Multiplex Serodiagnostic Protein Microarray

描述（由申请人提供）：我们开发了能够对传染病抗原的体液免疫反应进行快速、全面和高通量分析的技术，该技术正用于亚单位疫苗抗原发现和开发血清诊断测试。该方法基于用传染性微生物编码的蛋白质印刷微阵列芯片，这些微生物会导致人类医学上的重要疾病。目前，我们已经表达并打印了来自 6 种细菌、17 种病毒和 2 种寄生虫的 14,000 多个单独蛋白质。使用感染患者和健康对照者的血清对芯片进行探测，以识别显性抗体反应、确定免疫显性抗原谱并针对每种传染原识别一组 10-20 种血清诊断抗原。通过对每种传染原使用多种抗原，可以配置具有统计上改进的灵敏度和特异性的血清诊断测试。在这里，我们将制造伯内特立克次体、普氏立克次体、羊布鲁氏菌、伤寒沙门氏菌的完整蛋白质组微阵列，以及来自每种病原体相关菌株的非同源蛋白质。将使用来自充分表征的感染受试者和健康对照的血清来探测阵列，以识别免疫显性和血清诊断抗原组。我们还将制造一个包含来自 16 种虫媒病毒和 8 种汉坦病毒的 190 种蛋白质的芯片。该病毒芯片区分从感染不同病毒的人类和动物中收集的特征明确的血清的能力将被确定，并将结果与​​对相同样品进行的既定测定进行比较。该项目产生的成果包括质粒、模块化多重格式的定制蛋白质微阵列、纯化的重组蛋白和侧流（试纸）/免疫条装置，并且可以在充电的基础上从 PSWRCE 蛋白质微阵列核心访问。这些将通过许可协议提供给 RCE 调查人员、政府机构和私营企业。蛋白质微阵列核心还将提供芯片探测和分析服务。血清诊断抗原组将向有兴趣开发血清诊断测试的企业提供许可。免疫显性抗原组可能包含亚单位疫苗抗原，将通过许可协议向商业疫苗开发商提供。多重血清诊断芯片将有助于确定进入这些病原体流行地区的军事人员或平民是否接触过其中的任何一种。它们还将有利于公共卫生监测活动，以跟踪流行地区感染流行率的年度变化，并监测发展中国家的血液供应。这些芯片还可用于评估生物恐怖主义袭击后人群中暴露的传播情况。

DESCRIPTION (provided by applicant): We have developed technology that enables rapid, comprehensive and high-throughput analysis of humoral immune responses to infectious disease antigens that is being used for subunit vaccine antigen discovery and to develop serodiagnostic tests. The method is based on printing microarray chips with proteins encoded by infectious microorganisms which cause medically important diseases in humans. Currently, we have expressed and printed more than 14,000 individual proteins from 6 bacteria, 17 viruses, and 2 parasites. The chips are probed with serum from infected patients and healthy controls to identify the dominant antibody responses, to determine the immunodominant antigen profile and identify a set of 10-20 serodiagnostic antigens for each infectious agent. By using a multiplicity of antigens for each infectious agent, serodiagnostic tests with statistically improved sensitivity and specificity can be configured. Here we will fabricate whole proteome microarrays for Coxiella burnetii, Rickettsia prowazekii, Brucella melitensis, Salmonella typhi, and non-homologous proteins from related strains of each agent. The arrays will be probed with serum from well-characterized infected subjects and healthy controls to identify the immunodominant and serodiagnostic antigen sets. We will also fabricate a chip containing 190 proteins from 16 arboviruses and 8 hantaviruses. The ability of this virus chip to distinguish well-characterized sera collected from humans and animals infected with the different viruses will be determined and the results compared with established assays done on the same samples. Deliverables generated from this project include plasmids, customized protein microarrays in a modular multiplex format, purified recombinant proteins and lateral flow (dipstick)/immunostrips devices, and will be accessible from the PSWRCE Protein Microarray Core on a recharge basis. These will be available to RCE investigators, government agencies and to private businesses through licensing agreements. The Protein Microarray Core will also provide chip probing and analysis services. Serodiagnostic antigen sets will be available for licensing to businesses interested in developing serodiagnostic tests. The immunodominant antigen sets, which may contain subunit vaccine antigens, will be available to commercial vaccine developers through licensing agreements. Multiplex serodiagnostic chips will be useful for determining whether military personnel or civilians, who enter a region where these agents are endemic, are exposed to any of them. They will also benefit public health monitoring activities to track annual changes in the prevalence of infections in endemic regions, and for monitoring the blood supply in developing countries. The chips will also be useful for assessing the spread of exposure in a population following a bioterrorism attack.