Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72
- PMID: 31612854
- PMCID: PMC6794092
- DOI: 10.7554/eLife.48363
Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72
Abstract
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.
Keywords: C9ORF72; amyotrophic lateral sclerosis; antibody; cell biology; human; lysosome; macrophages; mouse; neuroscience; phagocytosis.
© 2019, Laflamme et al.
Conflict of interest statement
CL, PM, RK, JS, MK, CC, ZY, FB, OG, HM, TD, AE, LH, JR, PM No competing interests declared
Figures















References
-
- Andrews NP, Boeckman JX, Manning CF, Nguyen JT, Bechtold H, Dumitras C, Gong B, Nguyen K, van der List D, Murray KD, Engebrecht J, Trimmer JS. A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain. eLife. 2019;8:e43322. doi: 10.7554/eLife.43322. - DOI - PMC - PubMed
-
- Aoki Y, Manzano R, Lee Y, Dafinca R, Aoki M, Douglas AGL, Varela MA, Sathyaprakash C, Scaber J, Barbagallo P, Vader P, Mäger I, Ezzat K, Turner MR, Ito N, Gasco S, Ohbayashi N, El Andaloussi S, Takeda S, Fukuda M, Talbot K, Wood MJA. C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia. Brain. 2017;140:887–897. doi: 10.1093/brain/awx024. - DOI - PubMed
-
- Atanasio A, Decman V, White D, Ramos M, Ikiz B, Lee HC, Siao CJ, Brydges S, LaRosa E, Bai Y, Fury W, Burfeind P, Zamfirova R, Warshaw G, Orengo J, Oyejide A, Fralish M, Auerbach W, Poueymirou W, Freudenberg J, Gong G, Zambrowicz B, Valenzuela D, Yancopoulos G, Murphy A, Thurston G, Lai KM. C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice. Scientific Reports. 2016;6:1–14. doi: 10.1038/srep23204. - DOI - PMC - PubMed
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Medical
Molecular Biology Databases
Research Materials
Miscellaneous