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.

摘要 分析抗体对疾病相关抗原的反应对于癌症研究很重要。与之相对的是 历史上测试对单个蛋白质的反应的方法，筛查和诊断越来越依赖于 多重分析以阐明疾病和患者的异质性。蛋白质微阵列允许蛋白质组规模 具有低样本消耗但受表面结合蛋白的结合动力学限制的筛选， 非特异性结合，荧光检测的动态范围有限，临床上不易获得。 基于多肽的方法将分析限制为线性表位。在IMAT R21的支持下，我们拥有 开发了新一代液态蛋白质微阵列平台--“多路溶液蛋白质阵列” (MISPA)，它利用了下一代测序(NGS)的非凡动态范围 在研究和临床实验室中的适用性。我们定量分析了 口咽癌(OPSCC)患者和对照样本使用条形码人乳头瘤病毒(HPV) 12个HPV亚型NGS抗原库。该检测方法成功地检测到了 OPSCC样本，并显示出更高的信号背景比、重复性和动态范围。 随后，我们改进了MISPA，用于检测季节性SARS-CoV-2的抗体反应 冠状病毒和其他呼吸道病原体同时在1000多个样本中作为 NCI SeroNet与新冠肺炎临床诊断和商业诊断的总体符合率超过90% EUA血清学检测。在R33阶段，我们建议将MISPA平台进一步开发为完全 自动化研究平台，具有量化、健壮、高度可重复性、高通量和 对于早期癌症筛查来说，成本很低。我们将建立健全的蛋白质生产标准操作规程，稳定的蛋白质 库存储，试剂批次之间的差异最小。我们将通过以下方式展示MISPA的多功能性 通过包括来自不同亚型HPV的抗原，将条形码蛋白质库大小增加到192 病毒、细菌、真菌和肿瘤抗原。我们将通过端到端提高可重复性和吞吐量 MISPA平台的自动化，以支持需要进行数万次化验的大型项目 样本。我们将确定空白限值、检测限值、线性动态范围、精密度等 定量分析的绩效衡量标准。我们将用数百种抗体描述192种癌症相关抗体 OPSCC和宫颈癌患者以及1000多名无癌对照和基准 性能与目前黄金标准的酶联免疫吸附试验平台。我们在开发创新高技术方面的经验 使用实验室自动化和我们的初步数据质量的吞吐量免疫蛋白质组学平台 说明我们有能力实施我们提出的发展计划。一种可定量重复性的化验方法 在数千个个体中测量数百种针对全长正确折叠蛋白的抗体 同时将极大地有利于癌症血清流行病学、风险评估和筛查。