Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72

• DOI: 10.7554/elife.48363
• Publication Date: 2019-10-15T00:00:25Z
• Authors: Carl Laflamme; Paul M McKeever; Rahul Kumar; Julie Schwartz; Mahshad Kolahdouzan; Carol X Chen; Zhipeng You; Faiza Benaliouad; Opher Gileadi; Heidi M McBride; Thomas M Durcan; Aled M Edwards; Luke M Healy; Janice Robertson; Peter S McPherson
• Keywords: 0301 basic medicine; amyotrophic lateral sclerosis; QH301-705.5; Science; 610; C9ORF72; Gene Expression; Mice, Transgenic; Mice; 03 medical and health sciences; Phagocytosis; antibody; Cell Line, Tumor; Phagosomes; Animals; Humans; Biology (General); Antibody; Gene Editing; Osteoblasts; C9orf72 Protein; Macrophages; Q; Amyotrophic Lateral Sclerosis; R; phagocytosis; Antibodies, Monoclonal; Cell Biology; Amyotrophic lateral sclerosis; Lysosome; Immunohistochemistry; macrophages; 3. Good health; Mice, Inbred C57BL; HEK293 Cells; RAW 264.7 Cells; Frontotemporal Dementia; lysosome; Medicine; CRISPR-Cas Systems; Lysosomes; Biomarkers

Antibodies are a key resource in biomedical research yet there are no community-accepted standards to rigorously characterize their quality. Here we develop a procedure to validate pre-existing antibodies. Human cell lines with high expression of a target, determined through a proteomics database, are modified with CRISPR/Cas9 to knockout (KO) the corresponding gene. Commercial antibodies against the target are purchased and tested by immunoblot comparing parental and KO. Validated antibodies are used to definitively identify the most highly expressing cell lines, new KOs are generated if needed, and the lines are screened by immunoprecipitation and immunofluorescence. Selected antibodies are used for more intensive procedures such as immunohistochemistry. The pipeline is easy to implement and scalable. Application to the major ALS disease gene C9ORF72 identified high-quality antibodies revealing C9ORF72 localization to phagosomes/lysosomes. Antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over previously reported C9ORF72 properties.