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January 01, 2016 (Vol. 36, No. 1)

Reliable Antibodies for Reproducible Western Blots

New Line of Antibodies Raises Industry Standards for Quality

  • Techniques for investigating biological processes have advanced rapidly over the years. Yet, the humble Western blot—a technique first published in 19791—remains a laboratory standard. Despite their simplicity, Westerns continue to provide a unique window into the function of individual cells, signaling which proteins are present and in what abundance. Such findings have greatly advanced our understanding of human biology.

  • The Reproducibility Problem

    Western blots (or protein immunoblots) have high sensitivity, require no advanced equipment, and are relatively easy to perform. However, critical to their success is the primary antibody, a reagent that is all too well known for its unreliability2.

    In a sea of cellular proteins, the primary antibody must find and exclusively bind the target. Unfortunately the specificity of commercial antibodies is far from guaranteed.

    Two problems continue to plague the antibody industry and have greatly contributed to the so-called data reproducibility crisis3,4.

    The first is the variability of the manufacturing process, which draws on an animal immune response. As a result, antibody specificity can vary from animal-to-animal (i.e., batch-to-batch). While ongoing research seeks to improve this process, it’s likely to remain the industry standard due to its affordability.

    The second challenge is that antibodies have a huge scope of applications and their performance can be affected by a range of study variables such as cell or tissue type and sample-preparation methods. In a way, the utility and versatility of antibodies comes at the cost of reliability.

    Manufacturing variability can be controlled by rigorous screening and vendor validation. However the range of potential applications makes it impossible to guarantee product performance in the hands of each user.

  • Human Impact

    Click Image To Enlarge +
    Figure 1. Immunoblot results that invalidate several commercial anti-Epo-R antibodies. The 59-kDa protein detected by M-20 in MCF-7 cells is not bound by other anti-Epo-R antibodies.5

    Steven Elliott, Ph.D., former scientific executive director at Amgen, encountered antibody failure first-hand. For over a decade, Dr. Elliott studied every aspect of erythropoietin receptors (Epo-R) expressed on the surface of hematopoietic cells. In 2001, Dr. Elliott’s team successfully developed a new biologic—Aranesp—that stimulated Epo-R as a treatment for severe anemia.

    After Aranesp’s regulatory approval, a number of researchers published papers claiming that functional Epo receptors were present on a range of tissue types, including tumors.

    Dr. Elliott knew from experience that the published findings were incorrect. After extensive research and testing of the antibodies used in those findings, he determined that they were binding to multiple targets and generating false-positive results. His work showed no functional Epo receptors were present in significant levels outside of hematopoietic cells (Figure 1).

    Unfortunately by then the groundswell of incorrect findings had already been used to justify new clinical trials, which were ultimately unsuccessful. The failure of poorly validated antibodies had directly impacted the lives of patients who were given hope for a potential treatment.

  • Solution

    How can these negative outcomes be avoided? To ensure that antibodies are trustworthy and reproducible, manufacturers must work to produce quality reagents.

    In 2015, Bio-Rad Laboratories entered the antibody market with these challenges in mind. We released PrecisionAb™, a new line of primary antibodies that tackle the key issues of unreliability, insufficient vendor validation, and an overall lack of data.

  • Rigorous Quality Control

    Manufacturing is an imperfect process, but with proper gatekeeping vendors can ensure only top-quality products reach the market. Bio-Rad’s PrecisionAb Antibodies are screened using whole cell lysates from up to 12 different biologically relevant cell lines —by far the industry’s most rigorous validation process. Only antibodies that detect endogenous protein levels with high sensitivity and specificity are included in the product line. Each new lot is also screened before release to ensure batch-to-batch consistency.

  • Presenting More Data

    Click Image To Enlarge +
    Figure 2. Validation data for two antibodies (courtesy of Bio-Rad): A. Rabbit anti-Human ERK1 that failed validation due to non-specific binding and low signal-to-noise ratio. B. Rabbit anti-Human Annexin V that passed validation showing high specificity and sensitivity.

    It’s important for researchers to be able to make educated purchasing decisions when selecting from different antibody brands. However, there is currently no definitive way of determining how a reagent will perform before the vial is bought. This presents both a micro and macro problem:

    1. On an individual level the researcher is forced to “work with what they have” as few have the resources to purchase another product.
    2. At an industry level, there is little incentive for products to evolve. Scientists follow precedent, buying the antibody that is commonly cited in publications. With a lack of information, scientists cannot choose products based on performance, which would otherwise reward vendors for investing in quality.

    PrecisionAb Antibodies come with the industry’s most rigorously gathered and stringently quality controlled  validation data (Figure 2), allowing researchers to accurately assess product performance before committing to a purchase. Uniquely, Bio-Rad shows the entire gel image used for validation and includes the results from all cell lines used in validation, ensuring that researchers can clearly see relative protein expression levels and antibody performance in different cell types. The antibodies also come with positive controls, which allow researchers to readily optimize Western blotting conditions in their hands.

  • In the Hands of the User

    For Western blots, buying the right primary antibody is arguably the single-biggest factor for ensuring reproducibility and reliable results. However, much responsibility still lies with the end user. Whenever an antibody order arrives, scientists need to undertake systematic validation to ensure it works in their unique study environment. When possible, they should include positive and negative controls and blind the studies to remove bias. If these steps become routine, mistakes like the one that occurred with Epo-R will be less common. As such, Bio-Rad’s PrecisionAb Antibodies have set out to implement all these steps.

  • Damaging Science

    A range of antibody errors can occur in otherwise “routine”’ Western blots, each with different consequences:

    • Antibody failure: The antibody lacks the necessary sensitivity or affinity and produces no definable results. This can confuse early-career scientists while more experienced researchers can quickly identify the antibody as the source of the problem. In all cases, time and resources are wasted.
    • Dubious results: The antibody appears to bind the target of interest but without specificity. Multiple protein bands are present and results are not definitive. Scientists can spend their time repeating the experiment under different conditions in the hopes of achieving a better result, or crop the blot to make results look more compelling — a dangerous practice for data integrity.
    • False results: Possibly the worst outcome of all; the antibody specifically binds a protein that is not the target of interest. The researcher wrongly identifies that protein and band as the target and publishes the results accordingly, corrupting our collective scientific understanding in that field5.

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