A rapid, sensitive assay for quantitation of human prorenin in biological samples

描述（由申请人提供）：该I期提案将寻求通过使用我们实验室开发的一种独特的单克隆抗体来检测人肾素前段的表位，从而全面开发一种用于测量人肾素水平的原型测定方法。最近的文献和临床研究表明，血清prorenin水平升高与一些疾病有很强的相关性，包括心力衰竭、糖尿病视网膜病变和糖尿病肾病。预计到2010年，全世界约有3.66亿人将患2型糖尿病，使其成为世界上增长最快的流行病之一。其中三分之一的人将继续发展为糖尿病神经病变和肾病，许多人需要肾脏替代治疗。青少年糖尿病患者血清中prorenin水平的升高可能预示着视网膜病变和肾病的发生，这为预防性干预提供了一个很好的机会。因此，准确测量泌乳素可能对诊断和预测临床应用产生深远的影响。目前，检测prorenin水平的分析受到程序极其冗长、使用放射性同位素以及与prorenin的可变激活状态相关的不准确性的困扰。目前还没有简单可靠的方法来测量生物样本中的催乳素水平。虽然我们目前有一个工作原型分析方法，但目前形式的分析方法需要大量的重新设计和优化，以达到我们创建能够在早期阶段检测糖尿病的临床诊断分析方法的最终目标。具体来说，我们建议识别和替换原蛋白标准和免疫成分的原型分析，以获得更可靠和可重复的分析。我们将研究捕获和检测单克隆抗体的各种组合，以寻求最佳可能的配对。我们将研究几种标记单克隆的方法，以实现对最终用户具有非常高灵敏度和更大通用性的检测版本。我们将用市售的正常、糖尿病前期和糖尿病人血浆和尿液样本验证检测结果。最后，该检测将通过Luminex系统进行优化，以便在临床实验室中用于患者prorenin水平的高通量检测。在亚临床疾病状态的患者中，有效、早期检测异常的原肾素水平可能为早期干预和预防性护理提供机会。

DESCRIPTION (provided by applicant): This Phase I proposal will seek to fully develop a prototype assay for the measurement of human prorenin levels through the use of a unique monoclonal antibody developed in our laboratory that detects an epitope of the human renin prosegment. Recent literature, as well as clinical studies suggests a very strong correlation between elevated serum levels of prorenin and several diseases, including heart failure, diabetic retinopathy, and diabetic kidney disease. Approximately 366 million people worldwide are expected to develop type 2 diabetes by 2010, making this one of the fasted growing epidemics in the world. One third of these persons will go on to develop diabetic neuropathy and nephropathy, many requiring renal replacement therapy. Elevation of prorenin levels in serum of adolescent diabetics may be indicative of retinopathy and nephropathy several years before pathology actually occurs, providing an excellent opportunity for preventative intervention. Therefore, accurate measurement of prorenin may have profound impact on both diagnostic and predictive clinical applications. Currently, assays to detect prorenin levels are plagued by extremely lengthy procedures, use of radioisotopes, and inaccuracy related to the variable activation state of prorenin. There currently is no simple or reliable way to measure prorenin levels in biological samples. Although we currently have a working prototype assay, the assay in its current form requires extensive reengineering and optimization in order to reach our ultimate goal of creating a clinical diagnostic assay capable of detecting diabetes in its earliest stages. Specifically, we propose to identify and replace prorenin standards and immunological components of the prototype assay to obtain a much more reliable and reproducible assay. We will examine various combinations of capture and detection monoclonal antibodies to seek the best possible pairings. We will examine several methodologies for labeling the monoclonals to achieve versions of the assay that have very high sensitivity and greater versatility for the eventual end users. We will validate the assays with commercially available normal, pre-diabetic and diabetic human plasma and urine samples. Finally, this assay will be optimized for potential use in the clinical laboratory via the Luminex system for the high throughput detection of patient prorenin levels. Efficient, early detection of abnormal prorenin levels in patients with sub-clinical disease states may provide opportunity for early intervention and preventative care. PUBLIC HEALTH RELEVANCE: Elevated levels of prorenin in plasma have been shown to be indicative of a number of diseases, including preeclampsia, heart failure, diabetic retinopathy and kidney disease. For this Phase I proposal, we will develop and optimize a highly specific and rapid assay using a unique monoclonal antibody to detect human prorenin in biological samples, including human plasma and urine. This assay will then be adapted to the high throughput Luminex system for eventual use in the clinical laboratory, offering significant advantages of speed and sensitivity over current methods of prorenin quantitation.