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 对抗病相关抗原的抗体反应对癌症研究很重要。与 测试对单个蛋白质，筛查和诊断的回答的历史方法越来越依赖 多路复用测定法以阐明疾病和患者异质性。蛋白质微阵列允许蛋白质组规模 筛查样品消耗少，但受到结合蛋白的结合动力学的约束， 非特异性结合，有限的荧光检测动态范围，并且在诊所不容易获得。 基于肽的方法将测定限制为线性表位。在Imat R21的支持下，我们有 开发了下一代的液相蛋白微阵列平台，“溶液蛋白阵列中的多重） （MISPA），利用宽阔的下一代测序（NGS）的非凡动态范围 研究和临床实验室的适用性。我们定量介绍了 口咽（OPSCC）患者使用“条形码”人乳头瘤病毒（HPV）对照样品进行控制样品 12 hpv子类型的抗原库。该测定成功地检测了 OPSCC样品显示出更大的信噪比，可重复性和动态范围。 随后，我们提高了MISPA来测定针对SARS-COV-2的抗体反应，季节 冠状病毒和1000多个样本中的其他呼吸道病原体仅作为该样本的一部分 NCI Seronet与临床Covid-19诊断和商业的总体百分比一致性超过90％。 EUA血清学分析。在R33阶段，我们建议进一步开发MISPA平台 自动化的研究平台，具有定量，健壮，高度可重复，高通量和 早期癌症筛查便宜。我们将建立用于稳定蛋白质，稳定蛋白质的SOP 库存储和最小的试剂批次变化。我们将通过 通过包括来自HPV不同亚型的抗原，将条形码蛋白库的大小提高到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，以支持大规模项目，需要分析成千上万的项目 样品。我们将确定空白的极限，检测极限，线性动态范围，精度和其他 定量评估的绩效指标。我们将介绍数百种与癌症相关的抗体 OPSCC和宫颈癌的患者以及1000多个无癌症对照和基准测试 具有当前黄金标准ELISA平台的性能。我们在发展创新的高级经验 使用实验室自动化和我们初步数据的质量的吞吐量免疫蛋白质组学平台 代表我们实施我们提出的发展的能力。定量可重复的测定法 测量数百种针对数千个个体中正确折叠蛋白的抗体 同样，将非常有益于癌症血清流行病学，风险评估和筛查。