Antibody validation

Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA.
BioTechniques (Impact Factor: 2.95). 03/2010; 48(3):197-209. DOI: 10.2144/000113382
Source: PubMed


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.

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    • "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. "
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    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 · 3.05 Impact Factor
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    • "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. "
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    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 · 14.63 Impact Factor
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    • "Each antibody was validated by performing 1) titering, 2) reproducibility assessment on index arrays, and 3) verification of linearity with expression on cell line series, according to a previously described protocol [28]. The immunofluorescence staining was performed by using a standard protocol. "
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    ABSTRACT: Background We sought to determine the predictive value of in situ mRNA measurement compared to traditional methods on a cohort of trastuzumab-treated metastatic breast cancer patients. Methods A tissue microarray composed of 149, classified as HER2-positive, metastatic breast cancers treated with various trastuzumab-containing chemotherapy regimens was constructed. HER2 intracellular domain(ICD), HER2 extracellular domain(ECD) and HER2 mRNA were assessed using AQUA. For HER2 protein evaluation, CB11 was used to measure ICD and SP3 to measure ECD of the HER2 receptor. In addition, HER2 mRNA status was assessed using RNAscope assay ERRB2 probe. Kaplan – Meier estimates were used for depicting time-to-event endpoints. Multivariate Cox regression models with backward elimination were used to assess the performance of markers as predictors of TTP and OS, after adjusting for important covariates. Results HER2 mRNA was correlated with ICD HER2, as measured by CB11 HER2, with ECD HER2 as measured by SP3 (Pearson’s Correlation Coefficient, r = 0.66 and 0.51 respectively) and with FISH HER2 (Spearman’s Correlation Coefficient, r = 0.75). All markers, HER2 mRNA, ICD HER2 and ECD HER2, along with FISH HER2, were found prognostic for OS (Log-rank p = 0.007, 0.005, 0.009 and 0.043 respectively), and except for FISH HER2, they were also prognostic for TTP Log-rank p = 0.036, 0.068 and 0.066 respectively) in this trastuzumab- treated cohort. Multivariate analysis showed that in the presence of pre-specified set of prognostic factors, among all biomarkers only ECD HER2, as measured by SP3, is strong prognostic factor for both TTP (HR = 0.54, 95% CI: 0.31–0.93, p = 0.027) and OS (HR = 0.39, 95%CI: 0.22–0.70, p = 0.002). Conclusions The expression of HER2 ICD and ECD as well as HER2 mRNA levels was significantly associated with TTP and OS in this trastuzumab-treated metastatic cohort. In situ assessment of HER2 mRNA has the potential to identify breast cancer patients who derive benefit from Trastuzumab treatment.
    PLoS ONE 06/2014; 9(6):e99131. DOI:10.1371/journal.pone.0099131 · 3.23 Impact Factor
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