Estrogen-negative (ER-) breast cancer, is recognized as an aggressive subtype, more difficult to treat, with poor survival and prognosis. They are hormonally unresponsive, with no readily effective and specific target therapy. We have previously identified Nw-hydroxy L-Arginine (
NOHA) as a blood-based biomarker to distinguish between ER- and ER+ breast cancer tumors based upon disease burden, progression and molecular phenotype (U.S. Utility Patent 10,073,099). In this study we have demonstrated a competitive ELISA based assay for
NOHA measurement using a proprietary monoclonal antibody (mAb) specific for
NOHA (U.S. provisional patent 62/754,053). The ELISA assay was evaluated on sensitivity, selectivity, precision, dilution linearity and percent recovery parameters. The assay showed sensitivity at ≥60 pg/ml
NOHA antigen with 1 ng/ml
NOHA mAb, and maintained
NOHA antigen specificity even in the presence of other closely related cationic amino acids (i.e. L-Arginine, D-Arginine, l-Lysine, d-Lysine, L-Ornithine, and L-Citrulline). The reliability of the ELISA protocol was confirmed with the low percent-covariance, for all tested parameters of sensitivity (≤8.2%), selectivity (≤8.6%), precision (≤12.6%), dilution linearity (≤11.2%) and recovery (≤6.7%). Additionally, we can demonstrate
NOHA quantification by this ELISA assay to complement the sensitivity achievable with LC-MS (in both assay buffer and with patient plasma samples), thus suggesting it\'s utility as a simple yet sensitive methodology that might help in ER- breast cancer prognosis, and disease progression monitoring without the need for expensive analytical equipment (such as LC-MS), large lab space, or specialized technical training.