Sensitive detection of Aβ protofibrils by proximity ligation - relevance for Alzheimer's disease
© Kamali-Moghaddam et al; licensee BioMed Central Ltd. 2010
Received: 30 June 2010
Accepted: 5 October 2010
Published: 5 October 2010
Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of Aβ protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity.
For specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation.
The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.
In Alzheimer's disease (AD), brain deposits of extracellular amyloid-β (Aβ) and intracellular tau tangles are characteristic of the disease. Cerebrospinal fluid (CSF) is often investigated for levels of Aβ42, tau and phosho-tau in routine diagnostics of AD , where decreased Aβ42 and increased tau and/or phospho-tau (Thr181P) in CSF are indicative of the disease. These measures are reasonably good predictors of future conversion to AD among subjects with mild cognitive impairment, but they are not suitable to follow disease progression or to monitor drug intervention. Novel biomarkers are therefore needed, and evidence suggests that soluble, oligomeric aggregates of Aβ could be such a marker. For instance, levels of soluble forms of Aβ correlate more closely with disease severity than do the amounts of insoluble Aβ aggregates in the brain , and oligomeric Aβ has been shown to be neurotoxic, lead to synaptic dysfunction and to inhibit maintenance of hippocampal long-term potentiation [3–7]. Moreover, the so-called Arctic mutation causing early onset AD is located within the Aβ domain as are other mutations such as the Flemish, the Dutch and the Italian mutations, and this particular mutation has been shown to specifically enhance the formation of large soluble oligomers of Aβ (i. e. protofibrils), suggesting the notion that this Aβ species plays a central role in disease pathogenesis [8, 9]. We previously developed a sensitive sandwich ELISA where the protofibril-selective mAb158 was used both as capture and detecting antibody . Using this assay, the antibody used herein has been shown to detect Aβ protofibrils also in other, well-known, tg-mice such as PSAPP and tg2576 . Here, we demonstrate that the proximity ligation assay (PLA) can provide even more sensitive detection of synthetic Aβ protofibrils.
PLA is an affinity-based technology enabling sensitive and specific detection of proteins in which the detection of proteins by sets of antibodies results in the formation of a specific DNA sequence by ligation of two parts. This sequence can then be amplified and quantified by methods such as real-time, PCR [12, 13]. The technique makes use of affinity probes, typically antibodies coupled to oligonucleotides. Upon recognition of a common target molecule by a pair of such probes, the attached DNA strands are brought in proximity, allowing their free ends to be hybridized to a connector oligonucleotide that directs their joining by ligation. The reporter DNA strand that forms upon ligation can be amplified and quantified by methods such as real-time PCR. The assays can be performed in the homogenous phase with no need for washes or separations [12, 13]. Alternatively, a solid support-bound affinity reagent can be used that offers the possibility to search for target molecules in larger sample volumes and to remove excess probes and undesired sample components before the ligation and amplification steps. This approach also adds specificity by requiring simultaneous recognition of three epitopes on the targets [14, 15]. By using a single monoclonal antibody as the affinity reagent in all three affinity reagents required for solid-phase PLA (SP-PLA) we have achieved a highly sensitive assay that is specific for Aβ protofibrils, with excellent discrimination against monomers. We demonstrate that PLA is capable of detecting soluble Aβ aggregates in brains from mice transgenic for a pathogenic form of APP - the protein from which Aβ is derived.
Biologically derived Aβ protofibrils
Despite extensive efforts there remains an unmet need for highly specific and sensitive detection of soluble forms of the aggregated proteins that are found deposited in the brain in some neurodegenerative diseases. Sufficiently sensitive and specific methods could prove of great value in research, drug development, and for diagnostics and follow-up.
The proximity ligation assay has the advantage that it can be configured to require simultaneous binding to two or more epitopes in order to yield detectable signals. Herein we have used SP-PLA for specific detection of soluble Aβ aggregates via recognition by three reagents binding identical epitopes (Figure 1), thus, providing a stringent requirement for recognition of protein aggregates. This form of the assay involves one capture probe and two PLA probes, jointly ensuring low nonspecific background and exclusive detection of soluble aggregates where minimally three identical epitopes of the aggregated form of Aβ are recognized by the antibodies.
The other salient feature of PLA - the opportunity for DNA-based signal amplification using real-time PCR - serves to greatly enhance detection signals from the recognition reactions. The combination of highly specific recognition and amplified read-out allowed detection of protein protofibrils with increased sensitivity compared to our previously established ELISA (Figures 2B and 4). The SP-PLA allowed capture of Aβ protofibrils prior to extensive washes in a manner similar to a sandwich ELISA to remove excess detection reagents and components of the samples that might otherwise interfere with ligation, polymerization, or with fluorescence detection. We used five μl samples diluted to fifty μl in reaction buffer. SP-PLA served to further decrease background signals, providing for specific and near-linear detection of the protofibrils over a broad dynamic range.
The detection of different species of soluble Aβ aggregates in CSF could provide a means for unambiguous, potentially early diagnosis of AD. Other reported sensitive assays for detection of soluble aggregate proteins such as the bio-barcode assay rely on affinity binders specifically recognizing the oligomeric conformations of the target proteins , while in the SP-PLA-based approach the requirement for three recognition events also contribute to a high selectivity for protofibril conformations. A limited study using the SP-PLA tests for detection of Aβ aggregates in human CSF failed to reveal signals above background for either AD patients or controls, but this does not rule out protofibrils as a potential biomarker for the disease. It remains possible that further improved sensitivity will be required, or the lack of signal could be due to an inability of the antibody we used to detect the form of aggregated Aβ peptides in human CSF. Evidence has been presented that AD patients indeed do have aggregated forms of Aβ in CSF [18, 19]. The detection of endogenous Aβ aggregates spiked in human CSF (Figure 5) indicates that SP-PLA performs adequately in this biological matrix.
We have established that the PLA technique provides enhanced detection of aggregated Aβ proteins, offering high specificity, sensitivity and a wide dynamic range of detection. This makes PLA and further improvements thereof a promising tool for diagnostics in AD, and by extension also in other diseases characterized by increased levels of aggregated proteins.
Mouse brain homogenates and human CSF
Eight-month-old mice transgenic for human APPArc-Swe (n = 5), and nontransgenic littermates (n = 5)  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 a complete protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) 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 supernatant was aliquoted and stored at -80°C prior to analysis.
CSF samples were collected by lumbar puncture at the Memory Clinic, Uppsala University Hospital, Uppsala, Sweden, as approved by the local ethics committee at Uppsala University (decision number 2005:244 and Ö 48-2005). Samples were centrifuged at 1,800 × g for 10 min to eliminate cells and insoluble material, and kept at -80°C until analysis. The CSF used in this study was pooled from 4 healthy individuals.
The monoclonal antibody mAb158, having selective affinity for Aβ in its protofibrillar conformation, has been described previously . The monoclonal antibody 82E1, with affinity for a linear N-terminal Aβ neo-epitope, was purchased from IBL International (Hamburg, Germany). Synthetic Aβ42 was purchased from American Peptide (Sunnyvale, Ca, USA), and the Aβ protofibrils were prepared as previously described . Briefly, lyophilized synthetic Aβ42wt was dissolved in 10 mM NaOH to a concentration of 100 μM, and then further diluted 1:1 with 2 × PBS (50 mM phosphate buffer and 100 mM NaCl, pH 7.4), and incubated at 37°C over night (ON) in the presence of 50 μM Docosahexaenoic acid (DHA) to stabilize the protofibrils. To remove fibrillar material the sample was centrifuged for 5 min at 17,900 × g before analyses. As determined by density gradient ultracentrifugation the mass of the Aβ protofibrils in this preparation are approximately 100-400 kDa (unpublished data). Lyophilized synthetic Aβ1-16wt peptide (Bachem, Bubendorf, Switzerland) was dissolved in 10 mM NaOH, prior to use, and diluted in 2 × PBS to a final concentration of 50 μM.
Oligonucleotide-streptavidin conjugates SLC1 (5'-streptavidin CGCATCGCCCTTGGACTACGACTGACGAACCGCTTTGCCTGACTGATCGCTAAATCGTG-3') and SLC2 (5'-TCGTGTCTAAAGTCCGTTACCTTGATTCCCCTAACCCTCTTGAAAAATTCGGCATCGGTGA-streptavidin 3') were purchased from Solulink (San Diego, CA, USA), and treated prior to use with free streptavidin to reduce the numbers of oligonucleotides per streptavidin tetramer, as described .
The same PCR forward primer, Biofwd, 5'-CATCGCCCTTGGACTACGA-3', PCR reverse primer, Biorev, 5'-GGGAATCAAGGTAACGGACTTTAG-3', and connector oligonucleotide, 5'-TACTTAGACACGACACGATTTAGTTT-3' were used in all PLA tests. These oligonucleotides were purchased from Biomers (Germany). A TaqMan probe (5' FAM-TGACGAACCGCTTTGCCTGA-MGB 3') was obtained from Applied Biosystems.
Solid-phase proximity ligation assay
For all PLA reactions the oligonucleotide-streptavidin conjugates SLC1 and SLC2 were coupled to biotinylated antibodies by incubating identical volumes of 100 nM antibodies with 100 nM streptavidin-oligonucleotide conjugates for 1 h at room temperature. The antibody-oligonucleotide conjugates (PLA probes) thus obtained were used without purification after being separately diluted in PLA buffer (1 mM D-Biotin (Invitrogen), 0.1% purified BSA (New England Biolabs), 0.05% Tween 20 (Sigma-Aldrich), 100 nM goat serum IgG (Sigma-Aldrich), 0.1 μg/μl salmon sperm DNA (Invitrogen), 5 mM EDTA, 1 × PBS), and incubated for 15 min at room temperature prior to mixing the reagents to form a PLA probe mix.
Microparticle-based SP-PLA was carried out as described by Darmanis et al. , with some modifications as follows. Briefly, capture antibodies were bound to microparticles by using one mg of Dynabeads® MyOne™ Streptavidin T1 microparticles (Invitrogen) that had been washed twice with 500 μl washing buffer (1 × PBS, 0.05% Tween 20 (Sigma-Aldrich)), using a 96-well plate magnet (Perkin Elmer) for separation of microparticles. The microparticles were mixed with 200 μl of 50 nM (1.5 μg) of the same biotinylated monoclonal antibody that was used for PLA probe, and incubated for 1 h at RT under rotation, followed by washes as above. The antibody-coated microparticles were suspended in 200 μl of storage buffer (1xPBS, 0.1% purified BSA (New England Biolabs)), and stored at 4°C for up to 2 months.
For each assay the storage buffer of one μl of antibody-coated microparticles (≈5 μg of microparticles and 7.5 ng of antibody) was replaced by 5 μl of PLA buffer, and the microparticles were mixed with 45 μl samples to be investigated for Aβ protofibrils. The binding reactions were incubated ON at 4°C or for 1.5 h at RT under rotation with similar efficiencies (data not shown). The microparticles were washed twice, and 50 μl of PLA probe mix at a concentration of 30 pM for each probe was added to each well, and incubated for 1.5 h at RT with rotation, followed by washing. Finally, 50 μl of ligation/PCR mix (1 × PCR buffer (Invitrogen), 2.5 mM MgCl2 (Invitrogen), 0.2 μM of each primer Biofwd and Biorev, 0.4 μM TaqMan probe, 0.08 mM ATP, 100 nM connector oligonucleotide, 0.2 mM dNTPs (containing dUTP) (Fermentas), 1.5 units Platinum Taq polymerase (Invitrogen), 0.5 units T4 DNA ligase (Fermentas), 0.1 units uracil-DNA glycosylase (Fermentas)) were added, followed by a 5 min incubation at room temperature for the proximity ligation step, before a real-time PCR was performed on an Mx-3000 real-time PCR instrument (Stratagene), with an initial incubation for 2 min at 95°C, and then 45 cycles of 15 s at 95°C and 1 min at 60°C.
For higher volume samples, 10 μl of antibody-coated microparticles were transferred to a 1.5 ml tube, and after removing the storage buffer the particles were mixed with 0.8 ml samples to be investigated for the presence of Aβ protofibrils, and incubated ON at 4°C with end-over-end rotation. The microparticles were collected by spinning at 15,000 rpm for 30 s, and washed twice. 50 μl PLA probe mix was added followed by incubation for 1.5 h at RT. Next, the microparticles were washed twice and transferred to optical PCR tubes, 50 μl ligation/PCR mix was added, and the real-time PCR was performed as described above.
The mAb158 sandwich ELISA was carried out as previously described . In short, 96-well plates were coated with 200 ng/well of mAb158 at 4°C ON before being blocked with 1% BSA in PBS. 100 μl samples were added to the plate in triplicates and incubated for 2 h at RT. 0.5 μg/ml of biotinylated mAb158 was added and incubated for 1 h at RT, and then streptavidin-coupled horse radish peroxidase (Mabtech, Sweden) was added for 1 h at RT. K-blue enhanced (ANL produkter, Sweden) was used as a peroxidase substrate and the reactions were stopped with 1 M H2SO4. Wells were washed three times between each step after blocking the plates, and antibodies and samples were diluted in ELISA incubation buffer (PBS with 0.1% BSA, 0.05% Tween-20).
Amyloid-β precursor protein
Enzyme-linked immunosorbent assay
Phosphate buffered saline
Proximity ligation assay
This work was funded by the Knut and Alice Wallenberg Foundation, Uppsala Berzelii Centre for Neurodiagnostics, FORMAS (2006-2856, MKM), Alliance BioSecure, Åke Wiberg Foundation, European Science Foundation, the Swedish Alzheimer Foundation, the Swedish Brain Foundation, the Swedish Research Council for medicine (2004-2167, DS; 2009-4567, LL; 2009-4389, LN; 2007-2720, UL) and for natural sciences and technology (2006-5168, UL), and by the European Community's 6th and 7th Framework Programs.
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