Z. van Helmond et al. / Journal of Neuroscience Methods 176 (2009) 206–212 207
protein 104 (Hsp104) in the formation and elimination of self-
replicating Sup35 NM [PSI+] yeast prions (Shorter and Lindquist,
2004). Post-embedding A11 immunoelectron microscopy was also
used to investigate the ultrastructural distribution of oligomeric A␤
(Kokubo et al., 2005). A recent study suggested that A11 detects the
toxic oligomers only of ␣-synuclein and A␤1–42, and not non-toxic
aggregates (Ehrnhoefer et al., 2008).
The production of two further antibodies (M93 and M94)
raised against synthetic oligomeric A␤ was described by
Lambert et al. (2001). These were used for immunoﬂuores-
cence and immunoperoxidase detection of oligomers (Lacor et
al., 2004) and for immunoblots that demonstrated SDS-stable
oligomers in AD brain tissue (Gong et al., 2003; Lacor et al.,
Rapid, sensitive measurement of the levels of A␤1–42 oligomers
in brain tissue would be greatly facilitated by the development
of a speciﬁc enzyme-linked immunosorbent assay (ELISA). As far
as we are aware no such assay has been reported. Our initial
attempts at developing an assay that used A11 were unsuccess-
ful. Recently, two further oligomer-speciﬁc A␤ antibodies have
become available from New England Rare Reagents, Portland,
Maine: mouse monoclonal antibodies (clones 7A1a and 1G5) raised
against synthetic oligomers of A␤40. The immunogen had a pro-
prietary synthetic peptide structure that was designed to mimic
the three-dimensional structure thought to form within aggregated
A␤ (New England Rare Reagents, personal communication) and
the antibodies have been reported to detect low molecular weight
oligomers and protoﬁbrils of A␤. Our aims in the present study were
to examine the speciﬁcity of 7A1a and 1G5 for synthetic oligomers
of A␤1–42 and for oligomeric A␤1–42 in human brain homogenates,
and to develop a sandwich ELISA for measuring oligomeric
2. Materials and methods
2.1. Study cohort
Brain tissue from 15 cases of neuropathologically conﬁrmed
AD (CERAD classiﬁcation ‘deﬁnite AD’, Braak tangle stage V or VI)
(Braak and Braak, 1991) and 13 neuropathologically normal con-
trols without AD were studied by Western blot. These same cases
together with a further 94 AD and 29 control cases were sub-
sequently analysed by ELISA. The cases were selected from the
South West Dementia Brain Bank (SWDBB), University of Bristol.
The study was conducted with the approval of Frenchay Research
Ethics Committee. The AD cases ranged from 47 to 98 years in age
(mean = 79.3, S.D. = 9.6) and comprised 70 females and 39 males.
The post-mortem delays were between 4 and 101.5 h (mean = 44.8,
S.D. = 24.5). The controls were similarly aged, from 43 to 95 years
(mean = 76.6, S.D. = 10.3) and comprised 16 females and 26 males.
The post-mortem delays were between 3 and 106 h (mean = 41.4,
S.D. = 28.6).
2.2. Oligomerisation of synthetic Aˇ1–42
Lyophilised A␤1–42 peptide (Anaspec, CA) was dissolved in 35%
acetonitrile (Ryu et al., 2004) to a ﬁnal stock concentration of
1.6 mM. 10 l aliquots were stored at −20 ◦C until required.
Stock A␤1–42 peptide was diluted to 300 M in either
phosphate-buffered saline (PBS; 0.15 M sodium chloride, 1.9 mM
sodium dihydrogen orthophosphate 1-hydrate, 7.5 mM di-sodium
hydrogen orthophosphate 12-hydrate, pH 7.1) or Dulbecco’s Mod-
iﬁed Eagle Medium (DMEM; Gibco, Invitrogen, UK) and incubated
overnight at 37 ◦C.
2.3. Thioﬂavin S assay
This assay was adapted from a standard thioﬂavin T protocol
(LeVine, 1993; Maezawa et al., 2008). Brieﬂy, 50 l of oligomerised
A␤1–42 peptide was mixed with 150 l thioﬂavin S solution (5 M
in 50 mM glycine-NaOH, pH 8.5) and the ﬂuorescence measured
using a FLUOstar OPTIMA multidetection microplate reader (BMG
Labtech GmbH, Germany) with excitation at 450 nm and emission
at 490 nm. Fluorescence was measured immediately after mixing
and then every 2 h for 30 h, with incubation at 37 ◦C.
2.4. Brain homogenates
For each case, approximately 200 mg of frontal neocortex (Brod-
mann area 6) was homogenised in 1 ml lysis buffer (1% SDS, 10 mM
Tris, pH 6, 0.1 mM sodium chloride, 1 M phenylmethylsulphonyl
ﬂuoride, 1 g/ml aprotinin). Brain tissue was homogenised in a
Bertin Technologies Precellys 24 mechanical homogeniser (Stretton
Scientiﬁc, Stretton, UK) at 6000 rpm for 2 s × 30 s with approxi-
mately ten 2.3 mm Biospec Products zirconia-silica beads (Stratech
Scientiﬁc, Newmarket, UK) in a screw cap 2 ml centrifuge tube.
Samples were cooled on ice after homogenisation. Total protein was
measured using Total Protein kit (Sigma–Aldrich, Gillingham, UK)
and 20 l aliquots were stored at −80 ◦C until required.
2.5. Western blot
5 l of the 300 M synthetic A␤1–42 that had been incu-
bated overnight at 37 ◦C in either PBS or DMEM were diluted to
30 l in sample buffer (0.5 M Tris–HCl, pH 6.8, 0.25% glycerol,
10% (w/v) SDS, 0.5% (w/v) bromophenol blue) containing 5% ␤-
mercaptoethanol. Brain tissue homogenates containing 30 g of
total protein were diluted to 30 l in sample buffer containing 5%
␤-mercaptoethanol. 1.25 g tropomyosin-3 (TPM3) recombinant
protein (Abnova, TW) was, similarly, diluted to 30 l in sample
buffer containing 5% ␤-mercaptoethanol. Diluted samples were
loaded onto a 4–20% Tris–HCl pre-cast gel (Bio-Rad, Hemel Hemp-
stead, UK) and electrophoresed at 150 mV for 1 h. Proteins were
subsequently transferred to nitrocellulose membrane overnight at
30 mV. Membranes were blocked for 1 h at room temperature in
10% non-fat milk in Tris-buffered saline/Tween 20 (TBS-T; 20 mM
Tris base, 0.5 M NaCl, 0.05% Tween 20), incubated for 1 h at room
temperature in 5% non-fat milk in TBS-T with either 1G5 or 7A1a
antibodies (both 1:250), washed 3 min × 10 min in TBS-T and then
incubated for 1 h at room temperature with secondary antibody
horseradish-peroxidase (HRP)-conjugated horse anti-mouse IgG
(1:5000, Vector Laboratories, CA) in 5% non-fat milk in TBS-T. The
membrane was then washed 3 × 10 min in TBS-T and immunoreac-
tive proteins were detected by enhanced chemiluminescence (GE
2.6. Two-dimensional gel electrophoresis/mass spectrometry
Two-dimensional gel electrophoresis followed by mass spec-
trometry (2DGE/MS) was applied to crude homogenate of frontal
neocortex from a single AD case that had previously been anal-
ysed for oligomeric A␤ levels by Western blot. The sample was
precipitated using 2D Clean-Up Kit according to the manufac-
turer’s instructions (GE Healthcare, NJ). The resulting protein pellet
was resuspended in 2 l × 200 l of rehydration buffer (7 M urea,
2 M thiourea, 4% CHAPS, 60 mM DTT, 0.5% IPG buffer, 0.002% bro-
mophenol blue), sonicated for 15 min, left at room temperature
for 1 h and then loaded onto 2 cm × 11 cm Immobiline DryStrip
First Dimension IPG strips (GE Healthcare, NJ) by passive rehy-
dration. Following overnight rehydration, isoelectric focussing was
208 Z. van Helmond et al. / Journal of Neuroscience Methods 176 (2009) 206–212
performed using the Ettan IPGphor (GE Healthcare, NJ) accord-
ing to the manufacturer’s instructions (500 V for 1 h, 1000 V for
1 h and 8000 V for 2 h). The focussed IPG strips were then incu-
bated for 15 min in 10 ml SDS equilibration buffer (50 mM Tris–HCl,
pH8.8, 6 M urea, 30% (v/v) glycerol, 2% SDS, 0.002% bromophenol
blue) containing 100 mg DTT, and for a further 15 min in 10 ml
equilibration buffer containing 250 mg iodoacetamide. The equi-
librated strips were applied to the surface of 12.5% polyacrylamide
gels and the proteins separated in the second dimension in the
Ettan DALT ll separation unit (GE Healthcare, NJ) at 0.2 W/gel for
1 h, 0.4 W/gel for 1 h and then 20 W/gel until completion. Gels
were then either ﬁxed for an hour in 50% methanol, 10% acetic
acid and stained overnight using Sypro Ruby total protein stain
(Invitrogen, UK) or transferred to PVDF membrane for Western
blotting. Gels were destained in 10% methanol, 7% acetic acid
and imaged using a Typhoon 9200 scanner (GE Healthcare, NJ).
Western blots were stained with silver post-immunodetection to
give a total protein image which allowed matching of the spot
patterns between gel and blot. Immunopositive spots identiﬁed
on the Western blot were matched to those in the gel and the
relevant spots excised from the gel using the ProPic automated
spot picker and then digested with trypsin in the ProGest auto-
mated digestion unit (both from PerkinElmer Life Sciences, MA).
The resulting peptides were analysed by tandem mass spectrom-
etry in an Applied Biosystems 4700 mass spectrometer to give a
peptide mass ﬁngerprint and MSMS peptide sequence data; these
were used to identify the protein, in searches against the MSDB
database, performed with the Mascot search engine from Matrix
2.7. Oligomeric Aˇ1–42 ELISA
A Maxisorp ELISA plate (Nunc, NY) was coated with 5 g/ml
of rabbit polyclonal A␤1–42 antibody (Millipore, MA) (50 l/well)
in coating buffer (10 mM sodium carbonate, 30 mM sodium bicar-
bonate, pH 9.6) overnight at 4 ◦C. The plate was washed ﬁve
times with wash buffer (0.05% Tween 20, PBS) and incubated with
blocking buffer (5% Tween 20, 5% non-fat milk, 1% bovine serum
albumin (BSA)) for 2 h at room temperature. An 8-point standard
curve (0–125 g/ml) was prepared in duplicate using oligomerised
A␤1–42 peptide. Sample homogenates were spun for 10 min at
20,000 × g and, from the supernatant, 15 g total protein were
diluted in reagent diluent (1% BSA, 0.2% Tween 20, PBS) to a ﬁnal
volume of 50 l per well. Samples were tested in triplicate. To
some wells we added duplicate samples of TPM3 recombinant
protein (31.25 g/ml), either immediately after dilution or after
having been left overnight under the same conditions that were
used for polymerisation of synthetic A␤1–42 peptide (see above).
The plate was washed ﬁve times with wash buffer before sam-
ples, TPM3 and standards were added. Controls included wells from
which we omitted capture antibody, sample or detection antibody.
The plate was incubated at room temperature for 2 h, with gen-
tle shaking. The plate was washed ﬁve times with wash buffer,
and either 7A1a or 1G5 diluted to 10 g/ml in reagent diluent
(100 l/well) was added and incubated for 1.5 h at room tem-
perature with gentle shaking. The plate was washed ﬁve times
in wash buffer and HRP-conjugated horse anti-mouse IgG (1:500
in reagent diluent) was added (100 l/well) and incubated for
30 min at room temperature with gentle shaking. The plate was
washed ﬁve times with wash buffer and 100 l of ABTS sub-
strate (Vector Laboratories, CA) was added to each well. Protected
from light, the plate was incubated for 20–25 min at RT, and
then read at 450 nm. Reproducibility tests, run as two separate
experiments, were conducted on seven cases (four AD and three
2.8. Oligomeric Aˇ1–40 ELISA
This assay followed the protocol for the oligomeric A␤1–42 ELISA
but used a rabbit polyclonal A␤1–40 antibody (Alpha Diagnostic
International, TX) as the capture antibody (5 g/ml). 7A1a was used
as the detection antibody. An 8-point standard curve (0–10 g/ml)
was prepared with a commercially available oligomeric A␤40
standard (Maine Biotechnology Services, ME). The manufacturer’s
instructions were followed to prepare this standard. The assay was
performed in duplicate to test reproducibility. 20 AD and 14 control
cases were selected and analysed in duplicate for each assay run.
2.9. Statistical analyses
Inspection of the levels of oligomeric A␤1–42/A␤1–40 measured
showed that the distributions were skewed to the right. How-
ever, the data could be normalised by logarithmic transformation.
Oligomeric A␤1–42/A␤1–40 levels were analysed with respect to
diagnosis by use of an independent samples t-test, with the help
of Statistical Package for Social Science software (version 12.0.1
for Windows). Standard linear regression and both parametric and
non-parametric correlation analyses were used to assess (i) the
relationship between the levels of oligomeric A␤1–42 measured by
sandwich ELISA using either 7A1a or 1G5 as the detection antibody;
(ii) reproducibility of measurements of oligomeric A␤1–42/A␤1–40.
Values of p < 0.05 were considered to be statistically signiﬁcant.
3.1. 7A1a and 1G5 antibodies detect multimeric Aˇ1–42 species
Western blot analysis revealed that both antibodies to
oligomeric A␤ (7A1a and 1G5) detected a range of A␤1–42 oligomers
of synthetic A␤1–42 that had been allowed to polymerise overnight
(Fig. 1). Similar results were found after preparing the peptide in
either PBS or DMEM: a single band at approximately 10 kDa prob-
ably represents SDS-stable dimeric states, a double band in the
range of 15 kDa probably includes trimeric and tetrameric A␤1–42,
and high molecular weight species ranging from about 50 kDa to
greater than 150 kDa. A small amount of polymerised A␤1–42 did not
enter the gel; this amount was consistently higher after overnight
polymerisation in DMEM than in PBS.
3.2. Conﬁrmation of Aˇ1–42 oligomerisation by thioﬂavin S assay
Thioﬂavin analogues have previously been shown to detect
oligomerisation of A␤ (Maezawa et al., 2008). Here, an increase in
thioﬂavin S ﬂuorescence was observed for A␤1–42 peptide that was
incubated in either PBS or DMEM (Fig. 2), indicating that oligomeri-
sation was occurring. Thioﬂavin S ﬂuorescence was found to peak
at approximately 12 h when using PBS, and 18 h for A␤1–42 incu-
bated in DMEM. Thioﬂavin S ﬂuorescence subsequently declined as
ﬁbrillar A␤1–42 precipitated out of solution. Fibrillisation occurred
more quickly in DMEM, as evidenced by the earlier and more rapid
decline in ThS ﬂuorescence after it peaked at 18 h; the insoluble
A␤1–42 could be seen on the ﬂoor of the microplate wells.
3.3. Cross-reactivity with tropomyosin in human brain
Western blot analysis of homogenates from control and AD cases
yielded an unexpected dense band of apparent molecular weight
∼34 kDa (Fig. 3a; left panel). The labelling intensity of this band
did not vary considerably from case to case and did not differ
Z. van Helmond et al. / Journal of Neuroscience Methods 176 (2009) 206–212 209
Fig. 1. Western blot of synthetic A␤1–42 that had been allowed to polymerise
overnight. Anti-oligomeric A␤ antibody 7A1a (lanes 1 and 2) and 1G5 (lanes 4 and
5) used to probe the blot detected a range of multimeric A␤1–42 species. Similar
results were found when preparing the peptide in either PBS (lanes 1 and 4) or
DMEM (lanes 2 and 5). A single band at approximately 10 kDa probably represents
SDS-stable dimeric states. A double band in the range of 15 kDa probably includes
trimeric and tetrameric A␤1–42. High molecular weight species were also present,
ranging from about 50 kDa to greater than 150 kDa.
Fig. 2. Thioﬂavin S assay conﬁrms A␤ oligomerisation. 50 l of oligomerised A␤1–42
peptide was mixed with 150 l thioﬂavin S solution (5 M in 50 mM glycine-NaOH,
pH 8.5) and the ﬂuorescence measured. An increase in thioﬂavin S ﬂuorescence was
observed for A␤1–42 peptide that was incubated in both PBS and DMEM, indicating
that oligomerisation was occurring. Thioﬂavin S ﬂuorescence was found to peak
at approximately 12 h when using PBS, and 18 h for A␤1–42 incubated in DMEM.
Thioﬂavin S ﬂuorescence subsequently declined as ﬁbrillar A␤1–42 precipitated out
Fig. 3. Cross-reactivity with tropomyosin in human brain homogenates. (a) West-
ern blot of homogenates of frontal neocortex from several brains, probed with 7A1a
(probing with 1G5 yielded identical results). The dominant species detected had
an apparent molecular weight of approximately 34 kDa (left panel). Over exposure
revealed a range of other bands (right panel). (b) Two-dimensional gel electrophore-
sis of a representative homogenate from a case of AD. 7A1a was used here. The large
immunospot (arrow) represents the 34 kDa species detected by Western blot. Mass
spectroscopy showed this to be tropomyosin-3 (TPM-3). (c) Western blot of TPM3
recombinant protein probed with rabbit anti-TPM3 antibody (lane 1) or 7A1a (lane
210 Z. van Helmond et al. / Journal of Neuroscience Methods 176 (2009) 206–212
Fig. 4. Results of oligomeric A␤1–42 ELISAs. Box-and-whiskers plots show the range,
interquartile range and median values in homogenates of frontal neocortex from
control and AD brains. Oligomeric A␤1–42 levels were found to be signiﬁcantly
higher in AD than control cases (p = 0.041, independent samples t-test). The mea-
sured levels of oligomeric A␤1–42 differed approximately ten-fold between the
two detection antibodies, 7A1a giving higher values (0.12–28.40 g/ml) than 1G5
signiﬁcantly between the AD and control groups. Longer expo-
sure of the membranes revealed a range of other bands (Fig. 3a;
right panel), including a broad band of apparent molecular weight
For further investigation of the unexpected ﬁnding of a
dominant ∼34 kDa band, we performed two-dimensional gel elec-
trophoresis followed by mass spectrometry on one of the AD
brain homogenates (Fig. 3b). Results revealed that the 7A1a and
1G5 antibodies were cross-reacting with tropomyosins, particu-
larly tropomyosin-3 (TPM3), producing the predominant band at
∼34 kDa. Western blot analysis conﬁrmed that TPM3 recombinant
protein was detected by both of the anti-oligomeric A␤1–42 anti-
bodies (Fig. 3c) (The molecular weight of the recombinant TPM3
is shown to have a molecular weight of ∼60 kDa in Fig. 3c. This
is due to the recombinant protein (249 amino acids) including a
glutathione-S-transferase tag (total length: 482 amino acids).
3.4. Use of ELISA to measure oligomeric Aˇ1–42 levels in AD and
ELISAs using A␤1–42 capture antibody and either 7A1a or 1G5
detection antibody yielded signal above background in all sam-
ples of AD and control brain tissue (Fig. 4). TPM3 recombinant
protein, added either directly after dilution or only after being
left at 37 ◦C overnight, was not detected by this assay. Although
case-to-case variation was considerable, oligomeric A␤1–42 levels
were signiﬁcantly higher in AD cases than controls (p = 0.041, inde-
pendent samples t-test). Repetition of the ELISAs on a subset of
samples showed good reproducibility of the results (p < 0.001, Pear-
son correlation test, r2 = 0.984; data not shown). The amount of
oligomeric A␤1–42 detected by the two antibodies differed approxi-
Fig. 5. Scatterplot of results of ELISAs incorporating either 7A1a or 1G5 as the detec-
tion antibody. The best-ﬁt linear regression line (solid line) and 95% conﬁdence band
of the regression line (interrupted lines) are also shown. The results of the assays
with the two antibodies showed good correlation (p < 0.0001, r2
mately ten-fold, 7A1a giving higher values (0.12–28.40 g/ml) than
1G5 (0.13–3.16 g/ml). However, the results of the assays with the
two antibodies showed good correlation (p < 0.001, r2 = 0.51) (Fig. 5).
3.5. Use of ELISA to measure oligomeric Aˇ1–40 levels in AD and
Results conﬁrmed the feasibility of using speciﬁc A␤1–40 cap-
ture antibody and 7A1a detection antibody to measure oligomeric
A␤1–40 by ELISA, which yielded signal above background in
all of the samples analysed. Levels of oligomeric A␤1–40 mea-
sured were found not to be signiﬁcantly different in AD brains
(0.26–5.21 g/ml) compared to control brains (0.13–3.60 g/ml).
Repetition of the assay measurements showed good reproducibility
(p < 0.001, r2 = 0.789; data not shown).
We have demonstrated that antibodies 7A1a and 1G5 detect
oligomeric A␤1–42 but that they also bind to tropomyosin which
limits their utility for analysing oligomeric A␤ by dot blot. However,
combining of antibody to A␤1–42 with either 7A1a or 1G5 allows
measurement by sandwich ELISA of the levels of A␤1–42 oligomers
in homogenates of brain tissue. In this study, we have used the term
A␤ oligomers to include all soluble A␤ species with the exception
of monomers. This corresponds to the range of forms of A␤ that
we detected by Western blot with 7A1a and 1G5. Although there
was little direct evidence of labelling of insoluble A␤ in our stud-
ies, it is likely that these antibodies will detect, at least to a limited
degree, insoluble aggregated forms of A␤: A␤ readily binds to other
proteins and it is probable that soluble oligomers bind to insoluble
aggregated A␤ species.
Oligomers of A␤ are potent neurotoxins in vitro (Dahlgren et al.,
2002; Hartley et al., 1999; Lambert et al., 1998; Walsh et al., 2002).
Their role in AD is still under scrutiny and our development and
Z. van Helmond et al. / Journal of Neuroscience Methods 176 (2009) 206–212 211
validation of a simple and sensitive assay to quantitate the levels
of oligomeric A␤ in human brain tissue should prove very valu-
able. Stenh et al. (2005) demonstrated that oligomerisation of A␤
results in underestimation of total A␤ levels by ELISA. Because the
C-terminal part of the A␤ peptide is thought to be hidden inside
the hydrophobic core of the aggregate (Roher et al., 2000), the
immunological properties of monomeric and oligomeric A␤ dif-
fer. Stenh et al. (2005) and Roher et al. (2000) suggested that the
levels of A␤ detected by ELISA depend on the conformation speci-
ﬁcity of the antibodies used and recommended either denaturing of
samples prior to assay or the use of novel conformation-dependent
In vitro, 7A1a and 1G5 label a range of potentially toxic
oligomeric species of A␤. A␤ oligomers bind to synapses and affect
synaptic function. Synaptic A␤ ligands were shown to compose of
oligomers of between 50 and 100 kDa (Lacor et al., 2004, 2007). This
is consistent with the ∼55 kDa oligomer (A␤*56) that was reported
to cause cognitive impairment in vivo (Cleary et al., 2005) and with
a prominent oligomer found in AD brain tissue extract (Gong et al.,
2003). Smaller naturally secreted oligomers, particularly trimers,
were also shown to be toxic (Townsend et al., 2006; Walsh et al.,
2002). Monomeric A␤ (Mr 4.5 kDa) is not detected by either 7A1a
When used to probe Western blots of human brain homogenate,
both antibodies cross-reacted with tropomyosin. However, other
speciﬁc bands were detected by these antibodies, in particu-
lar a band with an approximate molecular weight of 55 kDa, as
found in other studies (Cleary et al., 2005; Gong et al., 2003). A␤
peptide and tropomyosin-3 both include the N-terminal amino
acid sequence DAEFR, but this region of the A␤ peptide was
not part of the immunogen used to raise 7A1a or 1G5 (New
England Rare Reagents, personal communication). Denaturation
of oligomers by SDS prior to polyacrylamide gel electrophoresis
could theoretically cause some loss of oligomer-speciﬁc confor-
mation and therefore reduce labelling of Western blots. This was
not, however, observed in Western blots of synthetic A␤1–42 pep-
To overcome the cross-reactivity issues using these antibodies,
a sandwich ELISA was designed using a pan A␤1–42 (for solu-
ble and insoluble A␤1–42 species) to capture target peptides, with
either 7A1a or 1G5 to detect oligomeric-speciﬁc conformations.
Both 7A1a and 1G5 were effective within this assay, but the lev-
els of oligomeric A␤ detected using these two antibodies differed
by about ten-fold. Perhaps 1G5 preferentially detects a narrower
range of speciﬁc oligomeric species. There was a suggestion from
our Western blot analysis that 1G5 is not as sensitive to low molecu-
lar weight oligomers as 7A1a (Fig. 1). However, the reasonably good
correlation between the results obtained with these two antibodies
suggests that both are likely to be useful for making comparative
measurements, e.g. between AD and control brains, or between
different regions of the same brain. Adaptability of the oligomeric
A␤1–42 ELISA to measure oligomeric A␤1–40 has also been demon-
In the past, sequential extraction techniques with ultracentrifu-
gation were used to isolate oligomeric A␤ from human brain tissue
(Kuo et al., 1996; Lue et al., 1999; McLean et al., 1999; Pitschke et
al., 1998; Wang et al., 1999). The isolated oligomeric A␤ species
were subsequently analysed by a number of methods, with varying
results. Kuo et al. (1996) reported that AD brains contain six-fold
more oligomeric A␤ (mostly A␤N–42 species) than controls do. In
contrast, Wang et al. (1999) found oligomeric A␤1–40 and A␤1–42
species to constitute the largest fractions of total A␤ in normal brain
but the smallest in AD. Increased levels of oligomeric A␤, particu-
larly A␤1–40, were noted by Lue et al. (1999) in AD compared to
The discrepancies within these studies probably reﬂect the small
numbers of subjects analysed, and variations in sampling, prepara-
tion and assay techniques. Our development of a simple sandwich
ELISA method, requiring neither sequential extraction nor ultracen-
trifugation to isolate and assay oligomeric A␤ species, should help
us to resolve these discrepancies by analysing a much larger num-
ber of brains than has previously been practicable. Additionally, this
assay is readily adaptable for measuring levels of other oligomeric
A␤ species such as truncated forms of A␤.
This work was supported by a grant from Alzheimer’s Research
Trust. Some of the equipment used in the study was purchased by
BRACE (Bristol Research into Alzheimer’s and Care of the Elderly).
We are grateful to New England Rare Reagents for allowing us access
to unpublished data and for providing antibodies.
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