Multiplexed immuno-SRM screening for primary immunodeficiencies

原发性免疫缺陷的多重免疫 SRM 筛查

• 批准号: 9392888
• 负责人: Sihoun Hahn
• 金额: $ 90.9万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-15 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9392888
• 关键词: Affect; Agammaglobulinaemia tyrosine kinase; Ataxia Telangiectasia; Biological Assay; Biological Markers; Blood; Blood Platelets; Blood Volume; Cessation of life; Child; Chronic Granulomatous Disease; Clinical; Collaborations; Common Variable Immunodeficiency; Congenital Disorders; Coupled; Coupling; DNA; DNA sequencing; Data; Detection; Diagnostic; Disease; Early Diagnosis; Early Intervention; Early treatment; Excision; Flow Cytometry; Genetic Diseases; Goals; Health Care Costs; Hepatolenticular Degeneration; Human Cell Line; Immune; Immune System Diseases; Immunologic Deficiency Syndromes; Immunology; Individual; Institutional Review Boards; Integral Membrane Protein; Laboratories; Leukocytes; Life; Link; MHC Class II Genes; Mass Spectrum Analysis; Measurement; Measures; Methodology; Methods; Monitor; Morbidity - disease rate; Neonatal Screening; Newborn Infant; Outcome; Paper; Patient Care; Patients; Peptides; Performance; Prediabetes syndrome; Procedures; Proteins; Proteomics; Protocols documentation; Reaction; Reproducibility; Research; Sampling; Severe Combined Immunodeficiency; Severities; Spottings; Symptoms; T-Cell Receptor; Test Result; Testing; Time; Wilson disease protein; Wiskott-Aldrich Syndrome; X-Linked Agammaglobulinemia; X-Linked lymphoproliferative disorders; base; biomarker panel; congenital immunodeficiency; cost effective; design; disability; effective therapy; experimental study; familial hemophagocytic lymphohistiocytosis; high throughput screening; improved; improved outcome; innovation; novel; novel strategies; premature; prevent; prospective; protein biomarkers; public health relevance; rapid detection; rapid diagnosis; research clinical testing; response; screening; screening panel; tandem mass spectrometry

 DESCRIPTION (provided by applicant): Primary immunodeficiency diseases (PIDDs) are a large group of genetic disorders of the immune system. These disorders vary in the severity and spectrum of symptoms, but without effective and early treatment, they can be fatal. The current approach to SCID utilizes quantitative PCR to detect T cell Receptor Excision Circles (TREC) which is applicable only to a subset of immunodeficiencies. The goal of our proposal is to develop and validate a specific and quantitative assay that will simultaneously identify multiple PIDDs using dried blood spots (DBS). We previously developed a novel proteomic screening method using Selected Reaction Monitoring-Mass Spectrometry (SRM-MS) to simultaneously identify specific signature peptides derived from the transmembrane protein cluster of differentiation 3 (CD3) and the intracellular proteins, Wiskott-Aldrich syndrome protein (WASP) and Bruton's tyrosine kinase (BTK) as markers of three life-threatening PIDDs; severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and X-linked Agammaglobulinemia (XLA). The objective of this application is to improve the sensitivity of our novel approach by developing peptide immunoaffinity enrichment coupled to SRM-MS (immuno-SRM-MS) to quantify a panel of biomarkers in DBS to facilitate the early detection and diagnosis of multiple life-threatening PIDDs. Our Aims are to: 1. Expand the existing panel of screenable PIDDs by identifying proteotypic signature peptides for 8 additional conditions using SRM-MS. These PIDDs include ADA-deficient SCID, MHC class II deficient SCID, DOCK8 deficiency, Common Variable Immunodeficiency, Ataxia Telangiectasia, Familial hemophagocytic lymphohistiocytosis 2, X-linked lymphoproliferative syndrome, and X-linked chronic granulomatous disease. We will use human cell lines to select "signature" peptides for these PIDDs and fully optimize SRM-MS conditions. 2. Increase sensitivity of the SRM-MS assay for PIDDs by coupling it with peptide immunoaffinity enrichment. We will employ immuno-SRM procedures for measurements of signature peptides for the target proteins in DBS for various PIDDs to improve the sensitivity of our assay. We will measure performance metrics for each assay by generating a response curve. 3. Evaluate the ability of a multiplex immuno-SRM approach to correctly identify patients with specific immunodeficiencies in a large set of clinical samples. Our multiplexed immuno-SRM assay will be tested on patient DBS samples collected by the Seattle Children's Immunology Diagnostic Laboratory. Positive newborn DBS from WA State will be retrieved and tested.

 描述（由申请人提供）：原发性免疫缺陷病（PIDDs）是免疫系统的一大组遗传性疾病。这些疾病的严重程度和症状范围各不相同，但如果没有有效的早期治疗，它们可能是致命的。目前的SCID方法利用定量PCR来检测T细胞受体切除环（TREC），其仅适用于免疫缺陷的子集。我们建议的目标是开发和验证一种特异性和定量的检测方法，该方法将使用干血斑（DBS）同时识别多种PIDD。我们开发了一种新的蛋白质组学筛选方法，利用选择反应质谱（SRM-MS）同时鉴定来自跨膜蛋白分化簇3（CD 3 β）和细胞内蛋白质Wiskott-Aldrich综合征蛋白（WASP）和布鲁顿酪氨酸激酶（BTK）的特异性特征肽作为三种危及生命的PIDDs的标志物;严重联合免疫缺陷（SCID）、Wiskott-Aldrich综合征（WAS）和X连锁无丙种球蛋白血症（XLA）。本申请的目的是通过开发与SRM-MS（免疫-SRM-MS）偶联的肽免疫亲和富集来量化DBS中的一组生物标志物，以促进多种危及生命的PIDD的早期检测和诊断，从而提高我们新方法的灵敏度。我们的目标是：1。通过使用SRM-MS鉴定8种额外条件的蛋白质型特征肽，扩展现有的可筛选PIDDs组。这些PIDDs包括ADA缺陷型SCID、MHC II类缺陷型SCID、DOCK 8缺陷型、常见变异型免疫缺陷、共济失调毛细血管扩张症、家族性噬血细胞性淋巴组织细胞增多症2、X连锁淋巴组织增生综合征和X连锁慢性肉芽肿病。我们将使用人类细胞系来选择这些PIDD的“签名”肽，并充分优化SRM-MS条件。2.通过将SRM-MS测定与肽免疫亲和富集偶联，提高PIDD测定的灵敏度。我们将采用免疫SRM程序来测量DBS中各种PIDD的靶蛋白的特征肽，以提高我们测定的灵敏度。我们将通过生成响应曲线来测量每个检测试剂盒的性能指标。3.评价多重免疫SRM方法在大量临床样本中正确识别特异性免疫缺陷患者的能力。我们的多重免疫SRM检测将在西雅图儿童免疫诊断实验室收集的患者DBS样本上进行测试。将检索并检测来自西澳州的阳性新生儿DBS。