Validation and in vivo characterization of research antibodies for Moesin, CD44, Midkine, and sFRP-1. [version 2; peer review: 2 approved with reservations]

• Published in: F1000 research 2023-01, Vol.12, p.1070
• Main Authors: Doolen, Suzanne, Ayoubi, Riham, Laflamme, Carl, Betarbet, Ranjita, Zoeller, Elizabeth, Williams, Sean-Paul G., Fu, Haian, Levey, Allan I., Sukoff Rizzo, S. J.
• Format: Article
• Language: English
• Subjects: Alzheimer's disease; Animal cognition; Antibodies; antibody characterization; Biomarkers; Brain research; Brief Report; CD44 antigen; Cell lines; eng; Frizzled protein; Frizzled-related protein; Frizzled-related protein 1; GTP-binding protein; Hypotheses; Laboratory animals; Midkine; Moesin; Molecular weight; Neurodegenerative diseases; Neuropathology; Pathogens; Proteins; sFRP-1; Therapeutic targets; Western blot
• ISSN: 2046-1402; 2046-1402
• DOI: 10.12688/f1000research.138354.2

Background A major goal of the Target Enablement to Accelerate Therapy Development for Alzheimer's disease (TREAT-AD) program is to develop and identify high-quality tools to test target or mechanistic hypotheses. As part of this initiative, it is important that commercial reagents including research antibodies being used to interrogate drug targets have confirmed validation data in knock-out cell lines. Ideally, these antibodies should also have utility for both in vitro and in vivo studies such that the levels of target proteins in target tissues can be quantified. Methods We evaluated commercial antibodies against TREAT-AD protein targets Moesin (Uniprot ID: P26038), CD44 (Uniprot ID: P16070), Midkine (Uniprot ID: P21741) and Secreted frizzled-related protein 1, referred to as "sFRP-1" (sFRP-1; Uniprot ID: Q8N474). Moesin, Midkine and sFRP-1, that were confirmed as selective based on data in knock-out cell lines. Western blot analysis was used to compare protein levels in brain homogenates from a mouse model with AD-relevant pathology (5XFAD) versus age-matched C57BL/6J control mice. Results Anti-Moesin ab52490 reacted in mouse brain homogenate with a predicted molecular weight of 68 kDa. Moesin protein expression was 2.8 times higher in 5xFAD compared to WT. Anti-CD44 ab189524 reacted with a band at the predicted size of 82 kDa. CD44 protein expression was 1.9 times higher in 5xFAD compared to WT. Anti-Midkine AF7769 reacted with a band ~16 kDa and a 17.8 times greater expression in 5xFAD compared to WT. Anti-sFRP-1 ab267466 reacted with a band at 35 kDa as predicted. sFRP-1 protein expression was 11.9 times greater in 5xFAD compared to WT. Conclusions These data confirm the utility of these selective commercially available antibodies against Moesin, CD44, Midkine, and sFRP-1 for in vivo studies in mice and provide insight into the use of 5XFAD mice for in vivo target engagement studies for these target proteins.