(M1530-09-54) Challenges in Development of Multiplex Biomarker Assays for Neurodegenerative Diseases

Purpose: Neurodegenerative diseases such as Parkinson’s Disease, Alzheimer’s Disease, Huntington’s Disease, Dementia and MCI (mild cognitive impairment) are some of the most debilitating age-related health issues on the rise due to increased life expectancy. Biomarker detection and quantification represents an invaluable tool to advance the development of therapeutics against neurodegenerative diseases. Therefore, there is an indispensable need for reliable and ultra-sensitive multiplex assays that require minimal sample volume to overcome the invasive and limited cerebrospinal fluid (CSF) available for biomarker detection. Here we describe several challenges encountered during method development and validation due to recent uptick in the quality of human CSF and associated analyte stability issues.

Methods: It’s a well-known fact that the concentrations of Aβ40/Aβ42 and total Tau in CSF indicate the cognitive status of the elderly. Therefore, we investigated the possibility of setting up the Single Molecule Array (SiMoA) digital ELISA assay utilizing the Quanterix Advantage kits (Item# 101995) to quantitate Aβ40/Aβ42 and Total Tau levels on HD-X analyzer. Manufacturer’s kit recommended instructions were followed in conjunction to our organization’s SOPs to process the samples. The Quanterix kits performance was acceptable for accuracy and precision of the calibrators and kit provided buffer quality controls, which were within the manufacturer’s predicted range. Two other matrix quality control pools were prepared from pooled human CSF samples to assess to precision, accuracy, and stability of the analytes.

Results: The accuracy and precision runs confirmed the calibration range of 1.09 pg/mL to 166 pg/mL for Aβ40, 0.221 pg/mL to 62.8 pg/mL for Aβ42 and 0.131 to 110 pg/mL for Total Tau for the three analytes. 64 apparently normal human CSF samples were screened, and the concentrations ranged from 12500-0 pg/mL for Aβ40, 977-0 pg/mL for Aβ42 and 1530-0 pg/mL for Total Tau. Significant differences in the quantifiable range of all three analytes were exhibited amongst different vendors and between multiple lots from the same vendor (Figure 1). Human CSF matrix pools prepared to track assay performance showed significant differences in intra-assay and inter-assay accuracy and precision (Table 1). The data also indicated that within the same pool, total Tau demonstrated acceptable precision while Aβ40 and Aβ42 showed higher variability in recoveries indicating that the differences could stem from analyte-specific properties increasing their sensitivity and vulnerability to the quality of matrices.

Conclusion: We show here that the basal endogenous analyte concentrations for the three biomarkers are significantly different across different lots/batches of CSF from apparently normal individuals (Figure 1). Additionally, the physiochemical and aggregation properties of these analytes are subject to alteration by temperature, storage containers and their relative concentration in the matrices can all impact recovery. These proteins are highly concentrated in CSF and are prone to polymerization and aggregation at higher concentrations (Meisl et al., PNAS 2014). Given these challenges, it is recommended to probe for these analytes in plasma or serum where the concentrations of these analytes are significantly lower compared to CSF. To this extent, we have developed and validated an ultra-sensitive Single Molecule Array (SiMoA)-based multiplex assay for the quantitative detection of Aβ40, Aβ42 and Total Tau in Human K₂EDTA plasma. The analytical performance of these analytes in plasma appears to be better in comparison to their performance in CSF (Table 2). Lastly, to circumvent the limited procurement of quality human CSF for matrix QC selection, we propose the utilization of bovine or porcine CSF as surrogate matrices (Dubiel et al, The AAPS Journal 2022) when comparable.

References: Meisl G, Yang X, Hellstrand E, Frohm B, Kirkegaard JB, Cohen SI, Dobson CM, Linse S, Knowles TP. Differences in nucleation behavior underlie the contrasting aggregation kinetics of the Aβ40 and Aβ42 peptides. Proc Natl Acad Sci U S A. 2014 Jul 1;111(26):9384-9.

Acknowledgments: The views and opinions expressed here do not necessarily reflect the position or official policy of PPD Labs (now part of ThermoFisher Scientific)

Figure 1: Box plot demonstrating biological variability among apparently healthy individuals for Aβ40 (a), Aβ42 (b) and total Tau (c).



Table 1: Inter-assay precision of AB40/42 and total Tau biomarkers in cerebrospinal fluid



Table 12: Inter-assay precision of AB40/42 and total Tau biomarkers in plasma.