Multiplex Test for Primary Immunodeficiencies by Affinity Column coupled to MS/MS

通过亲和柱与 MS/MS 联用对原发性免疫缺陷进行多重检测

• 批准号: 8896190
• 负责人: Sihoun Hahn
• 金额: $ 54.8万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8896190
• 关键词: Affect; Affinity; Agammaglobulinaemia tyrosine kinase; Ataxia Telangiectasia; Biological Assay; Biological Markers; Blood; Blood Platelets; Blood Volume; CD3 Antigens; Cessation of life; Child; Chronic Granulomatous Disease; Clinical; Common Variable Immunodeficiency; Coupled; Coupling; DNA; DNA Sequence; Diagnosis; Diagnostic; Disease; Early Diagnosis; Early Intervention; Early treatment; Excision; Flow Cytometry; Goals; Health; Health Care Costs; Hemophagocytic Lymphohistiocytoses; Hereditary Disease; Human; Human Cell Line; IgE; Immune; Immune system; Immunologic Deficiency Syndromes; Immunology; Individual; Infant; Infection; Integral Membrane Protein; Laboratories; Leukocytes; Life; Link; MHC Class II Genes; Mass Spectrum Analysis; Measurement; Measures; Methodology; Methods; Metric; Monitor; Morbidity - disease rate; Neonatal Screening; Outcome; Patient Care; Patients; Peptides; Performance; Plasma; Procedures; Proteins; Proteomics; Reaction; Recurrence; Reproducibility; Research; Sampling; Severe Combined Immunodeficiency; Severities; Symptoms; Syndrome; T-Cell Receptor; Testing; Time; Wiskott-Aldrich Syndrome; X-Linked Agammaglobulinemia; X-Linked lymphoproliferative disorders; base; congenital immunodeficiency; cost effective; disability; effective therapy; high throughput screening; improved; novel; novel strategies; premature; prevent; prospective; rapid diagnosis; research clinical testing; research study; response; screening; 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. Currently, there is no reliable screening assay for early diagnosis of PIDDs. The goal of our proposal is to develop and validate a specific and quantitative assay that will simultaneously identify multiple PIDDs using a small volume of blood. We previously developed a novel proteomic screening method using Selected Reaction Monitoring-Mass Spectrometry (SRM-MS) to simultaneously identify low-abundant 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 selected reaction monitoring- mass spectrometry (immuno-SRM-MS) to quantify a panel of biomarkers in infant blood to facilitate the early detection and diagnosis of multiple life-threatening PIDDs and validate these panels of biomarkers for clinical implementation. Our Aims are to: 1. Expand the existing panel of screenable PIDDs by identifying proteotypic signature peptides for 9 additional conditions using SRM-MS. These PIDDs include ADA- deficient SCID, MHC class II deficient SCID, Hyper IgE recurrent infection syndrome, 2 Common Variable Immunodeficiency Disorders (CVIDs) 3 and 8 , Ataxia Telangiectasia, Hemophagocytic lymphohistiocytosis, X-linked lymphoproliferative disease, 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 15 target proteins for various PIDDs to improve the sensitivity of our assay for clinical implementation. 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 larger set of clinical samples. Our multiplex immuno-SRM assay will be deployed on human clinical samples retrospectively and prospectively collected by the Seattle Children's Immunology Diagnostic Laboratory.

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，没有可靠的筛查方法用于PIDDs的早期诊断。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，该方法将使用少量血液同时识别多种PIDD。我们之前开发了一种新的蛋白质组学筛选方法，使用选择反应质谱（SRM-MS）来同时鉴定来自跨膜蛋白分化簇3（CD 3 β）和细胞内蛋白Wiskott-Aldrich综合征蛋白（WASP）和布鲁顿酪氨酸激酶（BTK）的低丰度特异性特征肽，作为三种危及生命的PIDD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肽免疫亲和富集结合选择反应监测-质谱法（免疫-SRM-MS）来量化婴儿血液中的一组生物标志物以促进多种危及生命的PIDD的早期检测和诊断并验证这些生物标志物组用于临床实施来提高我们的新方法的灵敏度。我们的目标是：1。通过使用SRM-MS鉴定9种额外病症的蛋白质型特征肽来扩展现有的可筛选PIDDs组。这些PIDDs包括ADA缺陷型SCID、MHC II类缺陷型SCID、高IgE复发性感染综合征、2种常见可变型免疫缺陷疾病（CVID）3和8、共济失调毛细血管扩张症、噬血细胞性淋巴组织细胞增多症、X连锁淋巴组织增生性疾病和X连锁慢性肉芽肿性疾病。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程序来测量各种PIDD的15种靶蛋白的特征肽，以提高我们用于临床实施的测定的灵敏度。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方法在较大临床样本集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检测将部署在人类临床样本回顾性和前瞻性收集的西雅图儿童免疫诊断实验室。