Novel One-step Immunoassays to Quantify α-Synuclein

Bidinosti, Michael; Shimshek, Derya R.; Mollenhauer, Brit; Marcellin, David; Schweizer, Tatjana; Lotz, Gregor P.; Schlossmacher, Michael G.; Weiss, Andreas

doi:10.1074/jbc.m112.379792

Cited by 46 publications

(27 citation statements)

References 48 publications

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“…The standard curve for h-asyn displayed a typical sigmoid curve, while the signal for m-asyn did not exceed the lower detection limit (LDL). We identified that the linear dynamic range of the h-asyn specific assay lies between 0.1 and 10 ng/ml in-well concentration, while the estimated detection limit is about 2.0 pg/ml and indicates a 3-times higher sensitivity than what previous asyn assays have reported [18–20]. Raw background counts from PBS solution (106.0 ± 11.3) was indifferent from rodent brain lysate readings (121.2 ± 16.9) demonstrating that the total asyn assay has essentially no unspecific signal.…”

Section: Resultsmentioning

confidence: 77%

“…Bidinosti and colleagues reported up to 3-fold differences in assay signal depending on the protein standards used [20]. Standards generated by phosphorylating recombinant h-asyn protein in vitro using PLK2, as is done here, have been reported to result in incomplete phosphorylation of the protein at the S129 residue [26], potentially with batch to batch variations, and present the risk for incorrectly calculated phosphorylation ratios.…”

Section: Discussionmentioning

confidence: 99%

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A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Landeck¹,

Hall²,

Ardah³

et al. 2016

Mol Neurodegeneration

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BackgroundAlpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson’s disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples.ResultsUsing our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41 % in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17 % in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples.ConclusionsThese results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0125-0) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 99%

A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Landeck¹,

Hall²,

Ardah³

et al. 2016

Mol Neurodegeneration

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“…Once the donor and acceptor fluoropheres are brought in close proximity to each other by the antibodies’ binding, a unique detectable fluorescent signal is generated. These immunoassays require small amount of sample, are run by incubating the labeled antibodies simultaneously to the analytes, involve simple one-step protocols that do not require separation steps, and are suitable to micro-plate high-throughput screening [65]. …”

Section: Time-resolved Fluorescence Resonance Energy Transfer Immumentioning

confidence: 99%

“…FMRP levels in peripheral blood mononuclear ear cells from control individuals were 6–7 times higher than those prepared from FXS patients. The assay had an elevated low limit of detection (10 pg/µL; 100 pmol/L) compared to a TR-FRET immunoassay for the detection of α-synuclein (0.1 ng/mL; 6.9 pmol/L) reported previously by the same group [65], and was not used for high-throughput screening of FMRP.…”

Section: Time-resolved Fluorescence Resonance Energy Transfer Immumentioning

confidence: 99%

Detection and Quantification of the Fragile X Mental Retardation Protein 1 (FMRP)

LaFauci¹,

Adayev²,

Kascsak³

et al. 2016

Genes

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The final product of FMR1 gene transcription, Fragile X Mental Retardation Protein 1 (FMRP), is an RNA binding protein that acts as a repressor of translation. FMRP is expressed in several tissues and plays important roles in neurogenesis, synaptic plasticity, and ovarian functions and has been implicated in a number of neuropsychological disorders. The loss of FMRP causes Fragile X Syndrome (FXS). In most cases, FXS is due to large expansions of a CGG repeat in FMR1—normally containing 6–54 repeats—to over 200 CGGs and identified as full mutation (FM). Hypermethylation of the repeat induces FMR1 silencing and lack of FMRP expression in FM male. Mosaic FM males express low levels of FMRP and present a less severe phenotype that inversely correlates with FMRP levels. Carriers of pre-mutations (55–200 CGG) show increased mRNA, and normal to reduced FMRP levels. Alternative splicing of FMR1 mRNA results in 24 FMRP predicted isoforms whose expression are tissues and developmentally regulated. Here, we summarize the approaches used by several laboratories including our own to (a) detect and estimate the amount of FMRP in different tissues, developmental stages and various pathologies; and (b) to accurately quantifying FMRP for a direct diagnosis of FXS in adults and newborns.

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“…Such a system can be thought of as a monoclonal affinity-reagent sandwich assay (MARSA) which is an evolution of the term single antibody sandwich ELISA (SAS-ELISA) meant to encompass other forms of antigen recognition elements [3] non-ELISA based methods of signaling [4], and to avoid confusion with single molecule analyses. MARSAs have already been used to detect several disease relevant oligomers, though sparingly for emerging viral pathogens; the scrapie prion protein isoform PrP Sc [5], Alzheimer disease amyloid-β protein [6], Parkinson's disease α-synuclein protein [4], [7], Plasmodium malariae sporozoites [8] and hemagglutinin content in influenza vaccines [9]. The polyvalent display of the antigenic epitope on large protein oligomers should ensure a very strong interaction with surface immobilized captor antibody increasing the sensitivity for even mediocre affinity antibodies through a Velcro like effect via massively parallel avidity.…”

Section: Introductionmentioning

confidence: 99%

Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

Sherwood

Hayhurst

2013

PLoS ONE

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BackgroundAntigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism.Methods and FindingsIn parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections.ConclusionsThe C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent driven antigen sandwich assays for the Ebolavirus genus.

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Novel One-step Immunoassays to Quantify α-Synuclein

Cited by 46 publications

References 48 publications

A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Detection and Quantification of the Fragile X Mental Retardation Protein 1 (FMRP)

Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

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