Qualification of classical biomarkers for Alzheimer’s Disease in a multiplex format in blood using a new technology

Abstract

BackgroundProtein biomarker profiling in biological fluids will soon become an established procedure in clinical diagnosis of neurodegenerative disease subtypes. Observed differences between technology platforms in performances can be attributed to technology principles and their development status, selection and characterization of antibody combinations, reproducibility of test results, and pre‐analytical factors and processing of samples. Combination of advantages of emerging technologies, together with availability of precision‐qualified assay formats and innovative sample processing procedures, will create additional possibilities for individual patient management using clinically validated protein biomarker panels.MethodThe Giant Magneto Resistance (GMR) technology platform was used to develop multiplex immunoassays for different biomarker proteoforms in blood (e.g., Aß, (phospho)Tau, NF‐L, and GFAP).ResultWe have developed and optimized performance of multiplex assays using a Design of Experiment (DOE) approach to generate reproducible results. The selectivity and specificity of each antibody combination was confirmed with multiplex assay designs. We have used clinical samples instead of calibrators in buffer to shorten the development time. Process optimization through DOE was done with biotinylated peptides. After optimization, reproducibility was improved by approximately 4‐fold to < 5% CV for each analyte. We have included 3D6 as a capture antibody on the printed circuit boards (PCBs), while analyzing Aß1‐42 & Aß1‐40 using 21F12 and 2G3 as capture antibodies, respectively, to identify the presence of Aß oligomers. PCB selection, printing conditions including environmental factors, buffer selections, and conjugation process were optimized in a stepwise manner while frequently checking biomarker levels in clinical samples. Sample processing is part of our roadmap to generate clinically valuable results. The diagnostic potential for each assay format to identify amyloidopathy in an early phase of the disease will be presented using blood samples from cognitively normal, MCI, and AD subjects, characterized by CSF biomarker profiles and/or amyloid PET status.ConclusionThe GMR technology allows screening of performance of antibodies in multiplex formats, shortening the assay development time. The biomarker panels and novel methods for analysis are uniquely positioned to help the healthcare community. This study was funded by Alzheimer’s Drug Discovery Foundation's Diagnostics Accelerator (DxA) Program.