Amyloid-β and phospho-Tau biomarkers immunoassay development and optimization to support the blood-based early and differential diagnosis of Alzheimer’s disease
by Barbara Morgado
Date of Examination:2025-01-20
Date of issue:2025-10-13
Advisor:Prof. Dr. Jens Wiltfang
Referee:Prof. Dr. Tiago Outeiro
Referee:Prof. Dr. Nils Brose
Referee:Prof. Dr. Oliver Wirths
Referee:Prof. Dr. Christine Stadelmann-Nessler
Referee:Prof. Dr. André Fischer
Persistent Address:
http://dx.doi.org/10.53846/goediss-11551
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Description:PhD thesis
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Abstract
English
The recent emergence of anti-amyloid treatments for early symptomatic Alzheimer’s disease (AD) require biomarker confirmation of amyloid pathology, increasing the need for biomarker testing. In the last few years, there has been enormous progress in the development of new ultrasensitive blood based biomarker assays with great analytical and clinical performances. Compared to amyloid PET or cerebrospinal fluid (CSF) tests, blood tests are less costly, more accessible and more scalable for widespread use than. The most promising tests consist of low Aβ42/Aβ40 ratio and phosphorylated Tau (pTau) in particular Tau phosphorylated at Threonine 217 (pTau217) that reflect brain Aβ-amyloid pathology. The overall goal of this thesis was to develop and validate blood-based biomarkers tests for the AD amyloid pathology. The thesis was divided in 3 independent parts, covering biomarker development, assay development and analytical/technical validation and clinical validation. Pre-analytical immunoprecipitation of Aβ peptides from blood plasma was previously shown to improve the diagnostic performance of MSD and Elecsys plasma Aβ assays for detecting amyloid pathology. In order to tentatively improve the previous stablished immunoprecipitation-immunoassay approach, this thesis investigated different antibody selection and immunoassay platforms in detecting low-abundant brain-derived biomarkers in blood plasma in proof of concept case-control cohorts. For extending the use of pre-analytical IP to additional AD biomarkers, the protocol was modified for the immunoprecipitation of Tau proteins for the subsequent quantification of plasma pTau181 and pTau217. Aβ- and Tau-IP were shown to be compatible with the Lumipulse G System, the currently best performing immunoassay platform for the measurement of plasma biomarkers Aβ1- 42/1-40, pTau217 and pTau181. However, as it turned out, the pre-analytical IP did not statistically significantly improve the diagnostic performance of the plasma AD biomarkers Aβ42/40 ratio and pTau217 compared to direct measurements in EDTA-plasma and following the manufacturer’s recommendations. In contrast, the proposed IP protocol worked reasonably well in improving the performance of plasma pTau181 in a small pre-selected sample. A clinical validation study was performed to demonstrate the clinical diagnostic value of the protocols developed in the first part of this thesis in samples from the DELCODE study of the German Center for Neurodegenerative Diseases (DZNE). This study cohort was assumed to have demographic characteristics representative of the patients presenting at memory clinics. Results indicated plasma pTau217 as the leading single plasma biomarker to detect brain amyloid pathology, supporting published findings. A novel composite biomarker was proposed, that combines the plasma measurements of Aβ1-40, Aβ1-42 and pTau in a single biomarker for detecting amyloid pathology. The Aβ1-40/Aβ1-42∗ pTau217 ratio showed an added diagnostic value relative to the more straightforward pTau217 but additional studies are needed to confirm these observations. Biomarkers results are often interpreted using a binary system with a single cut-off value, indicating either the presence (+) or absence (−) of specific pathogenic processes. With the aim to optimize the overall test accuracy, a previously proposed two-cut-point (three-category) approach was investigated. The results suggest that, for future use in clinical trials and routine, a two-cut-point approach provides a particularly useful way for the interpretation of biomarker measurements for predicting amyloid status, and thus decreasing the risk of patient misclassification. Finally, this thesis aimed to assess a novel blood biomarker candidate that was hypothesized to reflect brain amyloid deposition. The presence of N-terminally elongated Aβ-3-42 was confirmed for the first time in human CSF. In order to investigate the potential diagnostic value of the Aβ-3-42/-3-40 ratio, an ultra-sensitive 2-step immunoprecipitation-immunoassay approach was developed for blood plasma and CSF samples. On a small pilot study, the CSF Aβ-3-42/-3-40 ratio seemed to offer the same information as the well established CSF Aβ42/40 ratio. Moreover, the novel Aβ ratio was significantly altered in plasma samples of AD patients. Nevertheless, larger patient cohorts are needed to validate these results and investigate whether the Aβ-3-42/-3-40 ratio offers an added diagnostic information for the differential diagnosis of early diagnosis.
Keywords: Alzheimer’s disease; Amyloid beta; Tau; Blood plasma; Biomarker
Schlagwörter: Alzheimer’s disease; Amyloid beta; Tau; Blood plasma; Biomarker
