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Amyloid- oligomers are inefficiently measured by enzyme-linked immunosorbent assay.

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Amyloid-␤ Oligomers Are
Inefficiently Measured by
Enzyme-Linked
Immunosorbent Assay
Charlotte Stenh, MSc,1 Hillevi Englund, MSc,1
Anna Lord, MSc,1 Ann-Sofi Johansson, MSc,1
Claudia G. Almeida, MSc,2 Pär Gellerfors, PhD,1
Paul Greengard, PhD,3 Gunnar K. Gouras, MD, PhD,2
Lars Lannfelt, MD, PhD,1 and Lars N. G. Nilsson, PhD1
Amyloid-␤ (A␤) peptide levels are widely measured by
enzyme-linked immunosorbent assay (ELISA) in Alzheimer’s disease research. Here, we show that oligomerization of A␤ results in underestimated A␤ ELISA levels.
The implications are that comprehensive analysis of soluble A␤ requires either sample pretreatment at denaturing conditions or novel conformation-dependent immunoassays. Our findings might be of relevance for many
neurodegenerative disorders in which soluble protein aggregates are the main neurotoxic species.
Ann Neurol 2005;58:147–150
Enzyme-linked immunosorbent assay (ELISA) is widely
used for measurements of amyloid-␤ (A␤) peptides,
the main constituents of senile plaques in Alzheimer’s
disease (AD) brain. The A␤ ELISAs have been very
valuable in AD research and are today routinely used
in preclinical research and anti-A␤ drug evaluation.
Levels of A␤ are also measured with ELISA as a potential biomarker of AD, in which low levels of A␤42
is generally demonstrated in cerebrospinal fluid (CSF)
among AD patients.1,2 In plasma, elevated levels of
A␤42 are observed among familial AD cases.3 However, it has not been clarified whether A␤ levels in
plasma are changed in sporadic AD.
A␤, in particular, A␤42, has an inherent ability to
aggregate into different soluble A␤ oligomers, for example, A␤-derived diffusible ligands (ADDLs)4 and
From the 1Department of Public Health/Geriatrics, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden, 2Department of
Neurology and Neuroscience, Weill Medical College of Cornell
University; and 3Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, NY.
Received Feb 7, 2005, and in revised form Apr 18. Accepted for
publication Apr 18, 2005.
Published online Jun 27, 2005 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20524
Address correspondence to Dr Lannfelt, Department of Public
Health/Geriatrics, Rudbeck Laboratory, Uppsala University, Dag
Hammarskjölds väg 20, SE-751 85 Uppsala, Sweden. E-mail:
lars.lannfelt@pubcare.uu.se
protofibrils,5,6 and eventually into insoluble fibrils.
There is increasing evidence that soluble A␤ oligomers
are present in human brain,7 are highly neurotoxic,8 –10
and possibly are the main pathogenic A␤ species in
AD. Soluble A␤ oligomers might differ in their immunological properties as compared with A␤ monomers,
because the C-terminal part is thought to be hidden
inside the hydrophobic core of the aggregate.11 It
therefore is unclear to what extent A␤ ELISAs can detect soluble A␤ oligomers, because detection is highly
dependent on the conformation specificity of the antibodies used in the ELISA. A previous study drew attention to this problem by showing increased plasma
A␤ signals if samples were denatured before the immunoassay.12 Previous studies of the Arctic familial Alzheimer mutation (APP E693G)13 have indeed suggested that A␤ ELISAs may underestimate A␤ levels in
the presence of A␤ oligomers. The Arctic mutation
leads to accelerated oligomerization of monomeric A␤
into protofibrils13 and altered amyloid precursor protein (APP) processing by favoring ␤-secretase cleavage.14,15 The latter implies that the Arctic mutation
would cause increased A␤ levels. However, contrary to
other pathogenic AD mutations, ELISA measurements
have shown decreased levels of A␤40 and A␤42 in
plasma from carriers of the Arctic mutation,13 as well
as in cell culture media from transfected cells.13,14
Taken together, these findings suggest that the presence of A␤ oligomers may lead to underestimations of
A␤ levels measured by ELISA. Here, we investigated if
the decreased A␤ ELISA levels seen with the Arctic
mutation were caused by reduced ability to measure
oligomeric A␤, and if this hypothesis would be more
generally applicable by extending also to wild-type A␤
oligomers.
Materials and Methods
Transfections
HEK293 (human embryonic kidney) cells were cultured in
Dulbecco’s minimum essential medium (Invitrogen, La Jolla,
CA) supplemented with 10% fetal bovine serum. Cells were
transfected with plasmid vector pcDNA3 (Invitrogen) containing either the APP gene with both the Arctic and Swedish mutations (APPArc-Swe) or with the Swedish mutation
alone (APPSwe), using FuGENE 6 transfection reagent
(Roche, Basel, Switzerland). Transfected HEK293 cells were
analyzed 48 hours after transfection.
Animals and Tissue Preparation
cDNA clones coding for human APP695 carrying both the
Swedish and the Arctic mutations (APPArc-Swe) or the Swedish mutation alone (APPSwe) were microinjected (2␮g/ml)
into pronuclear oocytes from the hybrid mouse line C57BL/
6-CBA-F1 and implanted into pseudopregnant foster mothers at the two-cell stage. Founder mice were identified by
genotyping tail DNA with primer pairs 2577–2596 in
M12379.1 and 541–522 in Y00264 as well as 1858 –1877
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
147
Size Exclusion Chromatography
and 2299 –2281 in Y00264. Importantly, the APPArc-Swe and
APPSwe founder lines used for this study both had a threefold
human APP overexpression in the brain, as analyzed with
Western blot. The mice used in the biochemical analyses
were 2-3 months old on a C57BL/6-CBA background. The
animals were anesthetized with 0.4ml Avertin (SigmaAldrich, St. Louis, MO) (50mg/ml) and perfused intracardially with 0.9% saline solution. Brains were prepared and immediately frozen in liquid nitrogen and then stored at
⫺80°C. One brain hemisphere was extracted in 1% sodium
dodecyl sulfate (SDS)/0.1% Tween 20 in 1 ⫻ phosphatebuffered saline (PBS) containing Protease Inhibitor Cocktail
Tablet (Roche), with an extraction ratio of 1 to 10 (wt/vol).
The tissue was homogenized, sonicated for 30 seconds at a
defined setting, centrifuged at 100,000g for 1 hour at ⫹4°C
and the supernatant was saved.
Before each SEC analysis, 1.8% Tween 20 was added to the
aliquots to a final concentration of 0.6%, resulting in a final
peptide concentration of 35␮M. The samples were centrifuged for 5 minutes, 17,900g at 16°C before injection. Samples (10␮l) were analyzed on a Merck (Westchester, PA) Hitachi D-7000 HPLC LaChrom system with a diod array
detector. A Superdex 75 PC3.2/30 column (Amersham Biosciences, Arlington Heights, IL) was used for SEC. The samples were eluted with PBS–Tween 20 (50mM sodium phosphate, 0.15M NaCl, pH 7.4, 0.6% Tween 20) at a flow rate
of 0.08ml/minute at ambient temperature. All injected samples were subjected to a wavelength scan between 200 and
400nm, and data were collected from 214nm. Peak areas
were integrated using Merck Hitachi model D-7000 Chromatography Data station software.
Western Blot
Statistical Analysis
Transfected HEK293 cells were lysed in NP lysis buffer
(50mM Tris-HCl, pH 7.5, 150mM NaCl, 1% NP-40, 1
Protease Inhibitor Cocktail Tablet/25ml; Roche). Cell media
and lysates from transfected HEK293 cells, SDS-soluble
brain tissue extracts, and synthetic A␤1-42 wild-type aliquots
were run on either 10 to 20% Tris-tricine or 4 to 20% Trisglycine SDS gels (Invitrogen) followed by electrophoretic
transfer to nitrocellulose membranes (Schleicher and Schuell,
Keene, NH, or Bio-Rad, Richmond, CA). Membranes were
boiled in 1 ⫻ PBS for 5 minutes, blocked in 5% wt/vol
nonfat dry milk, and incubated with 6E10 (Signet Laboratories, Dedham, MA), anti–␤-actin (Sigma-Aldrich), or an
A␤40-specific C-terminal antibody (generously provided by
Jan Näslund) for up to 72 hours at ⫹4°C. Bands were visualized with horseradish peroxidase–conjugated anti–mouse
antibody (Pierce, Rockford, IL) and enzyme chemiluminescence detection. Levels of ␤-actin and A␤ were analyzed and
normalized for APP levels.
A␤ Enzyme-Linked Immunosorbent Assay
Conditioned cell media, SDS-soluble brain tissue extracts,
and synthetic A␤1-42 wild-type aliquots were analyzed for
A␤1-40 and A␤1-42 levels with ELISA using Amyloid Beta
1-40 and 1-42 ELISA kits (Signet Laboratories), according to
manufacturer’s instructions. To ensure equal epitope recognition between Arctic and wild-type A␤ by the antibodies
used in the ELISA, we analyzed dilution series of synthetic
A␤1-40 Arctic and A␤1-40 wild-type in their monomeric
form with the Amyloid Beta 1-40 ELISA kit.
Preparation of Synthetic Peptide
Synthetic A␤1-42 wild-type (PolyPeptide Laboratories,
Wolfenbüttel, Germany) was dissolved in 10mM NaOH to
a final concentration of 100␮M, followed by 2 minutes of
vortexing. This stock was diluted in 2 ⫻ PBS to a final concentration of 50␮M and vortexed for 1 minute. The 50␮M
peptide solution was divided into aliquots and incubated at
37°C. Aliquots were taken for analyses with size exclusion
chromatography (SEC), ELISA, or Western blot after 0, 70,
210, 280, or 350 minutes or 23 hours of incubation.
148
Annals of Neurology
Vol 58
No 1
July 2005
Data were statistically analyzed with unpaired Student’s t test
with or without Welch correction depending on population
distribution (GraphPad InStat 3.0, GraphPad Software, San
Diego, CA) and presented as mean ⫾ standard error of the
mean.
Results
A combination of the Swedish16 and the Arctic13 APP
mutation (APPArc-Swe) was expressed in cells and transgenic mice to generate high levels of oligomerized A␤
peptides. As a control, the Swedish APP mutation
(APPSwe), which produces wild-type A␤ peptides, was
expressed alone. A␤ levels in media from HEK293 cells
transfected with APPArc-Swe or APPSwe were analyzed
with both ELISA and Western blot. The ELISA demonstrated a 30% decrease in A␤1-40 levels and a 70%
decrease in A␤1-42 levels in media from APPArc-Swe
cells as compared with APPSwe (Fig 1A, B), similar to
earlier results.14 Remarkably, Western blot (antibody
6E10) of the same samples showed a 40% increase in
total A␤ levels in APPArc-Swe cells as compared with
APPSwe (see Fig 1C). Furthermore, brain tissue of
young APPArc-Swe and APPSwe transgenic mice was analyzed with both methods. When measured by ELISA,
a 50% decrease of both A␤1-40 and A␤1-42 levels was
found in APPArc-Swe transgenic mice as compared with
APPSwe (see Fig 1D, E). In contrast, A␤ levels were
increased by 400% when the same samples were analyzed by Western blot (see Fig 1F).
There was no significant difference in APP protein
levels (normalized to ␤-actin) between APPArc-Swe and
APPSwe cells or between APP Arc-Swe and APPSwe
transgenic mice, (see Fig 1G, H). Moreover, the
A␤1-40 ELISA detected monomeric Arctic and wildtype A␤40 peptides in a comparable way (data not
shown), demonstrating that the Arctic amino acid
substitution does not interfere with the antibody recognition in the ELISA. Furthermore, Western blot
analysis using an A␤40-specific C-terminal antibody
demonstrated results similar to when using 6E10
(data not shown), suggesting that the observed differences between Western blot and ELISA is not caused
by interference of the Arctic mutation with the
C-terminal A␤40-specific antibody used in the
ELISA.
We wanted to determine whether oligomerization
of wild-type A␤ also leads to underestimated A␤ levels by ELISA. Therefore, the oligomerization of wild-
Fig 1. Amyloid-␤ (A␤) levels in cell culture media (A–C) and
in 2- to 3-month-old transgenic mice brain (D–F). Enzymelinked immunosorbent assay (ELISA) measurements of A␤
levels in cell media showed (A) a 30% reduction of A␤1-40
and (B) a 70% reduction of A␤1-42 in APPArc-Swe cells in
comparison with APPSwe. (C) In contrast, Western blot of the
same samples demonstrated a 40% increased A␤ level in media from APPArc-Swe cells in comparison with APPSwe. (D)
Levels of A␤1-40 and (E) A␤1-42 were decreased by 50% in
APPArc-Swe mice compared with APPSwe when measured by
ELISA. (F) Western blot showed a 400% increase in A␤ levels in APPArc-Swe mice as compared with APPSwe. (G, H)
Representative Western blot gels showing APP and ␤-actin in
APPSwe and APPArc-Swe cells (G) and transgenic mice (H).
There was no significant difference in APP/␤-actin ratio between APPArc-Swe and APPSwe cells and transgenic mice indicating equal APP expression. All data were statistically analyzed with unpaired Student’s t test with or without Welch
correction and presented as means ⫾ SEM. *p ⬍ 0.05, **p
⬍ 0.01 and ***p ⬍.001. n ⫽ 10 –11 (cell culture media,
A–C), n ⫽ 6 –9 (transgenic mice, D–F).
type A␤1-42 was analyzed in parallel with SEC,
ELISA, and Western blot in a kinetic experiment.
Wild-type A␤1-42 was incubated at 37°C for up to
23 hours to generate soluble A␤ protofibrils, here defined as high-molecular A␤ species that remains in
Fig 2. Kinetic experiments of A␤1-42 wild-type oligomerization at 37°C. (A) Size exclusion chromatography (SEC) enables visualization of amyloid-␤ (A␤) oligomerization and
fibrilization.13 At time zero, approximately 50% of the
A␤1-42 wild-type peptide has oligomerized into protofibrils.
Over time, the level of monomers declines as more protofibrils
are formed. Moreover, the level of protofibrils starts to decline
after 70 minutes as fibrillization begins. (B) At time zero,
ELISA A␤1-42 wild-type levels are approximately 50% of the
theoretical level, and this level continues to decline in association with protofibril formation. This demonstrates the loss of
epitopes for the antibodies in the ELISA. (C) Western blot
demonstrates unchanged A␤1-42 wild-type levels over time,
despite the presence of oligomers. Centrifugation of the 23hour A␤1-42 wild-type sample before gel loading results in a
markedly weaker band, as compared with the sample that was
not centrifuged.
Stenh, et al: A␤ Oligomers and ELISA
149
the supernatant after centrifugation for 5 minutes at
17,900g and elutes in the void volume of a Superdex
75 PC3.2/30 column. The SEC analysis showed a
rapid aggregation of A␤ monomers into protofibrils,
leading to an almost 100% protofibril content in the
sample after 70 minutes of incubation. After 23 hours
of incubation, nearly all A␤ peptides partitioned into
the pellet upon centrifugation (Fig 2A, C). The same
samples analyzed by ELISA showed a decline in
A␤1-42 levels with time (see Fig 2B) but remained
essentially unchanged over time when assayed by
Western blot (see Fig 2C). This demonstrates that
samples containing soluble A␤ oligomers need to be
assayed in a denaturing environment to quantify total
A␤ content.
Discussion
In conclusion, our findings demonstrate that oligomerization of A␤ peptides complicates interpretations of ELISA measurements, because A␤ aggregates
are inefficiently detected by ELISA leading to an underestimation of A␤ levels. We believe that the decreased A␤ levels in plasma from patients with the
Arctic mutation13 could be explained by this effect.
Interestingly, the decreased plasma A␤ levels among
Arctic mutation carriers suggests that oligomeric
forms of A␤, that is, protofibrils, might exist in
plasma. Recent transgenic studies with the Arctic mutation reinforces the perception that A␤ oligomerization and fibrilization are central in AD pathogenesis.15,17 Our findings highlight the great need of
methods suitable to discriminate and quantify various
soluble A␤ species to gain a better understanding of
A␤ oligomers in AD. Protein misfolding and aggregation is a common theme among many neurodegenerative disorders, for example, Parkinson’s disease,
amyotrophic lateral sclerosis, and prion disorders.
Thus, novel ELISAs with conformation-specific antibodies might be useful for the analysis of neurotoxic
oligomeric species in various pathogenic conditions.
This work was supported by the Alzheimer Foundation (C.S., L.L.),
Swedish Society for Medical Research, Hjärnfonden (L.L.), Bertil
Hållstens Forskningsstiftelse (L.L.), The Swedish Research Council
(2003-5546, L.L., 2004-6203, L.N.), DIADEM (QLK3-CT-200102362, L.L.), APOPIS (contract no. LSHM-CT-2003-503330, L.L.),
and the NIH (National Institute of Aging, AG09464, P.G., G.K.G.).
We thank H. Basun and M. Ingelsson for valuable discussions, H.
van der Putten for supplying the 323-vector, and J. Näslund for
supplying the A␤40-specific antibody.
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