Immuno Biosensor for Real-Time Factor V Leiden Diagnosis

描述（由申请人提供）：该项目的全球目标是开发一种快速，准确，廉价的诊断免疫光学生物传感器，用于因子V Leiden（FVL）诊断。FVL是最常见的遗传性凝血障碍，占一般人群的3 - 7%，占血栓形成患者的20- 40%。 传感器将由两个光纤阵列组成，每个光纤固定有针对正常因子V（FVN）或FVL的单克隆抗体。样品中的FVN/FVL与相应的抗体反应后，将用荧光团标记的另一种抗体对其进行探测，以特异性定量样品中这两种分子的量。该传感器将以高特异性（相对于凝血测定）、更快（相对于DNA分析）和更便宜的方式诊断FVL，并且还通过具有FVN和FVL两者的量来提供关于血栓形成倾向程度的信息。这种早期诊断方法通过允许患者在需要时接受立即治疗来防止患者发生创伤性血栓栓塞并发症。启动研究目标的具体目的是： 目标1.从纯合FVL患者纯化FVL（0- 24个月）。FVL分子将从纯合FVL患者的血浆中纯化。 目标二。抗FVN/FV L抗体的鉴定和产生（4-12个月）。已经产生了针对FVN或FVL的单克隆抗体。将鉴定对天然形式的FVN或FVL的亲和力。产生适当抗体的杂交瘤细胞将在生物反应器中培养并纯化。 目标3：FVN和FVL传感器的开发（7-24个月）。为蛋白C生物传感器开发的协议将用于在两个单独的光纤传感器上检测FVN和FVL，并将开发双传感器的原型。

DESCRIPTION (provided by applicant): The Global Goal of the project is to develop a rapid, accurate, and inexpensive diagnostic immuno-optical biosensor for Factor V Leiden (FVL) diagnosis. FVL is the most common hereditary blood coagulation disorder, comprising 3 - 7 percent of the general population and 20-40 percent of individuals with thrombosis. The sensor will be composed of an array of two optical fibers, with each immobilized monoclonal antibodies against normal Factor V (FVN) or FVL. After FVN/FVL in the sample reacts with the corresponding antibodies, they will be probed with a fluorophore tagged another antibody to specifically quantify the amount of these two molecules in the sample. This sensor will diagnose the FVL with high specificity (vs. clotting assays), faster (vs. DNA analysis), and less expensive, and also provide the information on the degree of thrombophilia by having the amount of both FVN and FVL. This early diagnosis method prevents patients from having traumatic thromboembolic complications, by allowing them to receive immediate treatments, when needed. The specific aims to initiate the research goal are: Aim 1. Purification of FVL from homozygous FVL patients (months 0-2 4). FVL molecules will be purified from the plasma of homozygous FVL patients. Aim 2. Identification and Production of Antibodies against FVN/FV L (months 4-12). Monoclonal antibodies against either FVN or FVL have been generated. The affinity to the native form of FVN or FVL will be identified. Hybridoma cells producing proper antibodies wiII be cultured in bioreactors and purified. Aim 3. Development of Sensors for FVN and FVL (months 7-24). Protocols developed for the Protein C biosensor will be used to detect FVN and FVL on two individual fiber optic sensors and a prototype of dual sensor will be developed.