Sensitive ELISA detection of amyloid-? protofibrils in biological samples

Sensitive ELISA detection of amyloid-b protofibrils in biologicalsamples

Hillevi Englund,*,1 Dag Sehlin,*,�,1 Ann-Sofi Johansson,* Lars N. G. Nilsson,* Par Gellerfors,*,�Staffan Paulie,� Lars Lannfelt* and Frida Ekholm Pettersson*

*Department of Public Health/Molecular Geriatrics, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden

�Mabtech AB, Nacka Strand, Sweden

�BioArctic Neuroscience AB, Stockholm, Sweden

Abstract

Amyloid-b (Ab) protofibrils are known intermediates of the

in vitro Ab aggregation process and the protofibrillogenic

Arctic mutation (APPE693G) provides clinical support for a

pathogenic role of Ab protofibrils in Alzheimer’s disease (AD).

To verify their in vivo relevance and to establish a quantitative

Ab protofibril immunoassay, Ab conformation dependent

monoclonal antibodies were generated. One of these anti-

bodies, mAb158 (IgG2a), was used in a sandwich ELISA to

specifically detect picomolar concentrations of Ab protofibrils

without interference from Ab monomers or the amyloid pre-

cursor protein (APP). The specificity and biological signifi-

cance of this ELISA was demonstrated using cell cultures and

transgenic mouse models expressing human APP containing

the Swedish mutation (APPKN670/671ML), or the Swedish

and Arctic mutation in combination. The mAb158 sandwich

ELISA analysis revealed presence of Ab protofibrils in both

cell and animal models, proving that Ab protofibrils are formed

not only in vitro, but also in vivo. Furthermore, elevated Ab

protofibril levels in the Arctic-Swedish samples emphasize the

usefulness of the Arctic mutation as a model of enhanced

protofibril formation. This assay provides a novel tool for

investigating the role of Ab protofibrils in AD and has the

potential of becoming an important diagnostic assay.

Keywords: Alzheimer’s disease, amyloid-b protofibril, con-

formation-dependent antibody, protofibril-specific ELISA.

J. Neurochem. (2007) 103, 334–345.

Soluble oligomeric amyloid-b (Ab) is likely to be involved inthe pathogenesis of Alzheimer’s disease (AD) (Haass andSteiner 2001; Klein 2002; Caughey and Lansbury 2003) andmany types of in vitro formed oligomeric Ab species, amongthem protofibrils (Harper et al. 1997; Walsh et al. 1997;Johansson et al. 2006), have been described. Ab protofibrilsare described as >100 kDa curved linear structures with adiameter of �5 nm and a length of up to 200 nm whichremain soluble upon centrifugation at 16 000–18 000 g(Harper et al. 1997; Walsh et al. 1997; Paivio et al. 2004;Johansson et al. 2006). The neurotoxicity of in vitro formedAb protofibrils, as well as their ability to induce electro-physiological effects on neurons, have been demonstrated byseveral groups (Hartley et al. 1999; Walsh et al. 1999; Wardet al. 2000; Klyubin et al. 2004; Johansson et al. 2007a,b).Strong clinical evidence for Ab protofibrils as a neurotoxicagent in AD is provided by the Arctic mutation, found in aSwedish family with hereditary AD (Nilsberth et al. 2001).

This intra-Ab mutation (AbE22G) causes enhanced forma-tion of Ab protofibrils in vitro, both for Ab1–40 (Nilsberthet al. 2001) and Ab1–42 (Johansson et al. 2006) and

Received January 23, 2007; revised manuscript received May 14, 2007;accepted May 15, 2007.Address correspondence and reprints requests to Professor Lars

Lannfelt, Department of Public Health/Molecular Geriatrics, RudbeckLaboratory, Dag Hammarskjolds vag 20, SE-751 85, Uppsala, Sweden.E-mail: [email protected] contributed equally to this work.Abbreviations used: AD, Alzheimer’s disease; ALP, alkaline phos-

phatase; APP, amyloid precursor protein; Arc, the Arctic APP mutation;Ab, amyloid-b peptide; BSA, bovine serum albumin; cryo-TEM, cryo-transmission electron microscopy; HEK 293, human embryonic kidney293 cells; HPLC-SEC, high performance liquid chromatography-sizeexclusion chromatography; HRP, horseradish peroxidase; IAPP, islet a-myloid polypeptide; LMW-Ab, low molecular weight amyloid-b; PBS,phosphate-buffered saline; SDS, sodium dodecyl sulfate; Swe, theSwedish APP mutation; TBS, Tris-buffered saline.

Journal of Neurochemistry, 2007, 103, 334–345 doi:10.1111/j.1471-4159.2007.04759.x

334 Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 334–345� 2007 The Authors

accelerates intraneuronal Ab accumulation in vivo (Lordet al. 2006). Still, it is yet to be established if and how Abprotofibrils, alone or together with other soluble oligomericAb species, cause the neurodegeneration leading to AD.

At present there is no curative treatment of AD, thoughmany novel therapeutic strategies are being evaluated. Oneimportant aspect of AD treatment is the need for earlydiagnosis – preferably even before clinical symptoms areobserved. There is also a need for reliable biomarkers toevaluate effects of amyloid-directed treatment over time.Today decreased levels of Ab1–42 and increased levels of tauand phospho-tau in CSF measured by ELISA have beensuggested as possible biomarkers (Hansson et al. 2006). In arecent publication, we have shown that the presence of solubleAb aggregates, such as Ab protofibrils, are not well detectedwith conventional Ab1–40 and Ab1–42 ELISA measure-ments, leading to an incomplete analysis of soluble Ab and anunderestimation ofAb levels if samples are not denatured priorto analysis (Stenh et al. 2005). As oligomeric Ab, includingthe protofibril, is thought to be a primary neurotoxic agent inAD, and as oligomeric Ab has been shown to be present inhuman CSF (Pitschke et al. 1998; Georganopoulou et al.2005) and AD brain (Gong et al. 2003; Kayed et al. 2003;Barghorn et al. 2005), an emerging strategy within the ADfield is to use oligomeric Ab as a possible biomarker and atherapeutic target for the disease. An assay which allowsconformation-specific measurements of Ab protofibrils inbiological samples would be desirable and could have greatpotential as a diagnostic test. For that reason antibodies withhigh affinity for the Ab protofibril conformation were gener-ated and used to establish an Ab protofibril-specific sandwichELISA. This assay enables quantification of Ab protofibrils insamples from cell and mouse models but does not detectmonomeric Ab or the amyloid precursor protein (APP).

Materials and methods

Preparation of defined Ab peptide conformations

Due to uncertainties concerning the molecular weights of Abprotofibrils and fibrils, all Ab concentrations are expressed as

molarity of the monomer unit (i.e. 4514 g/mol for Ab1–42wt).

LMW-Ab (monomers–tetramers), Ab1–16 and Ab17–40

Lyophilized synthetic peptides; Ab1–40wt (PolyPeptide Laborator-

ies, Wolfenbuttel, Germany), Ab1–16wt (Bachem, Bubendorf,

Switzerland), and Ab17–40wt (Sigma-Aldrich, St Louis, MO,

USA), were dissolved in 10 mmol/L NaOH and diluted in 2 · phos-

phate-buffered saline (PBS; 1 · PBS: 50 mmol/L phosphate buffer

and 100 mmol/L NaCl, pH 7.4) to a final concentration of 50 lmol/L.

AbArc protofibrils

Lyophilized synthetic Ab1–42 (E22G) (PolyPeptide Laboratories)

was dissolved in 10 mmol/L NaOH, diluted in 2 · PBS to a final

concentration of 50 lmol/L and incubated for 60 min at 37�C.

Ab protofibrils

Lyophilized synthetic Ab1–42wt (PolyPeptide Laboratories) was

dissolved in 10 mmol/L NaOH, diluted in 10 · PBS to 443 lmol/L

(2 mg/mL), incubated overnight at 37�C and then diluted with PBS

to a final concentration of 50 lmol/L.

Ab fibrils

Lyophilized synthetic Ab1–42wt (rPeptide, Athens, GA, USA) wasdissolved in 10 mmol/L NaOH, diluted in 2 · PBS to a final

concentration of 50 lmol/L and incubated for >48 h at 37�C.All peptide solutions except Ab fibrils were centrifuged at

17 900 g for 5 min at 16�C to remove any insoluble aggregates from

the preparation. Purity and size of the Ab preparations were analyzed

by size exclusion chromatography (SEC) after centrifugation, as

previously described (Johansson et al. 2006). In short, samples were

run on a Superdex 75 PC3.2/30 (GE-Amersham, Uppsala, Sweden)

column using a Merck Hitachi D-7000 HPLC LaChrom (VWR,

Stockholm, Sweden) system with a diode array detector. Injected

samples were eluted with PBS-Tween (0.6% Tween-20) at a flow rate

of 0.08 mL/min at ambient temperature, and data were obtained at

214 nm. Peak areas were integrated using Merck Hitachi model

D-7000 Chromatography Data software (VWR).

Cryo-transmission electron microscopy

Ab protofibrils and AbArc protofibrils were prepared as described

above. For a more detailed description of the cryo-TEM (transmis-

sion electron microscopy) see (Almgren et al. 2000). In short,

samples were equilibrated at 25�C and approximately 99% relative

humidity within a climate chamber. A small drop (�1 lL) of sample

was deposited onto a copper grid covered with a perforated polymer

film and with a thin carbon layer on both sides. Excess liquid was

removed by means of blotting with a filter paper, leaving a thin film

of the solution on the grid. Immediately after blotting, the sample

was vitrified in liquid ethane, held just above its freezing point.

Samples were kept below )165�C and protected against atmo-

spheric conditions during both transfer to the transmission electron

microscope and examination. The cryo-TEM investigations were

performed with a Zeiss EM 902A transmission electron microscope

(Carl Zeiss NTS, Oberkochen, Germany). The instrument operated

at 80 kV and in zero loss bright-field mode. Digital images were

recorded under low-dose conditions with a BioVision Pro-SM Slow

Scan CCD camera (Proscan GmbH, Scheuring, Germany) and

analysis software (Soft Imaging System, GmbH, Munster, Ger-

many). In order to visualize as many details as possible, an

underfocus of 1–2 lm was used to enhance the image contrast.

Generation of monoclonal antibodies

Balb/c-mice, kept under pathogen-free conditions at the animal care

facilities of Rudbeck Laboratory, Uppsala University, Sweden, were

immunized with 30 lg Ab1–42Arc protofibrils mixed 1 : 1 (v/v)

with Freund’s complete adjuvant followed by three boosts with

antigen suspended in Freund’s incomplete adjuvant. Spleen cells

were isolated and fused with Sp2/0 myeloma cells (de StGroth and

Scheidegger 1980) and hybridomas were screened for anti-Abreactivity with direct ELISA (see below). Positive clones were

subcloned to certify monoclonality and isotypes and subclasses were

determined with an IsoStrip kit (Roche, Basel, Switzerland). Two

clones, producing mAb158 (IgG2a) and mAb1C3 (IgG1), were

Ab protofibril specific sandwich ELISA 335

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 334–345

expanded and antibodies were purified from the supernatants with

protein G-sepharose chromatography.

Direct ELISA

Ninety-six well EIA/RIA plates (Corning Inc., NY, USA) were coated

with 500 ng of synthetic peptide, low molecular weight Ab (LMW-

Ab) or Ab protofibrils, per well for 2 h at +4�C. Wells coated with

only PBS were used as negative control. After 1 h blocking with 1%

bovine serum albumin (BSA) in PBS, 0.15% Kathon (Rohm & Haas,

Philadelphia, PA, USA) at 22�C, serial dilutions of primary

antibodies were incubated on the plate for 1 h at 22�C. Alkalinephosphatase (ALP)-coupled anti-mouse-IgG/IgM (Mabtech AB,

Nacka Strand, Sweden) was used as secondary antibody and

incubated for 1 h at 22�C before addition of ALP substrate

p-nitrophenyl phosphate substrate (Sigma-Aldrich) in a solution of

10% dietholamine and 0.5 mmol/L MgCl2, pH 9.8. Plates were

analyzed with a spectrophotometer at 405 nm, using SpectraMAX

190 (Molecular Devices, Palo Alto, CA, USA) and data were

analyzed with SOFTMax Pro (Molecular Devices). Wells were

washed three times in ELISA-washing buffer (phosphate-buffered

NaCl with 0.1% Tween 20 and 0.15% Kathon) between each step of

the ELISA. All antibodies were diluted in ELISA incubation buffer

(PBS with 0.1% BSA, 0.05% Tween, and 0.15% Kathon).

Control antibodies

6E10 (IgG1; Signet, Dedham, MA, USA), which binds to amino

acid residues 3–8 of Ab and to APP, was used as control anti-Ab-antibody. Anti-a-synuclein antibody mAb211 (Santa Cruz Biotech,

Santa Cruz, CA, USA), the anti-islet amyloid polypeptide (IAPP)

polyclonal antibody pAb A110 (gift from P. Westermark, Uppsala

University) and the anti-medin polyclonal antibody pAb 179 (gift

from A. Larsson, Uppsala University) were used as positive controls

in experiments on amyloidogenic proteins other than Ab.

Inhibition ELISA

Ninety-six well EIA/RIA plates (Corning Inc.) were coated and

blocked as described in the direct ELISA section above. Triplicates

of biotinylated antibody (mAb158 and mAb1C3 – 15 ng/mL, 6E10 –

100 ng/mL) were mixed with serially diluted Ab preparations

of Ab1–16, Ab17–40, LMW-Ab, AbArc protofibrils, or Abprotofibrils. After 1 h pre-incubation at 4�C in low-binding 96-well

plates the antibody–antigen mix was incubated on the antigen-coated

plate for 10 min at 22�C. After incubation with streptavidin-coupled

ALP (Mabtech) plates were developed with ALP substrate and

analyzed as described in the direct ELISA section. Wells were

washed three times in ELISA-washing buffer between each step of

the ELISA and dilutions were made in ELISA incubation buffer

(PBS with 0.1% BSA, 0.05% Tween, and 0.15% Kathon).

Low-denaturing western blot

A total of 9 ng of synthetic Ab protofibrils or LMW-Abwere mixed

with sample buffer [50 mmol/LTris–HCl, 2% sodium dodecyl sulfate

(SDS), 1% mercaptoethanol, and 10% glycerol], loaded onto a

10–20% Tris–tricine SDS–polyacrylamide gel electrophoresis (Invi-

trogen, Carlsbad, CA, USA) and run at 95 V. Proteins were

transferred to a nitrocellulose membrane (BioRad, Hercules, CA,

USA) at 45 V for 2 h at 4�C and then blocked with 5% milk powder

(BioRad) in Tris-buffered saline (TBS) 0.1% Tween (TBS-T).

Membranes were incubated in primary antibody, 6E10, mAb158,

and mAb1C3 diluted in TBS-T, for 24 h at 4�C. Then membranes

were washed in TBS-T-milk followed by a 30 min incubation with

anti-mouse-IgG/IgM (Pierce, Rockford, IL, USA), diluted 1 : 4000

in TBS-T-milk. After washing in TBS-T, signals were developed with

WestPico ECL-substrate (Pierce) on Hyperfilm (GE-Amersham).

Dot blot

Three amyloid fibrils unrelated to AD; medin, IAPP, and a-synuclein (generously provided by A. Larsson, P. Westermark, and

J. Bergstrom, Uppsala University) were diluted in PBS and applied

onto a nitrocellulose membrane (BioRad). Ab protofibrils and Abfibrils were used as positive control. Membranes were blocked with

1% gelatine (Sigma-Aldrich) in PBS-T (0.1% Tween). mAb158

(0.1 lg/mL), mAb1C3 (0.1 lg/mL), 6E10 (0.1 lg/mL), mAb 211

(0.4 lg/mL), pAb A110 (1 : 500), and pAb 179 (1 : 2000) diluted

in PBS-T were added and incubated for 1 h at 22�C. Membranes

were washed in PBS-T 1% gelatine before addition horse radish

peroxidase (HRP) -coupled anti-mouse-IgG or anti-rabbit-IgG

(Pierce) diluted 1 : 5000 in PBS-T 1% gelatine. After washing in

PBS-T, signals were developed with WestPico ECL-substrate

(Pierce) and Hyperfilm (GE-Amersham).

Cell cultures

For analysis of conditioned cell culture media aliquots of previously

assayed samples (Stenh et al. 2005) were used. In short, 48 h

conditioned media from human embryonic kidney 293 cells

(HEK293) cells were collected from mock cells (n = 3) and from

cells transiently transfected with the APP gene with both the Arctic

and Swedish (Mullan et al. 1992) mutations (APPArc–Swe, n = 11) or

with the Swedish mutation alone (APPSwe, n = 8). Media were

aliquoted, snap-frozen in liquid nitrogen and stored at )80�C. Ablevels were normalized to APP levels, measured by western blot

(Stenh et al. 2005), to compensate for differences in transfection

levels between cultures.

Mouse brain homogenization

Ten-months-old APPSwe (n = 3), APPArc–Swe (n = 6), and non-

transgenic littermates (n = 6) (Lord et al. 2006) were anesthetized

with 0.4 mL Avertin (25 mg/mL) and intracardially perfused with

0.9% saline solution. Frontal cortex from the brains was extracted as

1 : 10 (tissue weight/extraction volume ratio) in TBS (20 mmol/L

Tris and 137 mmol/L NaCl, pH 7.6) with complete protease

inhibitor cocktail (Roche) using a tissue grinder with teflon pestle

(2 · 10 strokes on ice). The homogenates were centrifuged at

100 000 g at 4�C for 60 min to obtain a preparation of TBS-soluble

extracellular and cytosolic proteins. The supernatants were aliquoted

and stored at )80�C prior to analysis.

Immunoprecipitation and western blot

Mouse brain homogenates and conditioned cell media were

incubated overnight at 4�C with 20 lg/mL of biotinylated primary

antibody; mAb158 or 6E10. Streptavidin-coated dynabeads (Invi-

trogen) were added and incubated for 30 min. Immunoprecipitated

proteins were eluted with 10 mmol/L glycine pH 1.7 followed by

pH neutralization with 1 mol/L Tris pH 9. Samples were denatured

in sample buffer (50 mmol/L Tris–HCl, 2% SDS, 1% mercaptoeth-

anol, and 10% glycerol), boiled for 5 min and run at 95 V on

336 H. Englund et al.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 334–345� 2007 The Authors

10–20% Tris–tricine SDS–polyacrylamide gel electrophoresis (In-

vitrogen). Proteins were transferred onto a nitrocellulose membrane

(BioRad) at 45 V for 2 h at 4�C, which was boiled in PBS for 5 min

before blocking. Membranes were blocked with 5% milk powder

(BioRad) in TBS 0.1% Tween (TBS-T) and incubated with 6E10,

diluted to 0.4 lg/mL in TBS-T, for 48 h at 4�C. All subsequentsteps were performed as described for the low-denaturing western

blot above.

mAb158 sandwich ELISA

Ninety-six well EIA/RIA plates (Corning Inc.) were coated at 4�Covernight with 200 ng/well of mAb158 in PBS. Plates were

blocked with 1% BSA in PBS with 0.15% Kathon. A standard

series of synthetic Ab protofibrils and the biological samples to be

analyzed were added to the plates in triplicates and incubated for

2 h at 22�C. A total of 1 lg/mL of biotinylated mAb158 was

added and incubated for 1 h at 22�C, followed by 1 h at 22�Cincubation of streptavidin-coupled polyHRP (Mabtech). K-blue

enhanced (ANL produkter, Alvsjo, Sweden) was used as HRP-

substrate and plates were read in a spectrophotometer at 450 nm,

using SpectraMAX 190 and then analyzed with SOFTMax Pro.

Wells were washed three times in ELISA-washing buffer between

each step after blocking the plates and antibodies and samples

were diluted in ELISA incubation buffer. Regardless of their

previous preparation, all samples were centrifuged at 17 900 g for

5 min at 16�C immediately prior to analysis to remove insoluble

Ab fibrils formed during freezing of the biological samples. The

6E10-6E10 sandwich ELISA was performed following the protocol

for the mAb158 sandwich ELISA.

Aggregation kinetics

Aliquots of 50 lmol/L Ab1–42wt (PolyPeptide Laboratories) were

incubated at 37�C for durations of between 0 and 7.5 h, centrifuged

for 5 min at 17 900 g and stored at )20�C until analysis. The

aliquots were analyzed by HPLC-SEC as described above, Ab1–42-specific ELISA (Signet) according to manufacturer’s instructions

and with the Ab protofibril-specific mAb158 sandwich ELISA.

Statistical analyses

Data were statistically analyzed with unpaired Student’s t-test withWelch correction depending on population distribution (GraphPad

InStat 3.0; GraphPad Software, San Diego, CA, USA) and presented

as mean ± SEM. Significance levels were labeled as *p < 0.05,

**p < 0.01, and ***p < 0.001.

Ethical approvals

All experiments involving animals were performed in compliance

with the local animal ethics committee (decision numbers C258/6

and C242/5).

Results

Definition of Ab preparations with different

conformation

Synthetic Ab was aggregated according to the differentprotocols described above, and the Ab conformationsgenerated were LMW-Ab (Fig. 1a), insoluble Ab fibrils

(a) (b)

(c) (d)

(e) (f)

Fig. 1 Analysis of the different amyloid-b

(Ab) conformations. Four different prepara-

tions of synthetic Ab – low molecular weight

Ab (LMW-Ab; (a)), Ab fibrils (b), Ab proto-

fibrils (c), and AbArc protofibrils (d) were

analyzed on HPLC-size exclusion chroma-

tography and gave the following chromato-

grams. Analyses were made on 50 lmol/L

peptide solutions and chromatograms show

arbitrary units for the absorbance at 214 nm

on the y-axis and retention time in minutes

on the x-axis, with a retention time of

10 min for the protofibril peak and 22 min

for the LMW-Ab peak. Insoluble fibrils were

removed from all samples by centrifugation

before HPLC-size exclusion chromatogra-

phy analysis. Ab protofibrils and AbArc

protofibrils were also analyzed by cryo-

transmission electron microscopy in parallel

(e and f). Scale bar: 100 nm.



(Fig. 1b), Ab protofibrils (Fig. 1c), and AbArc protofibrils(Fig. 1d). These 50 lmol/L preparations were analyzed byHPLC-SEC using a Superdex 75 column to ensure purity ofthe preparations and representative SEC-chromatograms areshown in Fig. 1a–d. The preparations were shown to be>95% pure, as previously published (Johansson et al.2006). As Ab fibrils are insoluble they were pelleted bycentrifugation at 17 900 g prior to analysis. Exact sizedeterminations of the different Ab conformations usingSEC is associated with methodological problems, as Ab is ahydrophobic, linear peptide and its elution behavior makesit difficult to compare with globular proteins often used assize markers (Paivio et al. 2004). Hence, the size of the gel-included LMW-Ab peak was estimated to 4–20 kDa,corresponding to Ab monomers–tetramers, whereas thegel-excluded Ab protofibril and AbArc protofibril peakscould be estimated to be >100 kDa, based on the cutoff sizeof the Superdex 75 column. Cryo-TEM studies of the Abprotofibril (Fig. 1e) and AbArc protofibril (Fig. 1f) prepa-rations showed a curved linear structure, well in line withthe Ab protofibril (Harper et al. 1997; Walsh et al. 1999)and AbArc protofibril (Johansson et al. 2006) structuresdescribed.

Characterization of mAb158

Immunization of mice with AbArc protofibrils resulted inseveral monoclonal antibodies, among which one wasparticularly interesting, mAb158 (IgG2a). To characterizeits binding properties, the antibody was compared with thecommercially available anti-Ab antibody 6E10. An inhibi-tion ELISA was used to investigate the selectivity andaffinity of mAb158 for different Ab conformations. In thismethod the antibody–antigen interactions take place insolution and at low concentrations, which makes it moresuitable for affinity measurements than e.g. a direct ELISAwhere the antigen is in large excess. Binding of theantibodies to LMW-Ab or Ab protofibril coat was inhibitedby addition of serially diluted Ab in different conformations.The antigen concentration required to inhibit half of themaximum signal in the inhibition ELISA was defined asIC50, which can be used as an estimate of the antibody’saffinity for the investigated antigen (Neri et al. 1996). Asseen in Fig. 2a, inhibition of mAb158’s binding to LMW-Abcoat with Ab protofibrils resulted in IC50 values in the lownanomolar range, at least 200-fold lower than for LMW-Aband Ab1–16, showing that the binding of mAb158 to Ab isconformation dependent. This epitope is not mutation

(a) (b) (c)

(d) (e)

Fig. 2 mAb158 is a high affinity amyloid-b (Ab) conformation-

dependent antibody. Binding of mAb158, 6E10, and mAb1C3 to dif-

ferent Ab conformations was analyzed by inhibition ELISA on low

molecular weight Ab (LMW-Ab) coat (a–c) with the molar (mol/L)

concentration of Ab displayed on the x-axis. mAb158 displayed 200

times higher affinity for Ab in the protofibril conformation compared

with LMW-Ab and the N-terminal Ab1–16 fragment (a). 6E10 and

mAb1C3 did not show any conformation selectivity (b and c). Ab17–40

did not inhibit binding of any of the antibodies (a–c). Values are mean

of triplicates and graphs show representative data from repeated

experiments. Small differences in IC50 values (nmol/L) were obtained

when inhibiting mAb158’s binding to LMW-Ab coat and Ab protofibril

coat, respectively (d) (data presented as mean ± SEM, n = 3). The

binding of mAb158 to different Ab species was also analyzed with a

low denaturing western blot, where mAb158 bound to a smear of Ab

aggregates in the 50–200 kDa range, but not to LMW-Ab, whereas

6E10 and mAb1C3 bound both to the smear and to LMW-Ab (e).

Representative gel of n = 3.



dependent as inhibition with AbArc protofibrils did not differfrom inhibition with wild type Ab protofibrils. Ab1–16 andLMW-Ab inhibited the mAb158 binding equally, whereasAb17–40 had no inhibitory effect at all (Fig. 2a). IC50 ofmAb158 for Ab protofibrils and AbArc protofibrils wascalculated to �5 nmol/L both when inhibiting the binding toLMW-Ab coat and Ab protofibril coat (Fig. 2d). The IC50 ofmAb158 for LMW-Ab and Ab1–16 differed more betweenexperiments and coating peptide, however, it was always atleast 200 times higher than IC50 for protofibrils (Fig. 2d).Even though mAb158 has higher affinity for Ab protofibrilsthan LMW-Ab, the large excess of coat antigen allows theantibody to bind also to the LMW-Ab-coated plate. Takentogether with the short incubation time of this assay, whichdoes not disturb the antibody–antigen equilibrium in thesolution, the inhibition on LMW-Ab and Ab protofibril coatresulted in similar IC50 values. However, as a smallequilibrium shift could still occur, we have chosen to presentthe data for inhibition on LMW-Ab coat in Fig. 2a–c, as theLMW-Ab coat is least likely to disturb the equilibriumbetween the antibody and inhibitory peptide in the solution.

6E10 had equal affinity for Ab protofibrils, AbArcprotofibrils, LMW-Ab, and Ab1–16, and thus did not showany preference for a certain Ab conformation (Fig. 2b). Inaddition to mAb158, a high affinity IgG1 antibody –mAb1C3 – was obtained from the immunizations. Contraryto mAb158, mAb1C3 displayed a binding pattern moresimilar to 6E10 with small differences in IC50 for thedifferent Ab conformations (Fig. 2c). To further characterizethese antibodies Ab-binding patterns synthetic Ab protofi-brils and LMW-Ab were analyzed in a low-denaturingwestern blot, where most of the Ab in the protofibrilpreparation remained aggregated. This experiment revealedthat mAb158 bound to a smear of Ab aggregates in the rangeof 50–200 kDa but not to monomeric Ab, whereas 6E10 and

mAb1C3 bound both to the aggregate smear and tomonomeric Ab, again proving that mAb 158 is conformationspecific (Fig. 2e).

mAb158 does not recognize a generic amyloid epitope

Previously reported Ab conformation-dependent antibodieshave been shown to bind oligomers and fibrils of otheramyloidogenic proteins (O’Nuallain and Wetzel 2002; Kayedet al. 2003), suggesting a common epitope present on allamyloid aggregates. Due to technical difficulties in gener-ating oligomeric species of other amyloids than Ab, mAb158was instead tested against fibrils of other amyloidogenicproteins in a dot blot assay. The inhibition ELISA, where theantibody–antigen reactions take place in solution, was notused for these analyses as ELISA is not suitable for insolubleantigens like fibrils. Fibrils of medin, IAPP, and a-synucleinwere immobilized on a nitrocellulose membrane to maintaintheir native conformations. The antibodies 6E10 (Ab),pAb179 (medin), pAbA110 (IAPP), and mAb211 (a-synuc-lein) were used as positive controls (Fig. 3b). mAb158 didnot show reactivity for any amyloid other than Ab (Fig. 3a).The binding of mAb158 to Ab fibrils suggests that the Abprotofibril epitope is present also in the Ab fibril structure.As mAb158 binds both Ab protofibrils and fibrils it seemsreasonable that it would recognize both protofibrils andfibrils of other amyloidogenic proteins as well. mAb158 didnot bind to fibrils of other amyloids and is therefore unlikelyto bind to protofibrils of these proteins.

mAb158 does not bind APP

Levels of APP and soluble APP fragments commonly exceedthe levels of Ab in biological samples, and therefore an anti-Ab-antibody’s cross-reactivity to APP could disturb e.g.sandwich ELISA analyses of Ab. To elucidate whethermAb158 binds to native APP, immunoprecipitation experi-

(a) (b)

(c)

Fig. 3 mAb158 does not bind to other

amyloids or to amyloid precursor protein

(APP). The conformation selectivity of

mAb158 is exclusive for amyloid-b (Ab) as it

did not display any binding to other amyloid

fibrils, as tested by dot blot (a and b).

Reactivity against APP was analyzed by

immunoprecipitation followed by western

blot with 6E10 as detecting antibody (c).

mAb158 did not immunoprecipitate a-APPs

from cell media or full-length APP from

mouse brain homogenate, whereas 6E10

did. Both antibodies immunoprecipitated

synthetic Ab protofibrils (1 nmol/L) equally

well, as shown to the right. Representative

blots from repeated experiments (n = 3).



ments were performed. HEK-cell culture media (mock,APPSwe, and APPArc–Swe) and mouse brain homogenates(non-transgenic, APPSwe, and APPArc–Swe) were immunopre-cipitated with mAb158 or 6E10, followed by a denaturingwestern blot with 6E10 as detecting antibody (Fig. 3c). Asseen in Fig. 3c, mAb158 did not immunoprecipitate aAPPsfrom cell culture media or full-length APP from mouse brainhomogenates, whereas, as expected, 6E10 did. The 1 nmol/Lsynthetic Ab protofibrils used as positive control wereimmunoprecipitated equally well by both antibodies(Fig. 3c). Even though Ab protofibrils were immunoprecip-itated in the experiment, the 4 kDa Ab monomer band isshown in Fig. 3c, as Ab protofibrils, which are not SDS-stable, dissociate into monomers during SDS electrophoresis.

Establishment of an Ab protofibril-specific sandwich

ELISA

To enable specific measurements of Ab protofibrils inbiological samples a sandwich ELISA with mAb158 as bothcapturing and detecting antibody was established. To ensurethe protofibril specificity of the assay, all samples werecentrifuged at 17 900 g before analysis to avoid signals fromfibrillar Ab. Titrated synthetic LMW-Ab, Ab protofibrils andAb1–16 were used to validate the conformation specificity ofthe ELISA (Fig. 4a). The hydrophilic Ab1–16 peptide wasused as it is not expected to aggregate and an ELISAcomposed of two identical antibodies requires at least adimer of a protein to produce a signal. As predicted, the non-aggregating Ab1–16 was not detected with the mAb158sandwich-ELISA even at micromolar concentrations(Fig. 4a). When pre-treating the LMW-Ab and Ab protofi-brils with 70% formic acid, known to dissociate aggregatedAb into monomers, the sandwich ELISA signal wasmarkedly reduced (Fig. 4b). Hence, the signal obtained fromthe LMW-Ab at high concentrations (Fig. 4a) is probablydue to a small content of larger aggregates in the preparationand not due to detection of monomers.

A large excess of monomeric Ab, holoAPP, and APP-fragments, naturally occurring in biological samples, couldinterfere with the Ab protofibril analysis by occupyingbinding sites of the capture antibody, thus inhibiting theprotofibrils from binding. This issue was addressed byadding an increasing excess of Ab1–16 to a fixed concen-tration of Ab protofibrils (50 pmol/L, expressed as monomerunits) and analyzing it with both the mAb158 ELISA and a6E10-6E10 sandwich ELISA (Fig. 4c). A 500 000-foldmolar excess of Ab1–16, when compared with Ab protofi-brils, did not disturb the measurements with the mAb158sandwich ELISA, as expected as Ab1–16 binds poorly to thecapture antibody. In contrast, a 500-fold excess of Ab1–16was enough to decrease the signal in the 6E10-6E10 ELISA,where Ab1–16, as shown in Fig. 2b, binds with high affinityto the capture antibody (Fig. 4c) and thereby occupies thebinding sites of the coat antibody and inhibits the detection

antibody from binding and giving a signal. Moreover, whensynthetic Ab protofibrils were added to mock HEK cellculture media or non-transgenic mouse brain homogenates,90% of the signal was recovered (data not shown).

Measurement of Ab protofibrils in biological samples

The protofibril-specific mAb158 sandwich ELISA measuresAb protofibrils with a detection limit of 1 pmol/L and with alinear range up to 250 pmol/L (Fig. 5a). Due to uncertaintiesconcerning the size of the Ab protofibrils used in thestandard curve, the concentration 1 pmol/L is based on themolecular weight of one Ab monomer (4514 g/mol).Although, as the molecular weight of a protofibril has beenestimated to be at least 100 kDa (Paivio et al. 2004), the

(a)

(b)

(c)

Fig. 4 Amyloid-b (Ab) protofibril-specific sandwich ELISA. Analysis of

synthetic Ab protofibrils, with and without the Arctic mutation, com-

pared with low molecular weight Ab (LMW-Ab) and Ab1–16 with the

mAb158 sandwich ELISA shows the Ab protofibril specificity of this

method (a) (mean ± SEM, n = 3). When pre-treating Ab protofibrils

and LMW-Ab with formic acid, known to dissociate aggregated Ab, the

signal in the mAb158 sandwich ELISA was lost (b). Analysis of

50 pmol/L synthetic Ab protofibrils with the mAb158 sandwich ELISA

was not affected by addition of 500 000-fold molar excess of Ab1–16,

whereas a 500-fold excess of Ab1–16 was enough to disturb the

analysis with a 6E10-6E10 sandwich ELISA (c).



limit of detection calculated as molar Ab protofibrils couldbe as low as 50 fmol/L. A standard curve of AbArcprotofibrils gave a lower signal than wild type Ab protofi-brils, possibly due to differences in Ab protofibril size ordifferences in stability of the protofibril preparation (Figs 4aand 5a).

The presence of Ab protofibrils in cell and mouse modelscarrying the Arctic mutation have been suggested throughindirect measurements (Stenh et al. 2005; Lord et al. 2006),though until now there has been no method for directquantification of Ab protofibrils. The mAb158 ELISAtherefore provided the first opportunity to measure Abprotofibril levels in biological samples and to comparemodels with and without the intra-Ab Arctic mutation.Samples from cells and mice carrying only the Swedishmutation were compared with the wild type Ab protofibrilstandard curve, whereas samples from cells and miceexpressing Ab with the Arctic mutation were compared withAbArc protofibril standard curve (Fig. 5a). To ensure that allAb measured in the mAb158 sandwich ELISA was soluble,and to exclude any possible interference from Ab fibrils, allsamples were centrifuged for 5 min at 17 900 g right beforeanalysis, regardless of previous centrifugations during e.g.homogenization steps.

Groups of cell media from transiently transfected APPSweand APPArc–Swe HEK-cells were analyzed and compared withmock HEK-cell culture media. Ab protofibril levels werecalculated from the standard curves (Fig. 5a) as the meanvalue of triplicates and were then normalized to APP levelsto compensate for differences in transfection levels (Stenhet al. 2005). The Ab protofibril concentration in APPArc–SweHEK-cell culture media was 28 ± 2 pmol/L (mean ± SEM),significantly higher (p < 0.0001) than the 8.2 ± 0.3 pmol/Lseen in APPSwe (Fig. 5b). No Ab protofibrils could be

detected in mock media. Levels of Ab protofibrils were alsomeasured in brains from 10-months-old APPArc–Swe andAPPSwe transgenic mice. At this age, the mice display bothplaques and intraneuronal Ab pathology (Lord et al. 2006).Brains were homogenized in TBS without any detergents andultracentrifuged (1 h at 100 000 g) in order to recover thefraction of soluble Ab in the brain. Similar to the cell culturemedia, Ab protofibril levels differed significantly (p = 0.005)between the groups, with 397 ± 59 pmol/L (mean ± SEM)in APPArc–Swe and 108 ± 14 pmol/L in APPSwe transgenicmouse brains (Fig. 5c). The non-transgenic mice, althoughsignificantly lower than transgenic mice (p = 0.0015, whencompared with APPArc–Swe and p = 0.03, when comparedwith APPSwe), displayed background signals, a phenomenonobserved by others when measuring Ab in rodent brainhomogenates (Lanz and Schachter 2006). The difference inAb protofibril levels between APPSwe and APPArc–Swe groupswas not due to the approximately 1.5-fold difference betweenthe Ab protofibril and AbArc protofibril standard curves(Fig. 5a), as the differences were still significant whencalculations were based on the Ab protofibril standard curvealone.

Accumulation of Ab protofibrils is accompanied by a

reduced Ab1–42 ELISA signal

Alzheimer patients, particularly in the later stages, display adecline in CSF and plasma Ab1–42 ELISA levels, whencompared with healthy controls (Motter et al. 1995; Jensenet al. 1999). This decline is often explained by the massiveaccumulation of Ab in the brain which acts as a sink, leadingto drainage of Ab from CSF and plasma. We have previouslysuggested that the low Ab1–42 levels also could beexplained by a high content of aggregated Ab, leading tounderestimation of Ab1–42 levels as measured by ELISA

(a) (b) (c)

Fig. 5 The Arctic mutation enhances amyloid-b (Ab) protofibril for-

mation in cell culture and transgenic mice. The mAb158 sandwich

ELISA enables measurements of Ab protofibrils linearly down to

low picomolar concentrations (a) (lines indicate linear regression

of the standard curves). Ab protofibrils were measured by the

mAb158 sandwich ELISA in cell culture media from human embryonic

kidney 293 cells; mock cells (n = 3) and transiently transfected with

APPSwe (n = 8) and APPArc–Swe (n = 11). Levels of Ab protofibrils in

APPArc–Swe media were approximately 2.5-fold higher than in APPSwe

media, whereas mock media gave no signal (b). Measurements of

Ab protofibril levels in the Tris-buffered saline-soluble fraction of

non-transgenic mouse brain homogenates (n = 6) were compared

with transgenic mice (APPSwe, n = 3 and APPArc–Swe, n = 6) (c).

Similar to the cell culture media, Ab protofibril levels of APPArc–Swe

mice were 3.5-fold higher than in APPSwe mice. Data are presented as

mean ± SEM.



(Stenh et al. 2005). To further test this idea, we analyzedsynthetic Ab1–42 with SEC, mAb158 sandwich ELISA, andAb1–42 ELISA at different time points during the Abaggregation process (Fig. 6). A high Ab protofibril content inthe sample, as measured by SEC (Fig. 6a) and mAb158sandwich ELISA (Fig. 6c), was accompanied by a reducedAb1–42 ELISA signal (Fig. 6b), strengthening the sugges-tion that low Ab1–42 levels in plasma and CSF may be dueto high content of aggregated Ab, i.e. Ab protofibrils.

Discussion

Many novel therapeutic strategies to battle AD have beensuggested and even though they may target Ab in differentways, their common denominator is a need for earlydiagnosis and a reliable AD biomarker capable of monit-oring treatment effects. At the moment CSF levels of Ab1–42 combined with tau or phospho-tau are promisingbiomarkers (Hansson et al. 2006). However, when meas-uring levels of Ab in biological samples, the presence ofoligomeric Ab species complicates the interpretation ofAb1–40 and Ab1–42 ELISA data (Stenh et al. 2005). Astotal soluble Ab correlates with disease progression (Kuoet al. 1996; Lue et al. 1999; McLean et al. 1999; Naslundet al. 2000) and studies are beginning to show thatdifferent oligomeric Ab species are indeed present inCSF (Pitschke et al. 1998; Georganopoulou et al. 2005),the amyloid cascade hypothesis (Hardy and Higgins 1992)has been refined to focus on oligomeric Ab rather than Abplaques (Hardy and Selkoe 2002). Therefore, great effortshave been made to develop antibodies that could enableconformation selective measurements and therapeutic tar-geting of such Ab species in biological tissues (Gaskinet al. 1993; Lambert et al. 2001, 2007; O’Nuallain andWetzel 2002; Kayed et al. 2003; Barghorn et al. 2005; Leeet al. 2006).

Several oligomeric Ab species ranging from Ab dimers andtrimers, via globular�50 kDa species like amyloid-b deriveddiffusible ligands (ADDLs) and Ab*56, up to the larger>100 kDa Ab protofibril have been suggested as possiblepathogens in AD (Lambert et al. 1998; Lashuel et al. 2002;Walsh et al. 2002; Hoshi et al. 2003; Kayed et al. 2003;Barghorn et al. 2005; Lesne et al. 2006). Most of them havealso been shown to mediate neurotoxicity in vitro and toimpair long-term potentiation in rats (Lambert et al. 1998,2001; Hartley et al. 1999; Bucciantini et al. 2002; Dahlgrenet al. 2002; Walsh et al. 2002; Wang et al. 2002; Chromyet al. 2003; Hoshi et al. 2003; Kayed et al. 2003, 2004;Kim et al. 2003; Klyubin et al. 2004; Cleary et al. 2005).Strong clinical and experimental evidence for Ab protofibrilsas a neurotoxic agent in AD is provided by the protofibrill-ogenic Arctic mutation. Individuals carrying the Arcticmutation develop AD already in their mid-fifties (Nilsberthet al. 2001) and Ab peptides with the Arctic E22G mutationare prone to form toxic Ab protofibrils in vitro (Nilsberth et al.2001; Paivio et al. 2004; Johansson et al. 2006, 2007a).Therefore, a quantitative assay for measuring Ab protofibrilsin biological samples could be of great importance whentrying to understand the pathogenesis of AD.

To establish such an immunoassay, high affinity antibodieswith an Ab conformation-dependent epitope are necessary.Here, we describe a monoclonal antibody – mAb158 – withhigh affinity (nmol/L) for Ab protofibrils and an at least200-fold lower affinity for synthetic LMW-Ab and Ab1–16,as determined by inhibition ELISA. Another importantcharacteristic of mAb158 is the lack of affinity for nativeAPP and cleaved soluble APP fragments, a feature of greatimportance when analyzing biological samples. Comparedwith other both monoclonal and polyclonal conformation-dependent antibodies, mAb158 does not recognize a genericamyloid epitope (O’Nuallain and Wetzel 2002; Kayed et al.2003). However, like the monoclonal antibody NAB61, it

(a) (b) (c)

Fig. 6 Accumulation of amyloid-b (Ab) protofibrils is accompanied by

reduced Ab1–42 ELISA signal. Synthetic Ab1–42 was analyzed at

different time points of the aggregation process. Three analyses were

made in parallel; size exclusion chromatography (SEC; (a)), Ab1–42-

specific sandwich ELISA (b), and mAb158 sandwich ELISA (c). All

samples were centrifuged before analysis to remove any insoluble Ab.

The aggregation process of Ab1–42 can be followed by SEC (a),

where the decline in low molecular weight Ab (LMW-Ab) content is

accompanied by an increase in Ab protofibril content. The Ab proto-

fibril content decreases over time as insoluble Ab fibrils, which are

removed by centrifugation, are formed. Samples containing high

amounts of Ab protofibrils result in a low signal in the Ab1–42-specific

sandwich ELISA (b), whereas they display a high signal in the mAb158

sandwich ELISA (c).



binds to Ab fibrils in addition to oligomeric Ab (Lee et al.2006) and like the recently published ADDL-specificmonoclonals, mAb158 has some cross-reactivity with mono-meric Ab (Lambert et al. 2007).

Given that mAb158 discriminates aggregated Ab fromAb monomers, that it does not bind to APP, and thatinsoluble Ab fibrils can be removed by centrifugation at17 900 g the mAb158-mAb158 sandwich ELISA setup isvery well suited for specific quantification of Ab protofi-brils in complex biological samples. The use of the sameantibody as both capture and detection antibody in asandwich ELISA, requiring at least a dimer to produce asignal, has previously been employed to detect aggregatedproteins in solution (El-Agnaf et al. 2000, 2006; Sian et al.2000; DeMattos et al. 2002; LeVine 2004; Pan et al. 2005)and any monoclonal antibody could be used for such anoligomer ELISA. Although, when analyzing oligomeric Abwith a sandwich ELISA built on pairs of for example 6E10,the presence of monomeric Ab or APP in biologicalsamples disturb the analyses (Fig. 4d).

With the mAb158 sandwich ELISA, we were able tomeasure levels of protofibrils in cell culture media and mousebrain homogenates from models carrying the Swedishmutation alone or in combination with the Arctic mutation.To ensure that only soluble Abwas analyzed in the sandwichELISA, samples were centrifuged at 17 900 g prior toanalysis and thus cross-reactivity with Ab fibrils could beavoided. The Arctic mutation was used as a model forenhanced Ab protofibril formation and as predicted, thesesamples indeed displayed an increase in Ab protofibril levels.We were also able to show that wild type Ab protofibrils arepresent in both cell culture media and brain homogenates ofAPP transgenic mice at significant levels but hardly detect-able in the non-transgenic controls. This important findingproves that wild type Ab protofibrils, and not only Arctic Abprotofibrils, are actually formed in vivo and that both theseAb species can be detected with this immunoassay.

At present, Ab1–40 and Ab1–42 levels in plasma and CSFare proposed as markers of AD. In a recent publication fromour group, we suggested that the low CSF levels of Ab1–42seen in AD patients (Motter et al. 1995; Jensen et al. 1999)may in fact be a sign of increased levels of oligomeric Ab(Stenh et al. 2005). This suggestion was supported experi-mentally by the observation that levels of Ab1–40 and Ab1–42 in cell culture media from APPSwe and APPArc–Swetransfected HEK-cells, as well as APPSwe and APPArc–Swetransgenic mouse brains, did not correlate with Ab levelsmeasured by western blot. The results indicated that asignificant pool of soluble aggregated Ab was not fullydetected by such traditional Ab ELISAs. Here, by measuringthe levels of Ab protofibrils with the mAb158 sandwichELISA in the very same cell culture media samples used byStenh et al., the missing piece of evidence for this hypothesisis delivered: higher levels of Ab protofibrils in APPArc–Swe

cell culture media and lower levels in APPSwe media(Fig. 5a). The same pattern was observed for the TBS-soluble fraction of APPArc–Swe and APPSwe transgenic mousebrain homogenates (Fig. 5b). The observation that ADpatients carrying the Arctic mutation display lower plasmalevels of Ab when measured by traditional C-terminal-specific Ab ELISAs (Nilsberth et al. 2001) fits well with thehypothesis of increased Ab protofibril levels. Analyses ofaggregated Ab1–42 samples with SEC, mAb158 sandwichELISA, and Ab1–42 ELISA in parallel (Fig. 6) furtherstrengthened this hypothesis, as high Ab protofibril levelsdetected by SEC and mAb158 sandwich ELISA wereaccompanied by low signals in the Ab1–42 ELISA.

In conclusion, the mAb158 sandwich ELISA provides auseful method for quantification of Ab protofibrils inbiological samples, which offers opportunities to expandthe panel of suitable early diagnostic markers and biomarkersfor AD.

Acknowledgements

This work was supported by grants from Hjarnfonden and Bertil

Hallstens forskningsstiftelse (LL), Alzheimerfonden (HE and LL),

The Swedish Research Council (2003-5546, LL; 2004-6203,

LNGN; 2004-2167 DS), DIADEM (QLK3-CT-2001-02362; LL),

APOPIS (Contract no. LSHM-CT-2003-503330; LL), Stiftelsen

Gamla Tjanarinnor (HE, FEP and LNGN), and from Stohnes

stiftelse (HE, DS, FEP, and LNGN). We are grateful to A. Lord for

the help with immunizations and valuable comments on the

manuscript, G. Karlsson and K. Edwards for performing cryo-

TEM, C. Sahlin for HEK-cell media and western blot collaboration,

M. Hedlund and A. Svensson for technical assistance, P. Wester-

mark, A. Larsson, and J. Bergstrom for providing non-Ab amyloids

and antibodies against non-Ab amyloids, P. Larsson for valuable

help with ELISA protocols and reagents, K. Svensson and M.

Karlsson for production, purification, and biotinylation of antibod-

ies, and to P. O’Callaghan and H. Wigzell for comments on the

manuscript.

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Sensitive ELISA detection of amyloid-? protofibrils in biological samples