Detection of Soluble Amyloid- Oligomers and Insoluble High-Molecular-Weight Particles in CSF: Development of Methods with Potential for Diagnosis and Therapy Monitoring of Alzheimer's Disease
Figure 1
Summary of method types used for the detection of Aβ oligomers in CSF. Sandwich-ELISA methods provide oligomer specificity because the same antibody is applied as capture and for detection. Alternatively, oligomer-specific antibodies can be used in the detection process. In sandwich nanotechnology tools, two specific antibodies frame Aβ oligomers. The signal of detection is amplified, for example, by conjugated gold nanoparticles with hundreds of DNA barcodes attached in biobarcode assays. The DNA-magnet-sandwich complexes are extracted from the sample using a magnet. Subsequently, the total number of DNA barcodes is determined. In assays based on seeded polymerization, preexisting multimeric Aβ particles in body fluids are spiked by adding labeled Aβ peptides to the sample. Detection is performed via fluorescence correlation spectroscopy (FCS). Fluorescence resonance energy transfer (FRET) is a mechanism describing energy transfer between two chromophores in spatial proximity, which is given between two fluorophores attached to the same Aβ oligomer. FRET signals are detected by flow cytometry. Surface-FIDA is based on a laser focus scanning the surface of a specially prepared glass chip. Either a fluorescence correlation spectroscopy (FCS) device or a laser scanning microscope (LSM) can be used. Aβ aggregates or oligomers are concentrated in a two-dimensional surface by immobilizing them on a glass slide using Aβ capture antibodies. The aggregates are detected by adding at least two fluorescence-labeled anti-Aβ antibodies. At least, two laser beams are focused on the surface of the glass chip, and the fluorescence light which is emitted by the fluorescence antibodies is detected in a confocal way, enabling single aggregate detection. A quantitative value for specific colocalized fluorescence pixels is yielded by the summation of all cross-correlated pixels in the colocalization area above a threshold (cutoff) intensity value. Only double-labeled events are considered for the analysis.
