Development and Analytical Validation of a Rapid Three-Step NOHA Assay for Blood-Based Assessment of Estrogen Receptor Status in Breast Cancer

Estrogen receptor (ER) status is a key determinant of breast cancer prognosis and treatment selection, yet standard tissue-based methods such as immunohistochemistry (IHC) are limited by infrastructure demands, turnaround time, and poor suitability for longitudinal monitoring. Blood-based biomarkers offer a minimally invasive alternative. Nω-hydroxy-L-arginine (NOHA), a stable intermediate of nitric oxide synthase activity, has been associated with ER tumor biology and represents a potential circulating indicator. We report here the analytical validation of a simplified three-step, equipment-free colorimetric assay for NOHA and evaluate its performance against a validated NOHA enzyme-linked immunosorbent assay (ELISA). The assay employs a monoclonal NOHA antibody, horseradish peroxidase (HRP)-conjugated 10-mer NOHA, and a molecular weight cut-off filtration step. Validation was conducted using buffer samples (1–15 ng/mL) to assess sensitivity, selectivity, precision, dilution linearity, and recovery. Reproducibility was evaluated across operators and days. Selectivity was tested against structurally related amino acids. Plasma samples from breast cancer patients with known IHC-classified ER status (n = 50 per group) and healthy controls (n = 50) were analyzed and compared with ELISA-derived NOHA concentrations. A five-point visual color scale was developed for semi-quantitative classification. The assay demonstrated adequate sensitivity across the tested range, high selectivity with no detectable interference, and consistent intra- and inter-day reproducibility. Linearity and recovery were within acceptable limits. The color scale correlated with ELISA measurements and ER status, with strong concordance between methods (R² = 0.98, p < 0.05). Plasma analysis enabled accurate stratification of ER-positive and ER-negative cases consistent with IHC, with minor ambiguity at color category thresholds. This three-step NOHA assay provides a rapid, low-resource, and minimally invasive approach for ER status assessment. Its agreement with ELISA quantification and IHC classification supports its analytical validity. The simplicity and portability of the method suggest potential utility in decentralized and point-of-care settings, although further clinical validation and stability studies are required.