Although it's painful: The importance of stringent antibody validation

The process of making antibodies is costly and time-consuming. Commercial antibodies offer a convenient solution. However, recent concerns have resulted in a National Institutes of Health (NIH) mandate to vigorously test the specificity of antibodies used in publications (http://grants.nih.gov/reproducibility). Currently, there is no standard for validation and reference data that must be provided in publications [1, 2], and crucial specificity data are often unavailable. Multiple studies have focused on issues of antibody specificity towards proteins such as G-protein-coupled receptors, kinase receptors, and integrins [3–5]. To determine whether an antibody is suitable, the following three issues must be considered: ability to detect the target (specificity), detection of the target above background (sensitivity), and generation of consistent results (reproducibility). Sensitivity is especially problematic with low-abundance proteins, for which the antibody in question can only detect high levels of target. Problems with reproducibility often arise due to lot-to-lot variability and affect both polyclonal (pAbs) and monoclonal antibodies (mAbs). pAbs are a heterogeneous mixture of antibodies that recognize multiple epitopes of the same target protein but can also include nonspecific antibodies. Each lot, even when prepared from the same donor animal, contains diverse antibody clones and concentrations [6]. However, it is possible to reduce nonspecific binding of a pAb via immunoaffinity enrichment [7]. mAbs, although generally more consistent, are not exempt from variation. Hybridomas maintained in ascites can be contaminated with endogenous immunoglobulins and other proteins, especially if the mAb is not purified [8]. A hybridoma might also lose its antibody gene through continued passaging [7]. Additionally, epitopes that mAbs target are generally short sequences of amino acids that might exist on other proteins [8]. In one report, an mAb targeting the Met tyrosine kinase receptor—a marker of breast cancer diagnosis—revealed the target protein in the nucleus, while another lot showed membrane and cytoplasmic staining [5]. Our laboratory recently published a study focused on β6 integrin (β6), a small heterodimeric molecule involved in cell signaling [9]. The specificity of antibodies to receptors and proteins implicated in cell signaling is not well defined in the literature [6]. It is especially difficult to generate antibodies against specific integrins due to their similar structures [9], and many specification sheets report a small degree (10%–20%) of cross-reactivity with other integrins and proteins [3]. In our work, we purchased several commercial antibodies from different companies to evaluate specificity. We used antibody 1 for immunofluorescence (IFA) staining of mouse pulmonary tissues to detect β6. Initially, we detected a strong signal by IFA. However, we also detected a weaker yet concerning signal in confirmed β6 knockout (KO) mice (Fig 1A). Slides stained with only secondary antibody were negative, suggesting that the signal was not due to nonspecific binding