Multiplex In-Solution Protein Array (MISPA) for high throughput, quantitative, early profiling of pathogen-induced head and neck

多重溶液内蛋白质芯片 (MISPA) 用于对病原体引起的头颈部进行高通量、定量、早期分析

• 批准号: 10713928
• 负责人: JOSHUA LABAER
• 金额: $ 39.09万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10713928
• 关键词: 2019-nCoV; Agreement; Antibodies; Antibody Response; Antigens; Autoimmune Diseases; Automation; Bacteria; Bar Codes; Benchmarking; Binding; Binding Proteins; Biological Assay; COVID-19 assay; COVID-19 diagnosis; COVID-19 pandemic; COVID-19 patient; Cancer Biology; Cancer Burden; Cancer Diagnostics; Cancer Patient; Cervical Squamous Cell Carcinoma; Characteristics; Clinic; Clinical; Communicable Diseases; Competence; Consumption; DNA; DNA Sequence; DNA sequencing; Data; Detection; Development; Diagnosis; Diagnostic tests; Disease; Disease Progression; Educational process of instructing; Enzyme-Linked Immunosorbent Assay; Epidemiology; Epitopes; Etiology; FDA Emergency Use Authorization; Fluorescence; Head and Neck Squamous Cell Carcinoma; Head and neck structure; Hepatitis C; Heterogeneity; Human; Human Herpesvirus 4; Human Papillomavirus; Immune response; Immunoassay; Individual; Informatics; Kinetics; Laboratories; Legal patent; Length; Libraries; Liquid substance; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Measurement; Measures; Methods; Monitor; Noise; Oropharyngeal; Oropharyngeal Squamous Cell Carcinoma; Patients; Peptides; Performance; Phase; Play; Preparation; Procedures; Production; Protein Array; Protein Array Analysis; Protein Microchips; Proteins; Proteome; Protocols documentation; Quality Control; Reagent; Reproducibility; Research; Risk Assessment; Running; SARS-CoV-2 antibody; Sampling; Screening for cancer; Serology test; Serum; Signal Transduction; Surface; Technology; Testing; Tumor Antigens; Variant; Viral Antigens; Virus; antibody test; anticancer research; carcinogenesis; co-infection; comparison control; cost; data pipeline; detection limit; detection method; early screening; experience; fungus; improved; indexing; innovation; large scale production; malignant oropharynx neoplasm; microbial; microorganism; microorganism antigen; multiplex assay; next generation; next generation sequencing; pathogen; process optimization; programs; protein folding; respiratory pathogen; response; screening; seasonal coronavirus; tool

Abstract Profiling antibody response to disease-associated antigens is important to cancer research. In contrast to the historical approach of testing responses to individual proteins, screening and diagnosis increasingly rely on multiplexed assays to elucidate disease and patient heterogeneity. Protein microarrays allow proteome-scale screening with low sample consumption but are constrained by binding kinetics of surface-bound proteins, non-specific binding, limited dynamic range of fluorescence detection and not readily available in clinics. Peptide-based approaches limit the assay to linear epitopes. With support from IMAT R21, we have developed a next-generation, liquid-phase protein microarray platform, “Multiplex In Solution Protein Array” (MISPA), which exploits the extraordinary dynamic range of next generation sequencing (NGS) with wide applicability in both research and clinical labs. We quantitatively profiled the immune responses of oropharyngeal (OPSCC) patient and control samples using a “barcoded” human papillomavirus (HPV) antigen library for 12 HPV subtypes NGS. The assay successfully detected the positive responses in the OPSCC samples and demonstrated greater signal-to-background ratio, reproducibility, and dynamic range. Subsequently, we have advanced MISPA to assay antibody response against SARS-CoV-2, seasonal coronaviruses, and other respiratory pathogens in more than 1000 samples simultaneously as part of the NCI SeroNet with over 90% overall percent agreement with a clinical COVID-19 diagnosis and commercial EUA serological assays. In the R33 phase, we propose to further develop the MISPA platform to a fully automated research platform that is quantitative, robust, highly reproducible, high-throughput, and inexpensive for early cancer screening. We will establish SOPs for robust protein production, stable protein library storage, and minimal reagent lot-to-lot variations. We will demonstrate the versality of MISPA by increasing the barcoded protein library size to 192 by including antigens from different subtype of HPV, other viruses, bacteria, fungi and tumor antigens. We will improve reproducibility and throughput with end-to end automation for the MISPA platform to support large-scale projects requiring assaying tens of thousands samples. We will determine the limit of blank, limit of detection, linear dynamic range, precision, and other performance measures for quantitative assays. We will profile the 192 cancer related antibodies in hundreds of patients with OPSCC and cervical cancer and more than 1,000 cancer free controls and benchmark the performance with the current gold standard ELISA platform. Our experience with developing innovative high- throughput immunoproteomics platforms using laboratory automation and the quality of our preliminary data speak for our competency in implementing our proposed development. A quantitatively reproducible assay to measure hundreds of antibodies against full length properly folded proteins in thousands of individuals simultaneously will greatly benefit cancer sero-epidemiology, risk assessment and screening.

抽象的 与癌症研究相比，分析抗体对疾病相关抗原的反应非常重要。 测试对单个蛋白质的反应、筛选和诊断的历史方法越来越依赖于 多重分析可阐明疾病和患者的异质性，从而实现蛋白质组规模。 样品消耗量低的筛选，但受到表面结合蛋白的结合动力学的限制， 非特异性结合，荧光检测的动态范围有限，并且在临床上不易获得。 基于肽的方法将检测限制为线性表位。在 IMAT R21 的支持下，我们有了。 开发了下一代液相蛋白质微阵列平台“Multiplex In Solution Protein Array” （MISPA），它利用了下一代测序（NGS）的非凡动态范围，具有广泛的 我们定量分析了免疫反应在研究和临床实验室中的适用性。 使用“条形码”人乳头瘤病毒 (HPV) 采集口咽 (OPSCC) 患者和对照样本 12种HPV亚型的抗原库NGS成功检测到阳性反应。 OPSCC 采样并展示了更高的信号背景比、再现性和动态范围。 随后，我们推进了 MISPA 来检测针对 SARS-CoV-2 的抗体反应，季节性 作为该研究的一部分，同时检测 1000 多个样本中的冠状病毒和其他呼吸道病原体 NCI SeroNet 与临床 COVID-19 诊断和商业诊断的总体一致性超过 90% 在R33阶段，我们建议将MISPA平台进一步开发为全面的EUA血清学检测。 定量、稳健、高度可重复、高通量和 我们将建立用于稳定蛋白质生产的标准操作程序（SOP）。 文库存储和最小的试剂批次差异 我们将通过以下方式展示 MISPA 的多功能性。 通过包含来自不同 HPV 亚型的抗原，将条形码蛋白库大小增加到 192 个 我们将通过端到端提高重复性和通量。 MISPA 平台自动化，支持需要数万次检测的大型项目 我们将确定样品的空白限、检测限、线性动态范围、精度等。 我们将分析数百种癌症相关抗体中的 192 种性能指标。 的 OPSCC 和宫颈癌患者以及 1,000 多名无癌症对照患者，并对 我们在开发创新的高性能 ELISA 平台方面拥有丰富的经验。 使用实验室自动化的吞吐量免疫蛋白质组学平台和我们初步数据的质量 代表我们实施我们提议的开发的能力。 测量数千个个体中针对全长正确折叠蛋白质的数百种抗体 同时将大大有利于癌症血清流行病学、风险评估和筛查。