High-throughput proteomics using submicroliter amounts of plasma for comprehensive assessment of the immune status

使用亚微升血浆进行高通量蛋白质组学综合评估免疫状态

• 批准号: 10595062
• 负责人: Hanno Steen
• 金额: $ 117.82万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-09 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10595062
• 关键词: Adolescent; Adult; Agreement; Aliquot; Antibodies; Antibody titer measurement; Autoimmunity; Bacterial Infections; Biological Assay; Blood Plasma Volume; Blood Volume; Boston; C-reactive protein; Cells; Communicable Diseases; Communities; Complement; Consumption; Data; Development; Disease; Elderly; Erythrocyte Sedimentation Rate; Frail Elderly; Funding; Funding Opportunities; Future; Health; Hepatitis B Surface Antigens; Hepatitis B Vaccination; Hepatitis B Vaccines; Human; Human body; Hypersensitivity; Immune; Immune response; Immune system; Immunity; Immunoglobulin Domain; Immunoglobulins; Immunologic Deficiency Syndromes; Immunologic Monitoring; Immunology; Individual; Infant; Infection; Maps; Measures; Methods; Modality; Molecular; Monitor; Mycoses; National Institute of Allergy and Infectious Disease; Neonatal; Newborn Infant; Organism; Parasitic infection; Participant; Patients; Pediatric Hospitals; Persons; Plasma; Plasma Proteins; Population; Populations at Risk; Predisposition; Preparation; Prevention; Proteins; Proteome; Proteomics; Regulation; Research; Research Design; Resolution; Sampling; Serology; Serum; Set protein; Specimen; System; Systems Biology; Term Birth; Testing; Time; Transplantation; Vaccine Design; Virus Diseases; Vulnerable Populations; Work; age group; anti-hepatitis B; biomarker identification; clinical biomarkers; cohort; complement pathway; complement system; experimental study; gene product; immune function; immunological status; immunoregulation; improved; innovation; insight; interest; liquid chromatography mass spectrometry; milliliter; nanolitre; novel; premature; preterm newborn; procalcitonin; response; vaccine immunogenicity; vaccinology

SUMMARY Often, many different tests are performed to obtain a multiparameter assessment of the innate and adaptive immune status. Such tests often include erythrocyte sedimentation rate, C-reactive protein (CRP), procalcitonin, various complement pathway proteins, total antibody (Ab) isotypes and/or subclasses, and specific Ab titers. All these tests are performed on different analytical platforms requiring a separate aliquot for each test. Thus, even with modest sample consumption per test, there is a substantial cumulative volume required for multiple independent tests. While these sample requirements may not be prohibitive for healthy adolescents and adults, they are particularly challenging in especially vulnerable populations such as premature newborns, and frail and/or elderly individuals. For these populations, a wide range of different tests is often very important, however, samples are often limited. In agreement with the purpose of the funding opportunity “Development of Sample Sparing Assays for Monitoring Immune Responses”, we propose to move four different protein assays onto a common processing and analysis platform, which can be performed on the same submicroliter aliquot of plasma thereby significantly reducing the sample volume requirements. The four protein assays of interest include i) an expanded panel of complement components, ii) CRP, iii) antibody isotypes and subclasses including a novel and innovative immunoglobulin domain-resolved map of the IgOme-related protein repertoire (aka IgOme), and iv) the classical plasma proteome, i.e. ~400 well described proteins many of which of clear immunological function. The quantitative information from all these assays will provide us with an exquisite map of the immune status, a hypothesis that we will be tested by correlating our findings with the anti-hepatitis B titer measured before and after hepatitis B vaccination in >500 newborns. Samples will be provided by Boston Children's Hospital's NIAID-funded HIPC (Human Immunology Project Consortium), which will significantly increase the impact of this cohort beyond its original scope. Taking the request of this funding opportunity for sample sparing to a different level, we will advance our plasma proteomics pipeline to quadruple the throughput to at least 96 samples/day and to quarter the plasma volume requirements to <100 nanoliter. Thus, in the future, 1 microliter of plasma will be sufficient for tens of temporally independent LC/MS- based protein assays, resulting in quantitative information for hundreds of proteins such that even a miniscule amount of plasma from the smallest and/or most frail patients will be sufficient to establish an exquisitely detailed and unbiased molecular map of immune status of any person.

概括 通常，会进行许多不同的测试以获得先天和后天的多参数评估。 适应性免疫状态。此类测试通常包括红细胞沉降率、C反应蛋白 (CRP)、降钙素原、各种补体途径蛋白、总抗体 (Ab) 同种型和/或 亚类和特定抗体滴度。所有这些测试都是在不同的分析平台上进行的 每个测试都需要单独的等分试样。因此，即使每次测试的样本消耗量不大， 是多个独立测试所需的大量累积体积。虽然这些样本 对于健康的青少年和成年人来说，这些要求可能不会令人望而却步，但它们尤其是 对于早产儿、体弱者和/或老年人等特别脆弱的人群来说具有挑战性 个人。对于这些人群来说，广泛的不同测试通常非常重要，但是， 样本往往是有限的。 与资助机会“开发样品备用测定”的目的一致 监测免疫反应”，我们建议将四种不同的蛋白质测定转移到一个共同的 处理和分析平台，可在相同的亚微升血浆等分试样上进行 从而显着减少样品体积需求。四种感兴趣的蛋白质测定 包括 i) 补体成分的扩展组，ii) CRP，iii) 抗体同种型和亚类 包括 IgOme 相关蛋白的新型免疫球蛋白结构域解析图 谱库（又名 IgOme），以及 iv) 经典血浆蛋白质组，即约 400 种详细描述的蛋白质 其中具有明确的免疫功能。所有这些测定的定量信息将提供 为我们提供了一张精美的免疫状态图，这是一个假设，我们将通过关联我们的免疫状态来进行测试 乙型肝炎疫苗接种前后测得的抗乙型肝炎滴度>500的结果 新生儿。样本将由波士顿儿童医院 NIAID 资助的 HIPC（人类 免疫学项目联盟），这将显着增加该群体的影响力 原来的范围。将样品备用资金机会的要求提升到一个不同的水平，我们 将推进我们的血浆蛋白质组学管道，将吞吐量翻两番，达到每天至少 96 个样本 并将血浆体积要求四分之一至 <100 纳升。 因此，在未来，1微升血浆将足以进行数十次时间独立的LC/MS- 基于蛋白质测定，产生数百种蛋白质的定量信息，即使是 来自最小和/或最虚弱患者的极少量血浆就足以建立 任何人免疫状态的详细且公正的分子图谱。