An article published by Nature earlier this year pointed out that researchers now have to worry about the reproducibility of production antibodies. The fact of the matter is that off-the-shelf antibodies from large distributors can be unreliable tools, often functioning incorrectly or completely failing. Though it may not be the antibodies at fault, it does not change the fact that prudence must be observed when purchasing and using these reagents. Antibodies are finicky molecules due to their high binding specificity, and even one discrepancy in either production or application can completely alter the result of an experiment.
As a provider, the ProSci team feels the most important step to resolving this conflict is to educate customers on what causes problems with reproducibility and how to avoid letting them disrupt your work.
Nature outlined 3 possible causes of reproducibility complications:
Cross reactivity: Some antibodies’ paratopes have less specific binding ability than others, and are able to bind to several other types of proteins with similar epitopes. This can result in labeling of incorrect molecules.
Variability: Antibodies made from one group of animals can have slightly different structures than antibodies made from other groups. Even polyclonal antibodies isolated from the same animal at different bleed times may characterize differently. Both antibody types may bind to the same epitope which allows them to be isolated with the same reagents; however, the two molecules may not always bind to other similar epitopes. This may result in failure to mark desired epitopes.
Application: Different experimental conditions can change the shape of a protein, altering the epitope and preventing the antibody from binding. This can be one burden of highly specific antibodies: they may work well in one application and not in another, as the epitope can be presented differently and not be recognizable.
Each of these 3 possible causes can significantly impact an experiment, especially in the case of poorly studied antibodies that are not fully understood. But that does not mean that antibodies are poor reagents; it does mean that they should be understood so that you may select the desired product and avoid cross reactivity, variability, or application based errors.
When purchasing production antibodies, keep these questions in mind:Application
1. Are there any similar proteins that this product may have cross reactivity with? Are they present in my experiment?
2. What types of validations are listed for the product? Do they provide sufficient evidence to avoid cross reactivity in my experiment?
3. Does the company provide data on lot-to-lot consistency as evidence of reproducibility?
4. Am I running experiments with conditions that could change the shape or reactivity of my target? How will those different conditions affect the experiment?
5. Is the product I am purchasing coming from the original manufacturer? Is it possible that two different manufacturers could be producing the same antibody with slight variability?
6. Should I consider antibody development through recombinant antibody generation or custom antibody services to lower the risk of reproducibility issues? Recombinant antibodies are created with known sequences of DNA in vitro, the possibility of variability. Custom antibodies also less likely to be cross reactive or variable since they are specifically designed to target individual epitopes while avoiding others.
On top of these questions, be sure to consider additional validations that you can perform. Validation can be performed by testing antibodies with a sample purchase or additional Assay services like ELISA or western blot with the proper loading controls. Testing your antibody with these tools will allow you to see that the products you order function as expected before making a large purchase. ProSci offer 20ug sample sizes to validate your antibody ( View ProSci’s full range).
References:
Baler, Monya. “Reproducibility Crisis: Blame It on the Antibodies.” Nature.com. Nature Publishing Group, 19 May 2015. Web.
Harris, Richard. “U.S. Science Suffering from Booms and Busts in Funding.” NPR.org. NPR. 9 September 2014. Web