Journal of Alzheimers Disease & Parkinsonism
Open Access

Abstract

Alzheimer Disease (AD) is characterized by abnormal extracellular accumulation of ?-Amyloid peptide in the brains of patients. The pathological isoform A?1-42 (A?42) agregates magnitudes of order faster than A?40 or A?39. However the analysis of the only A?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? peptides. Thus, it may be better to consider the ratio between A?42/A?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?40 and A?42 in artificial cerebrospinal fluid (ACSF) has been developed. In preliminary tests, the commercial peptides A?42 and A?39 aggregation status were assessed by Circular Dicroism, demonstrating the stability of A?42 structure for at least 2 hours, while A?39 does not show any conformational changes, even at 24 hours. Thereafter, an optimal antibody matched pair for A?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?42. As these capture antibodies were against the peptide C-terminus, a different capture antibody was selected as specific for A?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?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?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?42 and 7 ng/mL for A?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?40/ A?42 ratio. However, in contrast to the use of standard A?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.