Detection of amyloid-beta oligomers in human cerebrospinal fluid by flow cytometry and fluorescence resonance energy transfer.

Abstract

The neuropathology of Alzheimer's disease (AD) has been linked recently to non-fibrillar forms of neurotoxic amyloid-beta (Abeta) oligomers of which high levels are observed in the brain of AD patients. This suggests that Abeta oligomers play a key role in the early events of AD, underlining their potential for the early diagnosis of the disease. We have developed an extremely sensitive assay for the detection of oligomeric and fibrillar structures of Abeta that is based on multiparametric analysis of data obtained by flow cytometry and fluorescence resonance energy transfer (Fret). The assay readily detects Abeta oligomers in human cerebrospinal fluid (CSF) as verified by dot blot of the isolated particles. By measuring 174 CSF samples of non-demented control patients with various neurological disorders a high reliability and reproducibility of the method could be demonstrated.

Full text

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Journal of Alzheimer’s Disease 11 (2007) 117–125
IOS Press
117
Detection of Amyloid-β Oligomers in Human
Cerebrospinal Fluid by Flow Cytometry and
Fluorescence Resonance Energy Transfer
Alexander Navarrete Santosa,∗, Sandra Torklera, Denny Nowaka, Claudia Schlittiga, Mirjam Goerdesa,
Thomas Laubera, Lothar Trischmanna, Michael Schauppa, Monika Penzb,
Friedrich-Wilhelm Tillerband Gerald B¨ ohma
aACGT ProGenomics AG, Halle, Germany
bLabor Dr. Tiller, Munich, Germany
Abstract. The neuropathology of Alzheimer’s disease (AD) has been linked recently to non-fibrillar forms of neurotoxic
amyloid-β (Aβ) oligomers of which high levels are observed in the brain of AD patients. This suggests that Aβ oligomers play
a key role in the early events of AD, underlining their potential for the early diagnosis of the disease. We have developed an
extremely sensitive assay for the detection of oligomeric and fibrillar structures of Aβ that is based on multiparametric analysis
of data obtained by flow cytometry and fluorescence resonace energy transfer (Fret). The assay readily detects Aβ oligomers in
human cerebrospinal fluid(CSF)as verified by dot blot of the isolated particles. By measuring 174 CSFsamples of non-demented
control patients with various neurological disorders a high reliability and reproducibility of the method could be demonstrated.
Keywords:Alzheimer’sdisease, amyloid-β oligomers, cerebrospinalfluid,flowcytometry, fluorescence resonanceenergytransfer
Introduction
Alzheimer’s disease (AD) is the most common neu-
rodegenerative dementia with an average death prog-
nosis of 7 years [7]. Ongoing clinical studies point
out promisingpossibilities forthe treatment of this dis-
ease [1]. For ideal therapy and timely conservation of
essentialcognitivefunctions,however,adiagnostictool
for the early detection of AD is a pre-requisite. Recent
studies show that oligomeric assemblies of amyloid-β
(Aβ) are neurotoxic in cell culture and in vivo as they
areable to inhibitlong-termpotentiation[13,22]. Such
low-molecular-weight Aβ oligomers were also shown
to induce transient deficits in cognitive function [4].
To further demonstrate the adverse effect of oligomers
∗Corresponding author:
ProGenomics AG, Weinbergweg 22, 06120 Halle, Germany. Tel.:
+49 345 557 3314; Fax: +49 345 557 3317; E-mail: alexander.
navarrete@medizin.uni-halle.de.
Alexander Navarrete Santos, ACGT
onnervecell function,immunotherapywas successful-
ly used to neutralize Aβ oligomers, thereby restoring
synaptic plasticity in vivo [11].Further evidencepoint-
ing to Aβ oligomers as the neurotoxic species in AD
is that the concentration of these structures in human
brain were found to be up to 70-fold higher in AD pa-
tientsthaninnon-dementedcontrols[9,12,18].Itcould
thus be demonstrated that the severity of the disease
correlateswith oligomerconcentrationratherthanwith
number of plaques [6,14,15] and the presence of glob-
ular Aβ oligomers in the brain is suggested to be an
early pathological event in AD [2]. Hence, the search
for Aβ oligomers was extended to human CSF and re-
cent research studies demonstratedthe presence of low
amounts of stable Aβ oligomers also in this body flu-
id [8,17,21,23]. As the concentrationof oligomerswas
consistently higher in CSF of AD patients compared
to non-demented age-matched controls, this points to-
ward a correlation between the levels of oligomers and
the state of the disease [8], making them a possible
ISSN 1387-2877/07/$17.00  2007 – IOS Press and the authors. All rights reserved

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A.N. Santos et al. / Detection of Amyloid-β Oligomers in Human Cerebrospinal Fluid
biomarker and suitable target for the early detection of
AD. A sensitive method for the detection and accurate
quantification of Aβ oligomers is thus required. One
such method is the recently described ultra-sensitive
bio-barcode assay for the measurement of amyloid-β-
derived diffusible ligands (ADDLs) in CSF [8]. Al-
though this method is very sensitive, the procedure in-
cludesarelativelyhighnumberofcriticalstepsthatmay
affect the general performance of the assay. We have
therefore developed a new assay format that consists
of only one step (dilution of the sample in buffer and
incubation with fluorescence labeled antibodies) and
allowsahighthroughputofsamples. Theassayutilizes
flow cytometry combined with fluorescence resonance
energy transfer (Fret), which is an excellent tool for
determiningdistancesandsupramolecularorganization
of biomolecules.
Materials and methods
Chemicals and antibodies
All reagents were purchased from Sigma Aldrich
(Hamburg, Germany) unless otherwise specified. The
anti-Aβ monoclonal antibodies 4G8 and 6E10 were
suppliedbyChemiconInternational.Theanti-oligomer
antibody A-11 was purchased by Biosource.
Collection of cerebrospinal fluid
The collectionof the CSF samples was doneaccord-
ing to a vote from the ethics committee of the state
of Bavaria. CSF was obtained from 174 neurological
patients (mean age 49.3 years; range 8–89) with diag-
noses such as multiple sclerosis, neuroboreliosis and
inflammation. All samples were obtained by lumbar
puncture,frozenwithin2h andstoredat −85◦C before
analysis; repeated freeze/thaw cycles were avoided.
Fluorescence labeling of antibodies
The anti-Aβ antibodies 4G8 and 6E10 were labeled
with the fluorescence dyes Alexa Fluor 488 or Alexa
Fluor 594 according to the manufacturer’s instructions
(applying the respective Monoclonal Antibody Label-
ing Kits; Invitrogen).
Sample preparation for flow cytometry
200 µl of CSF were diluted with 200 µl of 1 × RI-
PA buffer (5 × RIPA buffer contains 250 mM HEPES;
750 mM NaCl, 0.25% SDS, 1.25% Na-deoxycholate,
2.5% NP-40 alternative (Calbiochem) and 1 tablet of
protease inhibitor cocktail CompleteTMMini (Roche
Applied Science) per 2 ml of 5 × RIPA buffer). Then
the fluorescence labeled antibodies were added to con-
centrationsof2nMand8nMfordonorantibody(4G8,
labeled with Alexa-Fluor 488) and acceptor antibody
(6E10, labeled with Alexa-Fluor 594), respectively.
Afterincubation(90minutesat roomtemperature,pro-
tected from light), samples were analyzed on a FACS
Calibur flow cytometer (BD Biosciences) as described
below.
Flow cytometry and particles sorting
For the detection of Aβ oligomers a FACS Cal-
ibur flow cytometer equipped with a 15 mW 488 nm
air-cooled argon-ion laser (BD Biosciences) was used.
The oligomer particles were gated in logarithmic for-
ward/sideward scatter dot plots (FSC vs. SSC). The
green or red fluorescence of the dyes Alexa Fluor 488
and Alexa Fluor 594 was detected by the correspond-
ing FL1 and FL3 (logarithmic scale) photomultipliers
through530/30or670LPbandpassfilters, respectively.
To avoid differences in the measurement due to vari-
ation in the CSF samples, all samples were measured
in TruCount Tubes (BD Biosciences) and analysis was
stopped after 28,000 beads were counted. The number
of oligomers/volume of CSF analysed was determined
asFret events. OneFret eventcorrespondedtoonepar-
ticleconsistingofAβ peptideinwhichseveralpeptides
aggregatetoformanoligomer. Sortingoftheoligomer-
specific region was performed on a FACS Vantage cell
sorter (BD Biosciences), applying a threshold to the
FL1 channel. The population of interest was gated in
a dot plot of FL1 vs. FL3 and only events with a Fret
signal were sorted.
Western and dot blot analysis
20 µl of CSF samples were applied to 16% Tricine
gels (Invitrogen), run for 2 hours, and transferred on-
to PVDF membranes. Membranes were boiled for 5
min in PBS and blocked for 2 hours in 5% skimmed
milk powder in TBS-T (10 mM Tris-HCl, pH 7.6 con-
taining 150 mM NaCl and 0.1% Tween 20). Then
the monoclonal anti-Aβ antibody 6E10 was applied

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119
Fig. 1. Principle of the assay. Aβ oligomers are detected by two specific monoclonal anti-Aβ antibodies and Fret. The clone 4G8 is used as
donor antibody and is labeled with the Alexa Fluor 488 dye, the acceptor antibody 6E10 is labeled with Alexa Fluor 594.
and incubated over night. After washing the mem-
branes in TBS-T, the bound antibody was visualized
using HRP-conjugated secondary anti-mouse antibody
andECLdetection(SuperSignalWestFemtoSubstrate,
Pierce). For dot blot analysis the samples were di-
rectly applied to the nitrocellulose membrane and air-
dried. The membranewas then processed to determine
the presence of Aβ oligomers using the anti-oligomer
specificantibodyA-11accordingtothe manufacturer’s
protocol.
Isolation of Aβ oligomers from brain homogenates
Frontotemporalbraintissuefromcontrolpersonsand
AD patients was kindly provided by the German brain
bank. Oligomers from control and AD brains were iso-
lated according to Wiltfang et al. [24].
Preparation of seedless Aβ (1–42)
Aβ (1–42) was purchased from Bachem, seedless
treated as previously reported [5], further purified by
RP-HPLC (column: Source 5RPC 4.6/150 ST (Amer-
sham);solventA:0.1%NH4OH(25%)inH2O,pH9.0;
solvent B: 60% acetonitrile, 40% solvent A; gradient:
25–56% solvent B in 31 min; flow rate: 1.0 ml/min)
and lyophilized.
Generation of in vitro oligomers and fibrils
Oligomers of Aβ were prepared as described by
Lambert et al. [13]. For fibrils generation seedless Aβ
(1–42) was dissolved in DMSO to 1 mM, diluted to
100 µM into 10 mM HCl and incubatedfor 24 hours at
37◦C [5]. For storage, fibril preparations were diluted
to 5 µM into 100 mM Hepes, 2.5 mM DTT, 5 mM
EDTA, 250 mM NaCl, 2% glycerol, pH 7.6 and frozen
at −85◦C.
Statistical analysis
Statistical analysis was performed using the Med-
Calc software and the Spearman’s rank correlation co-
efficient.
Results
Fluorescence resonance energy transfer (Fret) is a
special phenomenonin fluorescence spectroscopy dur-
ing which energy is transferred from an excited donor
moleculetoanacceptormoleculeunderfavorablespec-
tral and spatial conditions [10]. In our approach, two
monoclonal anti-Aβ antibodies that recognize differ-
ent epitopes of the Aβ peptide sequence were labeled
withthefluorescencedyesAlexaFluor488(mAb4G8;
raised against Aβ 17–24) and Alexa Fluor 594 (mAb
6E10; raised against Aβ 4–13). Thus, the mAb 4G8-
Alexa Fluor 488 corresponds to the donor molecule
and the mAb 6E10-Alexa Fluor 594 functions as the
acceptor molecule (Fig. 1). With this donor/acceptor
combination, Aβ monomers are not detectable in our
assay, which is due to the low amount of fluorophores
that are able to bind to Aβ monomers (a maximum of
two antibodies per monomer) and the resulting very
low intensity of the emitted fluorescence and Fret sig-
nal (data not shown). In contrast, oligomeric struc-
tures of Aβ are able to bind sufficient amounts of an-
tibody molecules and give thus rise to a fluorescence
signal strong enough to be detected by flow cytometry
(Fig. 2A, B). The information content of the fluores-
cence signal is increased by Fret, as it allows a differ-
entiationofunspecificbindingofthemAb4G8toother
molecules. Hence, signals from the 6E10-Alexa Fluor
594antibodyare onlyobservedif the distance between
both antibodies is closer than 10 nm, the F¨ orster dis-

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Fig. 2. Detection of Aβ oligomers prepared in vitro by flow cytometry and sensitivity of the assay. In contrast to monomers of Aβ the detection
of in vitro assembled Aβ oligomers can easily be achieved by flow cytometry and Fret (A, B). The sensitivity of the assay was determined by
titration of in vitro assembled Aβ fibrils. All data points are represented as mean values (± standard deviation) of measurements performed in
triplicate. The assay is linear over a wide range of concentrations, ranging from at least 10 pM to 2.5 nM. (C).
tance that allows energy transfer between donor and
acceptor fluorophores.
In order to determine the sensitivity of the assay in
vitro assembled Aβ oligomers and fibrils were ana-
lyzedwiththeproceduredescribed.Forthedetectionof
oligomers by flow cytometry ADDLs (Amyloid beta-
derived diffusible ligands) were prepared as described
by Lambert et al. [13]. Such molecules are claimed
to be found in CSF by Georganopoulou et al. [8]. In
vitro assembled ADDLs were detected efficiently by

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121
our method. However, our ADDL preparations were
not stable (even not at −85◦C) and transformed to fib-
rils. As a consequence, the reproducibility of the mea-
surements was poor (data not shown). Therefore we
preferred to deal with fibril preparations which were
stable. Only differences in the size and Fret-signal in-
tensity were observed between Aβ oligomers and fib-
rils (smaller for the oligomers). The sensitivity in both
cases was the same. The assay is linear over a wide
rangeof concentrations,rangingfromat least 10 pMto
2.5nM(Fig.2C).Takingintoaccountthatthefibrilcon-
centration is expressed as concentration of monomers
used for fibril assembly and assuming an average fib-
ril size of several hundreds to thousands of monomers,
the actual concentration of fibrils is about two to three
orders of magnitude lower. The detection limit for the
currentsetupoftheassayisthereforeinthefemtomolar
range.
For the determination of the presence of oligomers
inhumanfluids200µl ofCSF weredilutedwith200µl
of 1 × RIPA buffer, followed by incubation with the
Fret antibody mixture. Although we used 200 µl of
each CSF sample, this volume is not limiting and can
be increased if a higher sensitivity is required. To bet-
ter visualize the Fret effect, we first incubated human
CSF with the antibody 4G8-Alexa Fluor 488 alone. In
the subsequent flow cytometric analysis, an oligomer-
specific population was detected showing events in the
fluorescence 1 channel only (Fig. 3A; FL1-H). Addi-
tion of the Fret acceptor antibody 6E10-Alexa Fluor
594 then induced an additional fluorescence signal in
the fluorescence 3 channel (Fig. 3B, C; FL3-H). This
gain of an Alexa Fluor 594 specific emission demon-
strates the binding of both antibodies in close proxim-
ity to each other (< 10 nm), allowing energy transfer
fromthe Alexa Fluor 488 donorto the Alexa Fluor 594
acceptor fluorophore. Although the specificity for the
detected signal increases upon addition of the acceptor
antibody, the overall number of events simultaneously
decreases,possiblyduetoastericalcompetitionofboth
antibodiesfortheir respectiveepitopesas well as a Fret
induced fluorescence quenching of the acceptor fluo-
rophores. Since the probability for non-specific bind-
ing of both antibodies within a distance closer than 10
nm is extremely low, these data suggest that the detect-
ed events are indeed Aβ oligomers. To furthervalidate
this assumption, a pool of CSF (6 ml) was prepared
and analyzed as described before. The events detected
in the region of interest, however, were sorted with a
FACS Vantage cell sorter and subsequently analyzed
by dot blot, resulting in a positive signal when probed
with the oligomer-specific antibody A-11 (Fig. 3D).
It is known that repeated freeze/thaw cycles of
CSF lead to a decrease in the concentration of Aβ
monomers [19]. The absence of a reliable detection
method, however, did render such studies impossible
for oligomers. We therefore examined the effect of
freeze/thawcycles using a pool of CSF that was frozen
at −85◦C and thawed consecutively three times. The
first value was measured before freezing and was set
as 100%.In contrast to the situation observed for
Aβ monomers [19], no significant effect was observed
for the overall amount of oligomers for all applied
freeze/thaw cycles (Fig. 4A). We further investigated
the reproducibility of our assay, exemplified for seven
different CSF samples in Fig. 4B. The standard devia-
tion for these samples is ± 2.1% (Fig. 4B) and below
5% for more than 200 measured samples. As can also
be seen from this figure, two of the CSF samples were
negative for Aβ oligomers, even though all samples
contained approximately equal amounts of monomers
as detected by Western blot (Fig. 4B, C, lane 3, 5).
This demonstrates that there is no correlation between
oligomer content and concentration of Aβ monomer
as detected by Western blot or ELISA [20] and under-
linestheadvantageoftheassayoverothermethods,i.e.
specificity for oligomers, scalable sample volume and
thus enhanced sensitivity (Fig. 2C).
In order to test the clinical assay performance, we
finally extended our CSF analysis to samples obtained
from 174 non-demented individuals with various neu-
rological disorders (Fig. 5). Evaluation of the data
shows a large variation in the concentration of the de-
tected oligomers. We found, however, a weak correla-
tion between the age of the individuals and amounts of
Aβ oligomers (rho = 0.22; p = 0.0036).
As flow cytometry is a common method in routine
diagnostics, the use of the assay described here could
be a low cost alternative to other methods, particularly
as it allows a high sample throughput and automation.
Discussion
In the present study we describe a new approach for
the detection of Aβ oligomers in cerebrospinal fluid.
One of the advantages of the assay is the possibility
to discriminate between Aβ monomers, oligomers and
fibrils. In addition, the use of a fluid system, i.e. flow
cytometry,allows a multiparametricanalysis of the de-
tected molecules and thus a better differentiationof the
signal. This is not possible with other methods such as
ELISAorfluorescencecorrelationspectroscopy. Stenh

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Fig. 3. Validation of the assay. Incubation of a CSF sample with donor antibody (4G8-Alexa Fluor 488) alone gives rise to a signal in the FL1
channel only (A). Addition of the acceptor antibody (6E10-Alexa Fluor 594) results in a strong Fret signal, i.e. gain of fluorescence 3 (FL1
and FL3) (B). The histogram analysis of the FL3 channel to visualize the Fret-induced fluorescence shift is shown (C; light grey: only donor
antibody; dark grey: donor/acceptor antibody pair). Dot blot analysis of the material isolated from a CSF pool after sorting and probed with the
anti-oligomer specific antibody A-11 showed the presence of oligomers in the preparation (D, lane 3). Brain homogenate from an AD patient (D,
lane 2) and a non-demented control person (D, lane 1) were used as positive and negative controls, respectively.
et al. [20] showed that the amount of Aβ oligomers
determined by ELISA was underestimated due to the
suboptimal binding of the antibodies to the oligomers.
Pitschke et al. [17] demonstrated the presence of Aβ
aggregates in cerebrospinal fluid by fluorescence cor-
relation spectroscopy. Most recently, Georganopoulou
etal.[8]showedthepresenceofA-β-deriveddiffusible
ligands in the same fluid using a bio-barcode assay.
Although both assays successfully detected Aβ aggre-
gates in cerebrospinal fluid, flow cytometry possesses
the advantages of a widespread availability in all lead-
ing biomedical research institutions and the potential

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123
Fig. 4. Stability of natural Aβ oligomers and reproducibility of the
assay. Theeffect offreeze/thaw cycles onthe detection ofnatural Aβ
oligomers inCSFwas tested. Theconcentration ofthe Aβ oligomers
detected in a CSF pool was not altered by the application of three
freeze/thaw cycles (A).Thereproducibility of the assay was assessed
by measuring seven different CSF samples by flow cytometry. All
samples were analyzed in duplicate and values are represented as
mean values and standard deviations (B). When a Western blot using
20 µl of CSF samples analyzed in B was carried out no correlation
betweentheamountsofAβ monomerasdetectedbyWesternblotand
oligomer content wasobserved (C).sAPP:soluble amyloid precursor
protein.
ofa highthroughputofsamples. Intheassay presented
here, the combination of flow cytometry and fluores-
cence resonance energytransfer resulted in an increase
in the specificity of the particles detected. Only par-
ticles with fluorescence in the FL1 and FL3 channels
were counted as a specific signal. Attempts to deter-
Fig. 5. Clinical performance of the assay. The scatter diagram of
Aβ oligomers (Fret events) versus age of 174 CSF samples from
non-demented control subjects reveals a positive correlation (rho =
0.22; p = 0.0036) between age and concentration of Aβ oligomers.
All values are mean values of two independent measurements. The
linear regression and 95% confidence intervals are represented as
solid lines.
minetheminimalsizeofthedetectedoligomersinCSF
by size exclusion chromatography were unsuccessful
due to the heterogeneity of the particles. Nevertheless,
the size of the detected molecules in CSF and the in-
tensity of the Fret signals corresponded to the size of
the in vitro assembled ADDLs. The result of the dot
blotdevelopedwiththeanti-oligomerspecificA-11an-
tibody also suggested that the detected molecules were
oligomers and not fibrils. Taking all these points to-
gether we conclude that the detected particles in CSF
are oligomers. Whether Aβ (1–40), Aβ (1–42), or
other forms of the peptide (N-terminal or C-terminally
truncated) are a constituent of the particles, will be ad-
dressed in future studies. The antibodies we used in
the present study do not allow distinguishing between
Aβ (1–40) and Aβ (1–42) because they bind in the N-
terminal and central region of the peptide. Antibod-
ies recognizingepitopes in the C-terminalregioncould
not be used since the C-terminus of the Aβ peptide is
highlyhydrophobicand thus solvent-inaccessible[16].
Solubleamyloidprecursorprotein(sAPP)didnotinter-
ferewiththeassay.Thedonorantibody4G8recognized
the mid-domainof the Aβ peptide (epitope17–24)and
sAPP lacks this domain. Thus, sAPP could be bound
by the acceptor antibody 6E10 but was not exited due
to the lack of the donor antibody 4G8 and hence was
not detected. Silver gels of the sorted material showed
bands not corresponding to multimers of the Aβ pep-
tide,suggestingthatotherproteinsarealsoconstituents
of the particles (data not shown). Such proteins could

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be albumin or ApoE which are known to bind to the
Aβ peptide [3].
In this study, samples from patients suffering from
various neurological disorders other than AD were
analysed. A weak correlation between age and the
amounts of oligomers was observed. However, the Aβ
oligomer concentrations can not actually be contem-
plated to augment with age. The measurement of sam-
ples from patients diagnosed with AD will assess the
potential of the assay as a diagnostic tool for AD and
the suitability of Aβ oligomers as a biological marker
of AD.
Acknowledgement
The work was financially supported by a research
grant of the state of Sachsen Anhalt.
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