Breast cancer is the leading form of diagnosed cancer in American women in 2009. HER-2/neu is a biomarker which is overexpressed in 25–30% of patients with aggressive breast cancer. The extracellular domain (ECD) of this HER-2/neu protein is shed from the surface of breast cancer cells and found in circulation. An increased level (>15 ng/mL) in the shed extracellular domain (sECD-HER 2/neu) is indicative of the potential presence and associated progression of this disease. A fluorescent ELISA incorporating the newly developed ALYGNSA antibody-orientation system revealed a 10-fold increase in sensitivity (≤0.63 ng/mL) of sECD-HER 2/neu when compared to a control standard ELISA kit (≤7.5 ng/mL) This enhanced mode of detection has the potential to not only address breast and other cancers per se but also permit an in depth evaluation of "shed extracellular domains", in general, and the role of these "proteolytic derived factors" in physiological signaling at normal levels. An additional breast cancer biomarker, CA 15-3 was also investigated. CA 15-3 is a large transmembrane glycoprotein encoded by the MUC-1 gene and the protein is frequently overexpressed in malignant breast tumors. An increased level of the shed extracellular domain (>35 U/mL) of the CA 15-3 protein in serum is used for surveillance of patients with diagnosed breast cancer and monitoring therapy in advanced disease. The newly developed sECD-CA 15-3 ALYGNSA assay revealed a 16-fold increase in sensitivity (≤0.94 U/mL) of CA 15-3 when compared to a commercial ELISA kit (≤15 U/mL). Partial work, including sECD-HER 2/neu ALYGNSA assay results have been previously published by Chourb S., Mackness BC., Farris LR., McDonald MJ. (2009) Enhanced immuno-detection of shed extracellular domain of HER-2/neu. HEALTH 1:325-329.