Point-of-care immunoassay panel for cardiotonic steroids

描述(申请人提供)：先兆子痫(PE)是一种严重的妊娠并发症，表现为高血压、蛋白尿和水肿，有时还会伴有脑病、癫痫发作和肝功能衰竭。PE导致5%至10%的妊娠并发症，是全世界母婴发病率和死亡率的主要原因。内源性类地高辛因子(EDLF)是一种与抗地高辛抗体交叉反应并抑制Na+/K+ATPase(NKA)的未知物质，在20世纪80年代首次被发现。大量文献支持EDLF水平升高可能是高血压发病的致病因素这一假说。近年来，一种内源性强心类固醇(CTS)马利诺蟾毒配基(MBG)被确定为EDLF。血浆MBG在妊娠合并PE时升高5倍以上，提示其在病理生理中的作用。DigiBind是一种与MBG交叉反应的抗地高辛抗血清，在51例严重PE患者中，相对于安慰剂，DigiBind显著改善了肾功能，没有不良反应。抗MBG的单抗正在进行PE的临床试验。缺乏商业上可用的分析严重阻碍了对MBG和至少另外两种强心类固醇在PE和其他疾病中的作用的研究。这个项目的总体目标是开发 MBG和其他可能重要的内源性强心类固醇的免疫分析小组作为研究工具，最终用于临床诊断血浆和尿液中的MBG水平。在第一阶段，我们将确定针对每个强心类固醇的特异性单抗，以展示其敏感性和特异性。我们将配置一种竞争性ELISA，并对其进行优化，以定量测定血浆和尿液中的每一种CTS。最后，我们将把我们的竞争性ELISA转化为一个新的、具有超灵敏电化学检测的自动化免疫分析平台。第二阶段的工作将获得提交510K所需的数据，以支持未来的商业化。

DESCRIPTION (provided by applicant): Preeclampsia (PE) is a serious complication of pregnancy manifested by high blood pressure, proteinuria, and edema, sometimes with encephalopathy, seizures, and hepatic failure. PE complicates from 5 to 10% of pregnancies, and is a major cause of maternal a fetal morbidity and mortality worldwide. Elevation in the circulating level of an endogenous "digoxin-like" factor (EDLF), an unknown substance that cross reacts with anti-digoxin antibodies and inhibits the NA+/K+ ATPase (NKA) was first noted in the 1980s. An extensive literature supports the hypothesis that increased levels of EDLF may be causative factor in the pathogenesis of hypertension. Recently, marinobufagenin (MBG), an endogenous cardiotonic steroid (CTS), has been identified as the EDLF. Plasma MBG is elevated over five-fold in pregnancies complicated by PE, suggesting a role pathophysiological role. Digibind, an anti-digoxin antiserum that cross-reacts MBG, provided significant in improvement in renal function relative to placebo among 51 patients with severe PE, with no adverse effects. Monoclonal antibodies to MBG are in clinical trials for PE. The lack of commercially available assays severely hampers research on the role MBG and at least two other cardiotonic steroids in PE and other diseases. The overall goal of this project is to develop an immunoassay panel for MBG and other potentially important endogenous cardiotonic steroids as a research tool and, ultimately, a clinical diagnostic for MBG levels in plasma and urine. In Phase I, we will identify monoclonal antibodies specific for each cardiotonic steroid demonstrating sensitivity and specificity. We will configure a competitive ELISA and will optimize it for the quantitation each CTS in plasma and urine. Finally, we will translate our competitive ELISA to a novel, automated immunoassay platform with ultrasensitive electrochemical detection. Phase II work will obtain data necessary for the submission of a 510k to support future commercialization.