: Can you share your perspective on the reproducibility issue associated specifically with the use of antibodies?
JS: Antibodies are key reagents in all cell analysis applications, but their widespread use has also resulted in them being considered commodity products. Thus, although researchers still spend a lot of money on antibodies, they don’t necessarily spend the required time in validating and transparently reporting the usage of antibodies. Also, the research antibody market has become extremely crowded with multiple vendors selling the same clone. However, there are no universal standards for assessing the quality of an antibody sold by different vendors. This leads to a situation where there are no checks on vendors as to whether they developed a product with the highest quality guaranteed to give specific, sensitive and highly reproducible results.
: 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?
JS: The validation approaches vary from antibody to antibody and are highly dependent on the marker and the expression pattern of the marker. To test the specificity of an antibody, we use various approaches like siRNA knockdown, overexpression of the target protein and related proteins in cell lines, or cross-blocking or staining with other antibodies that recognize nonrelated epitopes. In addition, all antibodies are tested on cells—wherever possible on primary cells—to evaluate the sensitivity in detecting endogenous levels of the antigen.
The majority of the mouse antibodies are also validated with several primary tissue samples such as organs or tumor tissues. This gives us an idea of whether the sensitivity might be compromised when the tissue undergoes a harsh dissociation process. Another test, which is integral to our production and QC processes, is lot-to-lot testing of the antibodies. During production, the performance of the new lot is set to match the performance of the last lot. At the quality control department the lot-to-lot performance is tested again, but after the product has gone through one round of storage at our inventory site. This mimics the conditions at the customer site.
: Do you have any advice, recommendations and/or best practices scientists should follow when selecting and validating an antibody?
JS: The scientist should definitely confirm when they are purchasing an antibody from a commercial vendor that the antibody has been validated for the intended application. It is always advisable to check the data on the vendor’s site and when in doubt one should request further validation data by the vendor. Once bought, the antibody might need further validation depending upon the context of the researcher’s application. The recently published article in Nature methods and the upcoming guidelines from GBSI could be a good start to validate the antibody on the user’s/researcher’s end.
: What are your thoughts on the current efforts that are underway to tackle the reproducibility problem? Do you think they are realistic and implementable?
JS: GBSI has set a very ambitious goal, as it is a tough task to define universal guidelines that are valid for different cell types, different experimental conditions and even different types of researchers. However, given the fact that there is an absence of any standards at all, the GBSI initiative is definitely a step toward improving reproducibility. It will provide companies and researchers a common framework to test and validate different antibodies. Also, it is just the beginning, and I am sure that once grant agencies and funding bodies start enforcing antibody validation guidelines, the current scattered efforts will become a global mission.
: What else is your company doing to address reproducibility?
JS: Miltenyi Biotec has shifted all the processes for generating antibodies for flow cytometry toward making them recombinantly. They are sold under the brand name of REAfinityTM Recombinant Antibodies. Recombinantly generated antibodies possess highly defined characteristics and are produced under standardized culture conditions leading to products that are reproducible in nature. This saves users the time associated with standardizing analysis and decreases the financial burden associated with working with inconsistent reagents. Recombinant technology also opens up myriads of possibilities to improve antibody properties. REAfinity Antibodies provided by Miltenyi Biotec are engineered to lack background binding and share a single isotype. This solves the two common problems associated with flow cytometry: nonspecific signals and complicated experiment planning with multiple antibody isotypes.