As a way to address the antibody validation and reproducibility issue, some researchers are developing protocols to rigorously assess antibody performance. In a recent eLife paper and separate The Scientist opinion piece, Dr. Peter McPherson and his team at McGill University present a streamlined process employing CRISPR/Cas9-generated knockout (KO) cell lines as a platform for testing antibody validity and specificity (1, 2). They demonstrated the utility of their system by evaluating sixteen antibodies against the amyotrophic lateral sclerosis (ALS)-related protein C9ORF72.
Fig. 1. Analysis of C9ORF72 antibodies on C9ORF72 knockout cells by immunoblot (Laflamme et al, 2019).
Of these sixteen products, two mouse monoclonal antibodies from GeneTex (GTX632041 and GTX634482) were found to be outstanding, with the former performing exceptionally well for immunofluorescence and immunoprecipitation and the latter for immunoblot and immunohistochemistry (Figs. 1-3).
Fig. 2. Analysis of C9ORF72 antibody (GTX632041) on C9ORF72 knockout cells by immunofluorescence and immunoprecipitation (Laflamme et al, 2019).
Fig. 3. Analysis of C9ORF72 antibody (GTX634482) on C9ORF72 knockout mouse tissue by immunohistochemistry (Laflamme et al, 2019).
Using these GeneTex C9ORF72 antibodies, the authors successfully address the controversies regarding the protein’s expression and subcellular distribution, leading to the discovery of a cytosolic pool of C9ORF72 in macrophages. Interestingly, C9ORF72 was also found to accumulate on late phagosomes and phagolysosomes in macrophages, a finding that may offer new perspective on ALS pathogenesis. Thus, in addition to describing a general validation mechanism applicable to any antibody against a target amenable to CRISPR-mediated knockout, McPherson and colleagues identify two superior C9ORF72 antibodies (GeneTex antibodies GTX632041 and GTX634482) to accelerate ALS research.