Development of Highly Multiplex Antigen Specificity Assays

高度多重抗原特异性检测的开发

• 批准号: 9095224
• 负责人: STEPHEN J ELLEDGE
• 金额: $ 56.51万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-22 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9095224
• 关键词: Acute; Address; Antibodies; Antibody Repertoire; Antigen-Presenting Cells; Antigens; Apoptosis; Autoantigens; Autoimmunity; Autologous; B-Lymphocytes; Bacteriophages; Benchmarking; Biocompatible Materials; Biological Assay; Blood; CD4 Positive T Lymphocytes; CD8B1 gene; Cells; Complex; Cytotoxic T-Lymphocytes; DNA Sequence; Data Set; Databases; Development; Ensure; Epidemiology; Foundations; Goals; Growth; Health; Helper-Inducer T-Lymphocyte; High-Throughput DNA Sequencing; Human; Immune response; Immune system; Immunoassay; Immunodominant Epitopes; Immunoprecipitation; Individual; Infection; Libraries; Measurement; Methods; Molecular Chaperones; Patients; Peptides; Performance; Positioning Attribute; Procedures; Process; Proteins; Proteome; Publications; Research Personnel; Sampling; Serologic tests; Serological; Serum; Signal Transduction; Solid; Sorting - Cell Movement; Specificity; Synthetic Antigens; System; T cell response; Technology; Testing; Time; Viral; Viral Antibodies; Virus; Virus Diseases; Work; base; cancer immunotherapy; clinically actionable; cost; epidemiology study; human virome; interest; invariant chain; new technology; next generation; novel; peptide I; research study; response; screening; synthetic biology; vaccine development

 DESCRIPTION (provided by applicant): There is an increasing unmet need for immunoassays that yield a maximal amount of information from a minimal amount of patient sample. One way to address this challenge is to develop very highly multiplexed, sample sparing assays. This proposal presents new methods that integrate synthetic biology and next generation DNA sequencing ("NGS") to characterize the specificities of humoral and cellular immune responses using genetically encoded antigen libraries. The multi-PI project builds upon a solid foundation, which has been established in recent years, for developing highly multiplex, sample sparing immunoassays. The platforms described in this proposal will be developed and tested on a new antigen library that encompasses the proteomes of all viruses known to infect humans ("the human virome"). Proof-of-concept studies confirm both the quality of this library, and its utility or developing sample sparing, highly multiplex antigen specificity assays. The following three Specific Aims have been developed to broadly analyze human immune responses to viruses, using an absolute minimal amount of sample. Specific Aim 1. Development of minimal human virome serologic assays Two novel sample sparing serologic assays are proposed: 1) a 384-well, simplified bacteriophage-NGS based assay, and 2) a rapid cytometric Luminex bead array assay. These technologies will comprehensively characterize anti-viral antibodies at low cost, and require less than a single microliter of blood. Specific Aim 2. Development of a minimal antigen library screening assay for cytotoxic T lymphocytes A new platform for profiling CD8+ cytotoxic T lymphocyte (CTL) specificities has been devised. The system employs lentiviral delivery of a genetically encoded antigen library to present MHC I-peptide complexes on autologous antigen presenting cells, followed by phenotypic library enrichment and NGS analysis. Specific Aim 3. Development of a minimal antigen library screening assay for T helper cells A new platform for profiling CD4+ T helper (Th) cell specificities has been devised. The system employs lentiviral delivery of a genetically encoded antigen library to present MHC II-peptide complexes on autologous antigen presenting cells, followed by phenotypic library enrichment and NGS analysis.

 描述（由适用提供）：对免疫测定的未满足需求越来越多，从最少的患者样本中产生了最大信息。解决这一挑战的一种方法是开发非常高度的多重样品保证测定法。该建议提出了新方法，这些方法将合成生物学和下一代DNA测序（“ NGS”）整合在一起，以使用基因编码的抗原文库来表征体液和细胞免疫调查的细节。 Multi-Pi项目建立在近年来建立的坚实基础上，用于开发高度多重的样本稀疏免疫测定。该提案中描述的平台将在一个新的抗原库上开发和测试，该库涵盖了所有已知感染人类的​​病毒的蛋白质组织（“人类病毒瘤”）。概念验证研究既证实了该库的质量及其效用或开发样本较高的，高度多重抗原特异性分析。已经开发出以下三个特定目的，以使用绝对最少的样品来广泛分析人类的免疫血液调查。具体目的1。提出了最小的人类病毒蛋白血清学测定法两种新样品的较新样品血清学测定：1）基于384孔，简化的基于细菌噬菌体-NGS的测定法，以及2）快速的细胞仪Luminex珠阵列阵列。这些技术将以低成本的抗病毒抗体进行全面的特征，并且需要小于单个微量的血液。具体目的2。针对细胞毒性T淋巴细胞的最小抗原库筛选测定法的开发已经设计了一个新的用于分析CD8+细胞毒性T淋巴细胞（CTL）规范的平台。系统雇员慢性病毒的植物递送在自体抗原呈递细胞上呈现MHC I肽复合物，然后进行表型库富集和NGS分析。特定目的3。针对T辅助细胞的最小抗原库筛选测定的开发，已经设计了一个新的分析CD4+ T助手（TH）细胞规范的平台。系统雇员慢性病毒透射术在自体抗原呈递细胞上呈现MHC II肽复合物，然后进行表型库富集和NGS分析。