sFIDA automation yields sub-femtomolar limit of detection for Aβ aggregates in body fluids.
OBJECTIVE
Alzheimer's disease (AD) is a neurodegenerative disorder with yet non-existent therapeutic and limited diagnostic options. Reliable biomarker-based AD diagnostics are of utmost importance for the development and application of therapeutic substances. We have previously introduced a platform technology designated 'sFIDA' for the quantitation of amyloid β peptide (Aβ) aggregates as AD biomarker. In this study we implemented the sFIDA assay on an automated platform to enhance robustness and performance of the assay.


METHODS
In sFIDA (surface-based fluorescence intensity distribution analysis) Aβ species are immobilized by a capture antibody to a glass surface. Aβ aggregates are then multiply loaded with fluorescent antibodies and quantitated by high resolution fluorescence microscopy. As a model system for Aβ aggregates, we used Aβ-conjugated silica nanoparticles (Aβ-SiNaPs) diluted in PBS buffer and cerebrospinal fluid, respectively. Automation of the assay was realized on a liquid handling system in combination with a microplate washer.


RESULTS
The automation of the sFIDA assay results in improved intra-assay precision, linearity and sensitivity in comparison to the manual application, and achieved a limit of detection in the sub-femtomolar range.


CONCLUSIONS
Automation improves the precision and sensitivity of the sFIDA assay, which is a prerequisite for high-throughput measurements and future application of the technology in routine AD diagnostics.
Forschungszentrum Jülich, ICS-6 Structural Biochemistry, 52428 Jülich, Germany.
Yvonne
Y
Herrmann
Forschungszentrum Jülich, ICS-6 Structural Biochemistry, 52428 Jülich, Germany.
Andreas
A
Kulawik
Forschungszentrum Jülich, ICS-6 Structural Biochemistry, 52428 Jülich, Germany.
Katja
K
Kühbach
Forschungszentrum Jülich, ICS-6 Structural Biochemistry, 52428 Jülich, Germany.
Maren
M
Hülsemann
Forschungszentrum Jülich, ICS-6 Structural Biochemistry, 52428 Jülich, Germany.
Luriano
L
Peters
Forschungszentrum Jülich, ICS-6 Structural Biochemistry, 52428 Jülich, Germany.
Tuyen
T
Bujnicki
Forschungszentrum Jülich, ICS-6 Structural Biochemistry, 52428 Jülich, Germany.
Kateryna
K
Kravchenko
Forschungszentrum Jülich, ICS-6 Structural Biochemistry, 52428 Jülich, Germany.
Christina
C
Linnartz
Forschungszentrum Jülich, ICS-6 Structural Biochemistry, 52428 Jülich, Germany.
Johannes
J
Willbold
Forschungszentrum Jülich, ICS-6 Structural Biochemistry, 52428 Jülich, Germany.
Christian
C
Zafiu
Forschungszentrum Jülich, ICS-6 Structural Biochemistry, 52428 Jülich, Germany; Heinrich-Heine-Universität Düsseldorf, Institut für Physikalische Biologie, 40225 Düsseldorf, Germany.
Oliver
O
Bannach
Forschungszentrum Jülich, ICS-6 Structural Biochemistry, 52428 Jülich, Germany; Heinrich-Heine-Universität Düsseldorf, Institut für Physikalische Biologie, 40225 Düsseldorf, Germany. Electronic address: d.willbold@fz-juelich.de.
Dieter
D
Willbold
D000544:Alzheimer Disease / Q000175:diagnosis / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D001826:Body Fluids / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D057230:Limit of Detection; D066329:Protein Aggregates; D012371:Robotics; D012680:Sensitivity and Specificity
D016229:Amyloid beta-Peptides; D066329:Protein Aggregates
Alzheimer's disease; Amyloid β peptide; Automation; Diagnostic biomarker; Silica nanoparticles; Surface-based fluorescence intensity distribution analysis (sFIDA)