Non‐denaturing affinity purification of soluble oligomeric Aβ from human brain using a novel calcium‐sensitive monoclonal antibody. (1st February 2022)

• Record Type: Journal Article
• Authors: Stern, Andrew; Liu, Lei; Liu, Wen; Reczek, David; Pradier, Laurent; Batson, Megan; Kathuria, Sagar; Sun, Tingwan; Selkoe, Dennis J
• Is Part Of: Alzheimer's & dementia. Volume 17(2021)Supplement 3
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-02-01
• Subjects: Alzheimer's disease -- Periodicals; Alzheimer Disease -- Periodicals; Dementia -- Periodicals; Démence; Maladie d'Alzheimer; Périodique électronique (Descripteur de forme); Ressource Internet (Descripteur de forme); 616.83
• DOI: 10.1002/alz.051725
• Languages: English
• ISSNs: 1552-5260

Abstract

Abstract: Background: The soluble fraction of human AD brain extracts contains the most bioactive oligomeric forms of Aβ (oAβ), which are low in abundance. Few methods exist to enrich for oAβ from soluble brain extracts without denaturing them. Purification of natural, human oAβ would allow further structural and biochemical study of this truly disease‐relevant species. Method: A panel of anti‐oAβ monoclonal antibodies was developed by immunizing Trianni mice with synthetic Aβ aggregates. Clones were screened for oAβ selectivity and protection against human oAβ‐induced injury in an in vitro neuritic integrity assay and in mouse hippocampal slice LTP recordings. Four antibodies with selectivity for oAβ over monomeric Aβ and protective effects were chosen for further study. Quantitative immunoprecipitations (IP followed by guanidine denaturation and monomer‐specific ELISA) were performed on soluble extracts obtained by soaking cortical fragments bits in TBS to enrich for bioactive diffusible oAβ species. One antibody, B24, could IP oAβ only from non‐dialyzed extracts. Iterative IPs were used to identify calcium as the soluble, dialyzable factor required for B24 binding to oAβ. Differential Scanning Fluorimetry (nano‐DSF), Octet biolayer interferometry (BLI), and immunohistochemistry were further used to characterize B24 calcium‐dependence and reversibility of binding to oAβ. Result: B24 showed complete dependence on calcium at low millimolar levels for binding to soluble human oAβ, plaques, and synthetic Aβ protofibrils. Calcium induced a conformational change in B24. Binding of human soluble brain extracts to B24, washing, and elution with EGTA resulted in enrichment of oAβ. The recovered oAβ accounted for 20‐40% of the initial input oAβ, accompanied by >400‐fold reduction in total protein content. The recovered oAβ required guanidine hydrochloride for denaturation and detection by Aβ monomer‐specific ELISA, implying that the recovered oAβ had not been denatured during purification. Calcium did not affect the size distribution of oAβ from human brain extracts by size exclusion chromatography. Conclusion: B24 is protective against natural oAβ‐induced neuronal injury and binds oAβ in a calcium‐dependent manner. This unique characteristic allows affinity purification of bioactive oAβ from human brain extracts for further study and drug development.