Abstract
Abstract Personalized cancer treatment strategies depend on comprehensive and detailed characterization of individual human cancers. Translation of biomarker research into successful and tailored medical therapy at tumor and patient level require the identification and development of informative biomarkers that could be used to improve early diagnosis and prognosis. In clinical pathology, particularly IHC evaluation of biomarkers in tissue is considered as gold standard for diagnostic and therapeutic decisions, having a direct influence on patient management and therapeutic treatment. Although antibody-based approaches are established and successfully integrated into both clinical and research applications, for personalized treatment regimens new demands have been placed on the quality, reproducibility and accuracy of antibody-based assays. There is an ongoing debate on implementation and standardization of validation guidelines to provide for reliable and reproducible antibody-based tools in clinical management and basic research. To overcome variations of epitope specificity in immunohistochemistry, we introduce a novel approach combining extraction of immunoreactive proteins from FFPE tissue with Western Blot analysis and IHC using the same archival tissue of interest for antibody validation. This tandem application allows for specific characterization of the antigen, taking advantage of the main properties of both technologies: localization of the protein of interest in tissue sections stained by IHC and specification of the biochemical identity by molecular weight analysis in immunoblot experiments. We assessed a panel of antibodies used in both, academic research as well as routine diagnostics for their usability across various validation platforms. While a significant proportion of the antibodies tested showed reliable specificity in both assays, some proved to be unsuited for dual application in IHC and WB, failing in either assay. The results presented here, reflect the heterogeneity in antibody specificity and emphasize the advantage of combining a series of suitable methods to ensure reproducibility and specific epitope detection. Based on our results presented here, we propose an improved step by step strategy to validate antibody immunoreactivity and control for variability across protocols. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4875. doi:10.1158/1538-7445.AM2011-4875
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