample in IPG sample buffer was applied to the rehydrated dry strip at pH 6.5 (cathodic site) using sample cups and IEF was performed for 30 min/300 V, 30 min/800 V, 30 min/1400 V and 5 h/2000 V (Σ12 500 V/h). Subsequently the strips were equilibrated for the second analytical dimension (Aβ-SDS–PAGE) in the following buffer for 10 min at room temperature: 6 m urea, glycerol 20% (w/v), SDS 2.0% (w/v), bistris 0.36 m, bicine 0.16 m, DTT 1.0% (w/v). DTT was added just prior to equilibration. Equilibrated IPG strips were placed on top of the Aβ-SDS–PAGE stacking gel (1 mm thickness) and embedded by a low gelling temperature agarose solution: agarose 1.0% (w/v), bicine 0.16 m, bistris 0.36 m, SDS 0.25% (w/v), bromophenol blue 0.002% (w/v). A Teflon tooth was inserted next to the IPG strip to form a track for synthetic standard Aβ peptides or a one-dimensional reference separation of the SDS/heat denatured sample. Subsequently, Aβ-SDS–PAGE/immunoblot was performed as described, but separation gels were run at constant voltage for 15 min/60 V and for 1 h 30 min/120 V.
Western blotting, immunostaining and quantification
Aβ peptides were transferred for 30 min at 1 mA/cm2 and room temperature under semidry conditions (Hoefer Semiphor) onto Immobilon-P PVDF membranes according to Wiltfang et al. (1997).
For immunostaining Immobilon-P PVDF membranes were washed for 30 s in H2Odd and boiled for 3 min in PBS (phosphate buffered saline) using a microwave oven (Ida et al. 1996; Wiltfang et al. 1997). Blocking was performed for 1 h at room temperature in the presence of RotiBlock (Carl Roth GmbH & Co. KG, Karlsruhe, Germany). Incubation with primary mAb, which was diluted 4000-fold (stock: 0.25 mg/mL), was done overnight at 4°C. After a brief wash in PBS-T (0.075% v/v Tween 20) membranes were further washed for 30 min, 15 min and 2 × 10 min. Next, membranes were incubated for 1 h at room temperature with an anti-mouse biotinylated IgG (H + L) antibody (1.5 mg/mL), which was diluted 3000-fold in PBS-T. A second PBS-T wash was done for 3 × 10 min at room temperature. The membranes were then incubated for 1 h at room temperature with streptavidin-biotinylated horseradish peroxidase complex diluted 3000-fold with PBS-T. Following a wash for 3 × 10 min at room temperature, the membranes were developed for 5 min at room temperature with ECLPlusTM solution according to the protocol of the manufacturer. Detection of the emitted light signal was performed by a CCD camera (FluorSMax MultiImager; Bio-Rad), using a series of 1, 5, 20, 60, 120, and 300 s for data acquisition. Band intensities were quantified relative to an internal five-step dilution series of the Aβ peptide standard mix using Quantity One software (version 4.1, Bio-Rad). Detection sensitivity was 0.6 pg and 1 pg for Aβ1–40 and Aβ1–42, respectively (data not shown). Signal acquisition was linear within a range of 3.8 magnitudes of order. The high detection sensitivity was due to the mAb 1E8 and an optimization of the former immunoblot procedure (Wiltfang et al. 1997), as we used a synthetic reagent (Roti-Block) instead of non-fat milk powder to block the PVDF membrane. The method allowed quantification of Aβ peptides in only 10 µL of CSF. The inter- and intra-assay coefficients of variation for 80 as well as 20 pg of synthetic Aβ peptides were below 10%.
MALDI-TOF mass spectrometry of immunoprecipitated Aβ peptides
Samples micropreparatively immunoprecipitated from CSF were resolved in 60% acetonitrile, 0.1% trifluoroacetic acid. An aliquot of 0.5 µL was mixed with 0.5 µL saturated α-cyano-hydroxycinnamic acid, 50% acetonotrile, 0.1% trifluoroacetic acid on the target, dried and analysed using a REFLEX III MALDI-TOF mass spectrometer (Bruker Daltoniks, Germany) in the positive reflectron mode.
We investigated 59 patients with various neuropsychiatric disorders under the guidelines and regulations of the Institutional Review Board of the University of Göttingen. Neuropsychiatric diagnosis was established by ICD-10 and DSM-IV criteria [American Psychiatric Association (APA), 1994]. Patients with probable Alzheimer's dementia (AD) had to satisfy DSM-IV criteria for dementia of the Alzheimer's type and the NINCDS-ADRDA criteria (McKhann et al. 1984).
A group of patients with a broad range of neuropsychiatric disorders but without dementia (non-demented disease controls, NDC; n = 47; age: 45.2 ± 15.8 y; mean ± SD) were differentiated from patients with AD (AD, n = 12; age: 73.0 ± 7.9 years; mean ± SD). In five of the NDC patients analysis of the CSF samples by immunoprecipitation (IP) with Aβ-SDS–PAGE/immunoblot was compared with SDS-heat denaturation with direct loading and Aβ-SDS–PAGE/immunoblot. The two respective NCD groups were termed as IP-CSF and SDS-CSF (see Table 1). The latter two NDC subgroups allowed us to compare the effects of different sample pretreatment (immunoprecipitation versus SDS-heat denaturation) on the concentrations of CSF Aβ peptides.
Table 1. Aβ peptides in the CSF of several patient subgroups relative to their MMSE performance and ApoE genotyping
|Groups ||Subjects number M F||MMSE* median p25 p75||ApoE no ε4 1 or 2 ε4 n.a.||Aβ1–37 (ng/mL) median p25 p75||Aβ1–38 (ng/mL) median p25 p75||Aβ1–39 (ng/mL) median p25 p75||Aβ1–40 (ng/mL) median p25 p75||Aβ1–42 (ng/mL) median p25 p75||total Aβ1 (ng/mL) median p25 p75||Aβ1–372 (%) median p25 p75||Aβ1–382 (%) median p25 p75||Aβ1–392 (%) median p25 p75||Aβ1–402 (%) median p25 p75||Aβ1–422 (%) median p25 p75|
|28||29|| 9||1.12||2.44||1.11|| 9.32||1.56||15.66||6.44||14.37||6.86||58.30|| 9.98|
|ADb||12||16.0|| 1||1.30||3.08||1.35||12.81||1.49||19.93||7.13||16.22||7.78||62.42|| 6.73|
| 3|| 9.5||11||1.02||2.41||1.15||10.32||0.91||16.07||6.38||15.43||6.80||61.54|| 5.80|
| 9||22.5|| 0||1.81||4.11||2.13||15.74||1.57||25.36||7.33||16.57||8.40||64.11|| 7.65|
|IP-CSFc|| 5||30|| 4||1.35||2.80||1.93||13.23||2.73||22.04||6.49||13.73||8.73||60.03||12.39|
|subgroup of NCD|| 3||29|| 1||1.27||2.69||1.54||12.19||2.04||19.59||6.10||13.58||7.88||53.35||10.77|
| 2||30|| 0||2.30||4.74||2.54||15.25||3.76||28.58||8.04||16.05||8.75||62.25||13.16|
|SDS-CSFd|| 5||30|| 4||1.62||4.53||2.28||18.40||3.40||30.23||5.93||14.99||8.04||60.87||11.24|
|subgroup of NCD|| 3||29|| 1||1.23||3.30||1.94||14.24||2.38||23.02||5.35||14.33||7.56||58.54||10.43|
| 2||30|| 0||1.93||5.09||2.58||18.81||3.72||32.13||6.01||15.83||8.44||60.95||11.58|
Mini mental state examination (MMSE) results (Folstein et al. 1975) at the time of sampling, ApoE genotyping, and routine CSF parameters (cell count, total protein, albumin, presence of oligoclonal bands, IgG, IgM, and CSF/serum ratios for albumin and immunoglobulins) were available for 12/12, 11/12, 10/12, and 42/47, 46/47, 46/47 of the AD and NDC patients, respectively.
The NDC group was further subdivided into patients with symptoms of chronic inflammatory CNS disease (CID, n = 10; age: 44.9 ± 14.2 years; mean ± SD) and patients with other neuropsychiatric disorders (OND, n = 37; age: 45.3 ± 16.4 years; mean ± SD). The heterogeneous diagnostic group of patients with CID was defined by clinical (e.g. prompt response to glucocorticoid therapy), technical (electrophysiology, cerebral MRI) and/or neurochemical parameters (intrathecal IgG synthesis). The CID group included five patients with multiple sclerosis and five patients with unknown aetiology of the chronic neuroinflammation.
The OND group included patients with cerebral transient ischaemic attacks (n = 6), subcortical arteriosclerotic encephalopathy (n = 5), epilepsy (n = 4), Meniere's disease (n = 1), benign paroxysmal positioning vertigo (n = 1), head trauma (n = 2), brain metastasis (n = 1), motor neuron disorder (n = 2), tension headache (n = 1), hemicrania (n = 1), unspecified neurological condition without dementia (n = 1), major depressive disorder (n = 5), bipolar I disorder (n = 2), psychotic disorder not otherwise specified (n = 1), anxiety disorder (n = 2), somatoform (conversion) disorder (n = 1), and benzodiazepine dependence (n = 1).
To control for a pronounced effect of the ApoE ε4 allele on the pattern of Aβ peptides we further subdivided the OND group into patients with one or two alleles of ε4 (ONDε4plus, n = 6) and without ε4 (ONDε4minus, n = 30). Since 11/12 AD patients had one or two ApoE ε4 alleles, the effect of ApoE genotype could not be eliminated in this group of patients, but to identify AD-specific effects on the Aβ peptide pattern in CSF we compared patients with AD, who carried the ε4 allele (ADε4plus, n = 11) to the ONDε4plus group (n = 6). MMSE scores, ApoE ε4 allele frequencies, absolute and relative Aβ peptide CSF concentrations for groups NDC, AD, IP-CSF, and SDS-CSF are summarized in Table 1.