Peptide-Based Serological Test for Cysticercosis

描述(申请人提供)：猪带绦虫现在越来越被认为是一种具有国际健康影响的寄生虫，并有能力传播到非流行地区。对这种蠕虫物种的日益关注主要与与幼虫期中枢神经系统感染相关的健康问题的严重性有关，这种感染导致脑囊虫病目前被归类为最重要的寄生原性神经系统疾病。尽管理论上有可能根除猪囊尾虫囊虫病，但在不久的将来，全球根除这种感染的可能性仍然不大。目前第二阶段应用的总体目标将是完成开发，通过临床试验进行验证，并开始生产检测猪带绦虫幼虫阶段循环抗原特异性抗体的血清学测试。测试将使用标准化的抗原性材料，如合成肽、重组蛋白及其组合，这些抗原性材料是从从自然感染的猪的包囊材料制备的Western blots(WB)诊断带中最近识别的抗原中挑选出来的。将开发两种类型的检测：一种是标准的微孔酶联免疫吸附试验格式，第二种是利用侧向流动原理的快速非仪器格式。一种创新但已经得到验证的免疫捕获检测技术将被应用于这两种新的测试，这将强调使用纯合成和重组抗原的优势。这种快速、简单的酶联免疫吸附试验将理想地满足在血清流行病学调查和社区研究中对多个样本进行实验室测试的要求，以及诊断个人囊虫病感染的要求。具有全血检测能力的横向流动格式将为目前的WB检测提供一种方便和更具成本效益的替代方案，并适用于实验室或现场条件下的小批量或个别检测。将在横向流动试验中实施的方法学创新将使其灵敏度等于甚至高于微板法的灵敏度。酶联免疫吸附试验和横向流动试验都将能够检测到来自人和猪这两个宿主的样本中的抗体。该项目的第二阶段包括完成合成和重组抗原的分析，以便选择抗原数量最少的组合(S)，提供与目前的WB检测相同或更好的敏感性，用于检测不同疾病阶段的患者的抗体，具有相当高的特异性。第二个主要目标将是验证制备抗原性材料和诊断试剂的技术，包括启动新测试的试生产。这一阶段的重大努力将集中在优化和制造高灵敏度的横向流动版本的血清学检测上。本项目的目的是利用合成肽和重组蛋白等标准化抗原材料，建立诊断猪带绦虫幼虫期感染人和猪的两种商品化血清学试验。第一次测试将是简单的Microwell ELISA格式，非常适合于使用临床实验室和配备的设施中的半自动或自动化设备对任何数量的样本进行测试。第二个测试将采用快速非仪器形式，利用侧向流动原理在现场条件下或在实验室中测试相对较小批次的样品。新的测试将取代昂贵的WB技术，后者需要经过专门培训的人员，并将使临床和参考实验室达到目前无法达到的标准化和准确性水平。这些检测的主要应用将是诊断人类脑囊虫病、猪囊虫病、血清流行病学调查和基于社区的研究。

DESCRIPTION (provided by applicant): The pork tapeworm Taenia solium is now increasingly recognized as a parasite with international health implications and the capability of spreading to non-endemic areas. The growing attention to this helminth species relates primarily to the severity of health problems associated with infection of the central nervous system with the larval stage, which causes neurocysticercosis currently classified as the most important neurologic disease of parasitic origin. In spite of the theoretical possibility for eradication of T. solium cysticercosis global eradication of this infection is still unlikely in the near future. The overall goal of the current Phase II application will be to finish the development, validate through clinical trials, and launch the manufacture of serological tests for detection of specific antibodies to circulating antigens of the larval stage of Taenia solium. The tests will utilize standardized antigenic material such as synthetic peptides, recombinant proteins and combinations thereof selected from antigens identified recently in diagnostic bands of Western blots (WB) prepared from cyst material of naturally infected pigs. Two types of tests will be developed: one will be in a standard microwell ELISA format, and the second will be a rapid non-instrumental format utilizing lateral flow principles. An innovative but already proven immune-capture assay technology will be applied in both new tests, which will emphasize the advantage of using pure synthetic and recombinant antigens. The quick and simple ELISA test will ideally meet the requirements for laboratory testing of multiple samples in sero-epidemiological surveys and community-based studies, as well as for diagnosis of cysticercosis infection in individuals. The lateral flow format with capability for whole blood testing will provide a convenient and more cost-effective alternative to the current WB test, and will be suitable for small batch or individual testing in labs or field conditions. Methodological innovations that will be implemented in the lateral flow test will make its sensitivity equal to or even higher than the sensitivity of the microplate ELISA version. Both ELISA and lateral flow tests will be able to detect antibodies in samples from two hosts of T. solium, humans and pigs. Phase II of this project includes finalizing the analysis of synthetic and recombinant antigens in order to select combination(s) with a minimal number of antigens providing equal or better sensitivity than the current WB test for detection of antibodies in patients with various stages of disease, with comparably high specificity. The second major goal will be validation of technology for preparation of antigenic material and diagnostic reagents including launching the manufacture of pilot lots of the new tests. Significant efforts at this stage will be directed towards optimization and manufacture of highly sensitive lateral flow versions of the serological tests. The objective of the present project is the development of the two types of a commercial serological tests for diagnosis of infection caused by larva stage of pork tapeworm Taenia solium in human and pigs utilizing standardized antigenic material such as synthetic peptides and recombinant proteins. The first test will be in simple microwell ELISA format well suited for testing of any number of samples, using semi-automated or automated equipment in clinical labs and equipped facilities. The second test will be in rapid non-instrumental format utilizing lateral flow principles for application in field conditions or in laboratories for testing relatively small batches of samples. The new tests will replace the expensive WB technique that requires specially trained personnel, and will make possible a level of standardization and accuracy in clinical and reference laboratories that is currently unattainable. The primary application of these tests will be the diagnosis of neurocysticercosis in humans, pig cysticercosis, seroepidemiological surveys and community-based studies.