Multiplex Serodiagnostic Protein Microarray

描述(由申请人提供)：我们开发的技术能够快速、全面和高通量地分析对传染病抗原的体液免疫反应，用于亚单位疫苗抗原的发现和血清诊断测试。该方法是基于打印含有感染微生物编码的蛋白质的微阵列芯片，感染微生物会导致人类重要的医学疾病。目前，我们已经表达和打印了来自6个细菌、17个病毒和2个寄生虫的14,000多个单独的蛋白质。这些芯片用来自感染患者和健康对照的血清进行探测，以确定显性抗体反应，确定免疫显性抗原图谱，并为每个感染源识别一组10-20个血清诊断抗原。通过对每个感染源使用多种抗原，可以配置具有统计上改进的灵敏度和特异度的血清诊断试验。在这里，我们将制作完整的伯氏柯克斯体、普氏立克次体、布氏杆菌、伤寒沙门氏菌以及每个病原体相关菌株的非同源蛋白的蛋白质组芯片。这些阵列将用特征良好的感染者和健康对照的血清进行探测，以确定免疫优势抗原和血清诊断抗原集。我们还将制作一个包含来自16种虫媒病毒和8种汉坦病毒的190种蛋白质的芯片。将确定该病毒芯片区分从感染不同病毒的人和动物收集的特征良好的血清的能力，并将结果与对相同样本进行的既定分析进行比较。该项目产生的可交付成果包括质粒、模块化多路复用形式的定制蛋白质微阵列、纯化的重组蛋白质和横向流动(试纸)/免疫条设备，并将在充电的基础上从PSWRCE蛋白质微阵列核心获得。这些将通过许可协议向RCE调查人员、政府机构和私营企业提供。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对开发血清诊断测试感兴趣的企业提供许可。免疫优势抗原集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到其中任何一种病原体。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。这些芯片还将有助于评估生物恐怖主义袭击后暴露在人群中的传播情况。

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.