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.
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.
