NeuroMET and biomarkers M. Quaglia, LGC, Queens Road, Teddington, UK

Back ground Alzheimer disease and Parkinson disease are the two most common neurodegenerative diseases Early diagnosis is essential for successful treatment, but no early diagnosis method is currently accepted Measurement variability of CSF biomarkers currently used in diagnosis is high and requires a painful lumbar puncture with rare but potentially serious side effects Improved anxiety and stress management are within the top 10 patient priorities

Overview Biomarkers in plasma for clinical diagnostics Reference methods Biomarkers for monitoring stress

Method for quantification in biologics Reference methods Measurement procedure accepted as providing measurement results fit for their intended use in assessing measurement trueness of measured quantity values obtained from other measurement procedures for quantities of the same kind, in calibration, or in characterising reference materials (VIM 3rd Ed. 2.7 (2012)) An analytic procedure sufficiently free of random or systematic error to make it useful for validating proposed new analytic procedures for the same analyse Primary calibrator Method for quantification in biologics

Reference methods Tau: recognised biomarker for AD with limited utility due to high measurement variability α-synuclein: target for PD treatments. No reliable method currently available

tau

Tau: primary calibrator Intact protein analysis Next steps: larger batch primary calibrator and development of a method for quantification in CSF Amino acid analysis

α-synuclein: primary calibrator Intact protein analysis Amino acid analysis Alternative purification methods

α-synuclein in biological fluids Isotopically labelled N15 protein Peptides IS to monitor phosphorylation and acetylation MDVFMK GLSK AK EGVVAAAEK TK QGVAEAAGK TK EGVLYVGSKTK EGVVHGVATVAEK TK EQVTNVGGAVVTGVTAVAQK TVEGAGSIAAATGFVK K DQLGK NEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA

Overview Biomarkers in plasma for clinical diagnostics Reference methods Biomarkers for monitoring stress

Measurement of stress Stress and anxiety are important clinical aspects for AD and PD patients which need to be addressed to improve their quality of life. Stress may also interfere with the efficiency of drugs administered To develop immunoassay based methods to measure free and total cortisol and compare results with results from Hospital Anxiety and Depression, Parkinson’s Anxiety and Rating Anxiety in Dementia Scale questionnaires

Measurement of stress On-going recruitment of patients Devised assays validated assays Streptavidin labelled MSD SULFO-TAG Biotin Biotinylated primary detection antibody (rabbit anti-HRP pAb) Tracer (cortisol-HRP) Capture primary antibody (mouse anti-cortisol mAb) On-going recruitment of patients MSD plate

Overview Biomarkers in plasma for clinical diagnostics Reference methods Biomarkers for monitoring stress

Plasma biomarkers Proteins miRNA Conventional methods (Aβ1-40, 1-42, tau) Improved immunoassays (Aβ1-40, 1-42, tau, NFL, cortisol) - α-synuclein Immunoassays dPCR

Plasma biomarkers The MSD V-PLEX Ab Peptide Panel 1 (4G8) kit is currently tailored for use to detect Ab42, Ab40 and Ab38, within human CSF and mouse plasma. The levels of Ab42 and Ab38 peptides are generally too low to be detected within human plasma using this kit. To overcome matrix effects and achieve duplex detection of Ab42 and Ab40 However, we have adapted the assays to overcome complex matrix effects and achieve duplexed detection of Ab42 and Ab40 within human plasma. Our novel assay provides a means for minimally invasive detection of Ab peptides in human plasma, as blood is more accessible than CSF.

Results for Aβ Intra-assay CV ≤ 6.7% Preliminary experiments were promising to improve performance of Aβ kits. NFL assay development is proving to be challenging

To generate data for statistical model and validation of PCOMs Summary Progress have been made to: Develop reference methods (tau and α-syn) Develop methods for measurement of stress Develop improved methods for analysis of plasma biomarkers To generate data for statistical model and validation of PCOMs