Assay for Detection of Loxosceles Envenomation

描述（由申请人提供）：棕色隐士蜘蛛（Loxosceles reclusa）的毒液中毒是美国地方病地区发病的重要来源，误诊很常见。对流行地区的医生进行的一项调查表明，对这些蜘蛛叮咬进行准确的诊断测试具有经济可行性。一个优化的Loxosceles毒液检测的开发和测试将提出重大挑战。与常规构建ELISA检测单一蛋白质不同，该ELISA将检测含有多种蛋白质的毒液，包括毒液中大量存在的独特生理活性蛋白-鞘磷脂酶D（SMD）。在初步研究中，我们的多克隆方法显示出良好的灵敏度和体外特异性。我们的研究表明，在临床毒液蛰入中，可识别数量的毒液至少存在7天。在兔子中，我们的多克隆测定允许鉴定表面上的毒液长达两周。灵敏度限度、体内特异性和检测持续时间未知。第一阶段应允许确定可检测的最小量的毒液以及测定的灵敏度和特异性的体内持续时间的临床时间限制。将对单克隆抗体进行同种型分析，并在生物反应器中进行定量，以进行棋盘格分析。该分析将允许确定L的最佳组合。Reclusa单克隆和多克隆抗体。试剂盒检测将从I期开始，允许在II期进行多中心检测。这些临床研究将允许开发侧流测定或微量滴定板测定，目标是获得FDA设备批准和营销。临床应用的优化测定将节省的发病率和费用，由于不适当的诊断和治疗的各种皮肤状况与类似的Loxosceles envenomations介绍。成功检测这种蜘蛛毒液的技术直接适用于S。美洲Loxosceles物种，负责每年额外的死亡。拭子毒液检测技术可适用于许多物种的毒液 公共卫生相关性：在中西部，棕色隐士蜘蛛（Loxosceles reclusa）的叮咬会导致相当大的发病率和偶尔的死亡率。许多病变可能看起来相似，模仿蜘蛛咬伤，包括细菌和真菌皮肤感染以及皮肤癌。该项目的目标是开发一种商业上可行的测试棕色隐士蜘蛛咬伤使用无痛和简单的拭子测试皮肤表面的蜘蛛毒液。

DESCRIPTION (provided by applicant): Envenomations by the brown recluse spider, Loxosceles reclusa, are a significant source of morbidity in endemic regions of the United States, and misdiagnoses are common. A survey of physicians in the endemic area has shown the economic viability of an accurate diagnostic test for these spider bites. Development and testing of an optimized Loxosceles venom assay will present significant challenges. Unlike the routine construction of ELISAs dedicated to the detection of a single protein, this ELISA will detect venom containing multiple proteins, including a unique physiologically active protein- sphingomyelinase D (SMD) abundantly present in the venom. In preliminary research, our polyclonal assay has shown good sensitivity and good in-vitro specificity. Our research shows that identifiable amounts of venom in clinical envenomations are present for at least seven days. In rabbits, our polyclonal assay allows identification of venom on the surface for as long as two weeks. The limits of sensitivity, in-vivo specificity, and the duration of detection are unknown. Phase I should allow determination of the smallest amount of venom detectable as well as the clinical time limits for in-vivo duration of sensitivity and specificity of the assay. Monoclonal antibodies will be isotyped and quantities raised in a bioreactor to perform checkerboard analysis. This analysis will allow determination of an optimal combination of L. reclusa monoclonal and polyclonal antibodies. A kit assay will result from Phase I, allowing multi-site testing in Phase II. These clinical studies will allow development of a lateral flow assay or microtiter plate assay, with the goal of FDA device approval and marketing. Clinical application of an optimized assay would save the morbidity and expense due to inappropriate diagnosis and treatment of various skin conditions with presentations similar to Loxosceles envenomations. Techniques used in the successful detection of this spider venom are directly applicable to bites from S. American Loxosceles species, responsible for additional deaths each year. The swab venom assay technique could be applicable to envenomations from numerous species PUBLIC HEALTH RELEVANCE: Bites of the brown recluse spider, Loxosceles reclusa, cause considerable morbidity and occasional mortality in the Midwest. Many lesions can appear similar and mimic spider bites, including bacterial and fungal skin infections as well as skin cancer. The goal of this project is to develop a commercially viable test for brown recluse spider bites using a painless and simple swab test for the spider venom on the surface of the skin.