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Alzheimer Disease (AD) is characterized by abnormal extracellular accumulation of 尾-Amyloid peptide in the brains of
patients. The pathological isoform A尾1-42 (A尾42) agregates magnitudes of order faster than A尾40 or A尾39. However
the analysis of the only A尾42 concencentration is not enough specific for the discrimination of AD from other kinds of
Dementia in subjects with constitutively high or low levels of total A尾 peptides. Thus, it may be better to consider the ratio
between A尾42/A尾40 for higher reliability. In this way, a method to detect simoultanously both analytes in cerebrospinal fluids
is needed. Because of their multiplexing capability, the low volume sample consumption and the efficient sample-to-result
time for population-wide screening, microarrays are ideal tools for the identification of individuals with preclinical AD who
are still cognitively healthy. In this work, a highly sensitive immunoassay based on a label/label-free microarray platform
that utilizes silicon/silicon oxide (Si/SiO2) substrates for the detection of the AD biomarkers A尾40 and A尾42 in artificial
cerebrospinal fluid (ACSF) has been developed. In preliminary tests, the commercial peptides A尾42 and A尾39 aggregation
status were assessed by Circular Dicroism, demonstrating the stability of A尾42 structure for at least 2 hours, while A尾39 does
not show any conformational changes, even at 24 hours. Thereafter, an optimal antibody matched pair for A尾42 detection was
selected with a label and label-free microarray platform, using the Interfermometric Reflectance Imaging Sensor (IRIS), to
determine binding yield on the silicon surface and the spot morphology. The selected antibodies were tested on copoly (DMANAS-
MAPS) coated silicon chips for Cy3-label microarray analysis. Two antibody matched pairs resulted specific and sensitive
for A尾42. As these capture antibodies were against the peptide C-terminus, a different capture antibody was selected as specific
for A尾40. Both of the two peptides were simultaneously detected on a multiplexed fluorescence microarray using the same
biotinilated antibody against the peptides N-termini and the respective LODs were extrapolated. The best condition developed
for A尾42 detection in ACSF requires 2 hours of dynamic incubation, resulting in a LOD of 73 pg/mL. In the same conditions
the LOD for A尾40 was 350 pg/mL. The obtained results are compatible for the use of the proposed assay in diagnostics, where
CSF controls show a concentration of about 800 pg/mL for A尾42 and 7 ng/mL for A尾40. Moreover, our tool has been applied
to detect these biomarkers in real human CSF samples, distinguishing between healthy and AD patients on the base of A尾40/
A尾42 ratio. However, in contrast to the use of standard A尾42 in ACSF, either static or dynamic overnight incubation resulted
in an increased signal to noise ratio. This is most likely because in real human samples, the aggregation phenomenon observed
in artificial CSF rarely occurs, and a longer incubation time allows the analyte to reach the equilibrium.
Biography
Paola Gagni is an Italian student graduated in 2011 in Pharmaceutical Biotechnology and now she is attending her PhD courses in Medicinal Chemistry at the
University of Milan. She works on label and label-free protein microarrays at the Institute of Chemistry of Molecular Recognition (ICRM) of the CNR. She collaborates
in a laboratory which uses Si/SiO2 supports, coated with appropriate polymers in order to increase binding capability and specificity of capture biomolecules.
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