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、准确和廉价的用于因子V莱顿(FVL)诊断的免疫光学生物传感器。FVL是最常见的遗传性凝血障碍，占总人口的3%-7%，占血栓形成个体的20%-40%。 该传感器将由两根光纤阵列组成，每根光纤固定抗正常因子V(FVN)或FVL的单抗。当样本中的FVN/FVL与相应的抗体反应后，将用标记了另一种抗体的荧光团进行探测，以专门定量样本中这两个分子的数量。这种传感器将以高特异性(与凝血试验相比)、更快(与DNA分析相比)和更便宜的价格来诊断FVL，并通过同时拥有FVN和FVL的量来提供关于血栓亲和性程度的信息。这种早期诊断方法允许患者在需要时立即接受治疗，从而防止患者出现创伤性血栓栓塞症并发症。启动研究目标的具体目标是： 目的1.从纯合子FVL患者(0~2.4个月)中分离纯化FVL。FVL分子将从纯合子FVL患者的血浆中提纯。 目的2.L口蹄疫/口蹄疫4~12月龄抗体的鉴定和制备已经产生了抗FVN或FVL的单抗。将确定与FVN或FVL原生形式的亲和力。产生适当抗体的杂交瘤细胞将在生物反应器中培养和纯化。 目的3.FVN和FVL传感器的研制(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.