: Can you share your perspective on the reproducibility issue associated specifically with the use of antibodies?
MS: I think researchers have known for some time that not all commercial antibodies are created equally, but this issue has not received serious public attention until recently. Some commercial antibodies are of very good quality. They are highly specific for their target, they are sensitive enough to be used in a meaningful way, and they have good lot-to-lot consistency. However, an alarmingly high percentage of commercial antibodies have poor specificity, low sensitivity, or both, and may not even work in any immune assay at all. Poorly performing antibodies can lead researchers to make erroneous conclusions based on data from off-target binding of the antibody. Even if an antibody works well in an immunoassay, poor lot-to-lot consistency may make it impossible to replicate experiments. Because antibodies are such commonly used yet critical reagents, poor performance can waste time, resources, and put the quality of life science research at risk.
: Can you tell us what your company is doing on a routine basis to make sure you are testing and validating the antibodies you manufacture or obtain from other sources?
MS: At Bio-Rad, we have been focusing on setting standards for validating antibodies for Western blotting. We decided to focus on Western blotting because it is one of the most widely used immunoassays in the life sciences. All of our antibodies are tested in Western blotting on cell or tissue lysates that express the native target at endogenous levels. In order for an antibody to pass our validation, it must detect its target protein with minimal non-specific binding. An antibody will not pass our validation if it strongly detects a protein in samples not expected to contain the target protein. Finally, the antibody must be sensitive enough that it doesn’t require overexpression or purification of the target protein in order for detection. Only 10–20% of the antibodies that we test pass these criteria, ensuring high specificity and sensitivity. Once we have validated an antibody for a particular assay, we validate every new batch of the antibody using the identical assay to ensure lot to lot consistency. For more information from Bio-Rad refer to this article: http://www.bioradiations.com/how-does-an-antibody-qualify-to-become-a-precisionab-antibody/
: Do you have any advice, recommendations, and/or best practices scientists should follow when selecting and validating an antibody?
MS: Scientists should first critically review any data provided by the supplier for the applications in which they intend to use the antibody. In the case of Western blotting data, scientists should look for antibodies that detect the endogenous target protein in the appropriate samples (i.e., only in cell or tissue lysates that are expected to express the target protein). No matter how good the data looks, scientists should then retest the antibody in their lab using both positive controls and negative controls. In order for scientist to easily replicate the supplier’s experiments, scientists should look for suppliers that provide sufficient information on the antibody, including physical properties (e.g., host species, clonality, purification) what target it recognizes and the protocol used to test the antibody.
: What are your thoughts on the current efforts that are underway to tackle the reproducibility problem? Do you think they are realistic and implementable?
MS: The reproducibility issue is a significant one that we must tackle immediately. It is a daunting task to execute uniform standards of quality to ensure accountability and reproducibility. I must commend current efforts from all sections of academia, pharma, and reagent vendors aimed at dealing with it. If we ignore this issue too long, it won’t go away on its own and is likely to get worse, leaving us with a growing number of irreproducible experiments and incorrect conclusions. Even though it is a huge challenge to come up with an effective system to monitor antibody quality that is realistic to implement, this is not something that we should avoid. I don’t think anyone believes that the reproducibility issue will be solved overnight. However, as we bring awareness to this issue and start to seek solutions, researchers will start to understand the value of highly validated antibodies and demand high quality reagents, which in turn will drive the market to meet this demand.
: What else is your company doing to address reproducibility?
MS: Several of my colleagues at Bio-Rad and myself are actively involved in the efforts of the Global Biological Standards Institute (GBSI) to solve the reproducibility crisis by trying to define antibody validation standards in qualitative and quantitative terms. We participate in regular meetings, discussions, and working groups led by the GBSI. We are also looking for ways in which we can improve our own antibody validation, for example by using gene disruption technologies like CRISPR to eliminate expression of the antibody target. Finally, we are working with our customers so that they are aware of the importance of using high-quality reagents, such as validated antibodies, in properly controlled experiments.
Author Bio: Mark Shulewitz, Ph.D., received his BS in Molecular Biology from the University of Pittsburgh and his Ph.D. in Molecular and Cell Biology from the University of California, Berkeley. He did post-doctoral research at the Weizmann Institute of Science and Genentech, Inc. and held several industry positions focused on developing and validating monoclonal antibodies before joining Bio-Rad Laboratories, Inc.