The antibody problem
The ability of antibodies to bind specifically to antigens has been harnessed across an array of scientific fields over the years, both in laboratories and clinics. This specific binding enables scientists to utilize antibodies as "probes" that can bind to proteins in in a sample, identifying precisely where these proteins are in cells and in what quantities.
As protein production can go awry as a result of genetic mutations, this tool has been crucial in aiding our understanding of human disease and for the development of diagnostic tools. Thus, a plethora of companies focus their efforts on manufacturing antibodies as commercial products that can be sold for both scientific research and for use in a clinical setting.
A key issue that has been highlighted as of late is that some commercially available antibodies do not specifically bind to the proteins they have been manufactured to detect. This contributes to the "reproducibility crisis" that science is facing, and research studies that have previously adopted such antibodies must be called into question.
How can we better validate antibodies?
Undoubtedly, better methods are required for antibody validation to ensure that laboratories across the globe can utilize this valuable tool in their work.
A team led by Peter McPherson at The Neuro (Montreal Neurological Institute and Hospital) believe they may have found a solution.
In a new study published in eLife, McPherson and colleagues decided to use a human protein that is encoded by the gene C9ORF72 as a test case for exploring antibody validation issues and to highlight a procedure that other laboratories can adopt to validate their own antibodies.
C9O4F72 is a gene in which mutations are the major cause of the condition amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Commercially available antibodies have been used to target the subsequent protein in a range of highly cited papers, therefore evaluating the specificity of these antibodies is key.
In the publication, the authors outline their approach to testing antibody validity:
"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," suggesting that this pipeline is "easy to implement and scalable".
“We owe it to funders and patients to do better”
The research team tested a total of 16 antibodies advertised by companies as specific for C9ORF72. Using immunofluorescence, a method in which antibodies are used to stain proteins so that they are visible under a microscope, the team found that only one of the 16 antibodies accurately detected C9ORF72. Of concern is the fact that the antibodies passing the validation criteria have not yet been adopted in scientific research, but the ones that failed the validation, have.
Consequently, the study findings call into question previous work that has adopted these antibodies to detect C9ORF72 and emphasize the dire need for better antibody validation protocols.
"As we worked on our C9ORF72 paper, it became less about one gene and more about a template other labs can use to validate antibodies," says McPherson. "The procedures we use are not revolutionary, and in fact this makes our approach widely applicable to any laboratory skilled in the art, yet to my knowledge this is one of the first papers to describe a streamlined process for antibody validation. A large part of the reproducibility crisis is because of poor antibody validation. We owe it to funders and patients to do better."
Reference: Laflamme et al. 2019. Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. eLife. DOI: 10.7554/eLife.48363.