e22099 Background: Growth in biomarkers as therapeutic targets and as surrogate markers for efficacy presents a need for increasingly sensitive immunoassays to expand biomarker applicability. Improved immunoassays will provide: (1) better evaluation and validation of new drug candidates, (2) better matching of patients to new therapies, (3) accelerated drug approval (4) earlier diagnosis of at-risk patients, and (5) a deeper understanding of cancer biology. Towards this end, Singulex has developed two ultra- sensitive VEGF Immunoassays for human and mouse vascular endothelial growth factor (VEGF). Here we report the preliminary validation of these two novel assays. Methods: Two novel assays were developed with the Erenna Immunoassay System for detecting VEGF: human (hVEGF) and mouse (mVEGF). Analytical sensitivity, cross-reactivity and precision were determined and compared to an ELISA based VEGF assay. Both the Singulex assay and ELISA assay were used to test a range of specimen types (plasma, cell lysates, conditioned media, and tissue specimens) from humans and mice. Preliminary assays with human plasma and tissue specimens were conducted to compare hVEGF levels between normal and breast cancer samples. Results: The Singulex hVEGF assay had an LOD of 0.1 pg/mL, an LLOQ of 0.3 pg/mL, and 84–107% spike recovery; 90X more sensitive than the ELISA assay. Human VEGF concentrations were quantified in all specimens tested compared to the ELISA, which quantified VEGF in only 8% of plasma samples, but all of the cell lysate samples. The Singulex mVEGF assay had an LOD of 3.5 pg/mL, LLOQ of 5 pg/mL, and 68–111% spike recovery; 3X more sensitive than the ELISA assay. Cross-reactivity for the two assays was minimal for all specimen types tested, except for human plasma samples where the mVEGF assay demonstrated 80–100% CR. Conclusions: We show that the Singulex hVEGF and mVEGF Immunoassays can detect VEGF at or below pg/mL levels, and can effectively quantify VEGF levels in plasma, cell lysates, conditioned media, and tissue samples from mice and humans. These novel assays are an important tool when used to assess tumor and normal breast cancer tissue and plasma. [Table: see text]