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AMYLOID OLIGOMERS IN BODY LIQUIDS ARE POTENTIAL BIOMARKERS AND
CAN BE QUANTIFIED BY SFIDA AT SINGLE PARTICLE SENSITIVITY
Dieter Willbold, Andreas Kulawik, Christian Zafiu, and Oliver Bannach
Heinrich-Heine-Universität Düsseldorf, Institut für Physikalische Biologie, 40225 Düsseldorf,
Germany and Forschungszentrum Jülich, ICS-6, 52425 Jülich, Germany
A pathological hallmark of neurodegenerative diseases (NDs) such as Alzheimer’s disease (AD)
and Parkinson’s disease (PD) is accumulation of protein aggregates. In AD brain samples,
deposits consisting mainly of amyloid-beta protein (Aβ) are found, while in PD alpha-synuclein
(α-Syn) aggregates are enriched in so called Lewy bodies. However, increasing evidence
indicates substantial overlap of clinical manifestations, pathological features and biomarker
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patterns across many NDs [1]. Up to half of all AD cases, for example, exhibit Lewy body
pathology leading to a more aggressive manifestation of the disease. On the molecular level, a
crosstalk between amyloid aggregates consisting for example of Aβ and α-Syn, has been
postulated to contribute to clinical heterogeneity [2,3]. Molecular interactions, cross-seeding
activity and hetero-aggregate formation has been also described for other ND-associated proteins
including Tau protein, prion protein (PrP), and TDP-43 [4-9]. The clinical diversity of NDs
emphasizes the need for biomarker development to improve diagnostic accuracy, differential
diagnosis, prognostic guidance and measures of target engagement in future clinical studies and
neuroprotection trials. The ideal biomarker reflects fundamental and early pathological features
of a disease. Since it is widely accepted that aggregate formation is the common key event in all
NDs, we hypothesize that homo- and hetero-aggregates are the most promising and direct ND
biomarkers.
We have previously developed sFIDA (surface-based fluorescence intensity distribution
analysis) to detect single protein aggregates and quantify them as biomarkers for NDs [10-12]. In
sFIDA, protein aggregates are immobilized to a capture-coated glass surface and then are
decorated with at least two different antibody probes labeled with different fluorescent dyes. The
hereby obtained surface is imaged by high-resolution microscopy, e.g. total internal reflection
fluorescence microscopy. In contrast to classical sandwich ELISA assays, which yield only one
readout value per sample, sFIDA yields several millions of read-out values, each of which can be
either attributed to signal or noise, respectively. Capture and detection antibodies with
overlapping epitopes guarantee insensitivity of the assay for monomeric proteins, which is
extremely important, because monomeric protein species are abundant also in healthy subjects.
Single particle sensitivity is an essential feature of the assay as well, as the concentration of
protein aggregates in body fluids is extremely low. The innovative sFIDA experimental setup
allows single particle detection sensitivity. Use of more than one detection probe, each with its
own detection dye and the respective detection colour channel allows unprecedented specificity
for homo-aggregates and even specific detection and characterization of hetero-aggregates
(Figure 1). In principle, this allows determination of the composition of each single particle,
which represents a unique feature of this technology. We applied sFIDA already for diagnostics
of AD in CSF samples and of prion diseases in blood, and published the results in the past years
[13-15]. In addition, sFIDA application was already extended for detection of aggregated Tau, α-
Syn, TDP-43, ApoA and SOD1 as well as single hetero-aggregates containing both, Aβ and PrP,
respectively.
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Aggregate-specific biomarkers are also essential for the clinical development of compounds that
target amyloid aggregates. To identify patients that will most likely respond to oligomer-directed
drug candidates, it is essential to recruit preferentially those patients that are high of the
respective biomarker to allow successful therapy based on target engagement, i.e. reduction of
the respective biomarker. For the example of Alzheimer’s disease, there are many substances
under clinical investigation that aim to eliminate the most harmful isoform of amyloid, which is
the neurotoxic oligomeric Aβ. Measures of fibrillary amyloid by PET imaging and measures of
monomeric Aβ in CSF are commonly used for biomarker-based therapy monitoring. However,
both biomarkers do not match the proposed mechanism of action of these drugs, i. e. the
reduction of Aβ oligomers, which are thought to be the most attractive treatment target. Only
sFIDA is in place as a technology platform to further develop and validate Aβ oligomers as a
novel biomarker for patient selection as well as measures of target engagement and clinical
outcome. The sFIDA principle features single particle sensitivity and absolute specificity for
oligomeric assemblies. As an innovative platform technology, sFIDA will be applied for
differential diagnostics to identify and exclude patients that are positive for other, non-Aβ
oligomers. Still, no causal therapy is available for AD. Many promising drug candidates have
failed in late stage clinical trials, which has been largely attributed to the lack of a predictive
biomarker as well as to an inaccurate selection of patients based on clinical diagnosis. Our fully
automated and standardized sFIDA technology can measure Aβ oligomers in body liquids as
presumably the most direct AD biomarker and can link this biomarker with pathological
processes, target engagement and clinical end points. Apart from the use in drug trials, sFIDA
will improve diagnostic accuracy, differential diagnosis, and prognostic guidance in the clinical
setting.
An unmet need for oligomer-based diagnostic tests is a suitable standard that allows calibration
of the assay readout [16,17]. For quantitative analysis of Aβ oligomers we have recently
introduced silica-based nanoparticles (SiNaPs) coated with peptides which mimic the properties
of native oligomers with regard to size and epitope load [18-20]. This strategy is easily
transferred to any amyloidogenic protein and even mixed and thus heterogenic amyloid
oligomers. We have shown that when such standards were spiked into CSF and buffer,
respectively, and were subjected to sFIDA analysis on an automated platform for which we
determined several assay parameters, i.e. linearity, coefficient of variation (CV), limit of
detection (LOD) and lower limit of quantification (LLOQ). Using such calibration standards we
have shown that LLOQs in the sub-femtomolar range are achievable, which corresponds to
single particle sensitivity within microliter sample volumes [18,19].
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In Summary, sFIDA is in place to explore and validate homo- and hetero-oligomeric aggregates
as useful biomarkers for early diagnosis of neurodegenerative diseases, the recruitment of
patients for clinical studies and for monitoring therapy success.
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