Article

Antibody validation

Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA.
BioTechniques (Impact Factor: 2.75). 03/2010; 48(3):197-209. DOI: 10.2144/000113382
Source: PubMed

ABSTRACT 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.

1 Follower
 · 
111 Views
  • Source
    • "Accordingly, the year 2014 saw a myriad of technical papers published, covering a wide array of research areas. Antibody characterization for immunohistochemical/ immunoelectron microscopic investigations continues to be a popular topic in many areas of cell biological research (see, for instance, Bordeaux et al. 2010). Griffiths and Lucocq (2014) provided a timely review of the major issues associated with the use and validation of primary antibodies for immunolabeling at both the light and electron microscopic level. "
    [Show abstract] [Hide abstract]
    ABSTRACT: This review encompasses a brief synopsis of the articles published in 2014 in Histochemistry and Cell Biology. Out of the total of 12 issues published in 2014, two special issues were devoted to "Single-Molecule Super-Resolution Microscopy." The present review is divided into 11 categories, providing an easy format for readers to quickly peruse topics of particular interest to them.
    Histochemie 03/2015; 143(4). DOI:10.1007/s00418-015-1313-7 · 2.93 Impact Factor
  • Source
    • "The central role of antibodies for the characterization of histone PTMs in chromatin research makes the quality and reliability of these reagents a very important scientific issue. In general, antibodies are very powerful and important reagents in biomolecular research, but the validation of commercial antibodies is not always sufficiently rigorous (Bordeaux et al. 2010). This is particularly important in the chromatin field, where specific recognition and discrimination of subtle epitopes defined only by the presence of distinct PTMs is required. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies.
    Genome Research 10/2014; DOI:10.1101/gr.170985.113 · 13.85 Impact Factor
  • Source
    • "The choice of using either monoclonal or polyclonal primary antibodies for IHC further complicates the issue of epitope specificity and determining which antibody would be more suitable for IHC (Bordeaux et al., 2003). Polyclonal antibodies are collection of antibodies targeted against multiple epitopes of a particular antigen. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The use of immunohistochemistry (IHC) in clinical cohorts is of paramount importance in determining the utility of a biomarker in clinical practice. A major bottleneck in translating a biomarker from bench-to-bedside is the lack of well characterized, specific antibodies suitable for IHC. Despite the widespread use of IHC as a biomarker validation tool, no universally accepted standardization guidelines have been developed to determine the applicability of particular antibodies for IHC prior to its use. In this review, we discuss the technical challenges faced by the use of immunohistochemical biomarkers and rigorously explore classical and emerging antibody validation technologies. Based on our review of these technologies, we provide strict criteria for the pragmatic validation of antibodies for use in immunohistochemical assays.
    Molecular Oncology 06/2014; 8(4). DOI:10.1016/j.molonc.2014.03.008 · 5.94 Impact Factor
Show more