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

2. 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

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

4. 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

5. 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

6. tau

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

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

9. 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

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

11. 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

12. 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

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

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

15. 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.

16. 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

17. 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