Development of a novel in-house blood biomarker panel for early diagnosis of Alzheimer’s disease

Background: Currently a definitive diagnosis for Alzheimer's disease (AD) is not readily available to the wider community as the gold standard markers of AD are either invasive and/or expensive. Therefore, there is an urgent need for a relatively low-cost blood test that can be readily used in clinical settings. This study aims to address this gap by developing a reliable and cost-effective in-house blood-based biomarkers panel for routine use in clinical labs for early detection of AD. Our team have shown that specific proteins in the blood, namely phosphorylated tau and Glial Fibrillary Acidic Protein (GFAP) as biomarker for early AD diagnosis. However, only a combination of selected blood biomarkers can reliably predict who is at risk of AD. To achieve a combination diagnostic assay based on robust blood biomarkers, this study will first develop singleplex diagnostic Single Molecule Array (Simoa) blood biomarkers assays for phosphorylated Tau: p -Tau217 and pTau205, as well as the N- and C-terminus of GFAP. A novel panel of the best 3 biomarkers will then be selected to establish a blood test for preclinical diagnosis of AD. Method: Monoclonal antibodies against p -Tau217, p -Tau205, N- and C-terminus of GFAP were generated in collaboration with GenScript, Singapore. These antibodies have undergone initial characterisation with controlled primary assessment of specificity and affinity (i.e immunostaining, immunoprecipitation-Western Blot, and immunoprecipitation-mass spectrometry) using various tissues such as human cells expressing phospho-site-deficient tau as negative controls, human brain, CSF, and plasma from healthy controls and AD. Finally, these antibodies were developed for Simoa assay in collaboration with University of Gothenburg. Result: We have successfully generated specific high affinity monoclonal antibodies against p -Tau217, p -Tau205, and N-terminus GFAP and have confirmed that these antibodies have the necessary specificity and sensitivity for the Simoa platform. Conclusion: The development of Simoa assays will be validated in a highly characterised control and Alzheimer cohort The Australian Imaging, Biomarker, and Lifestyle (AIBL) and a novel panel of the best 3 biomarkers established.