Antibodies are among the most frequently used tools in basic science research and in clinical assays, but there are no universally accepted guidelines or standardized methods for determining the validity of these reagents. Furthermore, for commercially available antibodies, it is clear that what is on the label does not necessarily correspond to what is in the tube. To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which it is to be used. In this review, we highlight the common pitfalls when working with antibodies, common practices for validating antibodies, and levels of commercial antibody validation for seven vendors. Finally, we share our algorithm for antibody validation for immunohistochemistry and quantitative immunofluorescence.
Antibodies are among the most commonly used research tools, routinely used for Western blot (WB), immunoprecipitation (IP), enzyme-linked immunosorbent assays (ELISA), quantitative immunofluorescence (QIF), and immunohistochemistry (IHC). They are also important tools in clinical management with extensive use in both laboratory medicine (ELISA assays and flow cytometry) and anatomic pathology (IHC). In anatomic pathology, IHC serves as a diagnostic, prognostic, and predictive method and IHC readings directly influence the management of patients in the clinical setting. For example, the assessment of estrogen receptor α (ER-α), and human epidermal growth factor receptor 2 (HER2) by IHC in breast cancer patients is the definitive test to determine whether or not a patient will receive therapies that can cost as much as $100,000 per year. Thus, in the clinic, as well as in the research laboratory, careful accurate validation of antibody reagents is critical for correct results.
The influence of antibody-based tests on clinical decisions has led to a number of publications that have highlighted the unmet need for standardization of such assays and development of antibody validation guidelines (1,2,3,4,5,6,7,8). Although many groups have enunciated the need, there are no universally accepted guidelines for best practice in antibody-based tests. There are a number of books on the topics by world leaders such as Clive Taylor and David Dabbs, and recently, an ad hoc group published a set of “recommendations” (2). However, these works focus on the clinical aspects of IHC, often using subjective criteria and often not taking advantage of recent scientific advances that allow more quantitative evaluation of antibodies. Conversely, there are other groups that have done biologically rigorous evaluation of antibodies using surface plasmon resonance (9) and even X-ray crystallization of antibodies bound to their antigens (10), methods that are unachievable in a routine research or clinical setting. The wide range of rigor and methodology in what is used for validation is probably responsible for a lack of consensus on a single method for antibody validation. Here we present an overview of antibody validation approaches and the pitfalls associated with the failures of validation. This work specifically focuses on assessment of prognostic and predictive cancer-related biomarkers on formalin-fixed paraffin embedded (FFPE) tissue.What is antibody validation?
The FDA defines validation as “the process of demonstrating, through the use of specific laboratory investigations, that the performance characteristics of an analytical method are suitable for its intended analytical use” (www.fda.gov/downloads/ Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.pdf).
For antibodies, one must demonstrate that they are specific, selective, and reproducible in the context for which they are used. When it comes to IHC, standardization can be quite challenging due to the number of pre-analytical, analytical, and post-analytical factors known to influence staining in FFPE tissue. Variable time to fixation, inadequate fixation period, differences in fixative used, and tissue processing can all affect tissue antigenicity (5,11). Antibody clone and dilution, antigen retrieval, detection system, and interpretation of results using different cutoff points are also important variables that regulate IHC measurements (3,12) (V.K.A., unpublished data). Here we focus on analytical factors and highlight the importance of proper antibody validation, especially for IHC or QIF use.Common pitfalls in antibody studies Nonspecific antibodies
A recent editorial by Michel et al. (13) emphasizes the lack of target specificity for 49 antibodies against 19 subtypes of G protein–coupled receptors calling for more stringent antibody validation criteria. Examples highlighted by the authors included double-knockout mice lacking the M2 and M3 subtypes of muscarinic receptors still staining positive for M2 and M3 receptor antibodies (14), and triple-knockout mice for the three α1-adrenoceptor subtypes demonstrating staining patterns similar to wild-type mice (15).
Determining the specificity of an antibody is in part dependent on the type of the immunogens: synthetic peptides or purified proteins. Synthetic peptides provide the advantage of knowing the amino acid sequence to which the antibody binds; however, these peptides do not necessarily recapitulate the 3-D structure or post-translational modifications of the native protein (16). As a result, antibodies generated against a synthetic peptide may not work well when a protein is in its native conformation with intact 3-D structure. Such antibodies may not be useful for IP or IHC experiments, but may bind the protein of interest after it is fully denatured when running SDS WB. The opposite could also be the case, especially if the immunogen was the purified protein, where the antibody works well for proteins in their native conformation, but not when denatured. Thus WB cannot be an absolute standardization for antibody binding in IHC or other assays where the antigen is in its native conformation.