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

Author:

Laflamme Carl1ORCID,McKeever Paul M23,Kumar Rahul1ORCID,Schwartz Julie1,Kolahdouzan Mahshad4,Chen Carol X1,You Zhipeng1,Benaliouad Faiza1,Gileadi Opher5ORCID,McBride Heidi M1ORCID,Durcan Thomas M1,Edwards Aled M16,Healy Luke M4,Robertson Janice23,McPherson Peter S1ORCID

Affiliation:

1. Tanenbaum Open Science Institute, Montreal Neurological Institute, McGill University, Montreal, Canada

2. Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada

3. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

4. Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Montreal, Canada

5. Structural Genomics Consortium, University of Toronto, Toronto, Canada

6. Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom

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.

Funder

ALS Society of Canada

ALS Therapy Alliance

Motor Neurone Disease Association

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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