Curbside Immunoassay Device for Quantitative Measurement of Cannabis Intoxication

 描述（由申请人提供）：执法官员，工作场所药物检测计划和药物滥用领域的研究人员缺乏快速，易于部署的工具来测量目前的大麻（大麻）中毒。酶联免疫吸附试验（ELISA）是一种用于检测尿液或血液中δ-9-四氢大麻酚（THC）水平的成熟大麻检测方法;然而，现场获取尿液或血液样本的物流是当前THC ELISA的主要限制，特别是对于驾驶员的路边测试。路边检测最可行的候选样本是口腔液，因为它是一种微创采样途径，并且口腔液中THC浓度的药代动力学（半衰期为1.5小时）与血清平行;然而，这种方法的主要障碍是缺乏快速，定量THC免疫测定。传统的基于板的ELISA测试可能需要1至3小时，并且需要实验室设置，并且侧流分析缺乏定量大麻中毒检测所需的准确性。BreviTest Technologies LLC正在开发一种便携式分析仪，可在10分钟内进行快速定量ELISA测试。将样品加载到不连续的微流体盒中，并且机器人机构控制抗体缀合的磁性纳米颗粒通过连续的流体威尔斯孔的移动，其中增强的混合模式实质上减少了每个ELISA步骤所需的时间。这种创新的方法能够实现快速、便携、全自动化的ELISA检测，其准确性可与标准实验室ELISA相媲美。在这个SBIR第一阶段项目中，BreviTest将与Cayman Chemical Company合作，使用BreviTest机器人分析仪开发THC ELISA测试，具体目标如下：1）开发和生产具有非专业人员可操作样品端口的不连续微流体盒。将确定磁性纳米颗粒的类型和数量，以获得最佳的试剂混合和运输参数，并将完成用于计量20 µL样品流体的采样工具的设计和验证。将对笔芯的设计进行优化，以实现稳定性、一致性和与 生物材料。2)建立ELISA检测口腔液中THC的方法。BreviTest将与Cayman Chemical合作，筛选商用克隆，以生成用于THC检测的优化抗体。抗体将通过链霉亲和素-生物素连接与磁性纳米颗粒偶联，用于BreviTest分析仪。将对试剂浓度和分析仪参数进行优化，同时沿着对动态范围、特异性和灵敏度进行统计学验证。这些概念验证研究的完成将证明BreviTest便携式ELISA分析仪在与路边中毒分析相关的浓度下测量口腔液样本中THC浓度的能力。这项工作将为进一步的II期研究奠定基础，包括快速THC中毒检测的现场和临床测试以及FDA规定的510（k）许可的额外验证测试。

 DESCRIPTION (provided by applicant): Law enforcement officials, workplace drug testing programs, and researchers in the field of drug abuse lack rapid, easily deployable tools to measure present cannabis (marijuana) intoxication. The enzyme-linked immunosorbant assay (ELISA) is an established cannabis testing method for detecting delta-9-tetrahydrocannabinol (THC) levels in urine or blood; however, the logistics of obtaining urine or blood samples on site represents a major limitation of current THC ELISAs, particularly for curbside testing of drivers. The most feasible candidate sample for curbside testing is the oral fluid, as it is a minimally invasive sampling route and the pharmacokinetics of THC concentration in oral fluid (half-life of 1.5 hours) parallels that of serum; however, the primary obstacle to this approach is the lack of a rapid, quantitative THC immunoassay. Traditional plate-based ELISA tests can take 1 to 3 hours and require a laboratory setting, and lateral flow assays lack the accuracy needed for quantitative cannabis intoxication detection. BreviTest Technologies LLC is developing a portable analyzer to perform a rapid, quantitative ELISA test within a 10-minute run time. The sample is loaded into a discontinuous microfluidics cartridge, and a robotic mechanism controls the movement of antibody-conjugated magnetic nanoparticles through successive fluid wells, where enhanced mixing patterns substantially reduce the time required for each ELISA step. This innovative approach enables the implementation of a rapid, portable, fully automated ELISA test with accuracy comparable to a standard laboratory ELISA. In this SBIR Phase I project, BreviTest will partner with Cayman Chemical Company to develop a THC ELISA test using the BreviTest robotic analyzer, with the following Specific Aims: 1) Develop and produce discontinuous microfluidic cartridges with layman-operable sample port. The type and quantity of magnetic nanoparticles will be determined for optimal reagent mixing and transit parameters, and the design and validation will be completed for a sampling tool for metering 20 µL of sample fluid. Design of the cartridge will be optimized for stability, consistency, and compatibility with biological materials. 2) Develop curbside ELISA test to quantitatively measure THC in oral fluid. BreviTest will collaborate with Cayman Chemical to screen commercially available clones to generate an optimized antibody for THC detection. Antibodies will be conjugated to magnetic nanoparticles via streptavidin-biotin linkages, for use in the BreviTest analyzer. Optimization of reagent concentrations and analyzer parameters will be performed, along with statistical validation of dynamic range, specificity, and sensitivity. The completion of these proof-of-concept studies will demonstrate the ability of the BreviTest portable ELISA analyzer to measure THC concentration in oral fluid samples, at concentrations relevant to curbside intoxication analysis. This work will lay the foundation for further Phase II studies including fied and clinical testing of a rapid THC intoxication assay and additional validation testing towards a defined 510(k) clearance by the FDA.