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Detection and quantification of alpha-synuclein aggregates in human blood plasma/serum

Detection and quantification of alpha-synuclein aggregates in human blood plasma/serum

BACHELOR project and MASTER project   (published januari 2019)

Introduction

Parkinsons isease (PD), the second most frequently occurring neurodegenerative disease in the world, is mainly characterized by the loss of dopaminergic neurons and the presence of insoluble proteic complexes, so-called Lewy bodies (LB), in the brain [1]. LB are largely composed of fibrillar aggregates (amyloid) of the protein alpha-synuclein (aSyn). This suggests that aSyn plays an important role in the pathogenesis of PD. In addition, aSyn gene mutations and duplication/triplication are associated with hereditary forms of PD [2].

The aSyn protein is small (14 kDa), intrinsically disordered, and mainly expressed in dopaminergic neurons in the brain [3]. Several studies showed that aSyn is secreted from neuronal cells in the brain and can be found in cerebrospinal fluid (CSF) and human blood plasma [5, 6]. Therefore aSyn is potentially an interesting diagnostic biomarker for PD [8]. In the future PD may be detected though the aSyn monomer and aggregate concentrations in body fluids.

 Description of the project:

Improvement of the quantification of aSyn in human blood plasma/serum by optimization of ELISA

Towards the detection of aSyn in blood we have, in previous work developed a sandwich ELISA assay. This assay can be used to specifically detect aggregated forms of aSyn in a human blood serum. In these in vitro experiments we made use of recombinantly expressed and purified aSyn protein. We included low concentrations of in vitro prepared aSyn fibrils in the serum and used a specific combination of capture antibody and primary antibody to detect only aggregated aSyn species (Figure 1).


Figure 1: ELISA for aggregated aSyn detection (from presentation of Josine Kothuis).

Using this method we were able to detect low concentrations (5 nM) of aSyn fibrils in buffer and higher concentrations (25-100 nM) in human blood serum.

The aim of this project is to improve the ELISA based assay to detect and quantify, in a reproducible way, (even) low(er) concentrations of aSyn fibrils/oligomers in blood plasma/serum by testing different buffers, antibodies, detection parameters, etc.

Techniques involved:

  • In vitro aSyn aggregation reactions
  • Thioflavin T fluorescence measurements
  • Turbidity measurements
  • ELISA
  • Luminescence detection

 

Contact:

Department: NanoBioPhysics (NBP) UTwente - https://www.utwente.nl/en/tnw/nbp/

Daily supervisor: MSc. Jonathan Vaneyck (PhD student) - j.vaneyck@utwente.nl, Dr. G.M.J. Segers-Nolten (Researcher) - g.m.j.segers-nolten@utwente.nl

Project leader: Prof.Dr. M.M.A.E. Claessens (Chair Nanobiophysics) - m.m.a.e.claessens@utwente.nl