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.

中文翻译：

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竞争性ELISA方法用于新型雌激素阴性乳腺癌生物标志物定量。

雌激素阴性（ER-）乳腺癌是公认的侵袭性亚型，较难治疗，生存和预后较差。它们对激素无反应，没有现成的有效和特异性靶标治疗方法。我们先前已经确定Nw-羟基L-精氨酸（NOHA）为基于血液的生物标记物，可根据疾病负担，病程和分子表型来区分ER-和ER +乳腺癌肿瘤（美国实用新型专利10,073,099）。在这项研究中，我们证明了使用针对NOHA的专有单克隆抗体（mAb）进行基于竞争性ELISA的NOHA测定的测定法（美国临时专利62 / 754,053）。ELISA分析的灵敏度，选择性，精密度，稀释线性和回收率参数得到了评估。该测定法显示对≥60 pg / ml NOHA抗原和1 ng / ml NOHA mAb的敏感性，甚至在其他紧密相关的阳离子氨基酸（例如L-精氨酸，D-精氨酸，L-赖氨酸，d-赖氨酸，L-鸟氨酸和L-瓜氨酸）存在的情况下也能保持NOHA抗原特异性。对于所有测试参数的灵敏度（≤8.2％），选择性（≤8.6％），精密度（≤12.6％），稀释线性（≤11.2％）和回收率，协方差百分比低证实了ELISA方案的可靠性。 （≤6.7％）。此外，我们可以通过该ELISA测定法证明NOHA定量，以补充LC-MS可获得的灵敏度（在测定缓冲液和患者血浆样品中），从而表明它作为一种简单而灵敏的方法，可能有助于ER乳腺癌无需昂贵的分析设备（例如LC-MS），较大的实验室空间，